Anti-Inflammatory Properties of Fructo-Oligosaccharides in a Calf Lung Infection Model and in Mannheimia haemolytica-Infected Airway Epithelial Cells.

Emerging antimicrobial-resistant pathogens highlight the importance of developing novel interventions. Here, we investigated the anti-inflammatory properties of Fructo-oligosaccharides (FOS) in calf lung infections and in airway epithelial cells stimulated with pathogens, and/or bacterial components. During a natural exposure, 100 male calves were fed milk replacer with or without FOS for 8 weeks. Then, immune parameters and cytokine/chemokine levels in the bronchoalveolar lavage fluid (BALF) and blood were measured, and clinical scores were investigated. Calf primary bronchial epithelial cells (PBECs) and human airway epithelial cells (A549) were treated with Mannheimia haemolytica, lipopolysaccharides (LPS), and/or flagellin, with or without FOS pretreatment. Thereafter, the cytokine/chemokine levels and epithelial barrier function were examined. Relative to the control (naturally occurring lung infections), FOS-fed calves had greater macrophage numbers in BALF and lower interleukin (IL)-8, IL-6, and IL-1ß concentrations in the BALF and blood. However, FOS did not affect the clinical scores. At slaughter, FOS-fed calves had a lower severity of lung lesions compared to the control. Ex vivo, FOS prevented M. haemolytica-induced epithelial barrier dysfunction. Moreover, FOS reduced M. haemolytica- and flagellin-induced (but not LPS-induced) IL-8, TNF-α, and IL-6 release in PBECs and A549 cells. Overall, FOS had anti-inflammatory properties during the natural incidence of lung infections but had no effects on clinical symptoms.

Pentraxin 3 expression in lungs and neutrophils of calves.

Bacterial lung disease caused by Mannheimia haemolytica inflict significant mortality and morbidity resulting in enormous economic losses to cattle industry. The use of antibiotics is becoming more challenging because of development of anti-microbial resistance. The innate immune system plays a critical role in the initiation of immune response in the lung. Pentraxin 3 (PTX3), a pattern-recognition receptor is produced at sites of inflammation by many cell types, recognizes and binds to many pathogens, activates the complement cascade, and has a role in the clearance of apoptotic and necrotic cells. Because there are very few data on the expression of PTX3 in the lungs, we examined PTX3 expression in lungs of normal and M. haemolytica-infected calves and normal and E. coli lipopolysaccharide-treated cattle neutrophils using light and electron microscopic immunochemistry and Western blots. Immunohistology showed the presence of PTX3 in airway epithelial cells, alveolar septa and macrophages in normal and inflamed lungs of calves and the blots showed a significant increase in the expression of PTX3 in lungs from infected calves. Immuno-gold electron microscopy showed PTX3 in the nuclei, cytoplasm, and vesicular organelles of alveolar macrophages, endothelial cells and pulmonary intravascular macrophages (PIMs). Immunohistochemical staining for PTX3 in peripheral blood neutrophils shows an altered staining pattern in neutrophils stimulated with lipopolysachharide (LPS). However, western blots no significant change in PTX3 amount in LPS-treated neutrophils compared to the controls. These are the first data on the expression of PTX3 in the lungs and the neutrophils of cattle which may add to our understanding of innate immunity in cattle lungs.

Development of Bacterial Therapeutics against the Bovine Respiratory Pathogen Mannheimia haemolytica.

