Sphingoid bases are taken up by Escherichia coli and Staphylococcus aureus and induce ultrastructural damage.

Sphingoid bases found in the outer layers of the skin exhibit antimicrobial activity against gram-positive and gram-negative bacteria. We investigated the uptake of several sphingoid bases by Escherichia coli and Staphylococcus aureus, and assessed subsequent ultrastructural damage. E. coli and S. aureus were incubated with D-sphingosine, dihydrosphingosine, or phytosphingosine at ten times their MIC for 0.5 and 4 h, respectively, to kill 50% of viable bacteria. Treated bacterial cells were immediately prepared for SEM, TEM, and analyzed for lipid content by QTLC. E. coli and S. aureus treated with sphingoid bases were distorted and their surfaces were concave and rugate. Significant differences were observed in the visual surface area relative to controls for both E. coli and S. aureus when treated with dihydrosphingosine and sphingosine (p < 0.0001) but not phytosphingosine. While sphingoid base-treated S. aureus exhibited disruption and loss of cell wall and membrane, E. coli cytoplasmic membranes appeared intact and the outer envelope uncompromised. Both E. coli and S. aureus cells contained unique internal inclusion bodies, likely associated with cell death. QTLC demonstrated extensive uptake of sphingoid bases by the bacteria. Hence, sphingoid bases induce both extracellular and intracellular damage and cause intracellular inclusions that may reflect lipid uptake.

Organization, barrier function and antimicrobial lipids of the oral mucosa.

As one moves from the skin across the vermilion region of the lip and into the oral cavity, the oral mucosa is encountered. The oral mucosa consists of connective tissue known as the lamina propria covered by a stratified squamous epithelium. In the regions of the hard palate and gingiva, the epithelium is keratinized like the epidermis. In the buccal region, the floor of the mouth and the underside of the tongue, the epithelium is non-keratinized. The epithelium on the dorsum of the tongue is a specialized epithelium, but can be approximated as a mosaic of keratinized and non-keratinized epithelia. The non-keratinized epithelial regions do not produce a stratum corneum. Nuclei with intact DNA are retained in the superficial cells. In all regions, the outer portions of the epithelium provide a protective permeability barrier, which varies regionally. Antimicrobial lipids at the surfaces of the oral mucosa are an integral part of innate immunity.

The emerging role of peptides and lipids as antimicrobial epidermal barriers and modulators of local inflammation.

Skin is complex and comprised of distinct layers, each layer with unique architecture and immunologic functions. Cells within these layers produce differing amounts of antimicrobial peptides and lipids (sphingoid bases and sebaceous fatty acids) that limit colonization of commensal and opportunistic microorganisms. Furthermore, antimicrobial peptides and lipids have distinct, concentration-dependent ancillary innate and adaptive immune functions. At 0.1-2.0 µM, antimicrobial peptides induce cell migration and adaptive immune responses to coadministered antigens. At 2.0-6.0 µM, they induce cell proliferation and enhance wound healing. At 6.0-12.0 µM, they can regulate chemokine and cytokine production and at their highest concentrations of 15.0-30.0 µM, antimicrobial peptides can be cytotoxic. At 1-100 nM, lipids enhance cell migration induced by chemokines, suppress apoptosis, and optimize T cell cytotoxicity, and at 0.3-1.0 µM they inhibit cell migration and attenuate chemokine and pro-inflammatory cytokine responses. Recently, many antimicrobial peptides and lipids at 0.1-2.0 µM have been found to attenuate the production of chemokines and pro-inflammatory cytokines to microbial antigens. Together, both the antimicrobial and the anti-inflammatory activities of these peptides and lipids may serve to create a strong, overlapping immunologic barrier that not only controls the concentrations of cutaneous commensal flora but also the extent to which they induce a localized inflammatory response.

A multiplex immunoassay demonstrates reductions in gingival crevicular fluid cytokines following initial periodontal therapy.

