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Characterisation of the outer membrane protein antigens of British field isolates of Moraxella bovis.

Outer membranes of a single isolate of Moraxella bovis were prepared and their purity evaluated by a comparison of the iodinated proteins from whole cells and on outer membranes. The protein patterns of the outer membranes of 10 isolates were examined by SDS-polyacrylamide gel electrophoresis, and the antigenic relationship between proteins of different isolates determined by immunoblotting, using antiserum produced against the outer membrane of the single isolate of M. bovis. The overall protein pattern of the different isolates was similar although not identical, but, significantly, only three separate immunogenic proteins were common to all the isolates under examination.

Animals↗

Chemical composition of lipopolysaccharide from Moraxella bovis.

Lipopolysaccharide from Moraxella bovis was isolated and its components characterized by paper and gas-liquid chromatography and mass spectrometry. The endotoxin showed a small amount of protein and contained 2-keto-3-deoxyoctonic acid glucose, mannose, glucosamine, heptose and phosphate. Six fatty acids (palmitic, palmitoleic, margaric, stearic, oleic and arachidic acids) were also identified. Margaric acid was the most prevalent fatty acid present in the lipid fraction.

Animals↗

A unified serotyping scheme for Moraxella bovis.

Fifty-three Australian, seven British, two American and two New Zealand isolates of Moraxella bovis were classified into seven serogroups on the basis of their variable fimbrial (pilus) antigens using whole cell slide agglutination (SA), enzyme-linked immunosorbent assays (ELISA) and tandem-crossed immunoelectrophoresis (TCIE). Although results of serogroup classification by SA and ELISA were identical in 68.7% of isolates, it was found necessary to resolve the discrepancies between the two systems using TCIE. Results suggest that world-wide variation in the potentially host-protective fimbrial antigens of M. bovis may be relatively limited. It is proposed that the previous numerical classifications of British and Australian serogroups are appropriately amalgamated as a result of this latest study and are designated as serogroups A to G inclusive. A protocol for the further serotyping of fresh, fimbriate isolates of M. bovis is suggested.

Agglutination Tests↗

The protective efficacy of pili from different strains of Moraxella bovis within the same serogroup against infectious bovine keratoconjunctivitis.

Three groups of ten calves were each immunised with a total of 400 micrograms pili prepared from three separate strains of Moraxella bovis in Alhydrogel-oil adjuvant as two divided, equal doses 21 days apart. Groups 1 and 2 each received a monovalent vaccine made from strain 4L and S276R respectively, which belonged to pili serogroup A. Group 3 received vaccine made from pili of strain Maff1, belonging to serogroup F. A further group of ten calves served as non-vaccinated controls. Calves in groups 1 and 2 had developed serogroup A-specific antibody and those in group 3 developed serogroup F-specific antibody, and some evidence of cross-reacting antibody was also detected when measured by an agglutination test using formalin-killed piliated cells of serogroup A strain 4L. Although antibody titres measured against purified pili by ELISA were highest with homologous serogroup antigens, cross-reactive titres to shared epitopes of M. bovis pili were also detected by this method. Ocular challenge of the 40 calves with virulent M. bovis of serogroup A strain S276R was carried out 14 days after the second vaccine dose. All non-vaccinated calves developed infectious bovine keratoconjunctivitis (IBK). The percentage protection in groups 1 (strain 4L) and 2 (strain S276R) was 60% and 80% respectively (P less than 0.05), with mean lesion scores of 0.7 and 0.3 out of a possible 6.0. The percentage protection of calves in group 3 (strain Maff1) was only 30%, with a mean lesion score of 1.4 compared with 2.2 for non-vaccinated controls. The present findings, together with other evidence indicating that immunity to IBK is serogroup-specific, suggest that inclusion of pili from one representative strain from each of the seven Australian and British serogroups in a polyvalent, subunit vaccine should effectively protect the majority of cattle against IBK caused by most field strains of M. bovis encountered in Australia and the United Kingdom.

Animals↗

A Moraxella bovis pili vaccine produced by recombinant DNA technology for the prevention of infectious bovine keratoconjunctivitis.

