Moraxella Bovis Research Articles

Cloning and characterization of theMbollrestriction-modification system

The two genes encoding the class IIS restriction-modification system MboII from Moraxella bovis were cloned separately in two compatible plasmids and expressed in E. coli RR1 delta M15. The nucleotide sequences of the MboII endonuclease (R.MboII) and methylase (M.MboII) genes were determined and the putative start codon of R.MboII was confirmed by amino acid sequence analysis. The mboIIR gene specifies a protein of 416 amino acids (MW: 48,617) while the mboIIM gene codes for a putative 260-residue polypeptide (MW: 30,077). Both genes are aligned in the same orientation. The coding region of the methylase gene ends 11 bp upstream of the start codon of the restrictase gene. Comparing the amino acid sequence of M.MboII with sequences of other N6-adenine methyltransferases reveals a significant homology to M.RsrI, M.HinfI and M.DpnA. Furthermore, M.MboII shows homology to the N4-cytosine methyltransferase BamHI.

Identification, cloning, and sequencing of piv, a new gene involved in inverting the pilin genes of Moraxella lacunata

Moraxella lacunata is a bacterium that is a causative agent of human conjunctivitis and keratitis. We have previously cloned the Q and I pilin (formerly called beta and alpha pilin) genes of Moraxella bovis and determined that an inversion of 2 kilobases (kb) of DNA determines which pilin gene is expressed. Using an M. bovis pilin gene as a hybridization probe to screen a lambda ZAP library of M. lacunata DNA, we have isolated a clone that not only contains the entire type 4 pilin gene inversion region of M. lacunata but inverts the 2-kb region on a plasmid subclone (pMxL1) in Escherichia coli. Deletion derivatives of pMxL1 yielded some plasmids that still had the entire inversion region but were phase locked into one or the other of the two potential orientations. Similarly, insertions of a 2-kb streptomycin-resistant element (omega) within some regions outside of the inversion also resulted in phase-locked plasmids. These deletions and insertions thus localize a probable invertase necessary for the inversion event. The region was sequenced, and an open reading frame with over 98% DNA sequence homology to an open reading frame that we previously found in M. bovis and called ORF2 appeared to be a strong candidate for the invertase. This conclusion was confirmed when a plasmid containing the M. bovis ORF2 supplied, in trans, the inversion function missing from one of the M. lacunata phase-locked inversion mutants. We have named these putative invertase genes piv(ml) (pilin inversion of M. lacunata) and piv(mb) (pilin inversion of M. bovis). Despite previously noted sequence similarities between the M. bovis sites of inversion and those of the Hin family of invertible segments and a 60-base-pair region within the inversion with 50% sequence similarity to the cin recombinational enhancer, there is no significant sequence similarity of the Piv invertases to the Hin family of invertases.

Effectiveness of two commercial infectious bovine keratoconjunctivitis vaccines

SUMMARY Two commercially available infectious bovine keratoconjunctivitis (ibk) vaccines were evaluated for their effectiveness in protecting cattle from disease caused by experimental challenge exposure and natural transmission of Moraxella bovis infections. The study was conducted as 2 experiments, using a total of 81 cattle that were culture-negative for M bovis prior to vaccination. In each experiment, young adult cattle were randomly allotted to 4 groups. Each calf in groups 1 and 2 was vaccinated according to the vaccine manufacturer's directions. Groups 3 and 4 were unvaccinated controls. Three weeks after the last vaccination, each calf in groups 1 and 3 was experimentally challenge exposed by dropping a suspension of viable cells of a virulent strain of M bovis directly onto the corneal surface of each eye. Calves in all 4 groups were then commingled in open pastures so that calves in groups 2 and 4 could be naturally exposed to the calves with experimentally induced infections. Each calf was examined for signs of ocular disease on a regular basis by 2 experienced clinicians who scored each eye for severity of disease on the basis of a prearranged scale. Neither clinician was aware of the vaccination or exposure status of the calf nor to which experimental group they belonged. Lacrimal secretions were collected regularly to determine the number of eyes in which the virulent organism became established. Moraxella bovis with bacterial cultural characteristics similar to those of the virulent strain placed in the eyes of groups 1 and 3 was cultured from ≥ 83% of the eyes of calves in all groups. The incidence of clinical ibk was ≥ 50% in each group. There was no significant difference in the ability of vaccinated calves to resist experimental or natural challenge infection. Compared with that in nonvaccinated controls, no decreased incidence or severity of clinical ibk was noticed in vaccinated calves. Though the challenge strain used was not homologous to those used to prepare the vaccine, it was no different than those that may be expected to cause disease under field conditions.

