Bovine Immune Responses to Moraxella bovis and Moraxella bovoculi Following Vaccination and Natural or Experimental Infections.

Studies have sought to develop effective vaccines against infectious bovine keratoconjunctivitis (IBK). Most research has focused on parenterally administered vaccines against Moraxella bovis antigens; however, researchers have also included Moraxella bovoculi antigens in vaccines to prevent IBK. Critical knowledge gaps remain as to which Moraxella spp antigens might be completely protective, and whether systemic, mucosal, or both types of immune responses are required for protection against IBK associated with Moraxella spp. Immune responses to commensal Moraxella spp residing in the upper respiratory tract and eye have not been analyzed to determine if these responses control colonization or contribute to IBK.

Antigenic characterization of Moraxella bovis, Moraxella bovoculi and Moraxella ovis strains with potential use in vaccines.

Moraxella bovis is historically known as the primary agent of infectious bovine keratoconjunctivitis (IBK). However, Moraxella bovoculi and Moraxella ovis are also reported to be involved in the pathogenesis of IBK, therefore, these three species should be included in the development of a new vaccine with a broad-spectrum protection against the disease natural challenge. In this study we investigated the antigenic properties of clinical isolates and reference strains of M. bovis, M. bovoculi and M. ovis using a novel in vitro approach for vaccine evaluation based on two techniques, flow cytometry and western blotting (WB). Here, we demonstrated that rabbit antisera produced against reference M. bovis strain and commercial bacterin showed low number of IgG with capacity to recognize a panel of heterologous strains composed by M. bovoculi and M. ovis. On the other hand, the antisera generated against two clinical isolates of M. ovis (Mov2 and Mov3) presented high cross-reactivity levels against all M. ovis and M. bovis strains evaluated. Similarly, the antisera against Mbv3 (clinical isolate of M. bovoculi) had high levels of IgG associated on the surface of all M. bovoculi strains and most of the M. ovis strains analyzed. The WB analysis demonstrated that Moraxella spp. has multiple immunogenic antigens and most of them are shared between the three species. Based on the cross-reactivity analysis and considering the relative number of IgGs associated on the bacterial surface, we suggest that a multivalent vaccine including Mbv3, Mov2 and Mov3 strains may provide a strong and broad protection against all strains involved in IBK outbreaks.

Randomized blinded controlled trial to assess the association between a commercial vaccine against Moraxella bovis and the cumulative incidence of infectious bovine keratoconjunctivitis in beef calves.

OBJECTIVE To assess the association between a commercially available vaccine against Moraxella bovis and cumulative incidence of infectious bovine keratoconjunctivitis (IBK) from processing to weaning (primary objective) and body weight at weaning (secondary objective). DESIGN Randomized blinded controlled trial. ANIMALS 214 calves (≥ 2 months of age) born in the spring of 2015 at an Iowa State University cow-calf research unit with no visible lesions or scars on either eye. PROCEDURES Calves were randomly allocated to receive SC administration of a single dose of a commercial vaccine against M bovis (112 enrolled and 110 analyzed) or saline (0.9% NaCl) solution (111 enrolled and 104 analyzed). Calves were monitored for signs of IBK from treatment to weaning, and body weight at weaning was recorded. People involved in calf enrollment and outcome assessment were blinded to treatment group assignment. Cumulative incidence of IBK and weaning weight were compared between vaccinated and unvaccinated calves; the effect measure was the risk ratio and mean difference, respectively. RESULTS IBK was detected in 65 (59.1%) vaccinated calves and 62 (59.6%) unvaccinated calves (unadjusted risk ratio, 0.99; 95% confidence interval, 0.79 to 1.24) during the study period. No significant difference in weaning weights was identified between vaccinated and unvaccinated calves (unadjusted effect size, 4.40 kg [9.68 lb]; 95% confidence interval, -3.46 to 12.25 kg [-7.61 to 26.95 lb]). CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the commercially available M bovis vaccine was not effective in reducing the cumulative incidence of IBK or increasing weaning weight in beef calves.

Systemic and ocular immune responses in cattle following intranasal vaccination with precipitated or partially solubilized recombinant Moraxella bovis cytotoxin adjuvanted with polyacrylic acid.

