Identification of a reliable fixative solution to preserve the complex architecture of bacterial biofilms for scanning electron microscopy evaluation.

Bacterial biofilms are organized sessile communities of bacteria enclosed in extracellular polymeric substances (EPS). To analyze organization of bacteria and EPS in high resolution and high magnification by scanning electron microscopy (SEM), it is important to preserve the complex architecture of biofilms. Therefore, fixation abilities of formalin, glutaraldehyde, and Methacarn (methanol/chloroform/acetic acid-6:3:1) fixatives were evaluated to identify which fixative would best preserve the complex structure of bacterial biofilms. Economically important Gram-negative Mannheimia haemolytica, the major pathogen associated with bovine respiratory disease complex, and Gram-positive Staphylococcus aureus, the major cause of chronic mastitis in cattle, bacteria were selected since both form biofilms on solid-liquid interface. For SEM analysis, round glass coverslips were placed into the wells of 24-well plates and diluted M. haemolytica or S. aureus cultures were added, and incubated at 37°C for 48-72 h under static growth conditions. Culture media were aspirated and biofilms were fixed with an individual fixative for 48 h. SEM examination revealed that all three fixatives were effective preserving the bacterial cell morphology, however only Methacarn fixative could consistently preserve the complex structure of biofilms. EPS layers were clearly visible on the top, in the middle, and in the bottom of the biofilms with Methacarn fixative. Biomass and three-dimensional structure of the biofilms were further confirmed spectrophotometrically following crystal violet staining and by confocal microscopy after viability staining. These findings demonstrate that Methacarn fixative solution is superior to the other fixatives evaluated to preserve the complex architecture of biofilms grown on glass coverslips for SEM evaluation.

Role of the stress-associated chemicals norepinephrine, epinephrine and substance P in dispersal of Mannheimia haemolytica from biofilms.

Bovine respiratory disease (BRD) is a major problem for the cattle industry that is triggered by various environmental stressors, pathogens and host responses. Mannheimia hemolytica, an important bacterial component of BRD, are present within the nasopharayngeal region of normal calves as commensal biofilm communities. However, following stress there are changes in the nasopharyngeal microenvironment that triggers the transition of the commensal M. haemolytica into a pulmonary pathogen. The factors responsible for this transition in- vivo are unknown. In this study we developed an in-vitro biofilm model and investigated the effect of three stress- related compounds: norepinephrine (NE), epinephrine (E), and substance P (SP) on M. haemolytica biofilms. Biofilm formation was demonstrated for 3 bovine nasal isolates of M. haemolytica by growing them in basal culture media, basal media with additional glucose, and basal media with a reduced pH. Increased glucose enhanced biofilm biomass for 2/3 isolates, but acidic media did not increase biofilm biomass when compared to biofilm biomass in basal media. When the biofilm was exposed to NE, E and SP, there was a dispersal of the biofilm which was most effective with E, followed by NE, and SP being the least effective. Using high - throughput scanning electron microscopy and confocal-imaging we confirmed our experimental data that treatment with NE, E and SP cause dispersion of M.haemolytica from biofilms.

Pasteurella multocida- and Pasteurella haemolytica-ghosts: new vaccine candidates.

Pasteurella multocida is an important animal pathogen. Bacterial ghosts produced by the expression of phage PhiX174 lysis gene E are empty cells devoid of cytoplasmic and genomic material. Lysis of P. multocida 7A and P. haemolytica A1 carrying Pasteurella-specific lysis vectors (pSR2 and pSON2) occurred 140 min after induction of gene E expression induced by temperature upshift. The E-mediated cell lysis and killing activity was the same in both Pasteurella species and no viable cells could be detected after lysis of P. multocida and P. haemolytica. Pasteurella ghosts were used for immunization of rabbits and mice. Rabbits immunized subcutaneously with either P. multocida- or P. haemolytica-ghosts developed antibodies reacting with the immunizating strain, as well as with other Pasteurella strains. The number of proteins in whole cell protein extracts recognized by the sera constantly increased during the observation period of 51 days. In addition, dose-dependent protection against homologous challenge was observed in mice immunized with P. multocida-ghosts. Animals which received 1.15 x 10(8) ghosts and a challenge dose of up to 60 cfu (LD90), showed 100% protection. According to these results, we suggest ghosts of P. multocida and P. haemolytica as new vaccine candidates.

