Interaction study of Pasteurella multocida with culturable aerobic bacteria isolated from porcine respiratory tracts using coculture in conditioned media.

BACKGROUND: The porcine respiratory tract harbours multiple microorganisms, and the interactions between these organisms could be associated with animal health status. Pasteurella multocida is a culturable facultative anaerobic bacterium isolated from healthy and diseased porcine respiratory tracts. The interaction between P. multocida and other aerobic commensal bacteria in the porcine respiratory tract is not well understood. This study aimed to determine the interactions between porcine P. multocida capsular serotype A and D strains and other culturable aerobic bacteria isolated from porcine respiratory tracts using a coculture assay in conditioned media followed by calculation of the growth rates and interaction parameters. RESULTS: One hundred and sixteen bacterial samples were isolated from five porcine respiratory tracts, and 93 isolates were identified and phylogenetically classified into fourteen genera based on 16S rRNA sequences. Thirteen isolates from Gram-negative bacterial genera and two isolates from the Gram-positive bacterial genus were selected for coculture with P. multocida. From 17 × 17 (289) interaction pairs, the majority of 220 pairs had negative interactions indicating competition for nutrients and space, while 17 pairs were identified as mild cooperative or positive interactions indicating their coexistence. All conditioned media, except those of Acinetobacter, could inhibit P. multocida growth. Conversely, the conditioned media of P. multocida also inhibited the growth of nine isolates plus themselves. CONCLUSION: Negative interaction was the major interactions among the coculture of these 15 representative isolates and the coculture with P. multocida. The conditioned media in this study might be further analysed to identify critical molecules and examined by the in vivo experiments. The study proposed the possibility of using these molecules in conditioned media to control P. multocida growth.

Role of aspartate ammonia-lyase in Pasteurella multocida.

BACKGROUND: Pasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. However, the pathogenesis of this disease is poorly understood. We recently identified an aspartate ammonia-lyase (aspA) in P. multocida that was significantly upregulated under iron-restricted conditions, the protein of which could effectively protect chicken flocks against P. multocida. However, the functions of this gene remain unclear. In the present study, we constructed aspA mutant strain △aspA::kan and complementary strain C△aspA::kan to investigate the function of aspA in detail. RESULT: Deletion of the aspA gene in P. multocida resulted in a significant reduction in bacterial growth in LB (Luria-Bertani) and MH (Mueller-Hinton) media, which was rescued by supplementation with 20 mM fumarate. The mutant strain △aspA::kan showed significantly growth defects in anaerobic conditions and acid medium, compared with the wild-type strain. Moreover, growth of △aspA::kan was more seriously impaired than that of the wild-type strain under iron-restricted conditions, and this growth recovered after supplementation with iron ions. AspA transcription was negatively regulated by iron conditions, as demonstrated by quantitative reverse transcription-polymerase chain reaction. Although competitive index assay showed the wild-type strain outcompetes the aspA mutant strain and △aspA::kan was significantly more efficient at producing biofilms than the wild-type strain, there was no significant difference in virulence between the mutant and the wild-type strains. CONCLUSION: These results demonstrate that aspA is required for bacterial growth in complex medium, and under anaerobic, acid, and iron-limited conditions.

Insights into Pasteurellaceae carriage dynamics in the nasal passages of healthy beef calves.

We investigated three bovine respiratory pathobionts in healthy cattle using qPCR optimised and validated to quantify Histophilus somni, Mannheimia haemolytica and Pasteurella multocida over a wide dynamic range. A longitudinal study was conducted to investigate the carriage and density of these bacteria in the nasal passages of healthy beef calves (N = 60) housed over winter in an experimental farm setting. The three pathobiont species exhibited remarkably different carriage rates and density profiles. At housing, high carriage rates were observed for P. multocida (95%), and H. somni (75%), while fewer calves were positive for M. haemolytica (13%). Carriage rates for all three bacterial species declined over the 75-day study, but not all individuals became colonised despite sharing of environment and airspace. Colonisation patterns ranged from continuous to intermittent and were different among pathobiont species. Interval-censored exponential survival models estimated the median duration of H. somni and P. multocida carriage at 14.8 (CI95%: 10.6-20.9) and 55.5 (CI95%: 43.3-71.3) days respectively, and found higher density P. multocida carriage was associated with slower clearance (p = 0.036). This work offers insights into the dynamics of pathobiont carriage and provides a potential platform for further data collection and modelling studies.

