Differentiation of Mannheimia haemolytica genotype 1 and 2 strains by visible phenotypic characteristics on solid media.

Genotype 2 Mannheimia haemolytica associate with the lungs of cattle with bovine respiratory disease more frequently than genotype 1 strains. Different colony colors and morphologies were identified between genotype 1 and 2 solid media cultures. Genotype of strains, and frequency differences between them in mixed cultures are discernable by visual inspection.

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.

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.

Pathogenic Mannheimia haemolytica Invades Differentiated Bovine Airway Epithelial Cells.

The Gram-negative bacterium Mannheimia haemolytica is the primary bacterial species associated with bovine respiratory disease (BRD) and is responsible for significant economic losses to livestock industries worldwide. Healthy cattle are frequently colonized by commensal serotype A2 strains, but disease is usually caused by pathogenic strains of serotype A1. For reasons that are poorly understood, a transition occurs within the respiratory tract and a sudden explosive proliferation of serotype A1 bacteria leads to the onset of pneumonic disease. Very little is known about the interactions of M. haemolytica with airway epithelial cells of the respiratory mucosa which might explain the different abilities of serotype A1 and A2 strains to cause disease. In the present study, host-pathogen interactions in the bovine respiratory tract were mimicked using a novel differentiated bovine bronchial epithelial cell (BBEC) infection model. In this model, differentiated BBECs were inoculated with serotype A1 or A2 strains of M. haemolytica and the course of infection followed over a 5-day period by microscopic assessment and measurement of key proinflammatory mediators. We have demonstrated that serotype A1, but not A2, M. haemolytica invades differentiated BBECs by transcytosis and subsequently undergoes rapid intracellular replication before spreading to adjacent cells and causing extensive cellular damage. Our findings suggest that the explosive proliferation of serotype A1 M. haemolytica that occurs within the bovine respiratory tract prior to the onset of pneumonic disease is potentially due to bacterial invasion of, and rapid proliferation within, the mucosal epithelium. The discovery of this previously unrecognized mechanism of pathogenesis is important because it will allow the serotype A1-specific virulence determinants responsible for invasion to be identified and thereby provide opportunities for the development of new strategies for combatting BRD aimed at preventing early colonization and infection of the bovine respiratory tract.

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.

Probiotic bacteria inhibit the bovine respiratory pathogen Mannheimia haemolytica serotype 1 in vitro.

This study evaluated the potential of probiotic bacteria to inhibit growth and cell adhesion of the bovine respiratory pathogen Mannheimia haemoltyica serotype 1. The inhibitory effects of nine probiotic strains (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Streptococcus thermophilus and two Paenibacillus polymyxa strains) against M. haemolytica were evaluated using a spot-on-lawn method. Probiotic strains were then tested for their adherence to bovine bronchial epithelial (BBE) cells and the ability to displace and compete against M. haemolytica on BBE. Except for S. thermophilus, all probiotic strains inhibited the growth of M. haemolytica, with zones of inhibition ranging between 12 and 19 mm. Lactobacillus strains and Lactococcus lactis displayed greater (P < 0·05) BBE adhesion compared with M. heamolytica (8·3%) and other probiotics (<2·2%). Strains of P. polymyxa and L. acidophilus caused the greatest reduction in M. haemolytica adherence, through both displacement and competition, compared with other probiotics. The results of this study suggest that probiotics may have the potential to colonize the bovine respiratory tract, and exert antagonistic effects against M. haemolytica serotype 1. SIGNIFICANCE AND IMPACT OF THE STUDY: A common method to control bovine respiratory disease (BRD) in feedlots is through mass medication with antibiotics upon cattle entry (i.e. metaphylaxis). Increasingly, antimicrobial resistance in BRD bacterial pathogens has been observed in feedlots, which may have important implications for cattle health. In this study, probiotic strains were shown to adhere to bovine respiratory cells and inhibit the BRD pathogen M. haemolytica serotype 1 through competition and displacement. Probiotics may therefore offer a mitigation strategy to reduce BRD bacterial pathogens, in place of metaphylactic antimicrobials.

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.

Comparison of Active Drug Concentrations in the Pulmonary Epithelial Lining Fluid and Interstitial Fluid of Calves Injected with Enrofloxacin, Florfenicol, Ceftiofur, or Tulathromycin.