OBJECTIVE To evaluate changes in systemic and ocular antibody responses of steers following intranasal vaccination with precipitated or partially solubilized recombinant Moraxella bovis cytotoxin (MbxA). ANIMALS 13 Angus steers with ages ranging from 318 to 389 days and weights ranging from 352 to 437 kg. PROCEDURES Steers were assigned to receive 500 µg of a precipitated (MbxA-P; n = 5) or partially solubilized (MbxA-S; 5) recombinant MbxA subunit adjuvanted with polyacrylic acid. A control group (n = 3) received the adjuvant alone. Each steer received the assigned treatment (1 mL/nostril) on days 0 and 28. Serum and tear samples were collected on days 0 (before vaccination), 14, 28, 42, and 55. Changes in MbxA-neutralizing antibody titers and MbxA-specific IgG concentrations in serum and tears and changes in MbxA-specific IgA concentrations in tears were measured. RESULTS Mean fold changes in MbxA-specific IgG concentration in serum and tears and MbxA-neutralizing antibody titer in tears for the MbxA-P group were significantly greater than those for the MbxA-S and control groups. Mean serum MbxA-neutralizing antibody titer did not differ among the 3 groups. Although the mean fold change in tear MbxA-specific IgA concentration differed significantly among the groups in the overall analysis, post hoc comparisons failed to identify any significant pairwise differences. CONCLUSIONS AND CLINICAL RELEVANCE Systemic and ocular immune responses induced by intranasal administration of the MbxA-P vaccine were superior to those induced by the MbxA-S vaccine. Additional research is necessary to determine whether the MbxA-P vaccine can prevent naturally occurring infectious bovine keratoconjunctivitis.

A systematic review and meta-analysis of the antibiotic treatment for infectious bovine keratoconjunctivitis: an update.

Infectious bovine keratoconjunctivitis (IBK) is a common and important disease of calves. Without effective vaccines, antibiotic therapy is often implemented to minimize the impact of IBK. This review updates a previously published systematic review regarding comparative efficacy for antibiotic treatments of IBK. Available years of Centre for Biosciences and Agriculture International and MEDLINE databases were searched, including non-English results. Also searched were the American Association of Bovine Practitioners and World Buiatrics Congress conference proceedings from 1996 to 2016, reviews since 2013, reference lists from relevant trials, and U.S. Food and Drug Administration New Animal Drug Application summaries. Eligible studies assessed antibiotic treatment of naturally-occurring IBK in calves randomly allocated to group at the individual level. Outcomes of interest were clinical score, healing time, unhealed ulcer risk, and ulcer surface area. A mixed-effects model comparing active drug with placebo was employed for all outcomes. Heterogeneity was assessed visually and using Cochran's Q-test. Thirteen trials assessing nine treatments were included. Compared with placebo, most antibiotic treatments were effective. There was evidence that the treatment effect differed by day of outcome measurement. Visually, the largest differences were observed 7-14 days post-treatment. These results indicate improved IBK healing with many antibiotics and suggest the need for randomized trials comparing different antibiotic treatments.

Ocular immune responses in steers following intranasal vaccination with recombinant Moraxella bovis cytotoxin adjuvanted with polyacrylic acid.

Infectious bovine keratoconjunctivitis (IBK) caused by Moraxella bovis is the most common eye disease of cattle. The pathogenesis of M. bovis requires the expression of pili that enable the organism to attach to the ocular surface and an RTX (repeats in the structural toxin) toxin (cytotoxin or hemolysin), which is cytotoxic to corneal epithelial cells. In this pilot study, ocular mucosal immune responses of steers were measured following intranasal (i.n.) vaccination with a recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid. Beef steers were vaccinated with either 500 µg (n = 3) or 200 µg (n = 3) of recombinant M. bovis cytotoxin plus adjuvant. Control group steers (n = 2) were vaccinated with adjuvant alone, and all steers were given a booster on day 21. Antigen-specific tear IgA and tear IgG, tear cytotoxin-neutralizing antibody responses, and serum cytotoxin-neutralizing antibody responses were determined in samples collected prevaccination and on days 14, 28, 42, and 55. Changes in tear antigen-specific IgA levels from day 0 to days 28, 42, and 55 were significantly different between groups; however, in post hoc comparisons between individual group pairs at the tested time points, the differences were not significant. Our results suggest that i.n. vaccination of cattle with recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid effects changes in ocular antigen-specific IgA concentrations. The use of intranasally administered recombinant M. bovis cytotoxin adjuvanted with polyacrylic acid could provide an alternative to parenteral vaccination of cattle for immunoprophylaxis against IBK.

