The most common ocular disease of dairy and beef cattle is IBK (commonly known as pinkeye). Clinical signs of IBK-affected cattle include corneal ulcers and edema, blepharospasm, photophobia, and excessive lacrimation. Severely affected eyes may perforate, which results in blindness. The etiologic agent of IBK is the gram-negative rod-shaped bacterium Moraxella bovis.1 Gram-negative cocci have also been isolated alone or in combination with M bovis from IBK-affected eyes.2–6 Before 2007, such gram-negative cocci were likely to be identified as Moraxella ovis, M ovis–like organisms, Branhamella ovis, or B ovis–like organisms. In 2007, a novel species of gram-negative coccus designated Moraxella bovoculi was isolated from ulcerated eyes of beef and dairy calves with IBK.7 In 1 field trial,8 an autogenous M bovoculi bacterin was not effective at preventing IBK. In a study9 in which investigators evaluated a recombinant M bovoculi cytotoxin vaccine, there was no significant reduction in the cumulative proportion of calves with ulcerated eyes in vaccinated versus control groups. Nevertheless, anecdotal reports that administration of autogenous M bovoculi bacterins ameliorates pinkeye in some herds of cattle suggest a role for M bovoculi in the pathogenesis of IBK.
Pili and cytotoxin (hemolysin and cytolysin) are the 2 most well-characterized pathogenic factors of M bovis. Pili help mediate the adherence of M bovis to the corneal surface.10–12 Pilus-based vaccines are reported to be effective at preventing IBK13–16; however, antigenic variation in M bovis pili exist, accounting for at least 7 pilus serogroups.17 The M bovis cytotoxin (designated MbxA) helps mediate the lysis of bovine neutrophils, erythrocytes, lymphocytes, and corneal epithelial cells that leads to corneal ulceration.18–22 The MbxA is a member of the RTX (repeats in the structural toxin) family of bacterial exotoxins.23 Similar to M bovis, M bovoculi also possesses an RTX toxin (designated MbvA) encoded in an RTX operon; MbvA has 83% amino acid sequence homology with MbxA.24
In 1 study,25 administration of a partially purified native M bovis cytotoxin vaccine protected calves against naturally occurring IBK, and the M bovis cytotoxin amino acid sequence is almost homologous among geographically diverse M bovis isolates.26 Rabbits vaccinated with the recombinant carboxy terminus of M bovis cytotoxin develop serum hemolysin-neutralizing antibodies.23 These findings have led to further investigations into the use of recombinant cytotoxin in vaccines to prevent IBK. The authors are aware of 3 experimental recombinant Moraxella spp cytotoxin subunit-ISCOM matrix adjuvanted vaccines that have been evaluated in randomized controlled field trials: the carboxy terminus of MbxA,27 the conserved amino terminus of M bovis pilin expressed as a fusion protein with the carboxy terminus of MbxA,28 and the carboxy terminus of M bovoculi MbvA.9 There is a reduction in the cumulative proportions of calves that develop IBK after inoculation with these vaccines; however, there are no overall differences in the cumulative proportions of control calves or calves inoculated with these vaccines that develop IBK. A complicating factor in at least 2 of these field trials was the presence of both M bovis and M bovoculi in the study herds. The purpose of the study reported here was to test the efficacy of an M bovis pilin-cytotoxin–M bovoculi cytotoxin recombinant subunit vaccine for prevention of naturally occurring IBK.
Materials and Methods
Animals—A convenience sample of 107 purebred and crossbred steers (age range, 4.8 to 7.6 months) were included in the study. Calves were managed as a single cohort at the University of California Sierra Foothills Field Station, Brown's Valley, Calif, and were maintained on mixed grass Sierra Foothill range pastures at 200 to 400 m above sea level. The trial began on April 17, 2007 (day 0), and ended on August 7, 2007 (day 112). Calves were weighed on the day of weaning (May 8, 2007; day 21) and on day 112. The historical prevalence of IBK at the Sierra Foothills Field Station herd was 40% to 70%, and both M bovis and M bovoculi had been isolated from IBK-affected cattle in the herd. Dams of steers included in the study had not been vaccinated against IBK. The experimental procedures were approved by the University of California Institutional Animal Care and Use Committee.
