• View in gallery
    Figure 1—

    Individual and mean ± SE treatment group fold changes in MbxA-neutralizing antibody titer in serum (A) and tear (B) samples at various intervals for steers that underwent IN administration of 500 μg of partially solubilized (MbxA-S; circles and striped bars; n = 5) or precipitated (MbxA-P; squares and gray bars; 5) recombinant Mbx-A subunit adjuvanted with polyacrylic acid or 2 mL of polyacrylic acid adjuvant (control; triangles and white bars; 3) on days 0 (D0) and 28 (D28). The fold change was calculated as the MbxA-neutralizing antibody titer (or IgA or IgG concentration) on days 14, 28, 42, or 55/MbxA-neutralizing antibody titer (or IgA or IgG concentration) on day 0. The fold change in MbxA-neutralizing antibody titer in serum did not differ among the 3 groups. The fold change in MbxA-neutralizing antibody titer in tears differed significantly (P = 0.014) among the 3 groups; the fold change for the MbxA-P group was significantly greater than that for the MbxA-S and control groups but did not differ significantly between the MbxA-S and control groups.

  • View in gallery
    Figure 2—

    Individual and mean ± SE treatment group fold changes in MbxA-specific IgG concentration in serum (A) and tear (B) samples at various intervals for the steers of Figure 1. The fold change in MbxA-specific IgG concentration in both serum (P = 0.007) and tear (P = 0.031) samples differed significantly among the 3 groups; in both samples, the fold change for the MbxA-P group was significantly greater than that for the MbxA-S and control groups but did not differ significantly between the MbxA-S and control groups. See Figure 1 for remainder of key.

  • View in gallery
    Figure 3—

    Individual and mean ± SE treatment group fold changes in MbxA-specific IgA concentration in tear samples at various intervals for the steers of Figure 1. Although the overall fold change in tear MbxA-specific IgA concentration differed significantly (P = 0.001) among the 3 groups, none of the pairwise comparisons between groups were significant. See Figure 1 for remainder of key.

  • 1. Henson JB, Grumbles LC. Infectious bovine keratoconjunctivitis. I. Etiology. Am J Vet Res 1960; 21: 761766.

  • 2. Iwasa M, Kawada A, Yorita M, et al. Prolonged occurrence of Moraxella bovis infection on a restricted heifer farm. J Vet Med Sci 1994; 56: 429432.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Conceição FR, Paolicchi F, Cobo AL, et al. Antigenic relationships of Moraxella bovis isolates recovered from outbreaks of infectious bovine keratoconjunctivitis in Argentina, Brazil, and Uruguay between 1983 and 2000. Can J Vet Res 2003; 67: 315318.

    • Search Google Scholar
    • Export Citation
  • 4. Alexander D. Infectious bovine keratoconjunctivitis: a review of cases in clinical practice. Vet Clin North Am Food Anim Pract 2010; 26: 487503.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Schnee C, Heller M, Schubert E, et al. Point prevalence of infection with Mycoplasma bovoculi and Moraxella spp. in cattle at different stages of infectious bovine keratoconjunctivitis. Vet J 2015; 203: 9296.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Nayar PS, Saunders JR. Infectious bovine keratoconjunctivitis I. Experimental production. Can J Comp Med 1975; 39: 2231.

  • 7. Yeruham I, Perl S, Elad D. Infectious bovine keratoconjunctivitis and lymphofollicular hyperplasia of the third eyelid in heifers. J Vet Med B Infect Dis Vet Public Health 2001; 48: 137141.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Barner RD. A study of Moraxella bovis and its relation to bovine keratitis. Am J Vet Res 1952; 13: 132144.

  • 9. Misiura M. Keratoconjunctivitis infectiosa in calves—attempt at elimination by active immunization. Arch Vet Pol 1994; 34: 187194.