Randomized controlled field trial to assess efficacy of a Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis.

OBJECTIVE: To evaluate efficacy of a recombinant Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK). ANIMALS: 107 beef steers. PROCEDURES: 2 groups of calves were inoculated SC with an immunostimulating complex (ISCOM) matrix adjuvant (control group; n = 54) or a recombinant M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit antigen with the ISCOM matrix adjuvant (vaccine group; 53); calves received booster injections 21 days later. Calves were examined once weekly for 16 weeks. Investigators and herd managers were not aware of the inoculum administered to each calf throughout the trial. Primary outcome of interest was the cumulative proportion of calves that developed IBK. Serum samples were obtained before inoculation (day 0) and on days 42 and 112. Serum hemolysin-neutralizing titers against native M bovis and M bovoculi cytotoxin were determined. RESULTS: No difference was detected between groups for the cumulative proportion of calves that developed IBK at weeks 8 and 16 after inoculation. Non-IBK-affected calves in the vaccine group had a significantly higher fold change in serum hemolysin-neutralizing titer against native M bovoculi cytotoxin from day 0 to 42 compared to control calves. CONCLUSIONS AND CLINICAL RELEVANCE: The M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit vaccine with the ISCOM matrix adjuvant was not effective at preventing naturally occurring IBK. It is likely that the incorporation of additional protective antigens in a recombinant Moraxella spp subunit vaccine will be required to yield a product that can be used for effective immunization of cattle against IBK.

Evaluation of cytokines as adjuvants of infectious bovine keratoconjunctivitis vaccines.

In this work two cytokines were used in combination with inactivated bacteria (bacterin) to test the bovine conjuctival immune response to the pathogen Moraxella bovis, the causative agent of Infectious bovine keratoconjunctivitis (IBK). Treatments using the bacterin vaccine combined with interleukin-2 and interferon-α as adjuvants (Group A), the bacterin vaccine only (Group B), and controls without treatment (Group C), were applied by ocular spraying to evaluate the local immune response in the corneal structure of cattle experimentally infected with M. bovis. Six weeks after infection, 14 out of a total of 34 animals presented different corneal lesions; 9 corresponding to the control group C, 4 to the group B and only one to the group A. According to the clinical manifestations, a numeric score was calculated. Control animals presented the highest score value (12 points), followed by group B (7.5 points) and group A (1 point). These results suggest that the addition of cytokines to M. bovis treatments can reduce not only eye injuries caused by IBK but also the number of diseased animals.

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.

Vaccine against infectious bovine keratoconjunctivitis: a new approach to optimize the production of highly piliated Moraxella bovis cells.

Pili are the principal antigens and virulence factors of Moraxella bovis, the etiological agent of infectious bovine keratoconjunctivitis (IBK). Although it has been reported that the low efficacy of whole cell vaccines against IBK is mainly due to the difficulties in keeping the cellular piliation level of M. bovis during the growth of bacteria in stirred bioreactors, the problem has not yet been overcome because the mechanisms involved in the loss of piliation are still not fully clarified. In this work we found that during the culture of M. bovis in liquid media, around 15% of the cells changed from piliated to non-piliated phenotypes at the end of the growth. Nevertheless, we demonstrated that the main cause of cellular piliation loss in M. bovis growing in stirred and/or sparged bioreactors is due to shear forces, which are a function of the volumetric gassed power drawn (P(g)V(-1)). Therefore, we tested here the use of bubble column bioreactors to protect M. bovis cell-bound pili from mechanical agitation damage effects. These bioreactors operated at a superficial air velocity of 0.0065 m s(-1) yielded a cellular piliation level of 25%, in contrast to 1% obtained for stirred bioreactors. The addition of carboxymethylcellulose (CMC) at 0.10% (w v(-1)) to culture medium proved to be suitable to improve the final piliation level (65%). We demonstrated by FT-IR spectroscopy and ELISA technique, that this chemical additive has a pili protective role interacting with the cells but without affecting pili antigenic properties.

Serologic cross-reactivity of Australian Moraxella bovis to vaccinal bacterin strains as determined by competitive ELISA.