Bovine respiratory disease (BRD) is a major cause of morbidity and mortality in beef cattle. Recent evidence suggests that commensal bacteria of the bovine nasopharynx have an important role in maintaining respiratory health by providing colonization resistance against pathogens. The objective of this study was to screen and select bacterial therapeutic candidates from the nasopharynxes of feedlot cattle to mitigate the BRD pathogen Mannheimia haemolytica In a stepwise approach, bacteria (n = 300) isolated from the nasopharynxes of 100 healthy feedlot cattle were identified and initially screened (n = 178 isolates from 12 different genera) for growth inhibition of M. haemolytica Subsequently, selected isolates were evaluated for the ability to adhere to bovine turbinate (BT) cells (n = 47), compete against M. haemolytica for BT cell adherence (n = 15), and modulate gene expression in BT cells (n = 10). Lactobacillus strains had the strongest inhibition of M. haemolytica, with 88% of the isolates (n =33) having inhibition zones ranging from 17 to 23 mm. Adherence to BT cells ranged from 3.4 to 8.0 log10 CFU per 105 BT cells. All the isolates tested in competition assays reduced M. haemolytica adherence to BT cells (32% to 78%). Among 84 bovine genes evaluated, selected isolates upregulated expression of interleukin 8 (IL-8) and IL-6 (P < 0.05). After ranking isolates for greatest inhibition, adhesion, competition, and immunomodulation properties, 6 Lactobacillus strains from 4 different species were selected as the best candidates for further development as intranasal bacterial therapeutics to mitigate M. haemolytica infection in feedlot cattle.IMPORTANCE Bovine respiratory disease (BRD) is a significant animal health issue impacting the beef industry. Current BRD prevention strategies rely mainly on metaphylactic use of antimicrobials when cattle enter feedlots. However, a recent increase in BRD-associated bacterial pathogens that are resistant to metaphylactic antimicrobials highlights a pressing need for the development of novel mitigation strategies. Based upon previous research showing the importance of respiratory commensal bacteria in protecting against bronchopneumonia, this study aimed to develop bacterial therapeutics that could be used to mitigate the BRD pathogen Mannheimia haemolytica Bacteria isolated from the respiratory tracts of healthy cattle were characterized for their inhibitory, adhesive, and immunomodulatory properties. In total, 6 strains were identified as having the best properties for use as intranasal therapeutics to inhibit M. haemolytica If successful in vivo, these strains offer an alternative to metaphylactic antimicrobial use in feedlot cattle for mitigating BRD.

Nanopore ultra-long read sequencing technology for antimicrobial resistance detection in Mannheimia haemolytica.

Disruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible strain of Mannheimia haemolytica, isolated from pneumonic bovine lung samples, were sequenced at 146× and 111× coverage, respectively with Oxford Nanopore Technologies MinION. De novo assembly produced a complete genome for the non-resistant strain and a nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance Database) and ResFinder databases identified genes conferring resistance to different classes of antibiotics including ß-lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, sulfonamides and macrolides. Resistance phenotypes of the M. haemolytica strains were determined by minimum inhibitory concentration (MIC) of the antibiotics. Sequencing with a highly portable MinION device corresponded to MIC assays with most of the antimicrobial resistant determinants being identified with as few as 5437 reads, except for the genes responsible for resistance to Fluoroquinolones. The resulting quality assemblies and AMR gene annotation highlight the efficiency of ultra-long read, whole-genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.

Behavioral changes in group-housed dairy calves infected with Mannheimia haemolytica.

Monitoring sickness behavior may improve identification, management, and welfare of sick animals. The objective of this study was to characterize components of sickness behavior in group-housed dairy calves, using an experimental disease challenge model with Mannheimia haemolytica (MH). Holstein bull calves (aged 3-7 weeks; 58.0 ± 12.0 kg of body weight) were group-housed based on age and body weight in sand-bedded pens (6 calves/pen, 6.6 m2/calf) and provided pasteurized waste milk (8 L/d) 2×/d and grain concentrate ad libitum. Within group, calves were randomly assigned to 1 of 2 treatments: (1) inoculation at the tracheal bifurcation with 3 × 109 cfu of MH suspended in 5 mL of sterile phosphate buffered saline (PBS) followed by a 120 mL wash PBS (MH; n = 12, 3/pen), or (2) inoculation with 5 mL + 120 mL of sterile PBS only (control; n = 12, 3/pen). Rectal temperature and health scores were collected from d 0 to +6 of the challenge. A range of behaviors, including feeding patterns and social interactions, were recorded from video from d 0 to +2. The challenge model resulted in calves experiencing a mild disease state: rectal temperatures of MH calves were elevated throughout the challenge compared with control calves, peaking at 12 h postinoculation (39.2 vs. 38.9°C; standard error = 0.14). Many behavioral responses were subject to treatment by day effects, with calves generally becoming less active following inoculation with MH and then returning to baseline. Affected behaviors surrounding feeding included milk feeding time, frequency of competitive displacements, and concentrate feeding time. Lying time was similarly subject to treatment by day effects, and MH calves also spent more time lying on their left side compared with their right (604 vs. 471 min/h; standard error = 32), whereas control calves expressed no laterality. Duration of social lying did not differ, but frequency of social lying bouts decreased in MH calves following inoculation (0.44 vs. 0.75 bouts/h; standard error = 0.04). Social grooming was initiated less by MH calves (0.78 vs. 1.96 min/h; standard error = 0.38), but they tended to receive more social grooming for a greater duration of time (1.59 vs. 1.25 min/h; standard error = 0.13). Overall, we found that infected calves exhibited reduced grooming, feeding, and social interactions, suggesting that these behavioral changes may be useful indicators of early stages of respiratory disease.