BACKGROUND AND OBJECTIVE: Cytokines and chemokines play an important role in the pathogenesis of periodontal diseases. The objective of this study was to quantitatively assess the effect of initial periodontal therapy on gingival crevicular fluid levels of a comprehensive panel of cytokines and chemokines, including several less extensively studied mediators. MATERIAL AND METHODS: Clinical examinations were performed and gingival crevicular fluid samples obtained from six subjects with generalized severe chronic periodontitis prior to initial periodontal therapy and at re-evaluation (6-8 weeks). Four diseased and two healthy sites were sampled in each subject. Twenty-two gingival crevicular fluid mediators were examined using a multiplex antibody capture and detection platform. Statistical analyses were performed by fitting mixed effects linear models to log-transformed gingival crevicular fluid values. RESULTS: Gingival crevicular fluid interleukin (IL)-1alpha and IL-1beta were the only cytokines to differ in initially diseased vs. initially healthy sites. Following initial therapy, 13 of the 16 detectable cytokines and chemokines decreased significantly in diseased sites, including IL-1alpha, IL-1beta, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12 (p40), CCL5/regulated on activation, normally T cell expressed and secreted (RANTES), eotaxin, macrophage chemotactic protein-1, macrophage inflammatory protein-1alpha and interferon-gamma. At healthy sites, only three of the 16 mediators were significantly altered following therapy. CONCLUSION: This is the first study, to our knowledge, to evaluate such an extensive panel of gingival crevicular fluid mediators within the same sample prior to and following initial therapy. The results confirm that periodontal therapy effectively reduces pro-inflammatory cytokines and chemokines, including less well-described mediators that may be important in initiation and progression of periodontitis. The multiplex assay will prove useful for future gingival crevicular fluid studies.

Response of the ruminant respiratory tract to Mannheimia (Pasteurella) haemolytica.

Pneumonia is a leading cause of loss to the sheep and cattle industry throughout the world. Mannheimia (Pasteurella) haemolytica is one of the most important respiratory pathogens of domestic ruminants and causes serious outbreaks of acute pneumonia in neonatal, weaned and growing lambs, calves, and goats. M. haemolytica is also an important cause of pneumonia in adult animals. Transportation, viral infections with agents such as infectious bovine rhinotracheitis virus, parainfluenza-3 virus or bovine respiratory syncytial virus, overcrowding, housing of neonates and weaned animals together and other stressful conditions predispose animals to M. haemolytica infection [1, 2]. This review assimilates some of the findings key to cellular and molecular responses of the lung from a pathologist's perspective. It includes some of what is known and underscores areas that are not fully understood.

Anionic antimicrobial peptide-lysozyme interactions in innate pulmonary immunity.

The respiratory tract contains numerous antimicrobial factors necessary for normal innate pulmonary defense. Although many of these molecules reside in airway surface liquid (ASL) simultaneously, little information exists concerning antagonistic, additive, or synergistic interactions. Since both cationic lysozyme and anionic antimicrobial peptides (AP) are found in high concentrations in ASL, the purpose of this study was to assess any interaction that might affect antimicrobial activity. For this, Pasteurella haemolytica, Micrococcus lysodeikticus, or Pseudomonas aeruginosa were added to egg white lysozyme (3.9-250.0 microg/ml) or human neutrophil lysozyme (0.8-50.0 microg/ml) and H-GADDDDD-OH (from 0.01 to 0.50 mM) mixtures in 50, 100, or 150 mM NaCl; incubated for 2 h; and then plated. In this assay, the MICs of AP for P. haemolytica, M. lysodeikticus, and P. aeruginosa varied slightly depending upon the concentration of NaCl and MICs generally increased slightly with increasing NaCl concentrations. The MIC of lysozyme for P. haemolytica and M. lysodeikticus also increased slightly with increasing NaCl concentrations. The MIC of lysozyme for P. aeruginosa was greater than 50 microg/ml and did not vary with increasing NaCl concentrations. When AP was combined with lysozyme in 50, 100, or 150 mM NaCl concentrations, there was no significant interaction that affected antimicrobial activity. In conclusion, the MICs of AP generally increased with increasing NaCl concentrations but lysozyme and AP appeared not to interact significantly at physiologically relevant concentrations.

Suppression of Mannheimia (Pasteurella) haemolytica serovar 1 infection in lambs by intrapulmonary administration of ovine antimicrobial anionic peptide.

In this study, the efficacy of ovine antimicrobial anionic peptide (AP) was assessed in a lamb model of acute pneumonia. A single intratracheal dose of the peptide, H-DDDDDDD-OH (0.5 mg) reduced pulmonary inflammation and the concentration of Mannheimia (Pasteurella) haemolytica in infected lung tissue. Administration of H-DDDDDDD-OH after infection was more effective in reducing the consolidation and lesion scores at the deposition site than its administration prior to infection. Hence, the in vivo effectiveness of AP suggests that it may have applications in the treatment of pulmonary infections. Further studies are needed to confirm these findings and also to determine the optimal doses and intervals of H-DDDDDDD-OH therapy.