OBJECTIVE: To conduct a serologic survey and define pili antigenic variability via the serologic cross-reactivity of Moraxella bovis isolates from naturally occurring infectious bovine keratoconjunctivitis (IBK) outbreaks in Australia. This project applies to the development of an M bovis pili-based vaccine targeting Australian strains originating from intensive cattle producing regions. PROCEDURE: Ocular swabs were collected from cattle affected with clinical signs of IBK from 25 veterinary practices. Standard criteria were used to identify 70 M bovis. Pure, piliated isolates were evaluated with a modified competitive enzyme-linked immunosorbent assay (ELISA) for cell-bound M bovis pili to determine their serologic cross-reactivity with pili of vaccinal bacterin strains EPP63, FLA64, and SAH 38. RESULTS: Sixty-four percent (45/70) of M bovis isolates demonstrated homologous pili antigens to a vaccinal strain. M bovis isolates homologous to one of the three vaccinal strains were obtained in 77% (34/44) of IBK outbreaks sampled. No IBK outbreak had isolates homologous to more than one vaccinal strain; however, 29% (10/34) of outbreaks with a cross-reacting strain had non-cross-reacting strains as well. CONCLUSION: The similar prevalence of pilus antigen homology to strain FLA64 was observed with isolates derived from NSW, Tasmania, and Victoria, compared with results of prior smaller serologic studies, suggests that the common pilus antigens in M bovis within Australia have been relatively stable over the last 20 years. The prevalence of a limited number of pilus antigens in M bovis suggest that the application of a vaccine containing the bacterial strains EPP63, FLA64, and SAH38 may provide a useful management tool for reducing production losses associated with IBK in Australia.

Assessment of methodological quality and sources of variation in the magnitude of vaccine efficacy: a systematic review of studies from 1960 to 2005 reporting immunization with Moraxella bovis vaccines in young cattle.

A review was conducted of all identified literature evaluating Moraxella bovis vaccines efficacy in preventing pinkeye in beef calves. From 292 publications identified by the search, data on 123 unique vaccine-to-control comparisons were extracted from 38 studies published in English from 1960 to 2005. Descriptive analysis was performed and an analysis of sources of variation evaluated. Use of methods to control bias such as randomization and blinding were associated with decreased vaccine efficacy. Only 15 trials reported using randomization and blinding. The authors conclude that when designing and reporting veterinary vaccination studies, researchers must include methodological quality information necessary to judge the evidence produced from the study.

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis pilin-Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine.

To evaluate the efficacy of a recombinant Moraxella bovis pilin-M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus+ISCOM matrix (MbxA), or recombinant M. bovis pilin-cytotoxin carboxy terminus+ISCOM matrix (pilin-MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the pilin-MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and pilin-MbxA. In addition, vaccination of calves with MbxA and pilin-MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus pilin+cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the pilin-cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.

Effectiveness of a cytolysin-enriched vaccine for protection of cattle against infectious bovine keratoconjunctivitis.

OBJECTIVE: To determine the immunogenicity of a Moraxella bovis cytolysin-enriched vaccine for prevention of infectious bovine keratoconjunctivitis (IBK). ANIMALS: 104 mixed-breed beef calves ranging between 4 and 8 months of age. PROCEDURE: Vaccines were prepared by the diafiltration of broth culture supernatant from hemolytic M bovis or sterile media. The diafiltered retentate was combined with Quil A adjuvant. Calves were randomly assigned to receive either the cytolysin vaccine (n = 35) or, as controls, adjuvant (35) or saline (0.9% NaCl) solution (34). Eyes of all calves were examined weekly for signs of IBK for 15 weeks. Calves that developed severe IBK were treated SC with florfenicol. RESULTS: Cytolysin vaccine contained 4 proteins with molecular masses ranging between 65 and 90 kd. Cytolysin-vaccinated calves had fewer instances of IBK than control calves. The time of onset of corneal lesions in cytolysin-vaccinated calves that developed IBK was delayed, compared with that of calves in either control group. The cytolysin-Quil A vaccine contained endotoxin, but calves did not have clinical signs of illness after vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: Calves that were vaccinated with a cytolysin-enriched vaccine had some resistance to IBK. Vaccines containing concentrated diafiltered M bovis cytolysin could protect beef calves against IBK.

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine.

