Biological reactivity of Moraxella bovis lipopolysaccharide

Lipopolysaccharide (LPS) was isolated from Moraxella bovis 118F and ATCC 10900, M ovis ATCC 33078, and M phenylpyruvica ATCC 23333 by hot phenol-water extraction. In silver-stained sodium dodecyl sulfate polyacrylamide electrophoresis gels, M bovis 118F LPS had a smooth profile, whereas the other Moraxella preparations appeared to be rough. The LPS preparations induced pyrogenicity and dermal Shwartzman reactions in rabbits, and induced production of tumor necrosis factor and interleukin-1 in vitro. Induction of tumor necrosis factor appeared to be among the most potent biological activities of M bovis LPS.     

Moraxella bovis hemolysin

Moraxella bovis hemolysin was readily filterable through polycarbonate membrane filters, but not through nitrocellulose filters. The hemolysin was filterable through polycarbonate filters with pore diameters of greater than or equal to 0.015 micron (APD). Of the hemolytic activity of cell-free filtrates, 74% could be pelleted by ultracentrifugation at 100,000 X g for 2 1/2 hours. Hemolytic activity could be demonstrated in preparations of outer membrane fragments isolated from log-phase cultures. Hemolysin in M bovis broth cultures reached a maximum concentration in late logarithmic phase (4.5 hours after inoculation) and declined thereafter. Hemolysin was inactivated by heat, trypsin, formalin, and lyophilization.     

Antimicrobial susceptibility of Moraxella bovis

The antimicrobial susceptibility of 55 isolates of Moraxella bovis to seven antibiotics was evaluated by broth microdilution procedures. The isolates had an MIC₉₀ of ≤1 mg/l to erythromycin, ceftiofur, and ampicillin; 4 mg/l to tilmicosin; 16 mg/l to tylosin and gentamicin; and had MIC₉₀s of ≥32 mg/l for oxytetracycline. The modal MIC values for these antibiotics were as follows: ampicillin, <0.25 mg/l; ceftiofur, ≤0.125 mg/l; tilmicosin, 2 mg/l; tylosin, 8 mg/l; erythromycin 1 mg/l; oxytetracycline, ≤0.5 mg/l; and gentamicin, ≤0.5 mg/l. This in vitro data showed most antibiotics have low MICs that are suggestive of clinical efficacy.     

Immunogenicity of Moraxella bovis hemolysin

Anti-Moraxella bovis hemolysin activity was observed in 35 cattle exposed to field infections of infectious bovine keratoconjunctivitis (IBK). All cattle in infected herds seroconverted with respect to antihemolysin whether or not clinical IBK was noted. Cattle previously exposed to IBK possessed higher antihemolysin titers than did younger, nonexposed cattle. Antihemolysin activity was noted in bovine sera up to 7 years after exposure to IBK. Sera from experimentally infected calves were found to possess antihemolytic activity against all 33 strains of M bovis tested. Antihemolytic activity could be demonstrated in random-bred mice inoculated with whole doxycycline-treated M bovis, frozen or lyophilized whole M bovis, and membrane fractions treated with sodium lauryl sarcosinate, Triton X 100, and Triton X 100 + EDTA, but not with formalin-treated whole M bovis.     

Growth requirements for Moraxella bovis

Moraxella bovis isolates do not necessarily require complex growth media. Minimal growth requirements were approached by selecting mutants that grew on progressively simpler solid media. Isolate 137T was selected until it could be cultured on a medium containing sodium L-glutamate, with thiamine, nicotinamide, pyridoxine and inorganic salts. All 5 field strains isolated in 1983 showed the same requirements and no prior selection was needed. One of these 5 field strains (H388) was selected to require nicotinamide as the only added vitamin, and culture 137H was selected to grow without added vitamins. In contrast, type strains NCTC 8561 and NCTC 9426 could not be cultured on glutamate medium supplemented with a mixture of 8 growth factors. However, 35 out of 38 M. bovis cultures collected in this laboratory grew on that medium. A more complex medium (TG) contained 8 g glutamate and 2 g tryptone per litre of basal salts. Medium TG supported growth by both type strains and all 38 laboratory cultures. Growth on TG medium was promoted by adding 0.1 mg l-1 of pyridoxine, nicotinamide and thiamine. M. bovis strains from type-culture collections or those that have been subcultured on complex media only, may not have retained their native nutritional requirements.     