Pili (fimbriae) were prepared from Moraxella bovis strain Dalton 2d (Dal2d) and from a derivative of Pseudomonas aeruginosa K/2PfS that contained a plasmid-borne Dal2d pilin gene and produced pili having serogroup-specific identity to Dal2d. Nine calves were vaccinated with two doses each of 30 micrograms authentic M. bovis Dal2d pili in oil adjuvant and 10 calves were vaccinated with a similar dose of P. aeruginosa-derived Dal2d pili in the same formulation. All 19 calves and 10 non-vaccinated controls were challenged by instillation of 1 x 10(9) virulent M. bovis Dal2d cells into both conjunctival sacs 19 days after the second vaccine dose. The serological response to vaccination and the degree of protection against experimentally induced infectious bovine keratoconjunctivitis (IBK) were assessed. None of the nine calves vaccinated with authentic M. bovis Dal2d pili developed IBK while two of those vaccinated with P. aeruginosa-derived Dal2d pili developed lesions which accounted for a mean group lesion score of 0.3. In contrast, 9 of the 10 non-vaccinated calves developed IBK lesions, the majority of which were progressive, required early treatment and accounted for a mean group lesion score of 1.5. These results demonstrate the potential of a relatively low dose of pili produced by recombinant DNA technology for development of an effective vaccine against IBK.

Animals↗

Partial characterization of a Moraxella bovis cytolysin.

Moraxella bovis (M. bovis) is the etiologic agent of infectious bovine keratoconjunctivitis and M. bovis hemolysin is believed to be an important virulence factor. Two strains of M. bovis were compared, Epp 63(300) (Epp), a known virulent and hemolytic strain, and IBH 63 (IBH), a known avirulent and nonhemolytic strain. Sterile 10-fold (10x) supernatant concentrates were obtained from cultures grown in TSB broth with 10 mM CaCl2. Supernatant hemolysin titers for Epp, were 1:1024 and 1:8192 for unconcentrated (1x) and 10x, respectively. Supernatant cytotoxin titers to bovine mononuclear cells were 1:32 and 1:128 for 1x and 10x, respectively, for Epp. Cytolytic (hemolytic and cytotoxic) activities declined 10-fold but were still measurable for > 1 wk at 4 degrees C. Both activities were inactivated by trypsin and by heating at 56 degrees C for 20 min. A cytotoxic effect was observed on cultured bovine and ovine corneal epithelial cells with Epp. All cytolytic effects were neutralized with antiserum to 10x Epp. No cytolytic activities were detected for 10x IBH. SDS-PAGE electrophoresis and related immunoblots indicate a high molecular weight protein at 110 kDa for the 10x Epp preparation when stained with silver or probed with monoclonal antibodies to the E. coli alpha hemolysin. No 110 kDa band is observed for 10x IBH. These data suggest that hemolytic and cytotoxic activities are important in the pathogenesis of infectious bovine keratoconjunctivitis and identify the protein as a possible RTX related toxin of 110 kDa. Stability of the M. bovis cytolysin for > 1 week should allow further characterization and purification of the protein.

Animals↗

The protective efficacy of cloned Moraxella bovis pili in monovalent and multivalent vaccine formulations against experimentally induced infectious bovine keratoconjunctivitis (IBK).

Calves were vaccinated with cloned Moraxella bovis pili of serogroup C (experiment 1) or B (experiment 2) either as a monovalent formulation or as part of a multivalent preparation with pili of six other serogroups. Within 4 weeks of the second vaccine dose vaccinated calves and non-vaccinated controls were challenged via the ocular route with either virulent M. bovis strain Dal2d (serogroup C) or M. bovis strain 3WO7 (serogroup B) in experiments 1 and 2, respectively. Calves vaccinated with multivalent vaccines had significantly lower antibody titres than those vaccinated with monovalent preparations. Nevertheless, the levels of protection against infectious bovine keratoconjunctivitis (IBK) achieved with multivalent vaccines were 72% and 83% for the groups challenged with M. bovis strains of serogroups B and C, respectively. The serogroup C monovalent vaccine gave 100% protection against experimentally induced IBK and M. bovis isolates cultured from the eyes 6 days post-challenge were identified as belonging solely to serogroup C. Unexpectedly, only 25% protection was achieved against homologous strain challenge of calves that received the monovalent serogroup B vaccine. Furthermore, the majority of M. bovis isolates recovered from calves in this group belonged to serogroup C, as did half of those isolates cultured from the multivalent vaccinates. The remaining bacterial isolates from the latter group, together with all isolates from the non-vaccinated controls, belonged to serogroup B. Results are consistent with the hypothesis that derivatives of the serogroup B challenge inoculum had expressed serogroup C pilus antigen within 6 days of the challenge, possibly as a result of pilus gene inversion occurring in response to the presence of specific antibody in eye tissues and tears.

Animals↗

Reproduction of bovine keratoconjunctivitis with a purified haemolytic and cytotoxic fraction of Moraxella bovis.