Characterization of the pilin gene of Moraxella bovis Dalton 2d and expression of pili from M. bovis in Pseudomonas aeruginosa

The pilin gene of Moraxella bovis Dalton 2d was isolated by cloning in Pseudomonas aeruginosa. The nucleotide sequence of this gene encodes a prepilin of 156 amino acid residues. When high levels of pilin were expressed from the gene in P. aeruginosa, by using the pL promoter of bacteriophage lambda inserted upstream of the coding sequence, pili which were indistinguishable from pili of M. bovis were produced.

Pili (fimbriae) of Branhamella species

purpose: Pili (fimbriae) have frequently been found to be involved in the attachment of bacteria to mucosal epithelial cells, an important initial step in the disease process. The purpose of this study was to determine if Branhamella catarrhalis expresses type 4 pili. materials and methods: Piliated B. catarrhalis phenotypic characteristics of colony morphology, agar corrosion, twitching motility, competence for deoxyribonucleic acid (DNA) transformation, autoagglutination, and pellical formation were observed. DNA was isolated from Branhamella spp. and used in genomic Southern hybridizations with a Moraxella bovis pilin gene as a probe. Electron microscopy of negatively stained bacteria was carried out to visualize pili. results: B. catarrhalis has several (but not all) of the phenotypic characteristics that are related to the presence of type 4 (MePhe) pili in closely related Moraxella spp., including competence for DNA transformation, autoagglutination, pellicle formation, colony morphology, and pitting of agar. The one phenotype we have not found that is generally characteristic of type 4 piliated bacteria is twitching motility. Genomic Southern hybridization analysis using a cloned M. bovis Q pilin gene as a probe reveals DNA homologous to the Q pilin gene in B. catarrhalis, Branhamella ovis, Branhamella caviae, and Branhamella cuniculi. Examination of B. catarrhalis strain ATCC25240 by electron microscopy reveals two different kinds of pili. One kind appears similar to other type 4 pili, whereas a second class is short pili extending outward from all portions of the bacteria. conclusion: Phenotypic, electron-microscopic, and hybridization data are all consistent with type 4 pili being present on some B. catarrhalis strains.

Topically applied benzathine cloxacillin for treatment of experimentally induced infectious bovine keratoconjunctivitis

SUMMARY The efficacy of an ophthalmic ointment containing benzathine cloxacillin for treatment of infectious bovine keratoconjunctivitis was determined in 2 experiments. In the first experiment, Holstein calves (n = 6/group) were inoculated with Moraxella bovis and treated on postinoculation days 3 and 6 with either topically applied benzathine cloxacillin (250 mg/eye) or long-acting oxytetracycline formulation (20 mg/kg of body weight, im). A third group of inoculated calves remained untreated as controls. For the second experiment, 4 groups of calves (n = 6/group) were inoculated and treated on postinoculation days 3 and 6 with 50, 125, 250, or 375 mg of benzathine cloxacillin; a fifth untreated group served as controls. Ocular specimens were obtained for microbiologie culture, and eyes were observed and assigned a clinical score daily. Eyes were photographed on alternate days. Ulcer surface area was measured, using a planimeter. In experiment 1, the week-2 ulcer surface area measurements for both groups of treated calves were smaller than those for controls. There was a greater frequency of M bovis isolation from the ocular secretions of controls than from those of benzathine cloxacillin-treated calves during postinoculation weeks 2 and 3. The number of M bovis isolations from the benzathine cloxacillin- and oxytetracycline-treated calves was not significantly different at any sample collection interval. On week 3, the scores of the benzathine cloxacillin-treated calves were smaller than those of controls. In experiment 2, calves of the 250- and 375-mg groups had smaller ulcer surface area measurements than did controls on week 2. By week 3, calves of the 375-mg group had smaller scores than did controls. During postinoculation weeks 1 through 3, M bovis was isolated less frequently from the ocular secretions of calves of the 250- and 375-mg groups than from those of the control calves.