Bacterial pneumonia is the most common reason for parenteral antimicrobial administration to beef cattle in the United States. Yet there is little information describing the antimicrobial concentrations at the site of action. The objective of this study was to compare the active drug concentrations in the pulmonary epithelial lining fluid and interstitial fluid of four antimicrobials commonly used in cattle. After injection, plasma, interstitial fluid, and pulmonary epithelial lining fluid concentrations and protein binding were measured to determine the plasma pharmacokinetics of each drug. A cross-over design with six calves per drug was used. Following sample collection and drug analysis, pharmacokinetic calculations were performed. For enrofloxacin and metabolite ciprofloxacin, the interstitial fluid concentration was 52% and 78% of the plasma concentration, while pulmonary fluid concentrations was 24% and 40% of the plasma concentration, respectively. The pulmonary concentrations (enrofloxacin + ciprofloxacin combined) exceeded the MIC90 of 0.06 µg/mL at 48 hours after administration. For florfenicol, the interstitial fluid concentration was almost 98% of the plasma concentration, and the pulmonary concentrations were over 200% of the plasma concentrations, exceeding the breakpoint (≤ 2 µg/mL), and the MIC90 for Mannheimia haemolytica (1.0 µg/mL) for the duration of the study. For ceftiofur, penetration to the interstitial fluid was only 5% of the plasma concentration. Pulmonary epithelial lining fluid concentration represented 40% of the plasma concentration. Airway concentrations exceeded the MIC breakpoint for susceptible respiratory pathogens (≤ 2 µg/mL) for a short time at 48 hours after administration. The plasma and interstitial fluid concentrations of tulathromcyin were lower than the concentrations in pulmonary fluid throughout the study. The bronchial concentrations were higher than the plasma or interstitial concentrations, with over 900% penetration to the airways. Despite high diffusion into the bronchi, the tulathromycin concentrations achieved were lower than the MIC of susceptible bacteria at most time points.

Growth of Mannheimia haemolytica: inhibitory agents and putative mechanism of inhibition.

Leukotoxin-producing Mannheimia haemolytica consistently causes fatal pneumonia in bighorn sheep (BHS) under experimental conditions. Surprisingly, by culture methods, it has been isolated from pneumonic BHS lungs less frequently than other bacteria. However, in one study PCR assays detected M. haemolytica from over 70% of the pneumonic lung samples that were negative for this organism by culture, suggesting that the growth of M. haemolytica is inhibited by other bacteria. Previously, we have shown that Bibersteinia trehalosi inhibits the growth of M. haemolytica. Herein we report that 100% of a diverse panel of B. trehalosi isolates (n=55) tested in a bacterial competition assay inhibited the growth of M. haemolytica, suggesting that the inhibitory phenotype is conserved. Further, no plasmids were isolated from any of the 30 B. trehalosi isolates tested, suggesting that the effectors are chromosomally encoded. An earlier study by us showed that Pasteurella multocida also inhibits the growth of M. haemolytica. However, M. haemolytica has not been isolated even from pneumonic BHS lungs that did not carry B. trehalosi or P. multocida. Consequently, we tested Staphylococcus spp., Streptococcus spp., and Escherichia coli, the bacteria that have been detected frequently in pneumonic BHS lungs, for possible inhibition of M. haemolytica. Neither the Staphylococcus spp. nor the Streptococcus sp. strains inhibited the growth of M. haemolytica. E. coli inhibited the growth of M. haemolytica by a proximity-dependent mechanism. Growth inhibition of M. haemolytica by several bacterial species is likely to contribute to the infrequent detection of this bacterium from pneumonic BHS lungs by culture.

Direct and indirect anti-inflammatory effects of tulathromycin in bovine macrophages: inhibition of CXCL-8 secretion, induction of apoptosis, and promotion of efferocytosis.

Recent evidence indicates that immunomodulation by antibiotics may enhance their clinical efficacy. Specifically, drug-induced leukocyte apoptosis and macrophage efferocytosis have been shown to promote the resolution of inflammation in a variety of disease settings. Tulathromycin is a new macrolide antibiotic for the treatment of bovine respiratory disease. The direct antimicrobial effects of the drug alone do not fully justify its superior clinical efficacy, and we hypothesize that tulathromycin may have immunomodulating properties. We recently reported that tulathromycin promotes apoptosis and inhibits proinflammatory NF-κB signaling in bovine neutrophils. In this study, we investigated the direct and indirect anti-inflammatory effects of tulathromycin in bovine macrophages. The findings indicate that bovine monocyte-derived macrophages and alveolar macrophages readily phagocytose tulathromycin-induced apoptotic neutrophils both in vitro and in the airways of Mannheimia haemolytica-infected calves. Moreover, tulathromycin promotes delayed, concentration-dependent apoptosis, but not necrosis, in bovine macrophages in vitro. Activation of caspase-3 and detection of mono- and oligonucleosomes in bovine monocyte-derived macrophages treated with tulathromycin was observed 12 h posttreatment; pretreatment with a pan-caspase inhibitor (ZVAD) blocked the proapoptotic effects of the drug. Lastly, tulathromycin inhibited the secretion of proinflammatory CXCL-8 in lipopolysaccharide (LPS)-stimulated bovine macrophages; this effect was independent of caspase activation or programmed cell death. Taken together, these immunomodulating effects observed in bovine macrophages help further elucidate the mechanisms through which tulathromycin confers anti-inflammatory and proresolution benefits. Furthermore, these findings offer novel insights on how antibiotics may offer anti-inflammatory benefits by modulating macrophage-mediated events that play a key role in inflammation.

