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.

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.

Characterization of hemolysin of Moraxella bovis using a hemolysis-neutralizing monoclonal antibody.

A concentrated bacterial culture supernatant from the hemolytic Moraxella bovis strain UQV 148NF was used to immunize mice and generate monoclonal antibodies (MAbs). One, MAb G3/D7, neutralized the hemolytic activity of M. bovis and recognized a 94-kDa protein by Western blot analysis in hemolytic M. bovis strains representing each of the different fimbrial serogroups. Exposure of corneal epithelial cells to M. bovis concentrated culture supernatants demonstrated a role for an exotoxin in the pathogenesis of infectious bovine keratoconjunctivitis, while neutralization of hemolytic and cytotoxic activities by MAb G3/D7 implies that these activities are related or have common epitopes. The action of M. bovis hemolysin was further characterized in sheep erythrocyte preparations with a binding step and Ca(2+) required for lysis to proceed, similar to the RTX family of bacterial exotoxins. Neutralization of lytic activity in vitro is evidence for the presence of M. bovis antigens, which may be capable of protecting cattle from the development of infectious bovine keratoconjunctivitis.

Toxins of Moraxella bovis: effect on substrates and cells

Culturas totais em meio líquido e filtrados, de 12 cepas de Moraxella bovis, foram testados para determinar a presença de gelatinase, DNAse, lecitinase e dermonecrotoxinas. Foi também estudado seu efeito citotóxico sobre 5 culturas celulares de linhagem. Nas culturas totais demonstrou-se a presença de gelatinase e DNAse. Os filtrados produziram dermonecrose em cobaias brancas e efeito citotáxico em células BHK 21 (Cl.13), mas näo nas outras linhagens. A atividade tanto das culturas totais quanto dos filtrados foi eliminada por aquecimento a 100oC. Discute-se a participaçäo destas toxinas na patogênese e no controle da ceratoconjuntivite infecciosa bovina

A haemolytic cell-free preparation of Moraxella bovis confers protection against infectious bovine keratoconjunctivitis.

Protection conferred by a cell-free preparation from a haemolytic Moraxella bovis isolate, UQV 148NF, was compared to an equivalent fraction from a non-haemolytic M. bovis isolate, Gordon 26L3, and to a recombinant DNA-derived pili vaccine. Three groups of ten calves were vaccinated twice with one of the three preparations and, together with ten non-vaccinated calves, challenged with virulent M. bovis isolate Dal 2d. Compared to the control group, significant protection was observed in the group receiving the pili vaccine and the group receiving the preparation from haemolytic isolate, UQV 148NF.

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.

Infection rates, disease frequency, pilin gene rearrangement, and pilin expression in calves inoculated with Moraxella bovis pilin-specific isogenic variants.

Pili have been implicated as virulence factors that result in increased infectivity of Moraxella bovis, the causative agent of infectious bovine keratoconjunctivitis (IBK). Healthy calves' eyes were inoculated with I- or Q-piliate or nonpiliate M bovis Epp63 to compare the pathogenicity of these isogenic variants. Pathogenicity was determined by the rate of persistent M bovis infection and the prevalence of clinical IBK. Inoculation with M bovis expressing the Q pili resulted in the highest frequency of infection and IBK, whereas I-piliate M bovis elicited a lower rate and nonpiliate M bovis did not result in infection. In vivo pilin gene rearrangement and pilin-type switching were evaluated by DNA hybridization and immunoblot. Gene rearrangement and type switching varied dependently, and were observed only in eyes inoculated with Q-piliate M bovis. This study suggests that Q pili are specific for colonization of bovine corneal epithelium, whereas I pili enable maintenance of an established infection.

Q pili enhance the attachment of Moraxella bovis to bovine corneas in vitro.

Moraxella bovis, the causative agent of infectious bovine keratoconjunctivitis, exhibits several virulence factors, including pili, haemolysin, leukotoxin, and proteases. The pili are filamentous appendages which mediate bacterial adherence. Prior studies have shown that Q-piliated M. bovis Epp63 are more infectious and more pathogenic than I-piliated and non-piliated isogenic variants, suggesting that Q pili per se, or traits associated with Q-pilin expression, promote the early association of Q-piliated bacteria with bovine corneal tissue. In order to better evaluate the role of Q pili in M. bovis attachment, several M. bovis strains and a recombinant P. aeruginosa strain which elaborates M. bovis Q pili but not P. aeruginosa PAK pili, were evaluated using an in vitro corneal attachment assay. For each strain tested, piliated organisms attached better than non-piliated bacteria. M. bovis Epp63 Q-piliated bacteria adhered better than either the I-piliated or non-piliated isogenic variants. Finally, recombinant P. aeruginosa organisms elaborating M. bovis Q pili adhered better than the parent P. aeruginosa strain which did not produce M. bovis pili. These results indicate that the presence of pili, especially Q pili, enhances the attachment of bacteria to bovine cornea in vitro.