Ocular and serum disposition kinetics of cloxacillin after topical administration of benzathine cloxacillin and intravenous administration of sodium cloxacillin to calves

SUMMARY Disposition kinetics of cloxacillin were examined in calves after topical administration of benzathine cloxacillin and single iv administration of sodium cloxacillin, and the susceptibility of 17 field isolates of Moraxella bovis was measured. For the IV pharmacokinetic phase, sodium cloxacillin was administered at dosage of 10 mg/kg of body weight to male Holstein calves (n = 6, weighing 146 to 170 kg), and serum concentration of cloxacillin was measured thereafter for 10 hours. For the ocular pharmacokinetic phase, 6 calves were given either of 4 benzathine cloxacillin topical formulations consisting of 50-, 125-, 250-, or 375-mg doses. Treatment was repeated every 10 days until all 4 benzathine cloxacillin dosages were tested in the same 6 calves. Blood and tears were collected for 72 hours after each benzathine cloxacillin formulation was administered, and the concentration of cloxacillin in each specimen was measured, using a bioassay. The minimal inhibitory concentration of cloxacillin for 17 field isolates of M bovis was determined by use of an agar pour-plate dilution assay. After single iv administration of sodium cloxacillin, its half-life, body clearance, and volume of distribution were 19.5 ± 12.8 minutes, 18.3 ± 2.2 ml/min·kg, and 496 ± 290 ml/kg, respectively. After topical administration of benzathine cloxacillin, cloxacillin concentration in lacrimal fluid peaked between 30 and 45 minutes and ranged between 963 μg/ml and 3,256 μg/ml for the 125- and 375-mg doses, respectively. There was no detectable cloxacillin activity in the lacrimal fluid of any calf by 36 hours after topical administration of benzathine cloxacillin, and cloxacillin was not detected in the serum at any time. The mean lacrimal fluid cloxacillin concentration for the 4 groups during the first 8 hours was not significantly different; however, by 12 hours, the cloxacillin concentration in tears from calves of the 250- and 375-mg groups was significantly (P < 0.05) greater than that in calves of the 50- and 125-mg groups. Cloxacillin concentration ≥ 3.13 μg/ml was maintained for a significantly (P < 0.05) longer time after treatment, using the 375-mg dose, compared with the 50-mg dose of benzathine cloxacillin. The minimal inhibitory concentration of cloxacillin for 1 isolate was 6.25 μg/ml, but was ≤ 3.13 μg/ml for 16 other M bovis isolates.

Comparative characterization of the leukocidic and hemolytic activity of Moraxella bovis

SUMMARY The cytotoxic effect of Moraxella bovis 118F on bovine neutrophils was evaluated and characterized by use of a 51Cr release assay. Neutrophils harvested from healthy adult cattle were labeled with 51Cr. The leukocidic activity produced by M bovis 118F, a hemolytic strain of M bovis, was heat-labile. A live culture of strain 118F, at a ratio of 100 bacteria/neutrophil, released 97.7% of the 51Cr from labeled neutrophils. Neither a heat-killed preparation of M bovis 118F nor a live or heat-killed preparation of M bovis IBH63 (a nonhemolytic and nonpathogenic strain) induced significant (P > 0.05) release of 51Cr. Moraxella bovis 118F broth culture filtrates prepared for evaluation of leukocidic activity also were evaluated for hemolytic activity. These 2 toxic activities had several characteristics in common. Both were filterable, heat-labile, produced by a hemolytic strain, and were released during early logarithmic phase growth from broth cultures. Leukocidic and hemolytic activities were protected from degradation by phenylmethyl sulfonyl fluoride, a serine protease inhibitor. Leukocidic and hemolytic activities were dependent on calcium ions. Filtrate resulted in 54.1% 51Cr release from labeled neutrophils and contained 646.7 hemolytic U/ml, respectively, when saline (0.85% NaCl) + 10 mM CaCl2 solution was used as diluent. Neither saline solution nor saline + 10 mM MgCl2 solution supported leukocidic or hemolytic activity. Serum, obtained from several calves 10 to 38 days after M bovis inoculation, substantially neutralized leukocidic and hemolytic activities, compared with paired preinoculation serum samples. In addition, no significant difference (P > 0.05) was detected when the ability of each calf's postinfection serum to neutralize leukocidic activity was compared with the ability of the serum to neutralize hemolytic activity.