Development of an ex vivo model to study adherence of Mannheimia haemolytica serovar 1 to mucosal tissues of the respiratory tract of cattle.

OBJECTIVE: To develop and validate an ex vivo model for study of adherence of Mannheimia haemolytica (formerly Pasteurella haemolytica) to respiratory tract mucosa of cattle and to use this model to confirm adherence of M haemolytica serovar 1 (Mh1) to several relevant respiratory mucosal surfaces. SAMPLE POPULATION: Excised nasal, nasopharyngeal, turbinate, and tonsillar mucosal tissue from the bovine upper respiratory tract. PROCEDURE: Mh1 was radiolabeled by use of tritiated leucine. Various concentrations of labeled bacteria were incubated with bovine upper respiratory tract tissues for various times. Tissue was washed to remove nonadherent bacteria, and percentage of bacteria adhered (percentage of adherence) was estimated using radioactivity. Using an optimal inoculum concentration and incubation time, percentage of Mh1 adherence was compared on nasal, nasopharyngeal, turbinate, and tonsillar mucosal tissue, and adherence to nasopharyngeal tissue was confirmed by scanning and transmission electron microscopy. RESULTS: The optimal Mh1 inoculum concentration was 1 X 10(7) colony forming units/ml and incubation time was 3 hours. Percentage of adherence of Mh1 to nasopharyngeal tissue was greater than adherence to other tissue types. CONCLUSIONS AND CLINICAL RELEVANCE: The ex vivo model maintained the functional and structural integrity of bovine upper respiratory tract mucosa, as confirmed by light and electron microscopy. Electron microscopy revealed participation of epithelial cell cilia and surface mucus in adherence of Mh1 to nasopharyngeal tissue. Adherence of Mh1 was confirmed in repeated assays, indicating that this organism adheres to upper respiratory tract mucosa of cattle.

Ultrastructure of Pasteurella haemolytica-induced changes in gnotobiotic calf lung.

Gnotobiotic calves born and maintained in a germ-free environment until inoculated with a pathogen are model animals for studying the progression of a specific disease, such as pneumonic pasteurellosis. To investigate early progression of pneumonic pasteurellosis, we compared the ultrastructure of regions of gas-exchange in the lungs of three challenge-exposed and three uninoculated control gnotobiotic calves. Three calves were inoculated endobronchially with a bolus of 7.9 x 10(10) CFU of Pasteurella haemolytica A1 and studied in a specific pathogen-free environment until severe respiratory distress occurred, at which time they were euthanized. Slices of lung tissue from the midregion of the right dorsal caudal lobe (area of lesion) of infected and control calves were fixed in glutaraldehyde and prepared for scanning (SEM) and transmission (TEM) electron microscopy. SEM revealed bacteria among long tangled strands of fibrin in pulmonary alveoli that became obliterated with cellular debris. TEM revealed areas of encapsulated and/or nonencapsulated bacteria among the cellular debris and patches of fibrin. Many neutrophils and macrophages that infiltrated the alveoli had phagocytosed bacteria and undergone degradation. Less cellular damage was present when encapsulated bacteria bordered the interalveolar septa than when nonencapsulated lysed bacteria were present. Where lysed bacteria were present, the pulmonary capillaries were dilated because of swollen, degranulated neutrophils, fibrin clots, and cellular necrosis. Both encapsulated and nonencapsulated bacteria were present in the lung tissue of gnotobiotic calves within the first 24 h after endobronchial inoculation of early log phase P. haemolytica. Loss of capsular material around individual and divided pairs of bacteria led to their consequential aggregation, lysing, and severe damage to the adjacent pulmonary capillaries and interalveolar septa.

In vivo effect of Pasteurella haemolytica infection on bovine neutrophil morphology.