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.

Rapid detection of tetracycline resistance in bovine Pasteurella multocida isolates by MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA).

Pasteurella multocida is notorious for its role as an opportunistic pathogen in infectious bronchopneumonia, the economically most important disease facing cattle industry and leading indication for antimicrobial therapy. To rationalize antimicrobial use, avoiding imprudent use of highly and critically important antimicrobials for human medicine, availability of a rapid antimicrobial susceptibility test is crucial. The objective of the present study was to design a MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) procedure for tetracycline resistance detection in P. multocida. This procedure was validated on 100 clinical isolates with MIC-gradient strip test, and a comparison with disk diffusion was made. Sensitivity and specificity of the MBT-ASTRA procedure were 95.7% (95% confidence interval (CI) = 89.8-101.5) and 100% (95% CI = 100-100), respectively, classifying 98% of the isolates correctly after only three hours of incubation. Sensitivity and specificity of disk diffusion were 93.5% (95% CI = 86.3-100.6) and 96.3% (95% CI = 91.3-101.3) respectively, classifying 95% of the isolates correctly. In conclusion, this MBT-ASTRA procedure has all the potential to fulfil the need for a rapid and highly accurate tetracycline susceptibility testing in P. multocida to rationalize antimicrobial use in outbreaks of bronchopneumonia in cattle or other clinical presentations across species.

Synthesis and antibacterial activities of novel pleuromutilin derivatives.

Pleuromutilin derivatives 4a-h, 5a-g, and 6a-d were synthesized and characterized by IR, 1 H NMR, and 13 C NMR. All synthetic compounds were screened for their in vitro antibacterial activity against Staphylococcus aureus (ATCC 25923), methicillin-resistant S. aureus (MRSA, ATCC 43300), Pasteurella multocida (CVCC 408), Escherichia coli (ATCC 25922), and Salmonella typhimurium (ATCC 14028). Most compounds with quaternary amine showed higher antibacterial activities against both Gram-positive and Gram-negative bacteria strains. Among the screened compounds, compound 5a bearing an N,N,N-trimethyl group at the C-14 side chain of pleuromutilin was found to be the most active agent. Furthermore, preliminary molecular docking was performed to predict the binding interaction of the compounds in the binding pocket.

Mystery solved for mass mortality event.

It's been almost three years since over 200,000 saiga antelopes died in central Kazakhstan, in a mass mortality event that shocked researchers and wildlife experts alike. But now, as Georgina Mills explains, research has revealed that there was more to the deaths than first thought.

A vapour phase assay for evaluating the antimicrobial activities of essential oils against bovine respiratory bacterial pathogens.

The objectives of this study were to develop a new assay for the evaluation of the antimicrobial activities of essential oils (EOs) in vapour phase and to demonstrate the antimicrobial activities of commercial EOs against BRPs. To achieve the first objective, a microtube cap containing 100 µl of EO was embedded in an agar plate. An agar plug (diameter 13 mm) inoculated with a bacterial suspension containing108  CFU per ml was then placed over the cap and incubated at 37°C for 24 h. Subsequently, bacteria were recovered from the agar plug by immersion in 5 ml of broth for 10 min, followed by vortexing for 30 s, and the broths were then plated for enumeration. To demonstrate the usefulness of the assay, nine commercial EOs derived from the following specific plants: ajowan, carrot seed, cinnamon leaf, citronella, fennel, ginger grass, lavender, rosemary and thyme were first evaluated for their vapour phase antimicrobial activities against Mannheimia haemolytica serotype 1. Selected EOs were further tested against Pasteurella multocida and Histophilus somni. The EOs of ajowan, thyme and cinnamon leaf completely or partially inhibited BRPs growth. This new assay provided reproducible results on the vapour phase antimicrobial activities of EOs against BRPs. These results support further study of EOs as a potential mitigation strategy against BRPs. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we present a new vapour phase assay for evaluating the antimicrobial activities of essential oils (EO) against bovine respiratory pathogens (BRPs). Using this assay, we identified EOs, such as ajowan, thyme and cinnamon leaf, that can effectively inhibit growth of the BRPs Mannheimia haemolytica serotype 1, Pasteurella multocida and Histophilus somni. This is the first study to demonstrate the vapour phase antimicrobial activity of EOs against BRPs.