    • Search Google Scholar
    • Export Citation
  • 10. Angelos JA, Dueger EL, George LW, et al. Efficacy of florfenicol for treatment of naturally occurring infectious bovine keratoconjunctivitis. J Am Vet Med Assoc 2000; 216: 6264.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Dueger EL, Angelos JA, Cosgrove S, et al. Efficacy of florfenicol in the treatment of experimentally induced infectious bovine keratoconjunctivitis. Am J Vet Res 1999; 60: 960964.

    • Search Google Scholar
    • Export Citation
  • 12. Dueger EL, George LW, Angelos JA, et al. Efficacy of a long-acting formulation of ceftiofur crystalline-free acid for the treatment of naturally occurring infectious bovine keratoconjunctivitis. Am J Vet Res 2004; 65: 11851188.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Moore LJ, Rutter JM. Attachment of Moraxella bovis to calf corneal cells and inhibition by antiserum. Aust Vet J 1989; 66: 3942.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Ruehl WW, Marrs C, Beard MK, et al. Q pili enhance the attachment of Moraxella bovis to bovine corneas in vitro. Mol Microbiol 1993; 7: 285288.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Annuar BO, Wilcox GE. Adherence of Moraxella bovis to cell cultures of bovine origin. Res Vet Sci 1985; 39: 241246.

  • 16. Beard MK, Moore LJ. Reproduction of bovine keratoconjunctivitis with a purified haemolytic and cytotoxic fraction of Moraxella bovis. Vet Microbiol 1994; 42: 1533.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Gray JT, Fedorka-Cray PJ, Rogers DG. Partial characterization of a Moraxella bovis cytolysin. Vet Microbiol 1995; 43: 183196.

  • 18. Kagonyera GM, George LW, Munn R. Cytopathic effects of Moraxella bovis on cultured bovine neutrophils and corneal epithelial cells. Am J Vet Res 1989; 50: 1017.

    • Search Google Scholar
    • Export Citation
  • 19. Lehr C, Jayappa HG, Goodnow RA. Serologic and protective characterization of Moraxella bovis pili. Cornell Vet 1985; 75: 484492.

  • 20. Jayappa HG, Lehr C. Pathogenicity and immunogenicity of piliated and nonpiliated phases of Moraxella bovis in calves. Am J Vet Res 1986; 47: 22172221.

    • Search Google Scholar
    • Export Citation
  • 21. Lepper AW, Atwell JL, Lehrbach 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: 129138.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Lepper AW, Elleman TC, Hoyne PA, et al. A Moraxella bovis pili vaccine produced by recombinant DNA technology for the prevention of infectious bovine keratoconjunctivitis. Vet Microbiol 1993; 36: 175183.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. 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: 310316.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Moore LJ, Lepper AW. A unified serotyping scheme for Moraxella bovis. Vet Microbiol 1991; 29: 7583.

  • 25. Marrs CF, Ruehl WW, Schoolnik GK, et al. Pilin-gene phase variation of Moraxella bovis is caused by an inversion of the pilin genes. J Bacteriol 1988; 170: 30323039.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Angelos JA, Ball LM. Relatedness of cytotoxins from geographically diverse isolates of Moraxella bovis. Vet Microbiol 2007; 124: 382386.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Ostle AG, Rosenbusch RF. Immunogenicity of Moraxella bovis hemolysin. Am J Vet Res 1985; 46: 10111014.

  • 28. Billson FM, Hodgson JL, Egerton JR, et al. A haemolytic cell-free preparation of Moraxella bovis confers protection against infectious bovine keratoconjunctivitis. FEMS Microbiol Lett 1994; 124: 6973.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. George LW, Borrowman AJ, Angelos JA. Effectiveness of a cytolysin-enriched vaccine for protection of cattle against infectious bovine keratoconjunctivitis. Am J Vet Res 2005; 66: 136142.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Angelos JA, Hess JF, George LW. Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine. Vaccine 2004; 23: 537545.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Kagonyera GM, George LW, Munn R. Light and electron microscopic changes in corneas of healthy and immunomodulated calves infected with Moraxella bovis. Am J Vet Res 1988; 49: 386395.