Proximity-dependent inhibition of growth of Mannheimia haemolytica by Pasteurella multocida.

Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 µm) but not when they were separated by a membrane that limited contact (pore size, 0.4 µm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures of P. multocida or from P. multocida-M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS.

Localization and functional characterization of pulmonary bovine odorant-binding protein.

Bovine odorant-binding protein (OBP) may function in olfaction and defense against oxidative injury, but its role in inflammation and defense against bacterial infection has not been investigated. Expression of OBP was discovered in the bovine lung and found to undergo changes in abundance during glucocorticoid administration and stress. OBP was localized to nasal, tracheal, and bronchial mucosal glands with immunohistochemistry, with faint expression in airway surface epithelium and none in bronchioles or alveoli. Two isoforms of OBP were identified, appearing to be differentially regulated during lipopolysaccharide-induced pulmonary inflammation, but differences between these isoforms were not revealed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Functional studies showed no effect of OBP on in vitro growth of Escherichia coli or Mannheimia haemolytica under iron-replete or iron-depleted conditions, nor did OBP opsonize bacteria for an enhanced neutrophil oxidative burst. However, OBP did reduce the ability of supernatants from lipopolysaccharide-stimulated macrophages to induce neutrophil chemotaxis. These findings indicate that OBP may inhibit neutrophil recruitment by inflammatory mediators, and they suggest an ability to bind macrophage-derived inflammatory mediators within the airways.

Pharmacokinetic and pharmacodynamic modelling of marbofloxacin administered alone and in combination with tolfenamic acid in goats.

In a four-period cross-over study, the fluoroquinolone antibacterial drug marbofloxacin (MB) was administered to goats intramuscularly (IM) at a dose rate of 2 mg/kg, both alone and in combination with the non-steroidal anti-inflammatory drug tolfenamic acid (TA), also administered IM at a dose rate of 2 mg/kg. Using a tissue cage model of inflammation, based on the irritant actions of carrageenan, the pharmacokinetics (PK) of MB and MB in combination with TA were determined. MB mean values of area under concentration-time curve (AUC) were similar for serum (5.60 microg h/mL), inflamed tissue cage fluid (exudate; 5.32 microg h/mL) and non-inflamed tissue cage fluid (transudate; 4.82 microg h/mL). Values of mean residence time (MRT) of MB in exudate (15.5 h) and transudate (15.8 h) differed significantly from serum MRT (4.23 h). Co-administration of TA did not affect the PK profile of MB. The pharmacodynamics of MB were investigated using a caprine strain of Mannheimia haemolytica. Integration of PK data with ex vivo bacterial time-kill curve data for serum, exudate and transudate provided AUC(24h)/minimum inhibitory concentration (MIC) ratios of 160, 133 and 121 h, respectively, for the strain of organism used. Modelling of the ex vivo time-kill data to the sigmoid E(max) equation provided AUC(24h)/MIC values required for bacteriostatic and bactericidal actions of MB and for virtual eradication of the organism of 27.6, 96.2 and 147.3 h, respectively. Corresponding values for MB+TA were 20.5, 66.5 and 103.0 h. These data were used to predict once daily dosage schedules of MB for subsequent clinical evaluation.

Bibersteinia trehalosi inhibits the growth of Mannheimia haemolytica by a proximity-dependent mechanism.