Breed susceptibility to ovine progressive pneumonia (maedi/visna) virus.

In this retrospective study of breed differences in susceptibility to disease caused by ovine progressive pneumonia (OPP) virus, 29 Border Leicester sheep were compared with 46 Columbia sheep. As judged by frequency and severity of clinical signs and lesions attributable to the infection, Border Leicester sheep were markedly more susceptible than Columbia sheep and experimentally infected sheep were slightly more susceptible than naturally infected sheep. Differences in susceptibility to infection by the virus were not determined.

Ovine progressive pneumonia (maedi-visna) in sheep.

Ovine progressive pneumonia (OPP) is a multi-systemic disease of sheep caused by a nononcogenic exogenous retrovirus belonging to the Lentiviridae subfamily. Characteristics of the disease are chronic lymphocytic pneumonitis, encephalitis, arthritis, mastitis and vasculitis associated with progressive wasting, dyspnea, lameness, indurated udder and, rarely, paralysis. Any one or all of the characteristics may be manifest. Transmission of the virus is predominantly through the colostrum to newborn lambs, however, transmission can occur by contact and in utero. Treatment of the disease is only symptomatic and prevention of infection is only by avoiding the virus.

Failure of experimental vaccines to protect against infection with ovine progressive pneumonia (maedi-visna) virus.

Cell culture medium was harvested from cells infected with ovine progressive pneumonia (OPP) virus and used to prepare killed virus vaccines. Virus was inactivated by either heat, formalin, or ethyleneimine and used either without adjuvant, with Freund incomplete adjuvant, or with aluminum hydroxide adjuvant to vaccinate sheep. The sheep produced precipitating antibody against the virus but were not protected against infection when challenged with live OPP virus.

Lesions in lambs experimentally infected with ovine adenovirus serotype 6 and Pasteurella haemolytica.

Twenty-five colostrum-deprived lambs reared in isolation were inoculated with a US variant of ovine adenovirus serotype 6 (OAV-6) strain RTS-151, Pasteurella haemolytica, or a combination of the 2 agents. Although severe pulmonary lesions were caused by each agent, the lesions were more severe and lasted longer with the combined infection. Lesions induced by OAV-6 alone developed 6-9 days after inoculation and lasted for 15 days, the length of the experiment. The lesions were characterized by suppurative inflammation at the junction of the terminal bronchioles and alveoli. Air spaces were filled with neutrophils and sloughed epithelial cells, which often contained large intranuclear inclusions. Lesions induced by P. haemolytica alone developed within 1 day and persisted for no more than 10 days and were characterized by severe pulmonary edema with variable amounts of fibrin. Lesions induced by the combined infection had aspects of each infection alone and resulted in severe disease in 4 of 8 lambs that were permitted to live more than 1 day after inoculation with bacteria. Early pulmonary lesions included edema, limited fibrin deposition, and slight purulent bronchiolitis and alveolitis. Later lesions included necrosis and more fibrin. For lambs inoculated with both pathogens, resolution was incomplete 15 days after inoculation of virus (10 days after inoculation of P. haemolytica). The results presented here corroborate previous findings indicating that the RTS-151 variant of OAV-6 is common in lambs and acts in concert with P. haemolytica to cause severe and often fatal pneumonia.

Ultrastructural location of the cross-protection factor on in vivo and in vitro grown Pasteurella multocida.

Pasteurella multocida from infected turkey tissues expresses a unique immunogen called cross-protection factor (CPF) that induces immunity to challenge by both homologous and heterologous serotypes. In this study, we used a monoclonal antibody (AMP MAb) to CPF and protein A-colloidal gold (PACG) to locate CPF on P. multocida. After incubation with AMP MAb and PACG, CPF was detected at the bacterial surface and cell periphery of P. multocida in infected turkey liver and P. multocida isolated from infected turkey blood. CPF was not detected on P. multocida incubated with control monoclonal antibody. Pasteurella multocida isolated from infected turkey blood and cultivated in the peptone-based medium did not express CPF consistently, and some cells contained more CPF than others. The location of CPF also varied, and CPF was detected both intracellular and extracellular on the cell surface. In the latter cells, CPF was heavily concentrated to a specific lateral site or detected sloughing from the cell surface. These results correlate with laboratory observations that CPF detected on P. multocida from infected turkey tissues, P. multocida isolated from infected turkey blood, and P. multocida cultivated in peptone-based medium is associated with outer membrane fractions.