    • Search Google Scholar
    • Export Citation
  • 32. Pedersen KB. The origin of immunoglobulin-G in bovine tears. Acta Pathol Microbiol Scand B Microbiol Immunol 1973; 81: 245252.

  • 33. Nielsen K, Duncan JR. Bovine IgM: does it fix guinea pig complement in the absence of bovine complement components? Vet Immunol Immunopathol 1987; 14: 335343.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Vaerman JP, Heremans JF. Effect of neuraminidase and acidification on complement-fixing properties of human IgA and IgG. Int Arch Allergy Appl Immunol 1968; 34: 4952.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Marnila P, Korhonen H. Milk proteins: immunoglobulins. In: Fuquay JW, Fox PF, McSweeney PLH, eds. Encyclopedia of dairy sciences. 2nd ed. Cambridge, Mass: Academic Press, 2011; 807815.

    • Search Google Scholar
    • Export Citation
  • 36. Angelos JA, Edman JM, Chigerwe M. Ocular immune responses in steers following intranasal vaccination with recombinant Moraxella bovis cytotoxin adjuvanted with polyacrylic acid. Clin Vaccine Immunol 2014; 21: 181187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Horwitz J. Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci U S A 1992; 89: 1044910453.

  • 38. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156159.

    • Search Google Scholar
    • Export Citation
  • 39. Studier FW, Rosenberg AH, Dunn JJ, et al. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 1990; 185: 6089.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40. Nagai K, Thøgersen HC. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol 1987; 153: 461481.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41. Wang G, Pan L, Zhang Y, et al. Intranasal delivery of cationic PLGA nano/microparticles-loaded FMDV DNA vaccine encoding IL-6 elicited protective immunity against FMDV challenge. PLoS ONE 2011; 6: e27605.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Boland KG, Hayles AN, Miller CB, et al. Regional immune response to immunization with Escherichia coli O157:H7-derived intimin in cattle. Clin Vaccine Immunol 2013; 20: 562571.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43. Stanley AC, Buxton D, Innes EA, et al. Intranasal immunisation with Toxoplasma gondii tachyzoite antigen encapsulated into PLG microspheres induces humoral and cell-mediated immunity in sheep. Vaccine 2004; 22: 39293941.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. Shen X, Lagergård T, Yang Y, et al. Systemic and mucosal immune responses in mice after mucosal immunization with group B streptococcus type III capsular polysaccharide-cholera toxin B subunit conjugate vaccine. Infect Immun 2000; 68: 57495755.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45. Mapletoft JW, Latimer L, Babiuk LA, et al. Intranasal immunization of mice with a bovine respiratory syncytial virus vaccine induces superior immunity and protection compared to those by subcutaneous delivery or combinations of intranasal and subcutaneous prime-boost strategies. Clin Vaccine Immunol 2010; 17: 2335.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46. Liu Z, Yuan F, Yang Y, et al. Partial protective immunity against toxoplasmosis in mice elicited by recombinant Toxoplasma gondii malate dehydrogenase. Vaccine 2016; 34: 989994.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47. Pugh GW Jr, Hughes DE. Bovine infectious keratoconjunctivitis: carrier state of Moraxella bovis and the development of preventive measures against disease. J Am Vet Med Assoc 1975; 167: 310313.

    • Search Google Scholar
    • Export Citation
  • 48. Angelos JA, Hess JF, George LW. An RTX operon in hemolytic Moraxella bovis is absent from nonhemolytic strains. Vet Microbiol 2003; 92: 363377.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 49. Misiura M. Estimation of fimbrial vaccine effectiveness in protection against keratoconjunctivitis infectiosa in calves considering different routes of introducing vaccine antigene. Arch Vet Pol 1994; 34: 177186.

