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

Mortality in organic free-range chickens and molecular characterization of the involved pathogens

Longitudinal investigations on causes of mortality were carried out at one organic layer farm with four flocks of Lohman Brown and Lohman White chickens producing table eggs. All flocks were housed separately. One flock of each breed were followed from September 2001 to August 2002. Post mortem examinations were performed on a total of 16% of the dead chickens over the entire period. Of these 346 (96%) of the Lohmann Brown and 315 (91%) of the Lohmann White chickens were subjected for bacteriology. High mortality rates, 91% and 63% were observed in Lohman Brown and Lohman White chickens, respectively and were found to be due to infections with mainly Pasteurella multocida, Erysipelothix rhusiopathia and Escherichia coli. E. rhusiopathia, P. multocida and E. coli were isolated from 46%, 19% and 17%, respectively of the Lohmann Brown chickens. In the flock of Lohmann White chickens P. multocida and E. coli were isolated from 46% and 15%, respectively while E. rhusiopathia was not recorded. P. multocida and E. rhusiopathia isolates were characterized by Restriction Endonuclease Analysis (REA), Restriction Fragment Length Polymorphism (RFLP) and Pulse Field Gel Electrophoresis (PFGE). It was demonstrated that all the P. multocida isolates were genotypic identical over time. The E. rhusiopathia isolates obtained were also identical. It was concluded that the outbreaks caused by P. multocida and E. rhusiopathia were clonal and these two pathogens may cause severe losses in free-range chickens

Pharmacokinetic–pharmacodynamic integration and modelling of oxytetracycline for the calf pathogens Mannheimia haemolytica and Pasteurella multocida

A calf tissue cage model was used to study the pharmacokinetics (PK) and pharmacodynamics (PD) of oxytetracycline in serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids. After intramuscular administration, the PK was characterized by a long mean residence time of 28.3 hr. Based on minimum inhibitory concentrations (MICs) for six isolates each of Mannheimia haemolytica and Pasteurella multocida, measured in serum, integration of in vivo PK and in vitro PD data established area under serum concentration–time curve (AUC0–∞)/MIC ratios of 30.0 and 24.3 hr for M. haemolytica and P. multocida, respectively. Corresponding AUC0–∞/MIC ratios based on MICs in broth were 656 and 745 hr, respectively. PK-PD modelling of in vitro bacterial time–kill curves for oxytetracycline in serum established mean AUC0–24 hr/MIC ratios for 3log10 decrease in bacterial count of 27.5 hr (M. haemolytica) and 60.9 hr (P. multocida). Monte Carlo simulations predicted target attainment rate (TAR) dosages. Based on the potency of oxytetracycline in serum, the predicted 50% TAR single doses required to achieve a bacteriostatic action covering 48-hr periods were 197 mg/kg (M. haemolytica) and 314 mg/kg (P. multocida), respectively, against susceptible populations. Dosages based on the potency of oxytetracycline in broth were 25- and 27-fold lower (7.8 and 11.5 mg/kg) for M. haemolytica and P. multocida, respectively

Pharmacokinetic/pharmacodynamic integration and modelling of oxytetracycline for the porcine pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida

Pharmacokinetic–pharmacodynamic (PK/PD) integration and modelling were used to predict dosage schedules of oxytetracycline for two pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined in broth and porcine serum. PK/PD integration established ratios of average concentration over 48 h (Cav0–48 h)/MIC of 5.87 and 0.27 µg/mL (P. multocida) and 0.70 and 0.85 µg/mL (A. pleuropneumoniae) for broth and serum MICs, respectively. PK/PD modelling of in vitro time–kill curves established broth and serum breakpoint values for area under curve (AUC0–24 h)/MIC for three levels of inhibition of growth, bacteriostasis and 3 and 4 log10 reductions in bacterial count. Doses were then predicted for each pathogen, based on Monte Carlo simulations, for: (i) bacteriostatic and bactericidal levels of kill; (ii) 50% and 90% target attainment rates (TAR); and (iii) single dosing and daily dosing at steady-state. For 90% TAR, predicted daily doses at steady-state for bactericidal actions were 1123 mg/kg (P. multocida) and 43 mg/kg (A. pleuropneumoniae) based on serum MICs. Lower TARs were predicted from broth MIC data; corresponding dose estimates were 95 mg/kg (P. multocida) and 34 mg/kg (A. pleuropneumoniae)

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

Comparison of standardised versus non-standardised methods for testing the in vitro potency of oxytetracycline against mannheimia haemolytica and pasteurella multocida

The in vitro pharmacodynamics of oxytetracycline was established for six isolates of each of the calf pneumonia pathogens Mannheimia haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and bacterial time-kill curves were determined in two matrices, Mueller Hinton broth (MHB) and calf serum. Geometric mean MIC ratios, serum:MHB, were 25.2:1 (M. haemolytica) and 27.4:1 (P. multocida). The degree of binding of oxytetracycline to serum protein was 52.4%. Differences between serum and broth MICs could not be accounted for by oxytetracycline binding to serum protein. In vitro time-kill data suggested a co-dependent killing action of oxytetracycline. The in vitro data indicate inhibition of the killing action of oxytetracycline by serum factor(s). The nature of the inhibition requires further study. The outcome of treatment with oxytetracycline of respiratory tract infections in calves caused by M. haemolytica and P. multocida may not be related solely to a direct killing action

Efforts towards the development of recombinant Vaccines against Pasteurella multocida

