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.     

Serologic response of vaccinated cattle to strains of Moraxella bovis isolated during epizootics of keratoconjunctivitis.

In studies to determine whether there were antigenic differences between strains (isolates) of Moraxella bovis, the sera from vaccinated calves were tested with isolates of M bovis while the calves were experiencing epizootics of infectious bovine keratoconjunctivitis (IBK). Before the epizootics of IBK, the calves were intramuscularly vaccinated with a formalin-killed autogenous M bovis bacterin. During the epizootics, the eyes were examined by cultural technique, and isolates which were obtained were categorized by catalase activity, source (diseased or nondiseased eyes), and reactivity with the various sera. The serum reactivity of the isolates was compared with that of the vaccinal strain. The vaccinal strain and 8 of the 1 5 selected isolates obtained during the 1974 epizootic were catalase negative. Seven of the 15 isolates from the 1974 epizootic and all of the selected isolates from the 1975 epizootic were catalase positive. A significantly higher (P less than 0.01) percentage of calf sera were serologically reactive with the vaccinal strain and other catalase-negative isolates (45.0%) than with catalase-positive isolates (34.8%). The results, although not definitive, suggest that there may be antigenic differences among strains of M bovis. These differences should be considered when cattle are vaccinated against IBK under natural conditions of exposure.     

Immunogenicity of a Moraxella bovis bacterin containing attachment and cornea-degrading enzyme antigens

An adjuvanted Moraxella bovis bacterin containing attachment antigens and cornea-degrading enzyme antigens protected cattle from infectious bovine keratoconjunctivitis (IBK) when experimentally challenged with homologous and heterologous challenge cultures of M. bovis. This bacterin also protected cattle against field exposure to M. bovis. Transmission electron microscopy and fluorescein labeled anti-M. bovis pili antiserum showed pili on the M. bovis bacterin strain. Scanning electron microscopy demonstrated a fibrillar glycocalyx. The bacterin strain of M. bovis, but not all strains of M. bovis, destroyed bovine corneal cell monolayers in vitro. Bovine corneal cells began to separate from each other within 5 min after M. bovis organisms were added and adhered to the cell monolayers. Moraxella bovis organisms remained attached to the disintegrating cells as the cell membrane separated and was digested. Vaccination stimulated bacterial agglutination antibodies. However, protection against experimental challenge was more closely related to the cornea-degrading enzyme content of the experimental bacterins. Twenty-two of 29 cattle (76%) vaccinated with bacterins containing a relative enzyme activity (REA) greater than 0.4 were protected in a rigorous challenge of immunity test. Only 1 of 21 non-vaccinated calves (5%) was free of IBK. Ninety-two percent (24/26) of calves vaccinated with a bacterin containing a REA greater than 0.29 remained free of IBK following field exposure, whereas 47% (8/17) non-vaccinated calves developed IBK. Only 8 of 12 calves (67%) vaccinated with a bacterin containing a REA of 0.09 remained free of IBK. In a larger field efficacy test consisting of 32 herds in six states, the incidence of IBK in individual herds ranged from 0% to 55%. The overall rate of infection was 11.2%. Vaccination of calves with an M. bovis bacterin that contained a REA of 0.63 reduced the incidence of IBK from 11.2% (217/1931) in the non-vaccinated controls to 4.3% (66/1520) in cattle vaccinated once and to 3.1% (48/1536) in cattle vaccinated twice.     

The protective efficacy of pili from different strains of Moraxella bovis within the same serogroup against infectious bovine keratoconjunctivitis.

