Moraxella Bovis Research Articles

Molecular diversity of Moraxella bovis isolated from Brazil, Argentina and Uruguay over a period of three decades

The molecular profile of 30 Moraxella bovis strains, recovered from outbreaks of infectious bovine keratoconjunctivitis in Argentina, Brazil and Uruguay between 1974 and 2001, was determined through randomly applied polymorphic DNA (RAPD) analysis. Molecular profiles of nine strains recovered after 1990 varied from those recovered before 1990. The profiles of 13 strains (48%) differed from those of three vaccinal strains extensively used since 1984 in Argentina and Uruguay. Eight Argentinean strains, one from Brazil and two from Uruguay had identical RAPD profiles. Strains belonging to different serogroups had identical RAPD profiles, demonstrating that this technique was not able to discriminate among strains with low cross-reactivity indices. RAPD may be helpful in the primary characterization of M. bovis strains, but it does not replace serological characterization.

Crystal structure of MboIIA methyltransferase.

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.

Molecular cloning and characterization of a 79-kDa iron-repressible outer-membrane protein of Moraxella bovis

Moraxella bovis expresses an iron-repressible 79-kDa outer-membrane protein, IrpA. DNA and N-terminal amino acid sequence analysis indicate that IrpA is closely related to FrpB of Neisseria meningitidis, FetA of Neisseria gonorrhoeae and CopB of Moraxella catarrhalis. The results of manganese mutagenesis and a gel-shift assay suggested that the transcription of irpA is negatively regulated by the ferric uptake regulator. The insertion of an antibiotic resistance cassette into the irpA gene affected the strain’s ability to utilize bovine transferrin and lactoferrin. IrpA was detected in geographically diverse clinical isolates, and the antigenicity of IrpA was conserved in all the isolates tested. Therefore, IrpA may have potential as a candidate vaccine.

Moraxella bovis: influência das características genotípicas e fenotípicas no controle da ceratoconjuntivite infecciosa bovina

A Ceratoconjuntivite Infecciosa Bovina (CIB) continua sendo a mais importante enfermidade ocular dos bovinos da região do MERCOSUL. O agente etiológico da CIB, a bactéria Moraxella bovis, apresenta diferenças genéticas que determinam variações antigênicas e de susceptibilidade aos fármacos, que dificultam seu controle. São discutidos, neste trabalho, resultados de estudos moleculares, antigênicos e de susceptibilidade a antimicrobianos realizados com cepas isoladas durante os últimos vinte anos na região e comparados com os de outras regiões.

Identification of aMoraxella catarrhalisOuter Membrane Protein Exhibiting Both Adhesin and Lipolytic Activities

The UspA1 and Hag proteins have previously been shown to be involved in the ability of the Moraxella catarrhalis wild-type strain O35E to bind to human Chang and A549 cells, respectively. In an effort to identify novel adhesins, we generated a plasmid library of M. catarrhalis DNA fragments, which was introduced into a nonadherent Escherichia coli strain. Recombinant E. coli bacteria were subsequently enriched for clones that gained the ability to bind to Chang and A549 cells, yielding the plasmid pELFOS190. Transposon mutagenesis of this plasmid identified the potential adhesin gene mcaP (M. catarrhalis adherence protein). Sequence analysis revealed that McaP is related to autotransporter proteins and has substantial similarity with the GDSL family of lipolytic enzymes, particularly the Moraxella bovis phospholipase B. Expression of the mcaP gene product by E. coli increased adherence to Chang, A549, and 16HBE14o(-) polarized human bronchial cells 50- to 100-fold. Spectrophotometric assays with p-nitrophenol derivatives also demonstrated that McaP is an esterase. Furthermore, thin-layer chromatography revealed that McaP cleaves both phosphatidylcholine and lysophosphatidylcholine. McaP releases fatty acids and glycerophosphorylcholine upon cleavage of phosphatidylcholine, thus exhibiting phospholipase B activity. The construction and characterization of isogenic M. catarrhalis O35E mutants demonstrated that the lack of McaP expression abolishes esterase activity and considerably decreases adherence to several human cell lines.

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.

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.

Bacterial lactoferrin receptors: insights from characterizing the Moraxella bovis receptors.

Moraxella bovis is the causative agent of infectious conjunctivitis in cattle. Moraxella bovis isolates were shown to specifically bind bovine lactoferrin (bLf) and bovine transferrin (bTf) and to use these proteins as a source of iron to support the growth of iron-limited cells. Affinity isolation experiments with immobilized bTf yielded two proteins readily resolved by SDS-PAGE analysis, whereas only a single band of approximately 100 kDa was detected when immobilized bLf was used as the affinity ligand. Using a novel cloning strategy, regions containing the genes encoding the lactoferrin (Lf) and transferrin (Tf) receptor proteins were isolated and sequenced, demonstrating that they both consisted of two genes, with the tbpB or lbpB gene preceding the tbpA or lbpA gene. The cloned lbp genes were used to generate isogenic mutants deficient in lactoferrin binding protein A and (or) B, and the resulting strains were tested in growth and binding assays. The isogenic mutants were deficient in their use of bLf for growth and had substantially diminished bLf binding capability. The predicted amino acid sequence from the segment encoding Lf binding protein B revealed an internal amino acid homology suggesting it is a bi-lobed protein, with a C-lobe enriched in acidic amino acids, but without the evident clustering observed in Lf-binding proteins from other species.