Plasmid content of piliated and nonpiliated forms of Moraxella bovis

SUMMARY 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.

Sequence analysis of the inversion region containing the pilin genes of Moraxella bovis

Moraxella bovis EPP63 is able to produce two antigenically distinct pili called Q and I pili (previously called beta and alpha pili). Hybridization studies have shown that the transition between the types is due to inversion of a 2.1-kilobase segment of chromosomal DNA. We present the sequence of a 4.1-kilobase region of cloned DNA spanning the entire inversion region in orientation 1 (Q pilin expressed). Comparison of this sequence with the sequence of the polymerase chain reaction-amplified genomic DNA from orientation 2 (I pilin expressed) allows the site-specific region of recombination to be localized to a 26-base-pair region in which sequence similarity to the left inverted repeat of the Salmonella typhimurium hin system was previously noted. In addition, 50% sequence similarity was seen in a 60-base-pair segment of our sequence to the recombinational enhancer of bacteriophage P1, an inversion system related to the hin system of S. typhimurium. Finally, two open reading frames representing potential genes were identified.

Vaccine against infectious bovine keratoconjunctivitis containing cyanogen bromide cleavage fragments of pili of a pathogenic strain of Moraxella bovis

Vaccine against infectious bovine keratoconjunctivitis containing cyanogen bromide cleavage fragments of pili of a pathogenic strain of Moraxella bovis

Identification of Moraxella bovis by using a monoclonal antibody to a lipopolysaccharide epitope

A monoclonal antibody to the lipopolysaccharide of Moraxella bovis is described. In an indirect fluorescent-antibody test, this monoclonal antibody reacted with 39 of 39 strains of M. bovis tested and did not react with 26 nonfermenting gram-negative coccobacilli other than M. bovis. When used in an indirect fluorescent-antibody test, it proved useful for rapid and easy identification of M. bovis.

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.

The concentration of penicillin in bovine conjunctival sac fluid as it pertains to the treatment of Moraxella bovis infection. (I) Subconjunctival injection.

Single dose, subconjunctival injections of procaine penicillin using either skin or conjunctival routes of administration were evaluated in order to compare the duration of antibacterial concentrations at the site of Moraxella bovis infection. Samples of conjunctival sac fluid (CF) were collected using blunted capillary tubes and were periodically assayed for penicillin by an agar-well-diffusion technique. Linear regression lines were constructed for concentration of penicillin-time profiles. Similarity of the regression slopes for each treatment enabled the construction of three common lines by the method of co-variance analysis in order to represent each treatment. Treatments were thereafter compared by the slopes of the common regression lines. A subconjunctival injection of procaine penicillin at a dose of 6 X 10(5) iu in 2 ml, administered either through the skin or through the conjunctiva, produced a peak penicillin concentration in the CF of approximately 8 iu/ml for either route and a duration of therapeutic concentration (DTC) of 67.6 +/- 4.9 and 40 +/- 2.7 h respectively. A dose of 3 X 10(5) iu in 1 ml, administered through the conjunctiva, produced a similar peak but the DTC was reduced to 35 +/- 4.2 h. The results of this study support field practices, as adequate penicillin concentrations were maintained by both techniques investigated.

The concentration of penicillin in bovine conjunctival sac fluid as it pertains to the treatment of Moraxella bovis infection. (II) Topical application.