Relatedness of cytotoxins from geographically diverse isolates of Moraxella bovis

To determine whether amino acid sequence variation exists in the Moraxella bovis (M. bovis) cytotoxin (MbxA) from geographically diverse M. bovis isolated in the United States, mbxA was amplified and sequenced. The MbxA deduced amino acid sequence from M. bovis originally isolated in California, Washington, North Carolina, and Georgia, as well as reference strains of M. bovis isolated at the National Animal Disease Laboratory, Ames, IA, USA, all encoded a nearly identical 927 amino acid protein. MbxA from two of the four California isolates (SFS 9a and SFS 100a) differed from all other isolates at two sites at which the polar amino acids glutamine (position 666) and asparagine (position 823) were replaced by ionized amino acids glutamic acid and aspartic acid, respectively. Rabbit antiserum to the expressed carboxy terminus (amino acids 590-927) of MbxA from M. bovis (Tifton I) neutralized the hemolytic activity of SFS 9a and SFS 100a. The M. bovis cytotoxin appears to be conserved amongst geographically diverse isolates of M. bovis from the USA. Antiserum against the carboxy terminus of MbxA common to the majority of isolates neutralized the hemolytic activity of two strains with a divergent MbxA deduced amino acid sequence. Vaccines against IBK that incorporate MbxA as antigen may offer protection against geographically diverse strains of M. bovis.     

Moraxella bovis pathogenicity: an update

Moraxella bovis is the etiologic agent of infectious bovine keratoconjunctivitis, the most important ocular disease affecting cattle worldwide. The severity of the cases varied from eyes that exhibited mild signs to severe clinical cases with profuse lacrimation, conjunctival swelling, corneal opacity, and ulceration. Although the mortality is low, there is a high morbidity and important economic loss in terms of significant reduction in production. This paper examines aspects such as the pathogenesis of the disease and the mechanisms by which this unique bacterium is able to disrupt the corneal epithelium and cause infection.     

Characterization of the plasmids of Moraxella bovis

Restriction endonuclease digestions were performed on plasmids purified from Moraxella bovis isolates GRS, Newport, and IBH64. It was determined from single and double digestions of plasmid DNA that GRS and Newport isolates carried 3 large plasmids having molecular sizes of 43.8, 41.3, and 32.8 kilobases (kb). Digestion of the 3 large plasmids and restriction endonucleases Hae III, HindIII, Nde I, and Ava I strongly indicated that these isolates shared structurally identical large plasmids. Timed single digestions with Ava I revealed that the IBH64 isolate carried 2 large plasmids having molecular sizes of 45 and 32.8 kb. The 32.8-kb plasmid was the only large plasmid that appeared to be shared by all 3 M bovis isolates. Two isolates, Newport and IBH64, carried small plasmids in addition to the large plasmids. Restriction maps were constructed for the 43.8-, 41.3-, and 32.8-kb plasmids.     

Outer membrane protein antigens of Moraxella bovis

Outer membranes were isolated from bovine isolates and type strains of Moraxella bovis, M phenylpyruvica, M lacunata, and M ovis by sodium N lauroyl sarcosinate extraction and differential centrifugation. Analysis of outer membranes from these organisms by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis revealed that all M bovis isolates shared a common polypeptide pattern that was readily distinguishable from other Moraxella spp. Nine major outer membrane protein bands were identified by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis analysis of M bovis. Immunoblotting of protein antigens of M bovis revealed several outer membrane proteins that seemed to be common antigens of all M bovis isolates.     

Serologic and protective characterization of Moraxella bovis pili

This study was conducted to determine the protective nature of purified M. bovis EPP 63 pili in controlling experimentally induced Infectious Bovine Keratoconjunctivitis, and to determine antigenic similarity of pili isolated from various M. bovis isolates. Ten calves were vaccinated twice, 28 days apart, with 5.0 mg (protein) EPP 63 purified pili. Ten calves were maintained as non-vaccinated controls. All calves were exposed to ultraviolet light prior to challenge. The calves were challenged by instilling approximately 2.0 X 10(8) CFU of EPP 63 piliated organisms into the conjunctival sac. Antisera to respective pili types were prepared by immunizing the rabbits with purified pili from M. bovis strains EPP 63, FLA 64, IBH 68, MED 72 and ATCC 10900. Rabbit serum was evaluated for cross reactivity by enzyme-linked immunosorbent assay (ELISA). Purity of pili preparations was demonstrated on SDS-PAGE gels. Molecular weight of pili subunit was determined to be approximately 20,000 for EPP 63, 19,500 for IBH 68 and ATCC 10900, and 17,500 for FLA 64 and MED 72. One of 10 (10%) calves vaccinated with EPP 63 purified pili, and 6 of 10 (60%) nonvaccinated controls developed IBK, respectively. Average eye scores for vaccinates and controls were 0.05 and 0.85, respectively. Significant cross-reaction was found between EPP 63 and MED 72 pili. FLA 64 and ATCC 10900 were similar; however, antiserum to IBH 68 pili showed some degree of cross reaction with other pili.     