Detection of shared magnetic antigenic determinants on whole Moraxella bovis pili by use of antisera to cyanogen bromide-cleaved M. bovis pilus protein.

To determine the ability of antisera against cyanogen bromide-cleaved pili from 4 strains of Moraxella bovis to react with whole or nondenatured pili. Antisera to 4 strains of M. bovis produced by New Zealand White rabbits. Pili from 4 strains of M. bovis were collected and purified. Pilus proteins (pilin) were cleaved, using cyanogen bromide. Whole pilus and cyanogen bromide-cleaved pilin were injected into rabbits. Antisera were serially diluted, reacted with 4 strains of M. bovis, and examined by immunoelectron microscopy and indirect immunofluorescence. Antisera to whole pili aggregated and distorted pili from homologous strains, but pili from heterologous strains were unaffected. Antisera to cleaved pilin fragments resulted in partial aggregation and thickening of homologous and heterologous pili, suggestive of heterospecific antibodies. Attachment of antibodies to pili was detected by indirect immunofluorescence, indicating a strong reaction of antisera to whole pili with homologous pili. Weak cross-reactions were evident with certain heterologous strains. In contrast, antisera to cleaved pilin fragments reacted strongly with pili from homologous and heterologous strains. We detected shared antigenic determinants on pili from various strains of M. bovis that were not immunogenic in intact pili. These sites were immunogenic after cleavage of pilus protein with cyanogen bromide, and antisera produced to protein fragments reacted with whole pili from heterologous strains of the organism. Vaccines produced from cyanogen bromide-treated pili may induce broader immunity against infectious bovine keratoconjuctivitis than that provided by currently available vaccines.

Immunoblot analysis of cyanogen bromide-cleaved Moraxella bovis pilin reveals presence of shared antigenic determinants on pili from heterologous strains

Moraxella bovis pilus proteins, collected and purified from four strains of M. bovis, were cleaved with cyanogen bromide. Two major fragments were produced. Antisera were produced in rabbits to the pilin protein fragments and to whole uncleaved pili from these strains. Immunoblots of whole and cyanogen bromide-cleaved pilin were reacted with the homologous and heterologous antisera to whole pili and cleaved pilin. Antisera to whole pili reacted strongly with homologous pilin. Weaker and inconsistent reactions were detected with heterologous pilin. Antisera produced to cyanogen bromide-cleaved pilin proteins reacted strongly with homologous and heterologous pilin fragments and uncleaved pilin proteins. These findings demonstrate the presence of conserved antigenic determinants on pili from heterologous strains that are non-immunogenic in the intact pilus but are immunogenic after treatment with cyanogen bromide. Cyanogen bromide-treated pilus preparation might have potential as a vaccine because antibodies are induced against heterologous strains of M. bovis, whether these cross-reactive antibodies are protective remains to be determined.

Abstract

Detection of cell detachment activity induced by Moraxella bovis Marrion, R M. M. & Riley, L. K. (2000) Detection of cell detachment activity induced by Moraxella bovis. American Journal of...

Infectious Bovine Keratoconjunctivitis and Lymphofollicular Hyperplasia of the Third Eyelid in Heifers

On a dairy cattle farm, infectious bovine keratoconjunctivitis was diagnosed in 29 (24%) calves and heifers aged from 2 weeks to 1 year old. The highest infection rate (18%) occurred in animals aged 3-6 months. The bacteriological examination of swabs from the affected animals yielded several species of bacteria: Moraxella bovis, Neisseria ovis, N. cuniculi, plasma coagulase-negative Staphylococcus spp., alpha-haemolytic Streptococcus spp., Arcanobacterium pyogenes and Escherichia coli. Moraxella bovis and N. ovis were the most common isolates. Hyperplasia of the lymphatic tissue of the third eyelid in the form of nodules 7-8 mm in diameter was diagnosed in two heifers aged 8 and 10 months.

Detection of cell detachment activity induced by Moraxella bovis.

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. Cultured hamster corneal epithelial cells, bovine epithelial cells, and several transformed cell lines exposed to culture filtrate from a pathogenic isolate of M bovis. 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. 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. 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.

Identification of type 4 fimbriae in Actinobacillus pleuropneumoniae.

Type 4 fimbriae have been identified on the cell surface of Actinobacillus pleuropneumoniae by electron microscopy and N-terminal sequencing analysis. A. pleuropneumoniae type 4 fimbrial subunit protein, purified from cell cultures and from outer membrane preparations, reacted with polyclonal antibody raised against type 4 fimbriae of Moraxella bovis on Western blots. N-terminal sequence analysis of the purified 17 kDa type 4 fimbrial subunit protein, named ApfA, revealed the first 12 amino acids to be identical to those of other type 4 fimbrial subunit proteins.