Sodium benzyl penicillin, procaine penicillin and benethamine penicillin were applied into the bovine conjunctival sac as an aqueous solution or in ointment form in order to study the concentration-time profiles. The series of treatments was repeated in five animals in a random sequence. Penicillin concentration in conjunctival sac fluid (CF) was determined using the agar-well-diffusion assay technique. The data obtained were transformed to linear regression slopes. Similarity of the slopes within treatments (in five different eyes) enabled the construction of four common lines by co-variance analysis to represent each treatment. The regression coefficients of the four common lines were then compared to study the difference between treatments. Topical application of 5,000 iu sodium benzylpenicillin in aqueous solution at a concentration isotonic with 0.9% saline, produced a duration of therapeutic concentration (DTC) in CF of 12.6 +/- 1.5 h. When the same salt or other less water-soluble ones were formulated at the same concentration in an ointment base, the DTC was significantly prolonged. For all treatments, peak concentrations in CF were recorded at the first sampling and ranged between 7 iu/ml and 14 iu/ml. Sodium benzylpenicillin or procaine penicillin, both in the ointment base, produced DTCs of 38.8 +/- 2.1 h and 37 +/- 4.0 h, respectively, while the ointment formulation of benethamine penicillin produced a DTC of 56 +/- 4.5 h. The prolonged duration observed in the eye ointments can be partly accounted for by the viscous nature of the base. Other differences may be dependent on relative water solubility of each penicillin product and complexity of the surface mucosae of the eye.

Attachment of Moraxella bovis to calf corneal cells and inhibition by antiserum

An in vitro assay was developed using calf corneal cells to assess the importance of fimbriae in the colonisation of the bovine ocular surface by Moraxella bovis, and the role of fimbrial antibodies in the bovine immune response and resistance to infectious bovine keratoconjunctivitis (IBK). Fimbriae promoted adherence of M. bovis to calf corneal cells in culture; 15 fimbriate isolates, representative of 6 fimbrial serogroups of M. bovis, adhered to the cells whereas 4 non-fimbriate isolates failed to do so. Fimbrial antibodies in hyperimmune rabbit serum inhibited attachment of all fimbriate strains of the homologous fimbrial serogroup but not those of 5 heterologous serogroups. The relevance of these results to the use of a polyvalent fimbrial vaccine in the control of IBK is discussed.

Facultative psychrophilic bacteria isolated from the tropical districts in Indonesia.

In this study the bacteria inverstigated were 34 strains (P-1∼P-34) isolated at 4∼5°C by enrichment culture in Indonesia. These strains were divided into 7 subgroups (Group I∼Group VII) from the results of cultural, morphological, temperature dependency of growth, biophysical investigations and cellular fatty acid composition. Group I includes 5 strains, of which one strain was mesophile and four strains were facultative psychrophiles. Group II includes 4 strains, which were concluded to be in the genus Sporosarcina and to have high salt concentration dependency of growth. Group III includes 2 strains, which showed rhizoid rough colony formation and were considered to be in the genus Alcaligenes or Flavobacterium. The relation with salt was considered to be interesting as a growth factor in this Group. Group IV includes 10 strains, of which only one strain was mesophile and remaining 9 strains were facultative psychrophiles. These strains were considered to be in the genus Alcaligenes or Pseudomonas. Their sources were distributed over very wide range.Group V includes 7 strains, and Group VI includes 3 strains, all of which were considered as facultative psychrophile. Their optimum growth temperature were low (4∼27°C). Group VII includes 9 strains, all of which were considered to be in the genus Pseudomonas. In conclusion, there were no obligate psychrophlile and 23 strains were facultative psychrophile. In the case of Group VI and VII, the growth investigation and analyses of fatty acid composition by gas chromatography were carried out. As the results of these precise examinations, Group VI was considered to be very near to the genus Moraxella or Acinetobacter, and Group VII near to the geuns Chromobacterium or Pseudomonas. On the level of species determination, it was concluded that P-26, P-27 and P-33 were the same species near to Pseudomonas caryophylli, and P-6, P-31 and P-29 were considered as Moraxella bovis or Moraxella-Like-species.