Three groups of ten calves were each immunised with a total of 400 micrograms pili prepared from three separate strains of Moraxella bovis in Alhydrogel-oil adjuvant as two divided, equal doses 21 days apart. Groups 1 and 2 each received a monovalent vaccine made from strain 4L and S276R respectively, which belonged to pili serogroup A. Group 3 received vaccine made from pili of strain Maff1, belonging to serogroup F. A further group of ten calves served as non-vaccinated controls. Calves in groups 1 and 2 had developed serogroup A-specific antibody and those in group 3 developed serogroup F-specific antibody, and some evidence of cross-reacting antibody was also detected when measured by an agglutination test using formalin-killed piliated cells of serogroup A strain 4L. Although antibody titres measured against purified pili by ELISA were highest with homologous serogroup antigens, cross-reactive titres to shared epitopes of M. bovis pili were also detected by this method. Ocular challenge of the 40 calves with virulent M. bovis of serogroup A strain S276R was carried out 14 days after the second vaccine dose. All non-vaccinated calves developed infectious bovine keratoconjunctivitis (IBK). The percentage protection in groups 1 (strain 4L) and 2 (strain S276R) was 60% and 80% respectively (P less than 0.05), with mean lesion scores of 0.7 and 0.3 out of a possible 6.0. The percentage protection of calves in group 3 (strain Maff1) was only 30%, with a mean lesion score of 1.4 compared with 2.2 for non-vaccinated controls. The present findings, together with other evidence indicating that immunity to IBK is serogroup-specific, suggest that inclusion of pili from one representative strain from each of the seven Australian and British serogroups in a polyvalent, subunit vaccine should effectively protect the majority of cattle against IBK caused by most field strains of M. bovis encountered in Australia and the United Kingdom.     

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)     

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.     

Experimentally induced infectious bovine keratoconjunctivitis: resistance of vaccinated cattle to homologous and heterologous strains of Moraxella bovis.

In studies to determine whether vaccination with one strain of Moraxella bovis would protect against challenge with virulent homologous or heterologous strains, calves were intramuscularly inoculated 3 times with formalin-killed M bovis, with 14 days between inoculations. Fourteen days after the 3rd vaccinal dose was given, all calves were exposed to homologous or heterologous virulent cultures of M bovis. The results indicated that vaccination with one strain of M bovis may induce protective immunity against homologous and heterologous challenge exposure; however, because vaccinated cattle resisted infection and disease produced by a homologous strain to a greater extent than they resisted those produced by heterologous strains, polyvalent vaccines or highly immunogenic common antigens may be needed to protect cattle against the numerous strains they might encounter under natural field conditions. There was minimal correlation between the presence of precipitating antibodies against the heterologous strains and the establishment of infection and disease.     

Experimental production of infectious bovine keratoconjunctivitis: comparison of serological and immunological responses using pili fractions of Moraxella bovis.

The effect of vaccinating cattle and mice on the development of keratoconjunctivitis was studied. Cattle were vaccinated with whole cells, disrupted cells and pili fractions of three strains of Moraxella bovis. Mice were vaccinated with pili fractions of three strains. The resistance of all vaccinated animals was challenged with virulent cultures of M. bovis. In an attempt to correlate the response seen after vaccination and challenge with a pili fraction of M. bovis, vaccinated cattle and mice were grouped on the basis of signs of disease manifested and compared on the basis of serological responses. Serum samples were tested for antibodies by a gel diffusion precipitin test. A greater number of the sera of resistant cattle had antibodies to the homologous pili antigen than those of vaccinated nonresistant cattle. Cattle vaccinated with disrupted cells were not resistant to infectious bovine kerato-conjuctivitis and their sera lacked antibodies against the pili antigens. Vaccinated mice were more resistant to infectious bovine kerato-conjuctivitis and their sera lacked antibodies against the pili antigens. Vaccinated mice were more resistant to challenge exposure by homologous than heterologous cultures. A greater number of the sera of resistant mice had antibodies to pili antigens than nonresistant mice.     

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

OBJECTIVE: To determine the ability of antisera against cyanogen bromide-cleaved pili from 4 strains of Moraxella bovis to react with whole or nondenatured pili. SAMPLE POPULATION: Antisera to 4 strains of M. bovis produced by New Zealand White rabbits. PROCEDURE: 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. RESULTS: 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. CONCLUSIONS AND CLINICAL RELEVANCE: 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.     

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.     

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.     

Molecular and phenotypic analysis of Moraxella spp. associated with infectious bovine keratoconjunctivitis in Uruguay

Infectious bovine keratoconjunctivitis (IBK) is a common ocular disease of cattle, which is generally thought to be caused by Moraxella bovis. However, a recently characterized Moraxella, M. bovoculi, has been isolated from animals with IBK. The aim of this study was to identify and characterize strains of Moraxella spp. obtained from IBK cases in different geographic locations within Uruguay. Ribosomal gene sequencing indicated that there were two groups of isolates that showed homology with either M. bovis or M. bovoculi. Phylogenetic analysis confirmed the presence of two species as the isolates grouped in different branches of the dendrogram. Conventional biochemical characterization did not distinguish between the species; only 9/25 isolates which had genetic homology with M. bovoculi showed any differences in biochemistry.     