Mannheimia (Pasteurella) haemolytica is the only pathogen that consistently causes severe bronchopneumonia and rapid death of bighorn sheep (BHS; Ovis canadensis) under experimental conditions. Paradoxically, Bibersteinia (Pasteurella) trehalosi and Pasteurella multocida have been isolated from BHS pneumonic lungs much more frequently than M. haemolytica. These observations suggest that there may be an interaction between these bacteria, and we hypothesized that B. trehalosi overgrows or otherwise inhibits the growth of M. haemolytica. Growth curves (monoculture) demonstrated that B. trehalosi has a shorter doubling time ( approximately 10 min versus approximately 27 min) and consistently achieves 3-log higher cell density (CFU/ml) compared to M. haemolytica. During coculture M. haemolytica growth was inhibited when B. trehalosi entered stationary phase (6 h) resulting in a final cell density for M. haemolytica that was 6 to 9 logs lower than expected with growth in the absence of B. trehalosi. Coculture supernatant failed to inhibit M. haemolytica growth on agar or in broth, indicating no obvious involvement of lytic phages, bacteriocins, or quorum-sensing systems. This observation was confirmed by limited growth inhibition of M. haemolytica when both pathogens were cultured in the same media but separated by a filter (0.4-microm pore size) that limited contact between the two bacterial populations. There was significant growth inhibition of M. haemolytica when the populations were separated by membranes with a pore size of 8 mum that allowed free contact. These observations demonstrate that B. trehalosi can both outgrow and inhibit M. haemolytica growth with the latter related to a proximity- or contact-dependent mechanism.

Kinetics of growth and leukotoxin production by Mannheimia haemolytica in continuous culture.

The growth and product formation kinetics of the bovine pathogen Mannheimia (Pasteurella) haemolytica strain OVI-1 in continuous culture were investigated. The leukotoxin (LKT) concentration and yield on biomass could substantially be enhanced by supplementation of a carbon-limited medium with an amino acid mixture or a mixture of cysteine and glutamine. Acetic acid was a major product, increasing to 1.66 g l(-1) in carbon-limited chemostat culture at intermediate dilution rates and accounting for more than 80% of the glucose carbon, whereas in amino acid-limited cultures high acetic acid concentrations were produced at low dilution rates, suggesting a carbon-overflow metabolism. The maintenance coefficients of carbon-limited and carbon-sufficient cultures were 0.07 and 0.88 mmol glucose g(-1) h(-1), respectively. LKT production was partially growth-associated and the LKT concentration was maximised to 0.15 g l(-1) and acetic acid production minimised by using a carbon-limited medium and a low dilution rate.

Serotypes A1 and A2 of Mannheimia haemolytica are susceptible to genotypic, capsular and phenotypic variations in contrast to T3 and T4 serotypes of Bibersteinia (Pasteurella) trehalosi.

Mannheimia haemolytica and Bibersteinia (Pasteurella) trehalosi are the most common bacterial isolates that cause pulmonary diseases in ruminants worldwide. The disease is determined by specific serotypes found in cattle and small ruminants. The molecular epidemiology of strains involved in disease is important in the control of outbreaks as well as in the preparation of vaccines. This study aimed to detect the instability and variations of bacterial strains that may affect the analysis of epidemic strains, or the stability of vaccinal strains. Eight strains of M. haemolytica belonging to serotypes A1 and A2 and three B. trehalosi strains of the T3 and T4 serotypes were used. Strains were subjected to pulsed field gel electrophoresis (PFGE) and capsular and phenotypic typing at each round of a total of 50 successive subcultures. Remarkable stability was found in all selected strains of B. trehalosi in contrast to M. haemoltyica, in which strains of both serotypes showed pattern variations produced by PFGE and capsular and phenotypic analysis. Objective criteria for M. haemolytica and B. trehalosi typing are consequently addressed.

Use of computed tomography to evaluate pathologic changes in the lungs of calves with experimentally induced respiratory tract disease.

OBJECTIVE: To optimize methods for the use of computed tomography (CT) to assess pathologic changes in the lungs of calves and to determine the effect of treatment on lung consolidation. ANIMALS: 10 male Holstein calves. PROCEDURES: Calves were anesthetized to facilitate CT imaging of the thorax. After initial images were obtained, pneumonia was induced in the calves by inoculation through a bronchoscope. Two calves were used in a preliminary study to refine the inoculation dose and optimize CT images. Four calves were administered florfenicol and 4 calves were untreated control animals. Serial images were obtained 24, 48, and 72 hours after inoculation. After final images were obtained, calves were euthanized, and lung consolidation was estimated by use of lung surface area scoring and water displacement. These estimates were compared with estimated lung consolidation obtained by use of CT. RESULTS: Calves had rapid disease progression. Percentage of lung consolidation was not significantly different between treatment groups for any of the estimation methods. Results of an ANOVA of the 3 assessment methods indicated significant differences among methods. Estimates of the percentage of lung consolidation obtained by use of surface area scoring and CT correlated well, whereas water displacement estimates correlated poorly with other methods of consolidation estimation. CONCLUSIONS AND CLINICAL RELEVANCE: Because of the correlation with other methods for estimation of lung consolidation, CT has the potential to be used to monitor disease progression in calves with experimentally induced respiratory tract disease.