A new serotype of Pasteurella multocida associated with fowl cholera.

Gel-diffusion precipitin tests demonstrated an additional Pasteurella multocida serotype, designated serotype 16. Isolate P-2723, antigenically distinct from the other (previously reported) 15 serotypes, was from a turkey affected with fowl cholera. This serotype is not widely distributed. Isolate P-2723 was of mild virulence in turkeys, resulting in local infections in the hock joint and sternal bursa of only 1 of 9 turkeys exposed.

Serologic types and physiologic characteristics of 46 avian Pasteurella anatipestifer cultures.

Forty-six strains of Pasteurella anatipestifer isolated from different avian species were examined to determine their serologic types and physiologic characteristics. Serologic types were determined by a gel-diffusion precipitin test. Antigens from 39 field isolates reacted with antisera prepared from seven P. anatipestifer reference strains representing serotypes 1, 2, 4, 6, and 7. Antigens from five isolates did not react and could not be typed with available reagents. Gel precipitin reactions involving serotype 1 (43.6%) and serotype 2 (25.6%) were the most prevalent. Generally, the physiologic characteristics from 40 tests were typical for P. anatipestifer, and variations were observed among the strains in urease production, hemolysin production, litmus milk reaction, and gelatin liquefaction.

Partial characterization of the hemagglutinin of Alcaligenes faecalis.

The hemagglutinin of Alcaligenes faecalis was partially characterized. Hemagglutination (HA) was blocked by enzymes inactivating proteins, by heat, and by antisera but not by sugar-blocking substances. Pili were not determined to be a factor in HA activity. There was no connection between virulence and HA activity.

Capsular hyaluronic acid-mediated adhesion of Pasteurella multocida to turkey air sac macrophages.

Serogroup A strains of Pasteurella multocida, the major cause of fowl cholera, are resistant to phagocytosis in nonimmunized birds. Adherence studies with a capsulated strain of P. multocida (serotype A:3) and turkey air sac macrophages in culture showed that the bacteria were capable of adhering in large numbers to the macrophages but were not internalized. A noncapsulated variant of the bacteria (serotype -:3) showed little or no adherence and was not internalized. These data indicated that the adhesive properties were caused by the presence of a capsule on the bacteria. The role of capsular hyaluronic acid in adherence to macrophages was investigated. Depolymerization of the bacterial capsule with hyaluronidase increased phagocytosis by macrophage cultures, and addition of hyaluronic acid to the macrophages inhibited bacterial adherence. Additionally, exposure of macrophages to chondroitin sulfate B, an anionic polysaccharide similar to hyaluronic acid, did not affect the adhesive properties and resistance to phagocytosis of capsulated organisms. Treatment of macrophages with sodium metaperiodate or trypsin suppressed bacterial binding. Collectively, these data indicate that P. multocida adhesion to air sac macrophages, but not internalization, is mediated by capsular hyaluronic acid and suggest that recognition of this bacterial polysaccharide is a result of a specific glycoprotein receptor.

Immunogenicity of Corynebacterium pseudotuberculosis and the effect of adjuvants in mice.

Intraperitoneal inoculation of CF1 mice with 100 micrograms, 200 micrograms, or 300 micrograms whole cell (WC) or 250 micrograms, 500 micrograms, or 1000 micrograms cell wall (CW) of Corynebacterium pseudotuberculosis induced varying degrees of protection after intravenous challenge of immunity with 7.2 X 10(4) CFU of C. pseudotuberculosis. Generally, the degree of protection increased with the dose of WC or CW. However, intraperitoneal inoculation of mice with 100 micrograms, 200 micrograms, or 300 micrograms heat-killed Mycobacterium bovis BCG; 50 micrograms, 150 micrograms, or 300 micrograms of either muramyl dipeptide (MDP) or trehalose dimycolate (TDM); or 350 micrograms, 700 micrograms or 1400 micrograms Corynebacterium parvum did not induce resistance to intravenous inoculation of 7.4 X 10(4) CFU of C. pseudotuberculosis. The protection induced by 500 micrograms CW was enhanced by adding 100 micrograms BCG, 150 micrograms MDP, or 350 micrograms C. parvum but protection induced in mice by 300 micrograms of WC was not enhanced by adding any adjuvants.