Genomic signatures of Mannheimia haemolytica that associate with the lungs of cattle with respiratory disease, an integrative conjugative element, and antibiotic resistance genes.

BACKGROUND: Mannheimia haemolytica typically resides in cattle as a commensal member of the upper respiratory tract microbiome. However, some strains can invade their lungs and cause respiratory disease and death, including those with multi-drug resistance. A nucleotide polymorphism typing system was developed for M. haemolytica from the genome sequences of 1133 North American isolates, and used to identify genetic differences between isolates from the lungs and upper respiratory tract of cattle with and without clinical signs of respiratory disease. RESULTS: A total of 26,081 nucleotide polymorphisms were characterized after quality control filtering of 48,403 putative polymorphisms. Phylogenetic analyses of nucleotide polymorphism genotypes split M. haemolytica into two major genotypes (1 and 2) that each were further divided into multiple subtypes. Multiple polymorphisms were identified with alleles that tagged genotypes 1 or 2, and their respective subtypes. Only genotype 2 M. haemolytica associated with the lungs of diseased cattle and the sequence of a particular integrative and conjugative element (ICE). Additionally, isolates belonging to one subtype of genotype 2 (2b), had the majority of antibiotic resistance genes detected in this study, which were assorted into seven combinations that ranged from 1 to 12 resistance genes. CONCLUSIONS: Typing of diverse M. haemolytica by nucleotide polymorphism genotypes successfully identified associations with diseased cattle lungs, ICE sequence, and antibiotic resistance genes. Management of cattle by their carriage of M. haemolytica could be an effective intervention strategy to reduce the prevalence of respiratory disease and supplemental needs for antibiotic treatments in North American herds.

The use of MALDI-TOF mass spectrometry for rapid identification of Mannheimia haemolytica.

Mannheimia haemolytica is the most important bacterial pathogen isolated from cases of Bovine Respiratory Disease (BRD). Routine identification of these bacteria is usually performed using phenotypic methods. Our study showed that MALDI-TOF MS is a reliable alternative to these methods. All of the strains analyzed were identified as M. haemolytica. The identification results were compared to those obtained using conventional methods commonly used in microbiological diagnostics, based on detection and analysis of biochemical properties of microorganisms. The degree of agreement between the two methods for identifying M. haemolytica was 100%.

Comparison of Mannheimia haemolytica isolates from an outbreak of bovine respiratory disease.