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).

A large potentiation effect of serum on the in vitro potency of tulathromycin against Mannheimia haemolytica and Pasteurella multocida.

The antimicrobial properties of tulathromycin were investigated for M. haemolytica and P. multocida. Three in vitro indices of antimicrobial activity, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves, were established for six isolates of each organism. Each index was measured in two growth media: Mueller-Hinton broth (MHB) and calf serum. It was shown that MICs and MBCs were markedly lower in serum than in MHB. MHB:serum ratios for MIC were 47:1 (M. haemolytica) and 53:1 (P. multocida). For both serum and MHB, adjustment of pH led to greater potency at alkaline compared to acid pH. Tulathromycin MIC was influenced by size of inoculum count, being 4.0- to 7.7-fold greater for high compared to low initial counts. It was concluded that for the purpose of determining dosages for therapeutic use, pharmacodynamic data for tulathromycin should be derived in biological fluids such as serum. It is hypothesized that in vitro measurement of MIC in broth, conducted according to internationally recommended standards, may be misleading as a basis for estimating the in vivo potency of tulathromycin.

Synthesis and characterization of chitosan/curcumin blends based polyurethanes.

In this work, new hexamethylene diisocyanate (HMDI) and hyroxylterminated polybutadiene (HTPB) based polyurethanes (PUs) were prepared following step growth polymerization by the introduction of varying mole ratio of chitosan (CH) and curcumin (CUR). Structural study of blends through infrared spectroscopy confirmed the incorporation of CH and CUR into the backbone of the PU. The scanning electron microscopic (SEM) study confirmed the well dispersion of incorporated chitosan/curcumin and homogeneity of surface of synthesized samples. Thermogravimetric analysis (TGA) of PU blends indicated a better thermal stability with 0.25M:0.75M of chitosan to curcumin. Mechanical properties such as modulus and tensile strength of PU blends were found to be better with higher contents of chitosan and curcumin. The same extender composition (1mol BDO, 075mol chitosan and 0.25mol curcumin) based PU showed higher substantial of antimicrobial activity as compared to the all other PUs. On the whole, this work is actually a step towards the generation of novel biocompatible materials preferably useful for biomedical applications.

Differential Analysis of the Nasal Microbiome of Pig Carriers or Non-Carriers of Staphylococcus aureus.

Staphylococcus aureus is presently regarded as an emerging zoonotic agent due to the spread of specific methicillin-resistant S. aureus (MRSA) clones in pig farms. Studying the microbiota can be useful for the identification of bacteria that antagonize such opportunistic veterinary and zoonotic pathogen in animal carriers. The aim of this study was to determine whether the nasal microbiome of pig S. aureus carriers differs from that of non-carriers. The V3-V5 region of the 16S rRNA gene was sequenced from nasal swabs of 44 S. aureus carriers and 56 non-carriers using the 454 GS FLX titanium system. Carriers and non-carriers were selected on the basis of quantitative longitudinal data on S. aureus carriage in 600 pigs sampled at 20 Danish herds included in two previous studies in Denmark. Raw sequences were analysed with the BION meta package and the resulting abundance matrix was analysed using the DESeq2 package in R to identify operational taxonomic units (OTUs) with differential abundance between S. aureus carriers and non-carriers. Twenty OTUs were significantly associated to non-carriers, including species with known probiotic potential and antimicrobial effect such as lactic acid-producing isolates described among Leuconostoc spp. and some members of the Lachnospiraceae family, which is known for butyrate production. Further 5 OTUs were significantly associated to carriage, including known pathogenic bacteria such as Pasteurella multocida and Klebsiella spp. Our results show that the nasal microbiome of pigs that are not colonized with S. aureus harbours several species/taxa that are significantly less abundant in pig carriers, suggesting that the nasal microbiota may play a role in the individual predisposition to S. aureus nasal carriage in pigs. Further research is warranted to isolate these bacteria and assess their possible antagonistic effect on S. aureus for the pursuit of new strategies to control MRSA in pig farming.

Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation.

BACKGROUND: Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae. RESULTS: For the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure. CONCLUSIONS: In conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by cross-feeding, which enables A. pleuropneumoniae to grow and form multi-species biofilms.

Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity.

BACKGROUND: Pasteurella multocida (P. multocida) is an important veterinary pathogen that can cause severe diseases in a wide range of mammals and birds. The global regulator crp gene has been found to regulate the virulence of some bacteria, and crp mutants have been demonstrated to be effective attenuated vaccines against Salmonella enterica and Yersinia enterocolitica. Here, we first characterized the crp gene in P. multocida, and we report the effects of a crp deletion. RESULTS: The P. multocida crp mutant exhibited a similar lipopolysaccharide and outer membrane protein profile but displayed defective growth and serum complement resistance in vitro compared with the parent strain. Furthermore, crp deletion decreased virulence but did not result in full attenuation. The 50 % lethal dose (LD50) of the Δcrp mutant was 85-fold higher than that of the parent strain for intranasal infection. Transcriptome sequencing analysis showed that 92 genes were up-regulated and 94 genes were down-regulated in the absence of the crp gene. Finally, we found that intranasal immunization with the Δcrp mutant triggered both systematic and mucosal antibody responses and conferred 60 % protection against virulent P. multocida challenge in ducks. CONCLUSION: The deletion of the crp gene has an inhibitory effect on bacterial growth and bacterial resistance to serum complement in vitro. The P. multocida crp mutant was attenuated and conferred moderate protection in ducks. This work affords a platform for analyzing the function of crp and aiding the formulation of a novel vaccine against P. multocida.

One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae.

Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%-4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.

Fatal multifocal Pasteurella multocida infection: a case report.

BACKGROUND: In humans, Pasteurella multocida infections are usually limited to the soft tissues surrounding a lesion. However, P. multocida can also cause systemic infections (such as pneumonia, lung abscess, peritonitis, endocarditis, meningitis and sepsis)-especially in patients with other underlying medical conditions. CASE PRESENTATION: We report on a case of fulminant P. multocida bacteremia at several sites (soft tissues, endocarditis and joints) on a white European man. Despite surgery and intensive medical care, the patient died. CONCLUSIONS: The present case emphasizes the importance of appropriate initial treatment of skin wounds. Patients at risk should be aware of the possible consequences of being bitten, scratched or licked by their pet.

Effects of dietary L-glutamine supplementation on specific and general defense responses in mice immunized with inactivated Pasteurella multocida vaccine.

Little is known about effects of dietary glutamine supplementation on specific and general defense responses in a vaccine-immunized animal model. Thus, this study determined roles for dietary glutamine supplementation in specific and general defense responses in mice immunized with inactivated Pasteurella multocida vaccine. The measured variables included: (1) the production of pathogen-specific antibodies; (2) mRNA levels for pro-inflammatory cytokines, toll-like receptors and anti-oxidative factors; and (3) the distribution of P. multocida in tissues and the expression of its major virulence factors in vivo. Dietary supplementation with 0.5 % glutamine had a better protective role than 1 or 2 % glutamine against P. multocida infection in vaccine-immunized mice, at least partly resulting from its effects in modulation of general defense responses. Dietary glutamine supplementation had little effects on the production of P. multocida-specific antibodies. Compared to the non-supplemented group, dietary supplementation with 0.5 % glutamine had no effect on bacterial burden in vivo but decreased the expression of major virulence factors in the spleen. Collectively, supplementing 0.5 % glutamine to a conventional diet provides benefits in vaccine-immunized mice by enhancing general defense responses and decreasing expression of specific virulence factors.