Preenrollment examination, group allocation, and inoculation—Calves were restrained in a hydraulic squeeze chute, and both eyes were examined for gross evidence of preexisting corneal lesions. Only calves with 2 normal-appearing corneas with no evidence of corneal opacification were enrolled.
Enrolled calves were allocated to the control or vaccine group by use of a blocked randomization scheme, with a block size of 6 to maintain approximate balance among the treatment groups during the trial. There were 54 and 53 calves in the control and vaccine groups, respectively. Each calf remained in the group to which it was initially enrolled throughout the duration of the trial. The randomization table was generateda by a statistician who was not an author of the study, and the block size was not disclosed to the investigators until completion of the data analysis.
On day 0, calves were inoculated SC in the cervical region with a 2-mL dose of ISCOM matrix adjuvant (control group) or recombinant M bovis pilin-cytotoxin–M bovoculi cytotoxin with the ISCOM matrix adjuvant (vaccine group); calves received booster inoculations on day 21. Study personnel were not aware of the treatments administered to each calf. On day 0, all calves also were vaccinated against respiratory viral pathogens,b orally administered a selenium bolus,c and dewormed.d On day 7, all calves received a multivalent Clostridium spp–Mannheimia haemolytica bacterin.e Recombinant M bovis pilin-cytotoxin and recombinant M bovoculi cytotoxin were prepared as described elsewhere.9,28 The vaccine was mixed 1:1 with ISCOM matrices that had been prepared as described,27 such that the final vaccine provided 250 μg of recombinant M bovis pilin-cytotoxin and 250 μg of recombinant M bovoculi cytotoxin in a 2-mL dose. The adjuvant control inoculum was 2 mL of a 1:1 mixture of ISCOM matrix solution and PBS solution (pH, 7.4).
Postenrollment examination—Each calf was restrained in a hydraulic squeeze chute, and both eyes were examined every 7 days for 16 weeks. Eyes were examined for gross evidence of corneal opacification by 1 investigator (JAA), who was not aware of the treatment that had been administered to each calf. If corneal opacification was present, the inferior subconjunctival cul-de-sac was swabbed with a sterile polyester swab, and the cornea was then stained with fluoresceinf to identify corneal ulcers. When the cornea retained fluorescein dye, swab specimens were submitted for bacterial culture. A corneal ulcer was considered to be associated with IBK unless the ulcer had evidence of mechanical trauma as indicated by a linear or stellate fluorescein staining pattern or was present in an eye from which a plant awn had been removed. A mechanically induced ulcer was considered to be associated with IBK if it was still present at the subsequent weekly observation; for such ulcers, the first day of the ulcer was assigned to be that subsequent weekly observation day. Corneal ulcer scores were assigned on the basis of the widest diameter of the ulcer (0 = no ulcer; 1 = diameter ≤ 5 mm, 2 = diameter > 5 mm, and 3 = perforated corneal ulcer). Ulcerated eyes were digitally photographedg with a ruler positioned next to the eye for subsequent determination of the corneal ulcer surface area. Calves with a corneal ulcer score ≥ 2 were treated once with florfenicolh (40 mg/kg, SC). Rubber gloves, plastic sleeves, and an apron were worn by the examiner; these items were rinsed with a 1% chlorhexidine solutioni between subsequent calves to prevent iatrogenic ocular infections.
Bacterial culture of ulcerated eyes—Ocular swab specimens from eyes with ulcers associated with IBK (ie, nonmechanically induced ulcers) were streaked onto 5% sheep blood agar plates. Plates were stored at 4°C during transport to our laboratory, where plates were further streaked for bacterial isolation. Plates were incubated at 35°C for 24 hours and examined for the presence of β hemolysis. β-hemolytic colonies were subcultured and identified via biochemical criteria established for M bovis29 and M bovoculi.7
Corneal ulcer SAM—Corneal ulcers in the digital photographs of ulcerated eyes were traced on a computer tabletj by use of image analysis software.k Differences in magnification among photographs were accounted for by standardizing the scale of each tracing with the ruler included in each photograph. The mean of 3 tracings of each ulcer was used to calculate the corneal ulcer surface area. For data analysis, the square root of the corneal ulcer surface area was used to represent the ulcer SAM. The limit of detection was 0.008 cm2, which was an area corresponding to a 1-mm-diameter circle. Ulcers with an area < 0.008 cm2 were considered nonulcerated corneas in the data analysis.