The objective of this study was to determine the clonal relatedness of Mannheimia haemolytica isolates responsible for an outbreak of bovine respiratory disease in a commercial feedlot. The isolates were obtained from the lungs of 21 calves with fatal pneumonia that were part of a group of 206 total calves. All isolates were serotyped and analyzed by pulsed-field gel electrophoresis (PFGE) and for antibiotic sensitivity patterns. ELISA and immunoblotting assays were performed to compare serum antibody levels to M. haemolytica antigens in calves with fatal pneumonia to those calves that survived the outbreak. Isolates were categorized into 14 different PFGE groups based on 90% similarity. Two Group D isolates (1 and 6), and 3 Group H isolates (14, 15, and 16) were characterized as 100% similar. Antimicrobial susceptibility profiles defined 8 groups based on differences in patterns of resistance between isolates. The two 100% similar isolates from PFGE Group D were both in susceptibility Group 1. All but isolate 14 from PFGE Group H (3, 15, 16, and 19) were in susceptibility Group 4a. Serum antibody levels to M. haemolytica antigens in the dead calves were not different than the antibody levels in the 185 calves that survived the outbreak. Immunoblots of selected isolates from each of the PFGE groups demonstrated only minimal differences in antigenic profiles between strains when reacted with serum from calves that either died from or survived the outbreak. Based on the characteristics of these isolates, multiple strains of M. haemolytica were responsible for fatal pneumonia during this outbreak.

Bactericidal effects of various concentrations of enrofloxacin, florfenicol, tilmicosin phosphate, and tulathromycin on clinical isolates of Mannheimia haemolytica.

OBJECTIVE: To determine bactericidal effects of enrofloxacin, florfenicol, tilmicosin, and tulathromycin on clinical isolates of Mannheimia haemolytica at various bacterial densities and drug concentrations. SAMPLE: 4 unique isolates of M haemolytica recovered from clinically infected cattle. PROCEDURES: Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined for each drug and isolate. Mannheimia haemolytica suspensions (10(6) to 10(9) CFUs/mL) were exposed to the determined MIC and MPC and preestablished maximum serum and tissue concentrations of each drug. Log10 reduction in viable cells (percentage of cells killed) was measured at various points. RESULTS: Bacterial killing at the MIC was slow and incomplete. After 2 hours of isolate exposure to the MPC and maximum serum and tissue concentrations of the tested drugs, 91% to almost 100% cell killing was achieved with enrofloxacin, compared with 8% growth to 93% cell killing with florfenicol, 199% growth to 63% cell killing with tilmicosin, and 128% growth to 43% cell killing with tulathromycin over the range of inoculum tested. For all drugs, killing of viable organisms was evident at all bacterial densities tested; however, killing was more substantial at the MPC and maximum serum and tissue drug concentrations than at the MIC and increased with duration of drug exposure. Rank order of drugs by killing potency was enrofloxacin, florfenicol, tilmicosin, and tulathromycin. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that antimicrobial doses that equaled or exceeded the MPC provided rapid killing of M haemolytica by the tested drugs, decreasing opportunities for antimicrobial-resistant subpopulations of bacteria to develop during drug exposure.

Immunohistochemical expression of nuclear factor erythroid-2-related factor 2 and heme oxygenase 1 in normal bovine lung and bovine lung infected with Mannheimia haemolytica.

Mannheimia haemolytica is an important cause of pneumonia in feedlot cattle. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor responsible for the induction of antioxidant enzymes, such as heme oxygenase 1 (HO-1), within the lung. The expression of Nrf2 and HO-1 was immunohistochemically evaluated in 4 calves 24 h after experimental infection with M. haemolytica. Calves receiving normal saline served as controls. In the infected lungs, cytoplasmic Nrf2 expression was high in macrophages and bronchioles and low in alveolar epithelium, whereas nuclear expression was high in endothelial cells, macrophages, and bronchioles and lowest in alveolar epithelium. Normal lung samples displayed only faint Nrf2 cytoplasmic staining within bronchiolar epithelium. Expression of HO-1 was detected within the cytoplasm of macrophages and bronchiolar epithelial cells in all infected lung samples, whereas normal lungs displayed only weak cytoplasmic staining in bronchiolar epithelial cells. These findings suggest that bronchiolar epithelial cells and macrophages up-regulate Nrf2 expression early in the course of infection, which results in increased expression of HO-1 within these cells.