Further data to the aetiology, pathogenesis and therapy of infectious bovine keratoconjunctivitis

Out of a total of 224 bovine eye secretions, 126 Moraxella bovis and 64 Neisseria ovis strains were isolated. The pathogenesis and histological lesions caused by Neisseria ovis have been studied on the eyes of three calves naturally affected with IBK, using electron microscopy. Neisseria ovis caused in 1–12 weeks old calves acute, transient and mostly benign serous conjunctivitis with only slight affection of the cornea. More rarely erosions and even ulceration of the cornea have been observed. Moraxella bovis and Neisseria ovis strains proved nearly unanimously sensitive in vitro to chloramphenicol, neomycin, oxytetracyclin, nitrofurantoin, erythromycin and cefoperazone. Other antibiotics and chemotherapeutics inhibited the growth of these agents only partly or were ineffective. Experimental therapy has been carried out using a single i.m. injection of Terramycin/LA inj. (Pfizer) in a dose of 20 mg/kg body mass, repeated if necessary after 72–96 h. This formulation proved more effective and practical than treatments used earlier.

Vaccination against infectious bovine keratoconjunctivitis: protective efficacy and antibody response induced by pili of homologous and heterologous strains of Moraxella bovis.

The protective effect of 2 Moraxella bovis pili vaccines against infectious bovine keratoconjunctivitis (IBK) experimentally induced by homologous or heterologous strain challenge with virulent, haemolytic M. bovis strain, Dal 2d, was measured in trials using weaned calves aged 3 to 7 months. Purified pili vaccines were prepared from haemolytic strain Dal 2d, (pilus serogroup IV), and haemolytic strain Epp 63, (pilus serogroup III). Calves were challenged by conjunctival instillation of 1 x 10(9) colony forming units of virulent M. bovis strain Dal 2d 14 days after the second of 2 subcutaneous doses of vaccine. Each consisted of 200 micrograms of pili in alum-oil adjuvant administered at an interval of 21 days. In trial 1 the level of protection against challenge with the homologous strain was 46.7% (p less than 0.01). Small, rapidly resolving lesions of IBK occurred in some vaccinates compared with a larger proportion of severe lesions that required treatment in non-vaccinated calves (p less than 0.025). In trial 2, the level of protection against IBK after exposure of vaccinates to the homologous Dal 2d strain was 72.7%, but no significant level of protection or reduction in the size and duration of lesions was apparent in similarly challenged calves vaccinated with Epp 63 pili when contrasted with susceptible, non-vaccinated controls. No marked reduction in the duration of infection with M. bovis Dal 2d following challenge resulted from vaccination with pili of either of the serogroups III or IV. Rising homologous serum IgG antibody titres to serogroups III and IV pili were recorded in response to vaccination with each antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification, characterization, and pathogenicity of Moraxella bovis pili.

Pilins composed of the alpha or beta pilins of Moraxella bovis strain Epp63 were purified, subjected to chemical or enzymatic cleavage, and the resulting fragments sequenced by automated Edman degradation. alpha Pilin was found to be a 155-amino-acid polypeptide with a single intramolecular disulfide bridge. The beta pilin amino acid sequence substantiated the previously reported structure derived from the beta pilin gene DNA sequence, and indicated that the alpha and beta pilins of this strain are approximately 70% homologous. DNA hybridization studies of genomic DNA from the alpha- and beta-piliated variants of strain Epp63 indicated that the expression of the two pilin types was governed by an oscillating mechanism of chromosomal rearrangement. The alpha and beta pili were evaluated serologically and found to exhibit approximately 50% shared antigenicity, indicating that regions of conserved and heterologous sequence specify both type-specific and crossreacting epitopes. The pathogenicity of the alpha- and beta-piliated variants was studied by ocular inoculation of calves eyes; beta-piliated organisms were significantly more infectious than alpha-piliated organisms, indicating that beta pili confer, or are associated with, a relative advantage during the first stages of ocular infection. Preliminary analysis of other M. bovis strains suggests that each strain produces two types of pilin, and that this property may be characteristic of the species.