Hemorrhagic septicemia is caused by gram-negative bacterium of Pasteurella multocida (P. multocida) strains. Most of the current vaccines against P. multocida have shortcomings. Presently, there is increasing efforts towards construction of recombinant clone for vaccine development against P.multocida. In this review an effort is made to look at some strong candidate genes, different protein bands from virulent strains and recent reported recombinant antigens. The possibility of developing a broad spectrum or cross protection vaccine for P. multocida is also discussed. It is hoped that withthe current development of three (3) completed genome sequence of P. multocida, stronger potential immunogens with broader spectrum will be identified. Furthermore whole genome sequence of other P. multocida strains will surely bridge the gap between diagnosis and vaccine development.Key words: Virulent, Vaccine, P. multocida, immunogens

Potency of marbofloxacin for pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida: Comparison of growth media

Pharmacodynamic properties of marbofloxacin were established for six isolates each of the pig respiratory tract pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Three in vitro indices of potency were determined; Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Mutant Prevention Concentration (MPC). For MIC determination Clinical Laboratory Standards Institute guidelines were modified in three respects: (1) comparison was made between two growth media, an artificial broth and pig serum; (2) a high inoculum count was used to simulate heavy clinical bacteriological loads; and (3) five overlapping sets of two-fold dilutions were used to improve accuracy of determinations. Similar methods were used for MBC and MPC estimations. MIC and MPC serum:broth ratios for A. pleuropneumoniae were 0.79:1 and 0.99:1, respectively, and corresponding values for P. multocida were 1.12:1 and 1.32:1. Serum protein binding of marbofloxacin was 49%, so that fraction unbound (fu) serum MIC values were significantly lower than those predicted by correction for protein binding; fu serum:broth MIC ratios were 0.40:1 (A. pleuropneumoniae) and 0.50:1 (P. multocida). For broth, MPC:MIC ratios were 13.7:1 (A. pleuropneumoniae) and 14.2:1 (P. multocida). Corresponding ratios for serum were similar, 17.2:1 and 18.8:1, respectively. It is suggested that, for dose prediction purposes, serum data might be preferable to potency indices measured in broths

The effect of concurrent infections with Pasteurella multocida and Ascaridia galli on free range chickens

Pasteurella multocida and Ascaridia galli are observed with high prevalences in free range chickens in Denmark, but the impact is unknown. A study was carried out to examine the interaction between A. galli and P. multocida in chickens and the impact on production. Five groups, each with 20 18-week-old Lohmann Brown chickens were infected. Group I was orally infected with 1000 +/- 50 embryonated A. galli eggs. Group 2 received 10(4) cfu p. multocida intratracheally. Group 3 was infected with A. galli and subsequently with P. multocida. Group 4 was infected with P. multocida followed by A. galli. Group 5 was the control. The study ran for I I weeks where clinical manifestations, weight gain and egg production were recorded. Excretion of P. multocida was determined on individual basis and blood smears were made for differential counts. At the end of the study pathological lesions and the number of adult worms, larvae and eggs in the faeces were recorded. The birds were more severely affected when infected with both pathogens compared to single infections with A. galli or P. multocida, respectively. A lower weight gain and egg production was observed with dual infections. A. galli infection followed by a secondary P. multocida infection resulted in more birds with pathological lesions and continued P. multocida excretion. In conclusion a negative interaction between A. galli and R multocida was observed and it is postulated that free range chickens are at higher risk of being subjected to outbreaks of fowl cholera when they are infected with A. galli

The isolation, serotyping and antimicrobial susceptibility of Pasteurella multocida strains in cattle in the region of Aydin and Izmir

Bu çalısmada, Pasteurella multocida izolasyonu amacıyla kullanılan toplam 570 adet sıgır intratrcheal svabının 350 adedi İzmir ilinde, 220 adedi ise Aydın ilinde bulunan mezbahalardan temin edildi. Arastırmada kullanılan 570 adet örnegin 28 (%4,9)'inden P. multocida izolasyon ve identifikasyonu yapıldı. İzmir ilinden alınan 350 örnekten 18 adet (% 5,14) ve Aydın ilinden alınan 220 örnekten 10 adet (% 4,54) P. multocida izolasyon ve identifikasyonu yapılmıstır. Yapılan çalısmada 28 adet saha susunun 15 (% 53,6) adedi tip B, 10 (% 35,7) adedi tip A ve 1 (% 3,5) adedi de tip D olarak tespit edildi. İzolatlardan 2 (% 7,2) adedi ise tiplendirilememistir. P. multocida suslarının % 93,0 flourphenicole'e, % 61,0 enrofloxacine'e, % 54,0 oxytetracycline'e duyarlı oldugu bulundu. P. multocida suslarının tümünün erytromycine ve sulphamethaxsazole ? trimethoprim'e % 82,0, gentamycine'e % 64,0 ve amoxycilline clavulanic acid'e ise % 61,0 oranlarında dirençli oldugu tespit edilmistir. In this study, a total of 570 intratracheal swabs were examined for the Pasteurella multocida isolation that were taken 350 of from zmir region slaughterhouse and were taken 220 of from Aydın region slaughterhouse. P. multocida was identified from 28 (4,9%) of 570 intratracheal swabs that were examined in this study. Pasteurella multocida was identified from 18 (5,14%) of 350 in zmir region and 10 (4,54) of 220 in Aydın region. In the study, a total of 28 field Pasteurella multocida strains were serotyped as ; 15 (53,6 %) type B, 10 (35,7%) type A and 1 (3,5%) type D, respectively. Of 2 Pasteurella multocida strains were untypeable. The Pasteurella multocida strains were found to be susceptible to Flourphenicol (93,0 %), Enrofloxacine (61,0 %), Oxytetracycline (54,0 %) and were found to be resistant to Erythromycine (82,0 %), Sulphamethaxsazole-Trimethoprim (82,0 %), Gentamycine (64,0 %) and Amoxycilline-Clavulanic acid (61,0 %)