Mannheimia haemolytica est une cause importante de pneumonie chez les bovins en parc d'engraissement. Le facteur érythroïde-2 nucléaire apparenté au facteur 2 (Nrf2) est un facteur transcriptionnel sensible au potentiel redox responsable de l'induction d'enzymes antioxidants, tel que l'hème oxygénase 1 (HO-1), dans le poumon. L'expression de Nrf2 et HO-1 fut évaluée par épreuve immunohistochimique chez quatre veaux 24 h après une infection expérimentale avec M. haemolytica. Les veaux témoins ont reçu de la saline. Dans les poumons infectés, l'expression cytoplasmique de Nrf2 était élevée dans les macrophages et les bronchioles et faible dans l'épithélium alvéolaire, alors que l'expression nucléaire était élevée dans les cellules endothéliales, macrophages et bronchioles, et à son plus faible dans l'épithélium alvéolaire. Les échantillons de poumons normaux montraient seulement une faible coloration cytoplasmique pour Nrf2 dans l'épithélium des bronchioles. L'expression de HO-1 fut détectée dans le cytoplasme des macrophages et des cellules épithéliales des bronchioles de tous les échantillons de poumons infectés, alors que les échantillons de poumons normaux ne montraient qu'une faible coloration cytoplasmique dans les cellules épithéliales des bronchioles. Ces données suggèrent que les cellules épithéliales des bronchioles et les macrophages régulent à la hausse l'expression de Nrf2 tôt lors de l'infection, ce qui résulte en une expression augmentée d'HO-1 à l'intérieur de ces cellules.(Traduit par Docteur Serge Messier).

Pharmacokinetic/pharmacodynamic integration and modelling of amoxicillin for the calf pathogens Mannheimia haemolytica and Pasteurella multocida.

The antimicrobial properties of amoxicillin were determined for the bovine respiratory tract pathogens, Mannheima haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves were established. Pharmacokinetic (PK)/pharmacodynamic (PD) modelling of the time-kill data, based on the sigmoidal Emax equation, generated parameters for three levels of efficacy, namely bacteriostatic, bactericidal (3log10 reduction) and 4log10 reduction in bacterial counts. For these levels, mean AUC(0-24 h) /MIC serum values for M. haemolytica were 29.1, 57.3 and 71.5 h, respectively, and corresponding values for P. multocida were 28.1, 44.9 and 59.5 h. Amoxicillin PK was determined in calf serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids, after intramuscular administration of a depot formulation at a dosage of 15 mg/kg. Mean residence times were 16.5 (serum), 29.6 (exudate) and 29.0 h (transudate). Based on serum MICs, integration of in vivo PK and in vitro PD data established maximum concentration (Cmax )/MIC ratios of 13.9:1 and 25.2:1, area under concentration-time curve (AUC0-∞ )/MIC ratios of 179 and 325 h and T>MIC of 40.3 and 57.6 h for P. multocida and M. haemolytica, respectively. Monte Carlo simulations for a 90% target attainment rate predicted single dose to achieve bacteriostatic and bactericidal actions over 48 h of 17.7 and 28.3 mg/kg (M. haemolytica) and 17.7 and 34.9 mg/kg (P. multocida).

Serum concentrations of haptoglobin and haptoglobin-matrix metalloproteinase 9 (Hp-MMP 9) complexes of bovine calves in a bacterial respiratory challenge model.

BACKGROUND: Serum haptoglobin (Hp) and haptoglobin matrix metalloproteinase 9 complexes (Hp-MMP 9) have been identified as biomarkers with diagnostic potential in cattle with conditions resulting in an acute inflammatory response. The purpose of this study was to evaluate potential diagnostic applications of serum Hp and Hp-MMP 9 concentrations in calves with BRD and establish a timeline for their detection in calves experimentally challenged with Bibersteinia trehalosi and Mannheimia haemolytica. Thirty-five cross bred dairy calves were inoculated via tracheal catheterization with either a PCR confirmed leukotoxin negative B. trehalosi isolate, a PCR confirmed leukotoxin positive B. trehalosi isolate, a Mannheimia haemolytica isolate, a combination of leukotoxin negative B. trehalosi and M. haemolytica, or a negative control. Serum samples were collected throughout the study. Calves were euthanized and necropsy performed on day 10 post inoculation. RESULTS: M. haemolytica inoculated calves had increased lung involvement. Serum Hp and Hp- MMP 9 concentrations were elevated compared to the other treatment groups. Increases in serum Hp and Hp-MMP 9 concentrations for the M. haemolytica group were significantly different from other study groups on day 7 of the study. B. trehalosi inoculated calves did not have increased lung involvement compared to control calves, but the leukotoxin positive B. trehalosi group demonstrated increased serum Hp-MMP 9 concentrations from day 3 to the end of the study compared to the pre-inoculation concentrations. CONCLUSION: Serum Hp-MMP 9 concentration is a useful diagnostic tool for detecting early pulmonary inflammation in calves challenged with B. trehalosi and M. haemolytica. Serum Hp-MMP 9 may also be a useful tool in detecting subclinical pulmonary inflammation in challenged calves.