Herd-specific strains of Mycoplasma bovis in outbreaks of mycoplasmal mastitis and pneumonia

Mycoplasma bovis causes severe economic losses in livestock production, particularly on the Northern American continent and more recently also in continental Europe. The aim of the current study was to evaluate whether the recently emerging outbreaks were due to a particular clone or strain of M. bovis or whether these outbreaks are due to multiple infectious strains of M. bovis. The study is based on the analysis M. bovis isolated from cattle of herds with outbreaks of mycoplasmal mastitis or pneumonia from geographically non related parts of Switzerland. M. bovis isolates were typed by insertion sequence (IS) element analysis based upon ISMbov1 and ISMbov2 southern-blot hybridization. We observed a strong divergence of M. bovis strains among affected herds which mostly were herd specific. This argues against the assumption that a recent infiltration of a particular clone of M. bovis is the cause of the perilous emerging outbreaks. The study suggests that transmission occurs from animal to animal most probably via milk.     

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.     

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 crossreactivity, 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.     

Infectious bovine keratoconjunctivitis epizootic associated with area-wide emergence of a new Moraxella bovis pilus type

Pilus-mediated adherence is a virulence attribute of Moraxella bovis. Several pilus types have been shown to exist among strains of this bacterium, but correlation between pilus type and specific field cases of the disease has not been done. During the summer of 1987, an epizootic of infectious bovine keratoconjunctivitis was reported in 7 Iowa counties. Eight isolates of M bovis were secured from 12 episodes studied. All 8 of the isolates were nearly homogeneous in biochemical properties and had the same plasmid biotype. Pilus typing performed by immunofluorescence and immunogold electron microscopy identified a single new pilus type among 5 of the 8 isolates. This pilus type was identified in field cases that developed within a narrow time frame and over large distances. The implication of these findings is that infectious bovine keratoconjunctivitis epizootics may be associated with emergence of a novel pilus type, and that rapid dissemination over wide distances can occur, presumably by transportation of carrier cattle.     

The use of pulsed-field gel electrophoresis to investigate the epidemiology of Mycoplasma bovis in French calf feedlots

Mycoplasma bovis is a major cause of respiratory outbreaks in cattle feedlots. In this study pulsed-field gel electrophoresis (PFGE) was used to trace field strains and provide information on M. bovis patterns of spread in calf feedlots. The suitability of KpnI, MluI and SmaI restriction enzymes was assessed on different sets of strains. The discriminative power of the first two enzymes was first assessed using 28 epidemiologically unrelated strains; stability was 100% on multiple isolates from in vivo experimental infection. Thirty-nine field isolates from six feedlots were then evaluated. In contrast to the unique fingerprints displayed by the unrelated strains, the isolates from the feedlots showed identical patterns at the time of the outbreak of respiratory disease and 4 weeks later. The PFGE typing results suggest that M. bovis strains follow a clonal epidemic spread pattern at the herd level and that the same strain persists in calves of the herd after the clinical signs have disappeared.     

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.     

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.     

Evaluation of anti-Moraxella bovis pili immunoglobulin-A in tears following intranasal vaccination of cattle

Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease of cattle caused by Moraxella bovis (Mb). Parenterally administered immunogens used to prevent the disease do not offer complete protection possibly because they stimulate a poor ocular mucosal secretory response, in which locally secreted immunoglobulin-A (sIgA) is one of the main components. The principal aim of this study was to evaluate by an indirect enzyme linked immunosorbent assay (ELISA), the local ocular mucosal sIgA response against Mb purified pili, produced after intranasal inoculation of experimental vaccines. Pili were adjuvanted by several different adjuvants (QuilA, Marcol Arlacel, Marcol Span, microencapsulated pili with PLGA polymers). Results were compared to sIgA response produced by adjuvant placebo inoculations and by IBK natural infection. Significantly higher anti-pili IgA response (p<0.05) was detected in calves vaccinated intranasally with pili QuilA and pili Marcol Span compared to control calves, although this specific immune response did not seem to be related to protection against Mb infection or typical IBK lesion development.