Isolation of Chlamydia psittaci and Moraxella bovis from infectious keratoconjunctivitis in lambs

Out of 189 lambs in the flock, 25 animals suffered from bilateral or unilateral conjunctivitis, or keratoconjunctivitis. By serological examination (RVK), positive levels of antibodies to the group-specific antigen of Chl. psittaci were found in three out of six lambs examined by laboratory methods. Bacteriological examination of eye smears of six lambs showed in four cases the infection by microorganisms of Moraxella bovis. Smears from the conjunctivas of these lambs were after preparation instilled in the yolk sacs of six to seven days old chicken embryos. One strain of Chlamydia psittaci was isolated from the same material as Moraxella bovis.     

An RTX operon in hemolytic Moraxella bovis is absent from nonhemolytic strains

Pathogenic isolates of Moraxella bovis express a calcium-dependent transmembrane pore forming cytotoxin that is an RTX toxin encoded by mbxA. The DNA flanking mbxA was cloned and sequenced to determine if M. bovis contained a classical RTX operon. Open reading frames (ORFs) with deduced amino acid sequence homology to putative activation (RTX C) and transport (RTX B and D) proteins were identified and have been designated MbxC, MbxB, and MbxD, respectively. Thus, hemolytic M. bovis contains a typical RTX operon comprised of four genes arranged (5'-3') mbxCABD. In addition, the deduced amino acid sequences of DNA flanking mbxCABD revealed ORFs with amino acid sequence similarity to transposases (5'). At the 3' end of the mbx gene cluster, an ORF with homology to bacterial tolC genes was identified. Thus, as with the cya RTX operon of Bordetella pertussis, M. bovis appears to have a secretion accessory protein linked to RTX genes. Analysis of genomic DNA isolated from 5 nonhemolytic M. bovis strains by PCR and Southern blotting revealed the absence of mbxCABD. These strains did, however, amplify with primers specific for the 5' region flanking mbxC. M. bovis harbors a classical RTX operon that is absent in nonhemolytic strains.     

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.     

Cloning and characterization of a Moraxella bovis cytotoxin gene

OBJECTIVE: To identify the Moraxella bovis cytotoxin gene. PROCEDURE: Hemolytic and nonhemolytic strains of M. bovis were compared by use of western blotting to identify proteins unique to hemolytic strains. Oligonucleotide primers, designed on the basis of amino acid sequences of 2 tryptic peptides derived from 1 such protein and conserved regions of the C and B genes from members of the repeats in the structural toxin (RTX) family of bacterial toxins, were used to amplify cytotoxin-specific genes from M. bovis genomic DNA. Recombinant proteins were expressed, and antisera against these proteins were produced in rabbits. RESULTS: Several proteins ranging in molecular mass from 55 to 75 kd were unique to the hemolytic strain. An open reading frame encoding a 927-amino acid protein with a predicted molecular mass of 98.8 kd was amplified from M. bovis genomic DNA. The deduced amino acid sequence encoded by this open reading frame was homologous to RTX toxins. Antisera against the recombinant carboxy terminus encoded by this open reading frame neutralized hemolytic and cytolytic activities of native M. bovis cytotoxin. CONCLUSIONS AND CLINICAL RELEVANCE: A gene was identified in M bovis that encodes a protein with sequence homology to other RTX toxins. Results of cytotoxin neutralization assays support the hypothesis that M. bovis cytotoxin is encoded by this gene and belongs in the RTX family of bacterial exoproteins. Identification of this gene and expression of recombinant cytotoxin could facilitate the development of improved vaccines against infectious bovine keratoconjunctivitis.