Hemolysis-neutralization assay—Blood samples were collected via jugular venipuncture into serum separator tubes on days 0, 42, and 112. Serum was harvested and stored at −20°C. Prior to use in hemolysis-neutralization assays, serum was heat inactivated at 56°C for 1 hour. Native cytotoxin in diafiltered retentate derived from culture supernatants of M bovis strain Tifton I or M bovoculi 237 were prepared in accordance with methods described for preparation of M bovis cytotoxin.23 The diafiltered retentate that contained native M bovis or M bovoculi cytotoxins was stored at −80°C until used in the hemolysis-neutralization assays, which were performed as described elsewhere for determination of M bovis hemolysin-neutralizing antibody titer28 and M bovoculi hemolysin-neutralizing antibody titer.9 The titer was defined as the last dilution at which no hemolysis was observed. Titers were measured in triplicate, and the final serum hemolysin-neutralizing antibody titer used in the data analysis was the geometric mean titer of 3 titer measurements. The respective day 42 and day 112 fold change in geometric mean titer against M bovis Tifton I- or M bovoculi 237-hemolysin was calculated as the ratio of the geometric mean titer on day 42 to the geometric mean titer on day 0 or the ratio of the geometric mean titer on day 112 to the geometric mean titer on day 0. Only calves with eyes that remained nonulcerated on days 42 and 112 were used in the statistical analyses of respective fold changes in neutralizing titers on days 42 and 112.
Statistical analysis—The primary outcome evaluated in the study was the cumulative proportion of calves with ulcerated eyes at weeks 8 and 16 (cumulative proportion ulcerated). Secondary outcomes evaluated in the study were the proportion of calves that required at least 1 florfenicol treatment, median time until the first ulcer was observed, median SAM of the first ulcer, median peak SAM of the first ulcer, median time for the first ulcer to heal (corneal ulcer score, 0), and weight change from weaning (day 21) to day 112. Differences in the cumulative proportion of calves that developed corneal ulcers by weeks 8 and 16 and differences in the proportion of calves requiring at least 1 treatment for IBK by week 16 were evaluated with the Pearson χ2 test. Risk ratios with 95% CIs for developing a first ulcer were calculated for the control and vaccine groups at weeks 8 and 16; risk ratios were considered significant if the CI did not include a value of 1. The Mann-Whitney test was used to evaluate differences between groups with respect to median time to first ulcer, median SAM for the first ulcer, median peak SAM for the first ulcer, median time for the first ulcer to heal, median weight change from weaning (day 21) to day 112, and median fold change in serum hemolysin-neutralizing titers from day 0 to 42 and day 0 to 112. To avoid possible influences of acquired immunity as a result of the development of IBK, fold changes in hemolysin-neutralizing titers were evaluated in calves with eyes that remained nonulcerated on day 42 (for fold changes in titer from day 0 to 42) and on day 112 (for fold changes in titer from day 0 to 112). A Kaplan-Meier survival curve was constructed for the time to first ulcer; differences between Kaplan-Meier curves were evaluated with the log-rank test. Time to development of a first ulcer was defined as the time of enrollment (day 0) to the time of first observed IBK-associated ulcer (corneal ulcer score > 0). In all comparisons, a value of P < 0.05 was considered significant. Statistical analyses were performed with commercially available statistics programs.l–n
Results
Characteristics for the control and vaccine groups were summarized (Table 1). All enrolled calves completed the trial, and there were no deviations from the study protocol. The cumulative proportion of calves with ulcerated eyes in the control and vaccine groups at week 8 was 15 of 54 (0.28) and 12 of 53 (0.23), respectively; these values did not differ significantly (P = 0.54). The cumulative proportion of calves with ulcerated eyes in the control and vaccine groups at week 16 was 17 of 54 (0.31) and 14 of 53 (0.26), respectively; these values did not differ significantly (P = 0.56; Table 2). The risk for ulcer development at week 8 in calves in the control group was 1.23 times (95% CI, 0.64 to 2.37) as high as the risk for ulcer development in calves in the vaccine group; however, these risk values did not differ significantly. The risk for ulcer development in calves in the control group at week 16 was 1.19 times (95% CI, 0.66 to 2.17) as high as the risk for ulcer development in calves of the vaccine group; these risk values did not differ significantly. No differences were observed between vaccine and control groups with regard to the secondary outcome variables of proportion of calves requiring at least 1 florfenicol treatment, median time to first ulcer, median SAM of the first ulcer, median peak SAM for the first ulcer, median time to heal for the first ulcer, or median change in body weight from weaning to day 112. No difference was found between calves in the control and vaccine groups in the time to development of the first ulcer (Kaplan-Meier survival analysis; P = 0.56; Figure 1).
