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.     

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.     

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.     

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.     

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.     

Detection of cell detachment activity induced by Moraxella bovis

OBJECTIVE: To characterize the effect that filtrate obtained from cultures of Moraxella bovis has on cultured corneal epithelial cells and other types of cultured mammalian cells. SAMPLE POPULATION: Cultured hamster corneal epithelial cells, bovine epithelial cells, and several transformed cell lines exposed to culture filtrate from a pathogenic isolate of M bovis. PROCEDURE: Moraxella bovis was cultured, and bacteria were removed by filtration. The resulting bacterial culture filtrate was incubated with various types of cultured cells, and effects of the filtrate on detachment of various mammalian cell types was quantified by the use of neutral red dye. Additionally, bacterial culture filtrate was treated with protease inhibitors as well as trypsin and heat prior to incubation with cultured mammalian cells. RESULTS: Bacterial culture filtrate of M bovis caused all types of cultured cells to detach from each other and from the substrate, with the maximal effect evident 2 hours after initiating incubation. Detached cells were alive, and detachment was reversible. Serine protease inhibitors (phenylmethylsulfonylfluoride and alpha2-macroglobulin) inhibited cell detachment attributable to bacterial culture filtrate. Heating and treatment with trypsin also inhibited cell detachment. CONCLUSIONS AND CLINICAL RELEVANCE: Moraxella bovis produces a soluble factor that causes reversible detachment of cultured cells. This activity may play a role in the pathogenesis of infectious bovine keratoconjunctivitis.     

Conjunctival lesions caused by Moraxella bovis in gnotobiotic calves

Hemolytic Moraxella bovis was instilled into the conjunctival sac of gnotobiotic calves, and conjunctivae were sampled serially after infection. Bilateral lesions developed in seven of eight infected calves. Histologically, M. bovis was first seen within swollen epithelial cells near the lid margins and occasionally within superficial epithelium in other areas. Conjunctival erosions and ulcers were seen in later stages. Scanning electron microscopy showed M. bovis in pits on surfaces of epithelial cells and in erosions on palpebral conjunctivae; lesions were prominent near lid margins. By transmission electron microscopy, M. bovis was seen within swollen epithelial cells near lid margins; many epithelial cells had undergone cytolysis. This study demonstrates that virulent M. bovis can invade bovine conjunctival epithelial cells and cause conjunctivitis in the absence of injurious ultraviolet irradiation or other predisposing environmental factors.     

Plasmid content of piliated and nonpiliated forms of Moraxella bovis

Plasmid profiles were compared between nonpiliated and piliated forms of Moraxella bovis isolates. The piliated form of M bovis isolate IBH64 contained 1 fewer plasmid than did the nonpiliated form. Piliated and nonpiliated cells of IBH64 contained plasmids having molecular size of 45, 32.8, 4.9, and 4.6 kilobases (kb). Single- and double-restriction endonuclease digestion by Ava I and Nde I indicated that the size of the additional plasmid carried by the nonpiliated form of IBH64 was approximately 43.6 kb. The M bovis isolates, Newport and GRS, contained the same number of plasmids in either their piliated or nonpiliated form.     

Adherence of Moraxella bovis to cell cultures of bovine origin

The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.     

Serological characterization of strains of Moraxella bovis using double immunodiffusion.

Sera were produced in rabbits against nine Moraxella bovis strains isolated in Brazil and three in the United States. Antigens were prepared for double immunodiffusion tests by thawing concentrated suspensions of the strains. Sera were tested against homologous and heterologous antigen preparations by the double immunodiffusion method. Sera showing precipitin bands with heterologous antigens were absorbed. Antigenic differences were detected between the strains and a provisional grouping of strains of M. bovis was suggested on the basis of antigenic composition. Differences between isolates from different geographical locations were found and some strains appeared antigenically more complex than others. The relevance of this work to vaccine production was suggested.     

Whole-bacterial cell enzyme-linked immunosorbent assay for cell-bound Moraxella bovis pili

Infectious bovine keratoconjunctivitis (IBK), caused by Moraxella bovis, is a disease of major importance in cattle industry. M. bovis has several virulence factors among which pili are crucial antigen for the protective capacity of vaccines against this disease. The production of vaccines against IBK therefore requires a reliable technique for cellular piliation level assessment on cells to be included as vaccine components. In this study we describe a specific whole-bacterial cell enzyme-linked immunosorbent assay (bact-ELISA) capable of detecting pili antigen on M. bovis cell surface. A sequential competitive bact-ELISA was developed using highly piliated M. bovis cells as antigen. Samples to be analyzed were allowed to react with anti-pilus serum prior to incubation in wells coated with piliated cells of M. bovis. This assay proved useful for the rapid, sensitive and reproducible evaluation of piliation on M. bovis cells, and represents an important tool for cellular piliation monitoring daburing M. bovis cells production in stirred bioreactors.     

Characterisation and quantitation of pilus antigens of Moraxella bovis by ELISA

Hyperimmune serums raised in rabbits to purified pili from 9 Australian and 2 American strains of Moraxella bovis from infectious bovine keratoconjunctivitis (IBK) affected herds were used to study the degree of binding between combinations of antigen and antiserum in a conventional enzyme linked immunosorbent assay (ELISA). With the aid of appropriate absorption tests major antigenic differences among pili were found permitting 6 distinct serogroups to be recognised. Further, production of specific antiserums to representative strains of each serogroup in goats facilitated the development of a double antibody sandwich ELISA which could be used to quantitate pilus expression of a given strain of M. bovis, or to differentiate pilus serogroups of 22 strains of M. bovis obtained from a total of 12 Australian herds. Most isolates were found to belong to serogroups designated IV and V. One strain from the United States of America showed total homology with Australian serogroup IV while the other showed some cross-reactivity with serogroups V and VI.