Influence of immunization on the pulmonary inflammatory response of rabbits induced by Pasteurella haemolytica A1 lipopolysaccharide.

Immune complex formation has long been thought to play a role in the pathogenesis of Pasteurella haemolytica pneumonia. This study in laboratory rabbits was designed to investigate immune-mediated damage in respiratory tissue caused by lipopolysaccharide (LPS). Severe lesions were induced by the intratracheal (IT) injection of P. haemolytica A1 LPS (50 micrograms) into rabbits previously immunized with P. haemolytica killed whole cells emulsified with Freund's incomplete adjuvant (FIA); these lesions included perivascular oedema and polymorphonuclear leucocyte (PMN) infiltration of the subintima, with degeneration and necrosis of the media. Smaller vessels were occluded by PMNs in various stages of degranulation. PMN counts in bronchoalveolar lavage (BAL) fluid were significantly elevated (P < 0.05). Lesions were also induced by the IT injection of LPS (50 micrograms) into rabbits pretreated with an emulsion consisting merely of FIA and formol-saline; these lesions included moderate to severe congestion, interstitial oedema, alveolar serofibrinous exudation and PMN infiltration. PMNs were also present in BAL fluid. Rabbits pretreated with FIA in formol-saline and given a later IT injection of saline, and rabbits pretreated with bovine serum albumin (BSA) in FIA and given a later IT injection of BSA, were included as negative and positive control groups. Cutaneous lesions were also induced by the intradermal injection of LPS into rabbits immunized against P. haemolytica and of BSA into rabbits immunized with BSA. Overall, the pulmonary and cutaneous lesions induced in vaccinated rabbits by antigen administration were more severe than those seen in non-vaccinated rabbits. The lesions in rabbits, which were similar to those seen in natural cases of P. haemolytica pneumonia in cattle, were characterized by a fibrinopurulent inflammatory process with extensive interstitial oedema, fibrinous exudate, and PMNs. This model may help to elucidate the pathogenesis of pneumonic pasteurellosis in immunized animals.

Changes in the lungs of lambs after intratracheal injection of lipopolysaccharide from Pasteurella haemolytica A1.

Ten lambs aged 8 weeks were inoculated intratracheally through the tracheal wall with lipopolysaccharide from Pasteurella haemolytica A1 and examined in chronological sequence by light and electron microscopy for pulmonary lesions. An acute fibrinopurulent pneumonia was produced, which resolved within 72 h but bore many resemblances to field cases of pneumonic pasteurellosis. Sequestration of neutrophils in the capillaries of the lungs and aggregation of surfactant in the alveoli occurred rapidly, followed by swelling of the alveolar and capillary endothelia, oedema, haemorrhage, and emigration of neutrophils into the interstitium and small air spaces of the lungs. Necrosis of isolated neutrophils was a constant feature. Alveolar, interstitial and intravascular macrophages and lymphoid cells increased slowly to become the predominant inflammatory cells at 72 h. A surprising feature was the transient appearance of multinucleated cells in the lungs at 2 and 6 h after inoculation. It is concluded that lipopolysaccharide makes a major contribution to the pathogenesis of P. haemolytica infection in the lungs of sheep.

Reduction of pulmonary mast cells in areas of acute inflammation in calves with Mannheimia (Pasteurella) haemolytica pneumonia.

Mast cells in the left cranial pulmonary lobe of colostrum-deprived neonatal calves were quantified 2 and 6 h after intrabronchial inoculation with Mannheimia (Pasteurella) haemolytica A1. The mast cells were detected (1) immunohistochemically with a mouse anti-human mast cell tryptase monoclonal antibody, and (2) by metachromatic staining with low pH toluidine blue. A greater number of mast cells was demonstrated by the second method than by the first. At 6 h after inoculation, but not at 2 h, the number of mast cells was significantly reduced at the site of the main lesions. Treatment of calves with a sialyl Lewis mimetic (TBC1269) did not appreciably affect the results at 6 h.