    • Search Google Scholar
    • Export Citation
  • 50. Zbrun MV, Zielinski GC, Piscitelli HC, et al. Evaluation of anti-Moraxella bovis pili immunoglobulin-A in tears following intranasal vaccination of cattle. Res Vet Sci 2012; 93: 183189.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51. di Girolamo FA, Sabatini DJ, Fasan RA, et al. Evaluation of cytokines as adjuvants of infectious bovine keratoconjunctivitis vaccines. Vet Immunol Immunopathol 2012; 145: 563566.

    • Crossref
    • Search Google Scholar
    • Export Citation

Advertisement

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

John A. AngelosDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

Search for other papers by John A. Angelos in
Current site
Google Scholar
PubMed
Close
 DVM, PhD
,
Munashe ChigerweDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

Search for other papers by Munashe Chigerwe in
Current site
Google Scholar
PubMed
Close
 BVSc, PhD
,
Judy M. EdmanDepartment of Medicine and Epidemiology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616.

Search for other papers by Judy M. Edman in
Current site
Google Scholar
PubMed
Close
 BS
, and
John F. HessDepartment of Cell Biology and Human Anatomy, School of Medicine, University of California-Davis, Davis, CA 95616.

Search for other papers by John F. Hess in
Current site
Google Scholar
PubMed
Close
 PhD

Abstract

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.

Abstract

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.

Infectious bovine keratoconjunctivitis, or pinkeye, is a common and debilitating infectious ocular disease caused by Moraxella bovis1 that has been described in cattle populations throughout the world.2–9 Corneal ulceration associated with IBK can result in ocular pain and, in some cases, permanent corneal scarring or blindness following rupture of the globe.10–12 The pathogenicity of M bovis is dependent on pili that allow the organism to adhere to the corneal surface13–15 and a cytotoxin that kills corneal epithelial cells, leading to the formation of corneal ulcers.16–18 Although pilus-based vaccines can reduce the incidence and severity of IBK,19–23 the presence of multiple pilus serogroups24 coupled with the potential for pilin gene inversions25 accounts for antigenic variation among M bovis strains and is the proposed mechanism by which M bovis evades the immune response in cattle vaccinated with pilus-based vaccines.21

Unlike pilin, MbxA is conserved across geographically diverse strains of the bacterium.26 Results of a study27 published in 1985 indicate that anticytotoxin antibodies against one M bovis strain neutralized hemolysins from other M bovis strains. In a subsequent study,28 calves vaccinated with partially purified MbxA were protected against IBK when challenged with heterologous M bovis. Calves parenterally administered native29 or recombinant MbxA30 vaccines were partially protected against naturally occurring IBK. Collectively, the results of those studies26–30 suggest that MbxA may be superior to pilin as a vaccine antigen.

Most M bovis vaccine studies have evaluated parenterally administered vaccines; however, some research suggests that a targeted isotypic antibody response may be an important consideration for the development of more effective vaccines against IBK. In a study31 in which corneas from healthy and immunosuppressed calves were experimentally infected with M bovis, the ulcers that developed in the corneas of the immunosuppressed calves were shallower than the ulcers that developed in the corneas of healthy calves. That finding suggests that a mucosal route of vaccination, which induces an ocular IgA response against M bovis antigens, might be superior to a parenteral route of vaccination, which stimulates a high systemic IgG response. Bovine ocular IgG arises primarily from plasma,32 and results of another study30 indicate that an increase in the systemic IgG response against MbxA results in a corresponding increase in the ocular IgG response. Although ocular IgG likely provides some protection against M bovis, it may also indirectly lead to ocular injury mediated by neutrophils that are attracted to the eye during complement fixation. Because IgA does not fix complement,33–35 mucosally delivered vaccines that stimulate an ocular IgA response against M bovis antigens might be more effective than traditional parenterally delivered M bovis vaccines.

Results of a study36 that evaluated an IN recombinant MbxA subunit vaccine are promising but suggest that additional refinements are necessary to stimulate more robust systemic and ocular antibody responses. The purpose of the study reported here was to evaluate changes in the systemic and ocular antibody responses of steers following IN vaccination with precipitated or partially solubilized recombinant MbxA.