Increased annexin A1 and A2 levels in bronchoalveolar lavage fluid are associated with resistance to respiratory disease in beef calves.

Strategies to control bovine respiratory disease depend on accurate classification of disease risk. An objective method to refine the risk classification of beef calves could be economically beneficial, improve welfare by preventing unexpected disease occurrences, refine and reduce the use of antibiotics in beef production, and facilitate alternative methods of disease control. The objective of this study was to identify proteins in bronchoalveolar lavage fluid (BALF) of stressed healthy calves that predict later disease outcome, serve as biomarkers of susceptibility to pneumonia, and play a role in pathogenesis. BALF was collected from 162 healthy beef calves 1-2 days after weaning and transportation. Difference in gel electrophoresis (DIGE) and mass spectrometry were used to compare proteins in samples from 7 calves that later developed respiratory disease compared to 7 calves that remained healthy. Calves that later developed pneumonia had significantly lower levels of annexin A1, annexin A2, peroxiredoxin I, calcyphosin, superoxide dismutase, macrophage capping protein and dihydrodiol dehydrogenase 3. Differences in annexin levels were partially confirmed by western blot analysis. Thus, lower levels of annexins A1 and A2 are potential biomarkers of increased susceptibility to pneumonia in recently weaned and transported feedlot cattle. Since annexins are regulated by glucocorticoids, this finding may reflect individual differences in the stress response that predispose to pneumonia. These findings also have implications in pathogenesis. Annexins A1 and A2 are known to prevent neutrophil influx and fibrin deposition respectively, and may thus act to minimize the harmful effects of the inflammatory response during development of pneumonia.

Pharmacodynamics of marbofloxacin for calf pneumonia pathogens.

The pharmacodynamic (PD) properties of the fluoroquinolone, marbofloxacin, were determined for the bovine respiratory tract pathogens Mannheima haemolytica and Pasteurella multocida. For six pathogenic isolates of each organism, three in vitro indices of efficacy and potency were determined, namely, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill curves. Each parameter was determined in two matrices, Mueller Hinton Broth (MHB) and calf serum. For serum, MBC:MIC ratios were 2.7:1 (M. haemolytica) and 2.4:1 (P. multocida). The killing action of marbofloxacin had the characteristics of concentration dependency against M. haemolytica and co-dependency (on time and concentration) against P. multocida. To confirm the characteristics of the time-kill profiles, growth inhibition produced by marbofloxacin was also established ex vivo in three biological fluids, calf serum, exudate and transudate, harvested from a tissue cage model. The in vitro time-kill data were modelled with pharmacokinetic properties of marbofloxacin, established by intramuscular administration in calves at a dose of 2 mg/kg; three levels of activity, namely bacteriostatic, 3 log10 reduction and 4 log10 reduction in bacterial counts were determined. Mean AUC(24h)/MIC values (with percentage coefficients of variation indicating inter-isolate variability) for M. haemolytica, based on serum MICs, were 31.3 (41.6), 57.7 (42.4) and 79.2 (44.6) h, respectively. Corresponding values for MHB were 20.5 (58.0), 40.5 (51.8) and 51.2 (24.30) h, respectively. When allowance was made for binding of marbofloxacin to serum protein, the AUC(24h)/MIC values for serum were similar to those for MHB. Numerical AUC(24h)/MIC values for P. multocida were slightly lower than those obtained for M. haemolytica. These data establish for the first time inter-isolate variability in AUC(24h)/MIC values required for three levels of bacterial kill for two pathogenic species and thereby provide an indication of variability in serum concentration that might be required to achieve efficacy in clinical subjects.