Evidence that the beta-haemolysin produced in vitro by Moraxella bovis is an important virulence determinant in vivo was strengthened by studies using a haemolytic preparation of greater purity than previously available. A concentrated haemolytic fraction containing outer-membrane bound vesicles was separated from the cell-free filtrate of a bacterial culture using a process comprising tangential flow ultrafiltration, ion-exchange and gel-filtration high-performance liquid chromatography and centrifugal-driven filtration. The cytotoxicity of haemolytic fractions for calf-corneal epithelial cells in vitro was investigated at progressive stages of this attempted haemolysin purification procedure and the results demonstrated a positive correlation for the levels of haemolytic and cytotoxic activity throughout. Further support for the role of the haemolysin was obtained in vivo following the intra-corneal injection of calves with a crude or a purified haemolytic fraction. The ocular damage caused by both preparations, together with the healing processes and microscopic pathology of the experimentally induced damage closely resembled published descriptions of naturally occurring infectious bovine keratoconjunctivitis. No effect was obtained in vitro or in vivo from equivalent fractions prepared from a non-haemolytic strain of M. bovis.

Animals↗

Iron repressible outer membrane proteins of Moraxella bovis and demonstration of siderophore-like activity.

Moraxella bovis (strain Epp 63), grown in RPMI 1640 medium supplemented with desferrioxamine mesylate (0.05 mg/ml) resulted in cell free culture supernatants with an increased chromeazurol-S response indicating the presence of high affinity iron binding ligand(s). Supernatants of cultures where growth occurred in tryptic soy broth, RPMI 1640, or RPMI 1640-desferrioxamine supplemented with ferrous sulfate (10 micrograms/ml) were negative on the chromeazurol-S test. Growth of M. bovis in RPMI 1640 or RPMI 1640-desferrioxamine medium induced the expression of previously unrecognized outer membrane proteins whose expression was repressed when the medium was supplemented with iron and which were not produced when growth occurred in tryptic soy broth.

Bacterial Outer Membrane Proteins↗

Neisseria meningitidis and Moraxella osloensis: dual infection in blood and peritoneal fluid.

The clinical course of a malnourished alcoholic in which Neisseria meningitidis was isolated from the blood and Moraxella osloensis from the peritoneal fluid is described. Following bacteriologic diagnosis, the patient was treated and responded to a course of penicillin therapy. To our knowledge, this represents the first case of peritonitis associated with M. osloensis. Clinical reports of the isolation of this organism are rare; its pathogenicity is not clearly established, and the presence of the organism may often be unrecognized.

Adult↗

In vitro activity of roxithromycin against Moraxella catarrhalis.

The in vitro activity of roxithromycin was compared with that of the other antimicrobial agents (erythromycin, tetracycline, ampicillin, and cotrimoxazole) against 188 distinct clinical isolates of Moraxella catarrhalis. Of these, 106 strains (66%) produced beta-lactamase. The MIC90 of roxithromycin was 0.25 micrograms/ml compared with values of 0.5, 1, greater than 8, greater than 8:0.4 micrograms/ml for erythromycin, tetracycline, ampicillin, and sulfamethoxazole-trimethoprim, respectively. These results, allied with its improved pharmacokinetic properties, suggest that roxithromycin should be an effective treatment in children and adults for upper and lower respiratory tract infections caused by M. catarrhalis.

Ampicillin↗

Moraxella septicemia in children with diarrheal disease.

We report 10 cases of Moraxella septicemia associated with diarrheal disease. Their clinical presentations and outcomes are discussed. Recognition of the pathogenicity of these microorganisms in appropriate clinical setting should result in prompt and specific therapy.

Anti-Bacterial Agents↗

Susceptibility testing interpretive criteria for levofloxacin when testing respiratory pathogens, Haemophilus influenzae and Moraxella catarrhalis.