Characteristics of calves inoculated SC with an ISCOM matrix adjuvant (control group; n = 54) or a recombinant Moraxella bovis pilin-cytotoxin–Moraxella bovoculi cytotoxin subunit antigen with the ISCOM matrix adjuvant (vaccine group; 53) on day 0 and that received booster injections 21 days later.
| Variable | Control | Vaccine |
|---|---|---|
| Breed (No. of calves) | ||
| Angus | 38 | 31 |
| Angus-Hereford crossbred | 11 | 17 |
| Hereford | 3 | 4 |
| Hereford crossbred | 2 | 0 |
| Red Angus | 0 | 1 |
| Weaning age (mo)* | 6.1 ± 0.5 | 5.9 ± 0.5 |
| Weaning weight (kg)* | 229.9 ± 29.7 | 224.4 ± 33.5 |
| Day 0 serum M bovis hemolysin-neutralizing titer† | 3.7 ± 3.3 | 3.3 ± 1.9 |
| Day 0 serum M bovoculi hemolysin-neutralizing titer† | 8.6 ± 3.5 | 9.4 ± 6.1 |
Values reported are mean ± SD.
Values reported are geometric mean titer ± SD.
Differences in outcome variables for assessment of efficacy of a recombinant M bovis pilin-cytotoxin–M bovoculi cytotoxin vaccine for the prevention of IBK.
| Variable | Control (n = 54) | Vaccine (n = 53) |
|---|---|---|
| Calves with ulcerated eyes at week 8 | 15 (28) | 12 (23) |
| Calves with ulcerated eyes at week 16 | 17 (31) | 14 (26) |
| Calves requiring at least 1 florfenicol treatment for IBK | 10 (19) | 13 (25) |
| Time to first ulcer (d)* | 42 (14 to 63) | 42 (28 to 112) |
| SAM of first ulcer* | 0.39 (0.09 to 1.29) | 0.54 (0.11 to 1.36) |
| Peak SAM of first ulcer† | 0.26 (0.09 to 0.39) | 0.11 (0.11 to 11) |
| Time for first ulcer to heal (d)† | 7 (7 to 14) | 7 (7 to 7) |
| Body weight change from weaning (day 21) to day 112 (kg) | 50.9 (28.2 to 88.2) | 52.7 (−40.0 to 75.5) |
Values reported are cumulative No. (%) or median (range).
Represents results for 17 and 14 calves in the control and vaccine groups, respectively.
Excludes treated calves; represents results for 8 and 2 calves in the control and treated groups, respectively.
Kaplan-Meier survival function curves for time to development of a corneal ulcer attributed to IBK in beef steer calves inoculated with a recombinant Moraxella bovis pilin-cytotoxin–Moraxella bovoculi cytotoxin vaccine with the ISCOM matrix adjuvant (vaccine group; n = 53 [dashed line]) or with adjuvant alone (control group; n = 54 [solid line]). Calves were initially inoculated on day 0 and received a booster inoculation on day 21.
Citation: American Journal of Veterinary Research 73, 10; 10.2460/ajvr.73.10.1670
Median fold change in M bovoculi hemolysin-neutralizing titers on day 42 for calves without ulcerated eyes in the vaccine group was twice the median fold change in M bovoculi hemolysin-neutralizing titers on day 42 for calves without ulcerated eyes in the control group; these titers differed significantly (P = 0.016; Table 3). No difference was found in the median fold change between calves without ulcerated eyes in both the vaccine and control groups for the M bovoculi hemolysin-neutralizing titer on day 112, M bovis hemolysin-neutralizing titer on day 42, or M bovis hemolysin-neutralizing titer on day 112 (Table 4).