Evaluation of protein expression in bovine bronchoalveolar fluid following challenge with Mannheimia haemolytica.

Proteomics analysis of bovine bronchoalveolar fluid (BAF) following induction of pneumonia with Mannheimia haemolytica using nanoflow liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) resulted in the identification of 88 unique proteins. Proteins detected in BAF included antimicrobial peptides (AMPs), complement factors, acute-phase proteins, protease inhibitors, and proteins involved in oxidation-reduction. Notwithstanding biological variation, differences in relative protein abundance, determined using normalized peptide counts, were detected for select proteins in BAF from genuinely infected versus sham-infected animals. To demonstrate the applicability of using normalized peptide counts to assess protein expression trends, LC-MS/MS data for the acute-phase protein haptoglobin (HPT) were compared with ELISA data, and statistical evaluation of the relationship between the data revealed a strong measure of association. Differences were detected between sham- and genuinely infected animals for haptoglobin, as well as the AMPs cathelicidin-1 and cathelicidin-4, and inter-α-trypsin inhibitor heavy chain-4, a fairly novel protein involved in the acute phase response. Though the small sample size limited the scope of the inferences, the results indicate the likely importance of AMPs and acute-phase proteins during respiratory infection, and provide additional information regarding potential mechanisms involved in the bovine mucosal barrier defense.

Demonstration of the metaphylactic use of gamithromycin against bacterial pathogens associated with bovine respiratory disease in a multicentre farm trial.

On five commercial cattle rearing sites across Europe, a total of 802 young cattle at high risk of developing bovine respiratory disease (BRD) associated with the bacterial pathogens Mannheimia haemolytica or Pasteurella multocida and/or Mycoplasma bovis were enrolled into a multicentre, controlled field trial. Half were treated with a single dose of gamithromycin at 6 mg/kg bodyweight by subcutaneous injection and half received an injection of a saline placebo as the control. All animals were observed daily for 14 days for signs of BRD as defined by set criteria. The proportion of metaphylactic preventive treatment successes, defined as animals surviving to day 14 without signs of BRD, in the gamithromycin-treated group (86 per cent) was significantly (P=0.0012) higher than in the saline-treated controls (61 per cent). Morbidity among the treated animals was reduced by 64 per cent compared with the controls.

Determination of the duration of antibacterial efficacy following administration of gamithromycin using a bovine Mannheimia haemolytica challenge model.

The antibacterial efficacy of gamithromycin administered once 1, 5, or 10 days prior to a challenge infection with Mannheimia haemolytica serotype A1 was evaluated. Forty calves were randomly allocated on day -11, restricted by body weight, to one of three treatment groups given gamithromycin at 6 mg/kg of body weight 10, 5, or 1 days before challenge or to an untreated control group. M. haemolytica A1 challenge infections were induced on day 0 by depositing 7.4 × 10(7) CFU at the bifurcation of the main bronchus using a bronchoscope. Clinical observations were made daily from the day of allocation to day 10, when necropsy was scheduled; three calves died or were euthanized in extremis on welfare grounds prior to scheduled necropsy. At necropsy the lungs were removed, pneumonic lesions were scored, and samples of lung tissue were cultured for M. haemolytica. The three groups of animals treated with gamithromycin before challenge had significantly lower lung M. haemolytica counts and fewer clinical signs of respiratory disease than did the saline-treated group. For most of the clinical parameters, the pattern of responses differed significantly (P < 0.05) between the gamithromycin-treated groups and the control group. There were no statistically significant differences between groups in the mean lung lesion scores, partly as a result of high individual variability, particularly within the control group. The administration of gamithromycin 1, 5, and 10 days prior to M. haemolytica A1 challenge resulted in a reduction in bacterial isolation from the lungs and a reduction in the severity of clinical disease.