The more active L-isomer, levofloxacin, of the racemic ofloxacin mixture has been under development for therapeutic use. In this study, we evaluated the activity of ofloxacin, levofloxacin, and D-ofloxacin against the fastidious respiratory tract pathogens Haemophilus influenzae and Moraxella catarrhalis. Levofloxacin was two-fold more active than ofloxacin against H. influenzae (MIC90, 0.015 microgram/ml), and D-ofloxacin was least active (MIC90, 1 microgram/ml). For M. catarrhalis the MIC90 values were 0.03 microgram/ml, 0.06 microgram/ml, and 2 micrograms/ml for levofloxacin, ofloxacin, and D-ofloxacin, respectively. For disk diffusion susceptibility testing, Chocolate Mueller-Hinton agar (CMH) was considered preferable to Haemophilus test medium (HTM) because it supported the growth of all of 105 H. influenzae strains whereas five strains failed to grow on HTM. In addition, the margins of the zones of inhibition were more distinct on CMH and the Haemophilus species strains with elevated fluoroquinolone MICs were readily distinguished. The superior growth on CMH was reflected in a reduction of inhibition zone diameters of 2-3 mm relative to the inhibition zone diameters on HTM. The previously proposed interpretive criteria for the 5 microgram disk diffusion susceptibility test (susceptible at > or = 17 mm) results in complete categorical agreement with the reference microdilution broth method for M. catarrhalis on Mueller Hinton agar and for H. influenzae on HTM and CMH. However, the minimum diameter of the zone of inhibition recorded for a member of the dominant population of either species was considerably greater (25 mm) than 17 mm on any of the media tested.

Culture Media↗

Development of a species-specific DNA probe for Moraxella (Branhamella) catarrhalis.

Moraxella (Branhamella) catarrhalis is an aerobic Gram-negative diplococcus that is now recognized as a pathogen of the respiratory tract. Rapid and direct identification of this bacterium has become important to the clinical microbiology laboratory. Recently, rapid tests for the identification of Neisseria species and M. catarrhalis have been commercialized but they are primarily for Neisseriae; in these kits, M. catarrhalis is always identified presumptively. We have developed a DNA probe of chromosomal origin that is 100% specific for M. catarrhalis. The oligonucleotide probe was derived from a cloned Ase I chromosomal DNA fragment of M. catarrhalis that did not react with Haemophilus influenzae DNA in hybridization experiments. Three of the first 17 Ase I clones were selected randomly to be tested by colony hybridization against several different species that colonize the human respiratory tract. One of these three, pLQ121, has a 550 bp fragment inserted into pBR322 and was determined to be 100% specific to M. catarrhalis. This fragment was partially sequenced and a 30-mer oligonucleotide was synthesized from the sequence data. This probe was also shown to be 100% specific to the species.

Bacterial Typing Techniques↗

Reactivity of asparagine residue at the active site of the D105N mutant of fluoroacetate dehalogenase from Moraxella sp. B.

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon-fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the alpha-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.

Amino Acid Substitution↗

Aqueous sol-gel encapsulation of genetically engineered Moraxella spp. cells for the detection of organophosphates.

An all-aqueous sol-gel method for encapsulation of bacterial cells in porous silicate matrices towards the development of a biosensor is described. The sol-gel encapsulation of cells is achieved at room temperature and neutral pH. Furthermore, use of sodium silicate as precursor avoids generation of alcohol that can be detrimental to cells in contrast to the traditional alkoxide sol-gel encapsulation process. Moraxella spp. cells engineered to express recombinant organophosphorus hydrolase (OPH) on the cell surface were encapsulated and OPH enzymatic activity was measured for paraoxon hydrolysis. Kinetic parameters (Km and Vmax) as well as pH behavior of surface-expressed OPH were determined to evaluate the effect of encapsulation. Cells encapsulated by the sodium silicate method displayed higher activity retention compared to those by the traditional alkoxide process. Time-course studies over a 2-month period indicate that immobilization through the sodium silicate process led to a reduction in activity of approximately 5% as compared to approximately 30% activity reduction in case of free cells in buffer indicating that immobilization leads to stabilization, a key parameter in biosensor development.

Biosensing Techniques↗

Identification of a capsular polysaccharide from Moraxella bovis.

The bacterium Moraxella bovis is the causative agent of an economically important disease of cattle: Infectious Bovine Keratoconjunctivitis (IBK), otherwise known as pinkeye. Little is known regarding the structure of the carbohydrates produced by M. bovis. The structure of a capsular polysaccharide from M. bovis (strain Mb25) has been determined using NMR and MS analysis. From these data it is concluded that the polysaccharide is composed of the unmodified chondroitin disaccharide repeat unit.

Bacterial Capsules↗

Identification of a novel glycosyltransferase involved in LOS biosynthesis of Moraxella catarrhalis.

Moraxella catarrhalis is an important human mucosal pathogen that contributes to otitis media in infants and exacerbates conditions such as chronic obstructive pulmonary disease in the elderly. This study describes the identification of a novel gene, lgt5 that encodes a glycosyltransferase involved in the LOS biosynthesis of M. catarrhalis. Analysis of NMR data of LOS-derived oligosaccharide from a Serotype A lgt5 mutant strain of M. catarrhalis indicate that lgt5 encodes an alpha-(1-->4)-galactosyltransferase.

Antigens, Bacterial↗