Median (range) fold change in geometric mean titer of serum M bovoculi hemolysin-neutralizing antibodies of calves with nonulcerated eyes between days 0 and 42 and between days 0 and 112.
| Variable | Control | Vaccine |
|---|---|---|
| Days 0 to 42 | ||
| No. of calves with nonulcerated eyes | 43 | 44 |
| Fold change in titer | 1.0 (0.5–8.0) | 2.0 (0.5–4.0)* |
| Days 0 to 112 | ||
| No. of calves with nonulcerated eyes | 37 | 39 |
| Fold change in titer | 1.4 (0.5–8.0) | 1.0 (0.2–4.0) |
Value differs significantly (P = 0.016) from the value for the control group.
Median (range) fold change in geometric mean titer of serum M bovis hemolysin-neutralizing antibodies of calves with nonulcerated eyes between days 0 and 42 and between days 0 and 112.
| Variable | Control | Vaccine |
|---|---|---|
| Days 0 to 42 | ||
| No. of calves with nonulcerated eyes | 43 | 44 |
| Fold change in titer | 2.8 (0.7–22.6) | 3.4 (0.25–16.0) |
| Days 0 to 112 | ||
| No. of calves with nonulcerated eyes | 37 | 39 |
| Fold change in titer | 4.0 (0.5–16.0) | 2.8 (1.0–16.0) |
Moraxella bovis and M bovoculi were isolated alone or as a mixed infection from 12 of 17 ulcers in calves of the control group. In calves of the vaccine group, M bovoculi was isolated from 12 of 14 ulcers and M bovis was isolated from 11 of 14 ulcers (Table 5).
Frequency of isolation of Moraxella spp from 31 ulcers (first ulcer detected) in eyes of beef calves in the control and vaccine groups.
| Organism | Control (n = 17) | Vaccine (n = 14) | Total |
|---|---|---|---|
| M bovoculi | 3 | 3 | 6 |
| M bovis | 3 | 2 | 5 |
| M bovoculi and M bovis | 9 | 9 | 18 |
| No Moraxella spp isolated | 2 | 0 | 2 |
Discussion
The results of the study reported here indicated that calves inoculated with subunits of M bovis cytotoxin, M bovoculi cytotoxin, and M bovis pilin were not protected from naturally occurring IBK. It is possible that individual-animal or herd immunity may have led to reduced prevalence of IBK and our ability to detect significant differences between the control and vaccine groups. Reduced survival rate of Moraxella organisms in and subsequent transmission from eyes of calves in the vaccine group to other calves in the vaccine group or to calves in the control group may have reduced opportunities for direct spread of infectious ocular secretions among calves as well as indirect spread via flies from calves in the vaccine group to other calves in both groups.
In early studies that addressed vaccination against IBK, investigators found shorter durations of shedding of M bovis from cattle challenged with M bovis after vaccination with a live or formalin-killed M bovis product.30,31 Subsequent studies32–34 with formalin-killed autogenous M bovis and M bovis pilus–based antigens revealed no difference in the duration of M bovis shedding between vaccinated and control calves. In the present study, culturing of ocular swab specimens was only performed initially when ulcers attributed to IBK were first identified. Also, calves with ulcers that exceeded a maximal diameter of 0.5 cm were treated with florfenicol. In 1 antimicrobial efficacy trial, IBK-affected calves that received florfenicol became culture negative for M bovis 8 days after treatment, whereas untreated control calves continued to shed M bovis for up to 29 days.35 Quantitating the duration of shedding of Moraxella spp in calves that received the recombinant M bovis pilin-cytotoxin–M bovoculi cytotoxin vaccine may have provided another outcome with which to measure the effect of this recombinant vaccine. Maintaining vaccine and control groups under similar environmental conditions while keeping the groups spatially separated may have helped to reduce effects that herd immunity could have had on reducing spread of Moraxella spp and development of IBK in the calves of the present study during the summer of 2007. The lower end of the range of historical prevalence of IBK in this herd was 40%, and this was higher than the cumulative proportion of calves with ulcerated eyes in the control group (31%) at week 16.