Materials and Methods

Animals

The study was approved by the University of California Institutional Animal Care and Use Committee. Study animals included 13 Angus steers with ages ranging from 318 to 389 days and weights ranging from 352 to 437 kg that were fed a standard feedlot ration at the University of California-Davis Department of Animal Science campus feedlot. All steers had received an oral selenium bolusa as calves prior to arrival at the feedlot. Prior to study enrollment (day 0), steers were vaccinated against respiratory viral pathogensb (bovine herpesvirus 1, parainfluenza virus type 3, and bovine respiratory syncytial virus), Clostridium spp,c Histophilus somni,c and Mannheimia haemolyticad; were dewormed with doramectine; and underwent a physical examination. Only steers with 2 healthy corneas without evidence of active inflammation or corneal opacification suggestive of active or previous IBK were enrolled in the study.

Antigen preparation

Cloning and expressing the recombinant carboxy terminus of MbxA (amino acids, 590 to 927) as inclusion bodies in Escherichia coli30 and the subsequent purification of recombinant MbxA from those inclusion bodies36 were performed as previously described except the final chromatographic purification of MbxA was performed in chromatography buffer that contained 8M urea, 10mM Tris-HCl, 200mM NaCl, and 1mM EDTA (pH, 7.2). The MbxA protein concentration was measuredf prior to dialysis and subsequent vaccine formulation.

To prepare the precipitated MbxA antigen (MbxA-P), MbxA in chromatography buffer was dialyzedg against water at 4°C for 48 hours. During dialysis, the protein precipitated. The MbxA-P antigen was harvested and stored at −20°C prior to adjuvanting with polyacrylic acid.

The partially solubilized MbxA antigen (MbxA-S) was prepared by combining MbxA that was solubilized in chromatography buffer with bovine αB-crystallin prior to dialysis. αB- crystallin is a molecular chaperone37 that reduces protein aggregation and was used to help prevent protein precipitation. Bovine αB-crystallin was prepared from bacterial expression constructs originally constructed by a reverse transcription–PCR assay. Briefly, RNA from bovine lenses was prepared by use of guanidine isothiocyanate denaturation and ethanol precipitation.38 Total RNA was primed with oligo dT primers and reverse transcribed.h The reaction was terminated by heating at 65°C. The cDNA pool was diluted to 500 μL with water, and 5 μL of that solution was used as input cDNA for an amplification reaction with αB-crystallin oligonucleotide primers.i For cloning into the expression plasmid, vector primers carried additional sequences for NdeI and EcoRI recognition sequences on the amino and carboxyl ends, respectively. A PCR product with a single band of expected size was obtained and cloned into pCR2.1.j Miniprep DNA was isolated from positive transformants and sequenced.k Inserts were excised with NdeI and EcoRI and cloned into pT7-7.39 The resulting transformants were screened by a PCR assay, and the DNA sequence of the expression construct was verified.k The verified αB-crystallin construct was transformed into E coli.l For production of recombinant αB-crystallin, transformed E coli were grown in Luria-Bertani broth with ampicillin (100 μg/mL) until the optical density (measured at a wavelength of 600 nm) was approximately 0.5 to 0.6, induced with arabinose, and then incubated for 5 to 6 hours. Cells were harvested by centrifugation, and the wet pellets were stored overnight (approx 18 hours) at −20°C. Cells were thawed and lysed as described40 and then centrifugedm at 27,000 × g for 10 minutes. The cleared bacterial lysate containing the αB-crystallin was filtered with a 0.22-μm syringe filter and then chromatographedn in buffer containing 10mM Tris-HCl (pH, 7.5), 1mM EDTA, and 100mM NaCl. Peak fractions were determined by SDS-PAGE. The peak fractions were pooled and stored at −80°C until use. The protein was quantitatedf prior to use.