Identification of type 4 fimbriae in Actinobacillus pleuropneumoniae

Type 4 fimbriae have been identified on the cell surface of Actinobacillus pleuropneumoniae by electron microscopy and N-terminal sequencing analysis. A. pleuropneumoniae type 4 fimbrial subunit protein, purified from cell cultures and from outer membrane preparations, reacted with polyclonal antibody raised against type 4 fimbriae of Moraxella bovis on Western blots. N-terminal sequence analysis of the purified 17 kDa type 4 fimbrial subunit protein, named ApfA, revealed the first 12 amino acids to be identical to those of other type 4 fimbrial subunit proteins.

Epidemiology of infectious keratoconjunctivitis in cattle in south-east Ethiopia.

The aim of the study was to determine the prevalence of infectious keratoconjunctivitis on cross-breed dairy farms and to evaluate factors that influence its distribution. A total of 11 dairy farms and one cattle cross-breeding farm in different parts of Arsi region, south-east Ethiopia were clinically and bacteriologically investigated from February 1989 to December 1990. Out of 5221 local zebu and cross-breed dairy animals, 110 were found to have different degrees of infectious bovine keratoconjunctivitis. Of these, 94 (85.5%) were unilateral and 16 (14.5%) were bilateral. The prevalence of the disease was significantly (P < 0.001) influenced by age and breed; high exotic blood level and younger age groups were mainly affected. However, severity of the disease was not significantly associated with age (P > 0.750). Moraxella bovis was isolated from 88 (80%) of the 110 bacteriologically positive cases. Of these isolates, 61 (69%) were haemolytic and 27 (31%) were non-haemolytic strains. Infection with M. bovis was unilateral. Concurrent infections with M. bovis included Actinomyces pyogenes 23 (21%), Staphylococcus aureus 17 (15%), Pasteurella haemolytica nine (8%), Escherichia coli eight (7%) and Proteus spp. four (3.6%).

Analyses of lipopolysaccharides, outer membrane proteins and DNA fingerprints reveal intraspecies diversity in Moraxella bovis isolated in Argentina

Intra-specific diversity within Moraxella bovis was investigated analysing DNA fingerprints, outer membrane proteins (OMP) and lipopolysaccharides (LPS) profiles. Three collection strains and 57 isolates of M. bovis, collected during 3 years from cattle with infectious bovine keratoconjunctivitis (IBK) symptoms, from diverse geographical locations of Argentina, were examined. The LPS and OMP profiles were studied through SDS–PAGE analysis and genotype was determined by PCR-DNA fingerprinting. Genotyping identified five DNA types while analysis of LPS and OMP profiles identified three rough LPS types and three OMP types among the 60 isolates of M. bovis including the three collection strains. None of the three methods employed to assess diversity was discriminating when used alone because the degree of heterogeneity in each group of surface structures was limited, but when data of each typing method were combined, 15 distinct subgroups were determined. This subgrouping was clearly able to differentiate isolates of the same genotype. These typing methods appear to be useful to assess different aspects of the disease such as the diversity within a population of M. bovis associated to epidemic conditions, track the causal agent in an outbreak of the disease, monitoring vaccination programs and studies on virulence.

Multiple DNA Binding Activities of the Novel Site-specific Recombinase, Piv, from Moraxella lacunata

The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the lambda Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.

Infectious bovine keratoconjunctivitis: a review.

The economic impact of infectious bovine keratoconjunctivitis (IBK) warrants continued investigation of the mechanisms by which Moraxella bovis survives on and colonizes the corneal surface. Virulent strains of M bovis produce hemolysin and exhibit different plasmid profiles than nonvirulent strains. Interactions among host, environment, vector, season, and concurrent infection influence the prevalence of IBK. Mycoplasma sp. or infectious bovine rhinotracheitis virus may enhance or hasten the disease process. The manifestations of IBK may range from mild conjunctivitis to severe ulceration, corneal perforation, and blindness. Treatment of IBK is dictated by economic considerations, intended animal use, and feasibility of administration. Antibiotic therapy is aimed at achieving drug concentrations in tears to meet or exceed the minimum inhibitory concentration for prolonged periods. At present, IBK is not a preventable disease. Affected animals must be separated from the herd and vector control vigorously instituted. Carrier animals must be identified and removed from the herd. Vaccination trials have been unsuccessful because of pili antigen cross-reactivity, variable strains, and uncontrolled environmental factors. Recent investigations have determined that M bovis may utilize host iron sources via iron-repressible outer membrane proteins and siderophores for growth. Elucidation of normal defense mechanisms of the bovine eye may lead to new strategies to enhance the immune response against M bovis.

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 MIC 90 of ≤1 mg/l to erythromycin, ceftiofur, and ampicillin; 4 mg/l to tilmicosin; 16 mg/l to tylosin and gentamicin; and had MIC 90s 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.