The efficacy of a recombinant Moraxella bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK) was evaluated in a randomized, blinded, controlled field trial. Ninety-three cross bred beef calves were vaccinated with either saline, ISCOM matrix (adjuvant control), or a recombinant M. bovis cytotoxin carboxy terminus peptide plus ISCOM matrix and boostered 21 days later. Ocular examinations were performed once weekly for 20 weeks. At week 12, the cumulative proportion of calves with ulcerated eyes in the recombinant vaccine group was significantly lower than in the saline control group. Throughout the 20 week trial, the cumulative proportion of ulcerated calves remained lowest in the recombinant vaccine group. By week 7, nonulcerated calves in the recombinant vaccine group had significantly higher changes in serum neutralizing titers and cytotoxin specific to total IgG ratios in serum and tears as compared to calves in the control groups. The trend for a reduced cumulative proportion of IBK in the vaccinated calves over the 20 week trial suggests that a recombinant M. bovis cytotoxin vaccine may be beneficial in helping to prevent naturally occurring IBK.

Antigenic relationships of Moraxella bovis isolates recovered from outbreaks of infectious bovine keratoconjunctivitis in Argentina, Brazil, and Uruguay between 1983 and 2000.

Cross-reactivity indices (CRIs) of 28 isolates of Moraxella bovis recovered from outbreaks of infectious bovine keratoconjunctivitis in Argentina (A, 11 isolates), Brazil (B, 7), and Uruguay (U, 10) between 1983 and 2000 were estimated. Hyperimmune sera were produced in rabbits and antibody titres determined with each isolate. Isolates showing CRIs3 70 were placed in the same group. Group I had 13 isolates (A, 1; B, 6; U, 6); group II had 6 isolates (A, 4; U, 2); groups III, IV, and V had 2 isolates each, recovered in Argentina; group VI had 2 isolates, from Uruguay; and group VII had 1 isolate, from Brazil. The CRIs3 70 between vaccine strains and isolates recovered before and after 1990 were 58% and 42%, 50% and 50%, and 33% and 67% with vaccine strains 2419, 2358, and 2439, respectively. Isolate 273, from Uruguay, showed CRIs > 70 with 78% of the isolates and is recommended as the vaccine strain.

A field trial of autogenous Moraxella bovis bacterin administered through either subcutaneous or subconjunctival injection on the development of keratoconjunctivitis in a beef herd.

The primary purpose of these experiments was to evaluate an autogenous Moraxella bovis bacterin administered through 2 separate routes of inoculation. An autogenous bacterin was manufactured by using M. bovis recovered from the herd. The bacterin was administered by subcutaneous injection or subconjunctival injection. In each experiment, unvaccinated animals served as controls. Random selection methods were used to place calves into a vaccination or control group. There was no statistical difference in development of infectious keratoconjunctivitis between the vaccinated and unvaccinated calves. There was a statistically significant difference between the sexes; heifers had a higher rate of keratoconjunctivitis.

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.

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.

The protective efficacy of cloned Moraxella bovis pili in monovalent and multivalent vaccine formulations against experimentally induced infectious bovine keratoconjunctivitis (IBK).

Calves were vaccinated with cloned Moraxella bovis pili of serogroup C (experiment 1) or B (experiment 2) either as a monovalent formulation or as part of a multivalent preparation with pili of six other serogroups. Within 4 weeks of the second vaccine dose vaccinated calves and non-vaccinated controls were challenged via the ocular route with either virulent M. bovis strain Dal2d (serogroup C) or M. bovis strain 3WO7 (serogroup B) in experiments 1 and 2, respectively. Calves vaccinated with multivalent vaccines had significantly lower antibody titres than those vaccinated with monovalent preparations. Nevertheless, the levels of protection against infectious bovine keratoconjunctivitis (IBK) achieved with multivalent vaccines were 72% and 83% for the groups challenged with M. bovis strains of serogroups B and C, respectively. The serogroup C monovalent vaccine gave 100% protection against experimentally induced IBK and M. bovis isolates cultured from the eyes 6 days post-challenge were identified as belonging solely to serogroup C. Unexpectedly, only 25% protection was achieved against homologous strain challenge of calves that received the monovalent serogroup B vaccine. Furthermore, the majority of M. bovis isolates recovered from calves in this group belonged to serogroup C, as did half of those isolates cultured from the multivalent vaccinates. The remaining bacterial isolates from the latter group, together with all isolates from the non-vaccinated controls, belonged to serogroup B. Results are consistent with the hypothesis that derivatives of the serogroup B challenge inoculum had expressed serogroup C pilus antigen within 6 days of the challenge, possibly as a result of pilus gene inversion occurring in response to the presence of specific antibody in eye tissues and tears.