Inhibition of growth and alteration of host cell interactions of Pasteurella multocida with natural byproducts.

Pasteurella multocida is a leading cause of fowl cholera in both free-range pasture and conventional/commercially raised poultry. Its infection is a serious threat to poultry health and overall flock viability. Organic poultry is comparatively more vulnerable to this pathogen. It is a significant cause of production loss and price increase of poultry products, specifically organic poultry products. Some plant products are well documented as sources of natural antimicrobials such as polyphenols found in different berry pomaces and citrus oil. Pomace, a byproduct (primarily of seeds and skins) of fruits used for juice and wine production, and citrus oil, the byproduct of citrus juice production, show promising antimicrobial activity against various pathogens. Here, we showed for the first time that blackberry and blueberry pomace extracts and citrus oil inhibited P. multocida growth. Minimum bactericidal concentrations were determined as 0.3 and 0.4 mg/mL gallic acid equivalent for blackberry and blueberry pomace extracts, respectively. Similarly, only 0.05% citrus oil (vol/vol) completely inhibited P. multocida growth. Under shaking conditions, the antimicrobial activity of both pomace extracts and citrus oil was more intensive. Even citrus oil vapor also significantly reduced the growth of P. multocida. In addition, cell surface hydrophobicity of P. multocida was increased by 2- to 3-fold and its adherence to chicken fibroblast (DF1) and bovine mammary gland (MacT) cells was reduced significantly in the presence of pomace extracts only. This study indicates that these natural products might be good alternatives to conventional antimicrobial agents, and hence, may be used as feed or water supplements to control fowl cholera and reduce production loss caused by P. multocida.

Multiple-class antimicrobial resistance surveillance in swine Escherichia coli F4, Pasteurella multocida and Streptococcus suis isolates from Ontario and the impact of the 2004-2006 Porcine Circovirus type-2 Associated Disease outbreak.

The objective of this work was to describe trends in multiple-class antimicrobial resistance present in clinical isolates of Escherichia coli F4, Pasteurella multocida and Streptococcus suis from Ontario swine 1998-2010. Temporal changes in multiple-class resistance varied by the pathogens examined; significant yearly changes were apparent for the E. coli and P. multocida data. Although not present in the E. coli data, significant increases in multiple-class resistance within P. multocida isolates occurred from 2003 to 2005, coinciding with the expected increase in antimicrobials used to treat clinical signs of Porcine Circovirus Associated Disease (PCVAD) before it was confirmed. Prospective temporal scan statistics for multiple-class resistance suggest that significant clusters of increased resistance may have been found in the spring of 2004; months before the identification of the PCVAD outbreak in the fall of 2004.

Mouse model of haemorrhagic septicaemia: dissemination and multiplication of Pasteurella multocida B:2 in vital organs after intranasal and subcutaneous challenge in mice.

Haemorrhagic septicaemia (HS) is an endemic disease of bovines, occurring in most tropical regions of Asia and Africa. In the present study, the suitability of using mice to study pathogenesis of HS was assessed using mortality, mean death time and bacterial multiplication in vital organs after infection with live P multocida. Mice were infected with 10(5), 10(3) and 10(1)cfu of P. multocida B:2 via intranasal and subcutaneous routes along with control groups. Bacterial multiplication in lung, liver and spleen of mice were determined at 24 h interval after intranasal and subcutaneous challenge. More than 80 % of challenged mice died within 48 h of inoculation, irrespective of the dose and route of inoculation. A heavy bacterial load (up to 10(8)cfu) was observed in lung, liver and spleen of mice titrated at 24 h and following death of mice. Results of the present study indicate that even ten bacteria are enough to cause mortality in mice and the organism multiplies rapidly in respiratory epithelium and disseminated to other vital organs viz liver and spleen suggesting the important role of mouse model in investigating the pathogenesis and challenge studies during vaccine development.