Effect of pH, temperature and nutrient limitations on growth and leukotoxin production by Mannheimia haemolytica in batch and continuous culture.

AIMS: Quantification of the effects of pH, temperature and nutrient limitations on the growth and leukotoxin (LKT) production parameters of Mannheimia haemolytica in batch and chemostat culture. METHODS AND RESULTS: Mannheimia haemolytica strains OVI-1 and PH12296 were grown aerobically in two semi-defined media. In amino acid-limited cultures, the LKT concentration and yield in terms of biomass (Y(LKT/x)) were up to eightfold greater than in carbon-limited cultures. Supplementing amino acid-limited chemostat cultures with cysteine, glutamine, ferric iron and manganese further enhanced the Y(LKT/x) values up to threefold. Supplementation of an amino acid-limited batch culture of M. haemolytica strain OVI-1 with these nutrients resulted in an LKT concentration of 1.77 g l(-1) that was 45-fold greater than that obtained in RPMI 1640 medium. Aerobiosis enhanced LKT production. High acetic acid concentrations were produced under carbon-sufficient conditions. The highest maximum specific growth rates were recorded in the range of pH 6.8 to 7.8 and 37 to 40 degrees C. CONCLUSIONS: An amino acid-limited culture medium greatly improved LKT production in aerobic batch culture, which could be further enhanced by supplementation with cysteine, glutamine, ferric iron and manganese. SIGNIFICANCE AND IMPACT OF THE STUDY: It was demonstrated that LKT production by M. haemolytica could be dramatically increased through manipulation of the culture medium composition, which could benefit the production of LKT-based vaccines against bovine shipping fever pneumonia.

The response of Mannheimia haemolytica to iron limitation: implications for the acquisition of iron in the bovine lung.

Mannheimia haemolytica is the major causative agent of shipping fever, a severe pneumonia in cattle causing high morbidity and mortality. A prerequisite of successful lung colonization by M. haemolytica is the necessity to adapt to the paucity of iron. The lack of genome information has precluded an assessment of the genetic repertoire available to M. haemolytica to adapt to low iron environments. To close this knowledge-gap, we have determined 90% of a virulent M. haemolytica serotype A1 genome sequence and produced a microarray in order to study gene expression under iron-limiting growth for 15, 30 and 60 min. M. haemolytica responded to iron limitation by the up-regulation of transcripts coding for receptors and ABC-type transporters of transferrin, haemoglobin, haem and siderophores. Real time PCR analysis of lung tissue from Mannheimia-infected calves demonstrated the in vivo transcription of two potential haemoglobin receptors, hmbR1 and hmbR2. The relative hmbR1 and hmbR2 transcript levels in the infected lung tissue were comparable to the induced levels observed under iron-limiting growth, demonstrating in vivo induction of receptor transcription in the context of an infection. When the iron response of M. haemolytica was compared to the iron response of Pasteurella multocida, another pathogen colonizing the bovine lung, only few homologous genes were induced in both organisms. These included the haemoglobin receptor hmbR2 and the periplasmic transport systems yfeABCD and fbpABC. The comparative analysis suggests that the two pathogens use different strategies to adapt to the iron-limiting environment in the bovine host.

Antimicrobial susceptibility patterns of respiratory tract pathogens from sheep and goats.

OBJECTIVE: To determine the antimicrobial susceptibility of common respiratory tract pathogens from sheep and goats. DESIGN: Cross-sectional study. SAMPLE POPULATION: 41 respiratory tract isolates from sheep and 36 isolates from goats. PROCEDURES: Disk diffusion assay was used to determine antimicrobial susceptibility of isolates to amoxicillin-clavulanic acid, ceftiofur, ciprofloxacin, florfenicol, and tetracycline. Minimum inhibitory concentrations of florfenicol for these isolates were determined by use of the microbroth dilution technique. RESULTS: The most common isolates were Pasteurella multocida (n = 28) and Mannheimia haemolytica (39). All isolates were susceptible to amoxicillin-clavulanic acid, ceftiofur, ciprofloxacin, and florfenicol. Five percent (4/77) of isolates were resistant to tetracycline. CONCLUSIONS AND CLINICAL RELEVANCE: Susceptibility of respiratory tract pathogens isolated from sheep and goats to commonly used antimicrobial drugs in this study was high. Treatment of these species for bacterial respiratory tract disease is likely not complicated by antimicrobial resistance.