OBJECTIVE: To determine whether characteristic changes in neutrophil morphology caused in vitro by Pasteurella haemolytica leukotoxin (LKT) can be observed in vivo by electron microscopic examination of infected tissue chamber fluids and pneumonic lungs. ANIMALS: 7 mixed-breed beef calves. PROCEDURE: Tissue chambers were implanted subcutaneously in 3 calves and were inoculated with P haemolytica or phosphate-buffered saline solution. Chamber fluid samples, obtained at 8 and 32 hours after inoculation, were examined, using electron microscopy. Experimental pneumonia was induced in an additional 4 calves by transthoracic inoculation with P haemolytica. These calves were euthanatized at 6, 12, 24, and 36 hours after inoculation and lung sections were examined, using transmission electron microscopy. RESULTS: On examination, using transmission electron microscopy, neutrophils in lung sections and tissue chamber fluids had cytoplasmic and nuclear changes indicative of irreversible cell injury, including cell swelling, loss of plasma membrane ruffling, mitochondrial swelling, autolytic vacuolation, disruption of plasma membrane, nuclear pyknosis, karyolysis, and karyorrhexis. On examination, using scanning electron microscopy, leukocytes obtained from tissue chambers did not have their typical convoluted surfaces, but appeared rounded and swollen or shrunken with pitted surfaces. CONCLUSIONS: Pasteurella haemolytica-induced changes in neutrophil morphology in vivo were similar to those previously induced by in vitro exposure of neutrophils to LKT. Changes were suggestive of injury initiated by damage to the plasma membrane, which is consistent with the mechanism of action of pore-forming cytolysins. CLINICAL RELEVANCE: Pasteurella haemolytica LKT appears to be an important virulence factor in vivo; a fact that should be addressed in the development of vaccines.

Increase of glycocalyx and altered lectin agglutination profiles of Pasteurella haemolytica A1 after incubation in bovine subcutaneous tissue chambers in vivo or in ruminant serum in vitro.

Pasteurella haemolytica serotype A1 (bovine strain OK) was incubated for 2 and 6 h in bovine subcutaneous tissue chambers in vivo, and ovine strain 82-25 and bovine strain L011 were incubated in vitro for 2 h in heat-inactivated ovine or bovine serum from which gamma globulin had been depleted by protein G affinity chromatography to assess changes in morphology and lectin agglutination profiles (strains 82-25 and L101 only). Cells, removed from chambers after 2 h, were covered with an extensive, dense glycocalyx extending approximately 0.5 microm from the cell surface. In many cells, the glycocalyx was separated from the cell surface by a clear, electron-transparent area. Cells, removed at 6 h, were covered with a sparse glycocalyx of fine fibers 0.2 to 0.3 microm from the cell surface. Strains 82-25 and L101, incubated for 2 h in heat-inactivated ovine or bovine serum or in heat-inactivated ovine or bovine serum depleted of gamma globulin by protein G affinity chromatography, were also covered with a glycocalyx. The glycocalyx did not bind protein A-colloidal gold and therefore did not contain aggregates of accumulated antibody. Strains 82-25 and L101 were incubated individually for 2 h in 10 mM sodium phosphate buffer (pH 7.2) containing 0.14 M NaCl, 0.5 mM CaCl2, and 0.15 mM MgCl2 or with this buffer and either 25% heat-inactivated, gamma globulin-depleted ovine serum or 25% heat-inactivated, gamma globulin-depleted bovine serum. Agglutination profiles were then determined with 17 lectins in 10 mM HEPES-buffered saline (pH 8.4) with 0.1 mM CaCl2 and 0.08% sodium azide. Profiles did not vary with 10 of 17 lectins. However, profiles did vary with peanut agglutinin, Phaseolus vulgaris leucoagglutinin, Sophora japonica agglutinin, Maackia amurensis lectin II, Narcissus pseudonarcissus (daffodil) lectin, Griffonia simplicifolia lectin I, and Pisum sativum agglutinin. Altered profiles indicate a change in the bacterial cell surface, possibly by adsorption or alteration of surface carbohydrate moieties by serum constituents.

Morphological and functional disturbances in pulmonary vascular endothelium following exposure of sheep to an "avirulent" strain of Pasteurella haemolytica.

Sheep were vaccinated with a live attenuated strain of Pasteurella haemolytica and killed 3 days later. Segments of main intrapulmonic artery and vein were removed for biophysical and scanning electron microscopic studies. In the pulmonary artery, vaccination with Pasteurella haemolytica caused an increase in the number of endothelial cell surface blebs and, in some cases, those blebs appeared to be splitting open, suggesting cell damage or irritation. There was a surprising lack of platelet adherence to the lesions, suggesting that an antiplatelet factor is released by the damaged endothelium. The endothelial-dependent relaxant response to bradykinin was enhanced following vaccination. In the pulmonary vein, ultrastructural lesions similar to those in the artery were present in vaccinated animals. Bradykinin caused a contraction, an effect that was reduced following vaccination with Pasteurella haemolytica. These experiments demonstrate that a live, vaccine-derived strain of Pasteurella haemolytica causes both morphological and functional changes in the pulmonary vascular endothelium.