Bacterial pathogens of the bovine respiratory disease complex.

Pneumonia caused by the bacterial pathogens discussed in this article is the most significant cause of morbidity and mortality of the BRDC. Most of these infectious bacteria are not capable of inducing significant disease without the presence of other predisposing environmental factors, physiologic stressors, or concurrent infections. Mannheimia haemolytica is the most common and serious of these bacterial agents and is therefore also the most highly characterized. There are other important bacterial pathogens of BRD, such as Pasteurella multocida, Histophulus somni, and Mycoplasma bovis. Mixed infections with these organisms do occur. These pathogens have unique and common virulence factors but the resulting pneumonic lesions may be similar. Although the amount and quality of research associated with BRD has increased, vaccination and therapeutic practices are not fully successful. A greater understanding of the virulence mechanisms of the infecting bacteria and pathogenesis of pneumonia, as well as the characteristics of the organisms that allow tissue persistence, may lead to improved management, therapeutics, and vaccines.

Comparison of repetitive PCR and pulsed-field gel electrophoresis for the genotyping of Mannheimia haemolytica.

Mannheimia haemolytica is an opportunistic pathogen that can cause fibrinonecrotic pneumonia in cattle and is the main bacterial agent implicated in bovine respiratory disease-complex (BRD). Despite its economic importance to the cattle industry, few studies have characterized the genetic nature of M. haemolytica and none have genotyped isolates from feedlots. Identifying and monitoring genetic variants of M. haemolytica is important to understanding the etiology of BRD in cattle. We investigated the capacity of three genotyping techniques (BOX-PCR, (GTG)(5)-PCR and PFGE analysis of SalI-restricted DNA) to discriminate among 24 reference strains from the family Pasteurellaceae and 40 M. haemolytica isolates collected from feedlot cattle. From cluster analysis of the M. haemolytica isolates, PFGE was revealed as most discriminating, followed by BOX-PCR and then (GTG)(5)-PCR (Simpson's diversity index >0.98, 0.82, and 0.72, respectively). Of these methods, PFGE also had the greatest mean repeatability (0.96). The PFGE and BOX-PCR assays grouped all M. haemolytica in a single cluster but only BOX-PCR and (GTG)(5)-PCR grouped the Mannheimia glucosida and Mannheimia ruminalis strains together. Refinement of genotyping procedures for M. haemolytica could offer new insight into the etiology of this pathogen in BRD.

Expression and activity of N-myristoyltransferase in lung inflammation of cattle and its role in neutrophil apoptosis.

N-myristoyltransferase (NMT) attaches a 14 carbon fatty acid, myristic acid, to the N-terminal glycine residue of proteins. NMT exists in two isoforms NMT1 and NMT2. Myristoylated proteins play critical roles in protein-protein interactions, cell signaling and oncogenesis. Although elevated expression of NMT1 has been described in colorectal carcinoma, its expression and roles in normal and inflamed lungs of the cattle are unknown. Therefore, we investigated the expression and activity of NMT in a bovine model of lung inflammation induced with Mannheimia hemolytica and in vitro in neutrophils and macrophages. Western blots revealed increased expression of NMT1 in lungs from infected animals compared to control animals. Total NMT activity was reduced in inflamed lungs compared to control animals (p < 0.05) along with increased expression of enolase, a putative inhibitor of NMT. NMT1 staining was observed in the septum, vascular endothelium and the epithelium in the lungs from control as well as infected calves. NMT1 expression was intense in neutrophils in the necrotic areas in the inflamed lungs. Immuno-electron microscopy localized NMT1 in cytoplasm and nuclei of endothelium, pulmonary intravascular macrophages and airway epithelium. Total NMT activity and NMT1 expression were increased in neutrophils and macrophages exposed to Escherichia coli LPS in vitro. NMT knockdown increased apoptosis in activated neutrophils. This is the first report demonstrating expression of NMT in normal and inflamed lungs and a novel role for NMT in regulation of neutrophil lifespan.