In the present study, no difference was found in calves without ulcerated eyes in the vaccine versus control groups with regard to the fold change in M bovis serum hemolysin-neutralizing titers on day 42 and 112, relative to the titer on day 0. A significantly higher fold change was identified in the M bovoculi serum hemolysin-neutralizing titer on day 42 (but not day 112), relative to the titer on day 0, in calves without ulcerated eyes in the vaccine group, compared with that for calves without ulcerated eyes in the control group. The exact causes for the observed differences in duration and specificity of the immune responses to M bovoculi and M bovis cytotoxins are not known. It is possible that these changes reflected differences in cross-reactivity of immune responses to the recombinant Moraxella spp cytotoxins. In 1 study,24 rabbit antiserum against the recombinant M bovis cytotoxin carboxy terminus (amino acids 590 to 927) partially neutralized hemolytic activity of M bovoculi; however, rabbit antiserum against the equivalent region of M bovoculi cytotoxin did not neutralize hemolytic activity of M bovis. In the present study, it is likely that antibodies against the recombinant M bovis cytotoxin contributed to M bovoculi hemolysin-neutralizing responses on day 42. In contrast, rabbit antibodies against the recombinant M bovoculi cytotoxin may not have contributed to M bovis hemolysin-neutralizing responses, even on day 42.
Investigators in 1 study27 found that calves inoculated with 500 μg of recombinant M bovis cytotoxin with the ISCOM matrix adjuvant had significantly greater changes in M bovis serum hemolysin-neutralizing titers from days 0 to 49, compared with the changes in titer for saline (0.9% NaCl) solution–inoculated calves. Analysis of results of the present study suggests that antigen mass in recombinant vaccines that incorporate M bovis and M bovoculi cytotoxin–based antigens with ISCOM adjuvants may need to exceed 250 μg for adequate stimulation of serum hemolysin-neutralizing titers that persist for > 6 weeks. In another study,9 a 500-μg dose of recombinant M bovoculi cytotoxin resulted in a significant increase in serum neutralizing titers against M bovoculi hemolysin between days 0 and 42 but not between days 0 and 105 in calves without ulcerated eyes in the vaccinated group, compared with results for calves without ulcerated eyes in the control group.
In the present study, we did not measure anti-pilin antibody responses and thus cannot draw conclusions regarding the ability of the recombinant vaccine to stimulate antibodies against M bovis pili. As little as 30 μg of recombinant M bovis pili with an oil adjuvant can stimulate agglutinating antibody titers against M bovis16 In the present study, the calculated antigenic mass of pilin used in each vaccine dose was approximately 38 μg; whether this was sufficient to stimulate an anti-pilin antibody response with an ISCOM matrix adjuvant is not known.
Analysis of results of the present study and other trials in which investigators evaluated recombinant M bovis cytotoxin,27 M bovis pilin-cytotoxin,28 and M bovoculi cytotoxin9 with ISCOM matrix adjuvants suggests that these vaccines are not effective at preventing IBK. It is possible that a mucosal route of administration for these antigens may improve local ocular immune responses that could improve vaccine efficacy. Recently, intranasal administration of M bovis pili with Quil A adjuvant was found to stimulate increased ocular mucosal IgA responses against purified M bovis pili36; however, in that study,36 there was no correlation between high ocular mucosal anti-pilin IgA concentrations and the development of IBK. The use of the full-length M bovis cytotoxin or additional protective antigens that were copurified with cytotoxin might account for the reported efficacy of native M bovis cytotoxin vaccine administered SC.25 It is likely that investigations of alternate routes of administration, novel adjuvants, and use of additional protective antigens will help to improve the efficacy of recombinant Moraxella spp subunit vaccines against IBK.
ABBREVIATIONS
| CI | Confidence interval |
| IBK | Infectious bovine keratoconjunctivitis |
| ISCOM | Immunostimulating complex SAM Surface area measurement |
Spotfire S+, Tibco Software Inc, Somerville, Mass.
Bovi-Shield Gold 5, Pfizer Animal Health, New York, NY.