To prepare the MbxA-S, MbxA in chromatography buffer and αB-crystallin were thawed, mixed together in a 2:1 mass ratio, and incubated at 37°C for 1 hour. The protein mixture was then dialyzedg for 24 hours at 4°C against buffer containing 2M urea, 200mM NaCl, 1mM EDTA, and 10mM Tris-HCl (pH, 7.2). A final 24-hour dialysis against water at 4°C was performed prior to harvest. The amount of MbxA that remained in solution in the retentate was estimated to be approximately 50% as determined by comparing the intensityo of Coomassie-stained MbxA protein on SDS-PAGE gels before and after centrifugationp at 20,000 × g for 5 minutes. The MbxA-S antigen was stored at 4°C until it was adjuvanted with polyacrylic acid.

Vaccine formulation

The vaccines containing MbxA-P or MbxA-S were formulated to deliver 500 μg of MbxA in a 2-mL volume. To each of those antigens, polyacrylic acid was added to 10% v-v in accordance with the manufacturer's instructions.q Following the addition of polyacrylic acid, the vaccines were mixed on a rotator for 1 hour at room temperature (approx 22°C). The final pHr of each mixture was incrementally adjusted to 7 with 5N NaOH. Vaccines were incubated overnight at 4°C and then warmed to room temperature; the pH was readjusted to 7 if necessary as described previously. To reduce the viscosity of the final vaccines, 1.54M NaCl was added to achieve a final salt concentration of approximately 5mM. At that salt concentration, the vaccine could be injected easily through a nasal vaccine applicator yet retained enough viscosity to adhere to a smooth vertical surface without running off. The control vaccine consisted of sterile water adjuvanted with polyacrylic acid as described previously.

Vaccination and sample collection

Steers were randomly allocated to 3 treatment groups (MbxA-P group [n = 5], MbxA-S group [5], or control group [3]) on the basis of drawing ear tag numbers from a hat without replacement. On day 0, each steer was administered 1 mL of the assigned vaccine or control solution in each nostril (total treatment dose, 2 mL) with a disposable 15-cm flexible nasal cannulas attached to a 3-mL syringe; the cannula was inserted in each nostril up to the level of the Luer-lock attachment with the syringe. Each steer's head was maintained in an elevated position with nylon halters for approximately 1 to 2 minutes immediately following treatment administration to maximize retention of the treatment solution in the nasal cavity. A second (booster) dose of the assigned treatment was administered in the same manner to each steer on day 28. Each treatment group was housed in separate, nonadjacent pens throughout the duration of the study.

Blood and tear samples were collected immediately prior to vaccination (day 0) and on days 14, 28, 42, and 55. From each steer on each sample acquisition day, a blood sample (10 mL) was collected by venipuncture from the tail vein into a serum separator tube,t allowed to clot, and then centrifuged at 2,000 × g for 20 minutes, after which the serum was harvested. Tear samples were collected from both eyes by placement of a cotton dental rollu under each lower eyelid until it was saturated. Once removed, the rolls were processed and tear fluids were collected as described.30 Tears collected from left and right eyes were pooled on each sample acquisition day. All serum and tear samples were stored at −80°C until analyzed.

The tear total protein concentration was determined in thawed tear samples following dilution (1:8 and 1:16) in Tris buffered saline–calcium chloride buffer (50mM Tris, 150mM NaCl, and 1.5mM CaCl2; pH, 7.4) by use of a commercial protein assay kit.f The final tear protein concentration was calculated from the mean of triplicate measurements at each dilution.

Tear and serum hemolysin neutralization assays

A diafiltered retentate containing native MbxA was prepared as described36 for use in tear and serum hemolysin neutralization assays. Tear and serum samples were heat inactivated at 56°C for 1 hour prior to initiation of the hemolysin neutralization assays. Serum neutralization assays were performed as described.36 The geometric mean of 2 dilution end points was used as the final serum MbxA-neutralizing antibody titer.