Ovine pulmonary surfactant induces killing of Pasteurella haemolytica, Escherichia coli, and Klebsiella pneumoniae by normal serum.

Pulmonary surfactant has been shown to play an increasingly important role in bacterial clearance at the alveolar surface in the lung. This study describes a bactericidal mechanism in which ovine pulmonary surfactant induces killing of Pasteurella haemolytica by normal serum. To demonstrate killing, six bacterial species were incubated first with pulmonary surfactant for 60 min at 37 degrees C and then with serum for an additional 60 min at 37 degrees C. P. haemolytica type A1 strains 82-25 and L101, a P. haemolytica type 2 strain, Escherichia coli, and Klebsiella pneumoniae were susceptible and Pasteurella multocida, Serratia marcescens, and Pseudomonas aeruginosa were not susceptible to killing by ovine pulmonary surfactant and normal serum. No bacteria incubated with bovine pulmonary surfactant were killed by normal serum. Although the species origin of pulmonary surfactant was selective, the species origin of serum was not. P. haemolytica incubated with ovine pulmonary surfactant was killed by fetal calf serum, gnotobiotic calf serum, pooled normal sheep serum, pooled normal rabbit serum, and pooled guinea pig serum. Ultrastructurally, killed P. haemolytica suspensions contained dead cells and cells distorted with vacuoles between the cytoplasmic membrane and the cytoplasm. The mechanism of killing did not correlate with concentrations of complement or lysozyme or titers of residual antibody in either the pulmonary surfactant or the serum, and killing was reduced by preincubation of surfactant with P. haemolytica lipopolysaccharide. Preliminary characterization of both surfactant and serum implicate a low-molecular-weight proteinaceous component in the surfactant and serum albumin in the serum. This mechanism may help clear certain gram-negative bacteria from the lungs of sheep as a part of the pulmonary innate defense system.

A characterization of monoclonal antibodies prepared against Pasteurella haemolytica serotype 1 surface antigens.

The production and characterization of monoclonal antibodies against Pasteurella haemolytica serotype 1 is described. Ten monoclonal antibodies were produced and divided, on the basis of their properties, into six different groups. One produced bacteria agglutination only of P. haemolytica serotype 1. Three antibodies bound with P. haemolytica serotypes 1, 5-8 and 12 and the antigen was identified in immunoblots as lipopolysaccharide. Two antibodies bound P. haemolytica serotypes 1, 2, 5-8 and 12 and P. multocida serotypes 1-7, 9, 12, 15 and 16, recognizing an epitope present on a 29 kDa outer membrane protein. One antibody bound all P. haemolytica and P. multocida serotypes. The antigen was a hexosamine less than 30 kDa which contained a formalin sensitive epitope. One antibody bound only to P. haemolytica serotype 1 and the antigen was identified as a 66 kDa outer membrane protein. Two antibodies bound P. haemolytica serotypes 1, 2, 5-9 and 12 and the antigen, while not identified, was localized on the outer membrane. This study identified antigens which contribute to the cross-reactions among P. haemolytica and P. multocida serotypes and the antibodies may be useful in investigating the pathogenesis of pneumonic pasteurellosis.

Characterisation and biological activity of monoclonal antibodies specific for Pasteurella haemolytica A1 capsule and lipopolysaccharide.

Monoclonal antibodies (mAb) against both Pasteurella haemolytica A1 capsule and lipopolysaccharide (LPS) were produced. Anti-capsule mAb reacted with the homologous A1 serotype only, whereas mAb against LPS reacted with P. haemolytica serotypes A2, A5, A8, A12, A14 and A16 but not with 33 bacterial species or rough LPS mutant strains tested. Both capsule and LPS antigens were visualised on the surface of bacteria by immunogold electron microscopy. Neither of the mAbs demonstrated antibody-dependent complement-mediated killing in vitro but both facilitated phagocytosis in vitro.