Se 365 bolus selenium supplement, Pacific Trace Minerals Inc, Sacramento, Calif.
Dectomax pour-on, Pfizer Animal Health, New York, NY.
One Shot Ultra 8, Pfizer Animal Health, New York, NY.
Sigma, St Louis, Mo.
Canon EOS D30, Canon USA Inc, Lake Success, NY.
Nuflor, Intervet/Schering Plough Animal Health, Summit, NJ.
Nolvasan solution, Fort Dodge Animal Health, Fort Dodge, Iowa.
Wacom Cintiq 15X LCD tablet, Wacom Technology Corp, Vancouver, Wash.
ImageJ, version 1.40a, National Institutes of Health, Bethesda, Md. Available at: rsbweb.nih.gov/ij/index.html. Accessed Apr 7, 2008.
SPSS, version 16.0, SPSS Inc, Chicago, Ill.
Epi Info, version 3.5.1, CDC, Atlanta, Ga. Available at: www.cdc.gov/epiinfo/. Accessed Aug 4, 2008.
Minitab, version 13.1, Minitab Inc, State College, Pa.
References
- 1.↑
Henson JBGrumbles LC. Infectious bovine keratoconjunctivitis. I. Etiology. Am J Vet Res 1960; 21:761–766.
- 2.
Fairlie G. The isolation of a haemolytic Neisseria from cattle and sheep in the North of Scotland. Vet Rec 1966; 78:649–650.
- 3.
Spradbrow PB. A microbiological study of bovine conjunctivitis and keratoconjunctivitis. Aust Vet J 1967; 43:55–58.
- 4.
Wilcox GE. Bacterial flora of the bovine eye with special reference to the Moraxella and Neisseria. Aust Vet J 1970; 46:253–256.
- 5.
Pugh GW JrHughes DE. Infectious bovine keratoconjunctivitis induced by different experimental methods. Cornell Vet 1971; 61:23–45.
- 6.
Elad DYeruham IBernstein M. Moraxella ovis in cases of infectious bovine keratoconjunctivitis (IBK) in Israel. Zentralblatt Veterinarmed B 1988; 35:431–434.
- 7.↑
Angelos JASpinks PQBall LM, et al. Moraxella bovoculi sp. nov., isolated from calves with infectious bovine keratoconjunctivitis. Int J Syst Evol Microbiol 2007; 57:789–795.
- 8.↑
Funk LO'Connor AMMaroney M, et al. A randomized and blinded field trial to assess the efficacy of an autogenous vaccine to prevent naturally occurring infectious bovine keratoconjunctivis (IBK) in beef calves. Vaccine 2009; 27:4585–4590.
- 9.↑
Angelos JALane VMBall LM, et al. Recombinant Moraxella bovoculi cytotoxin-ISCOM matrix adjuvanted vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis. Vet Res Commun 2010; 34:229–239.
- 10.
Moore LJRutter JM. Attachment of Moraxella bovis to calf corneal cells and inhibition by antiserum. Aust Vet J 1989; 66:39–42.
- 11.
Ruehl WWMarrs CBeard MK, et al. Q pili enhance the attachment of Moraxella bovis to bovine corneas in vitro. Mol Microbiol 1993; 7:285–288.
- 12.
Annuar BOWilcox GE. Adherence of Moraxella bovis to cell cultures of bovine origin. Res Vet Sci 1985; 39:241–246.
- 13.
Lehr CJayappa HGGoodnow RA. Serologic and protective characterization of Moraxella bovis pili. Cornell Vet 1985; 75:484–492.
- 14.
Lepper AW. Vaccination against infectious bovine keratoconjunctivitis: protective efficacy and antibody response induced by pili of homologous and heterologous strains of Moraxella bovis. Aust Vet J 1988; 65:310–316.
- 15.
Lepper AWAtwell JLLehrbach PR, et al. The protective efficacy of cloned Moraxella bovis pili in monovalent and multivalent vaccine formulations against experimentally induced infectious bovine keratoconjunctivitis (IBK). Vet Microbiol 1995; 45:129–138.
- 16.↑
Lepper AWElleman TCHoyne PA, et al. A Moraxella bovis pili vaccine produced by recombinant DNA technology for the prevention of infectious bovine keratoconjunctivitis. Vet Microbiol 1993; 36:175–183.
- 17.↑
Moore LJLepper AW. A unified serotyping scheme for Moraxellabovis. Vet Microbiol 1991; 29:75–83.
- 18.
Beard MKMoore LJ. Reproduction of bovine keratoconjunctivitis with a purified haemolytic and cytotoxic fraction of Moraxella bovis. Vet Microbiol 1994; 42:15–33.
- 19.
Gray JTFedorka-Cray PJRogers DG. Partial characterization of a Moraxella bovis cytolysin. Vet Microbiol 1995; 43:183–196.
- 20.
Hoien-Dalen PSRosenbusch RFRoth JA. Comparative characterization of the leukocidic and hemolytic activity of Moraxella bovis. Am J Vet Res 1990; 51:191–196.
- 21.
Kagonyera GMGeorge LWMunn R. Cytopathic effects of Moraxella bovis on cultured bovine neutrophils and corneal epithelial cells. Am J Vet Res 1989; 50:10–17.
- 22.
Kagonyera GMGeorge LMiller M. Effects of Moraxella bovis and culture filtrates on 51Cr-labeled bovine neutrophils. Am J Vet Res 1989; 50:18–21.
- 23.↑
Angelos JAHess JFGeorge LW. Cloning and characterization of a Moraxella bovis cytotoxin gene. Am J Vet Res 2001; 62:1222–1228.
- 24.↑
Angelos JABall LMHess JF. Identification and characterization of complete RTX operons in Moraxella bovoculi and Moraxella ovis. Vet Microbiol 2007; 125:73–79.
- 25.↑
George LWBorrowman AJAngelos JA. Effectiveness of a cytolysin-enriched vaccine for protection of cattle against infectious bovine keratoconjunctivitis. Am J Vet Res 2005; 66:136–142.
- 26.↑
Angelos JABall LM. Relatedness of cytotoxins from geographically diverse isolates of Moraxella bovis. Vet Microbiol 2007; 124:382–386.
- 27.↑
Angelos JAHess JFGeorge LW. Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine. Vaccine 2004; 23:537–545.
- 28.↑
Angelos JABonifacio RGBall LM, et al. Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis pilin-Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine. Vet Microbiol 2007; 125:274–283.
- 29.↑
Weyant RSMoss CWWeaver RE, et al. Identification of unusual pathogenic gram-negative aerobic and facultatively anaerobic bacteria. 2nd ed. Baltimore: Williams & Wilkins, 1996.
- 30.
Hughes DEPugh GW Jr. Experimentally induced bovine infectious keratoconjunctivitis: effectiveness of intramuscular vaccination with viable Moraxella bovis culture. Am J Vet Res 1971; 32:879–886.
- 31.
Hughes DEPugh GW. Experimentally induced infectious bovine keratoconjunctivitis: relationship of vaccination schedule to protection against exposure with homologous Moraxella bovis culture. Am J Vet Res 1975; 36:263–265.
- 32.
Smith PCBlankenship THoover TR, et al. Effectiveness of two commercial infectious bovine keratoconjunctivitis vaccines. Am J Vet Res 1990; 51:1147–1150.
- 33.
Hughes DEPugh GW JrKohlmeier RH, et al. Effects of vaccination with a Moraxella bovis bacterin on the subsequent development of signs of corneal disease and infection with M bovis in calves under natural environmental conditions. Am J Vet Res 1976; 37:1291–1295.
- 34.
Pugh GWMcDonald TJKopecky KE. Infectious bovine keratoconjunctivitis: effects of vaccination on Moraxella bovis carrier state in cattle. Am J Vet Res 1980; 41:264–266.
- 35.↑
Dueger ELAngelos JACosgrove S, et al. Efficacy of florfenicol in the treatment of experimentally induced infectious bovine keratoconjunctivitis. Am J Vet Res 1999; 60:960–964.
- 36.↑
Zbrun MVZielinski GCPiscitelli HC, et al. Evaluation of anti–Moraxella bovis pili immunoglobulin-A in tears following intranasal vaccination of cattle. Res Vet Sci 2012; 93:183–189.