Tear neutralization assays were performed in 96-well plates as described,36 except that the samples were assayed in duplicate. The protein concentration in all tear samples was standardized to 2.8 mg/mL by dilution in Tris buffered saline–calcium chloride buffer prior to performance of serial 2-fold dilutions, and the volume of supernatant used for final measurement of the optical density at a wavelength of 455 nm (OD455) was 150 μL. The tear cytotoxin-neutralizing antibody titer was defined as the last serial dilution for which the OD455 remained < 25% of the mean OD455 for the 2 lysis-positive control wells, and the geometric mean of the 2 dilution end points was used as the final tear titer.

MbxA-specific immunoglobulin ELISA

The concentration of MbxA-specific IgA and IgG in tears and IgG in serum was determined by use of an ELISA. Assays were performed in flat-well 96-well platesv at room temperature. Wells were coated with either 100 μL of MbxA (5 μg/mL) diluted in coating buffer (0.05M sodium carbonate; pH, 9.6) for determination of MbxA-specific IgA or IgG concentration or affinity-purified sheep anti-bovine IgAw or IgGw diluted 1:100 in coating buffer for determination of the IgA or IgG standard curve. Plates were incubated for 1 hour on a platform shaker, then washed 5 times in ELISA buffer (50mM Tris, 0.14M NaCl, and 0.05% Tween 20; pH, 8.0). To each well, 200 μL of blocking buffer (ELISA buffer plus 2% fish gelatinx) was added. Plates were incubated for 1 hour and washed 5 times in ELISA buffer. Then, 100 μL of diluted tears (for measurement of MbxA-specific IgA and IgG concentrations), serum (for measurement of MbxA-specific IgG concentration), or bovine reference serumw (diluted to generate a standard curve) was added to each well. For the measurement of serum MbxA-specific IgG concentrations, serum samples were diluted 1:200 in ELISA buffer before being added to the wells, and some samples had to be diluted 1:400 in ELISA buffer so that the results fell within the standard curve. For the measurement of tear MbxA-specific IgA and IgG concentrations, tear samples were diluted in ELISA buffer to attain protein concentrations of 1.0 and 2.25 mg/mL, respectively, before being added to the wells. For the tear IgA assay, if the initial result for the 1.0 mg/mL protein dilution of a sample did not fall within the range established by the standard curve, an additional 1:20 dilution of that sample was made, and the assay was repeated. Duplicate and triplicate wells were created for each respective standard curve value and unknown tear and serum sample. Diluted tear or serum samples obtained from a steer that was vaccinated IN with MbxA during a previous study36 were used for positive-control wells on each plate. Plates were incubated for 1 hour on a platform shaker and washed 5 times in ELISA buffer. Then, 100 μL of either sheep anti-bovine IgG (1:50,000 in ELISA buffer) conjugated to horseradish peroxidasew or sheep anti-bovine IgA conjugated to horseradish peroxidasew (1:35,000 in ELISA buffer) was added to each well, and plates were incubated for 1 hour on a platform shaker. Following incubation, plates were washed 5 times in ELISA buffer, and 100 μL of an enzyme substratey was added to each well. Reactions were stopped with 100 μL of 0.1N HCl. The OD of each well was measured at a wavelength of 450 nm by an automated ELISA plate reader.z The final tear MbxA-specific IgA or IgG concentration was determined from the slopes of the standard curves generated by use of commercial ELISA analysis software.aa If the OD of a particular well differed by > 10% from the mean of the 3 replicates for that tear or serum sample, that value was excluded from the data analysis. To correct for interplate variation in the IgA or IgG concentrations from the positive control wells, the calculated sample IgA or IgG concentration was corrected by the following formula: (calculated sample IgA or IgG concentration) × (mean of all test plate positive-control IgA or IgG concentrations/positive-control IgA or IgG concentration for the test plate of interest).

Statistical analysis

Analysis of the data with the Shapiro-Wilk test revealed that the data were not normally distributed; thus, nonparametric analyses were used. The Friedman test was used to analyze the variance of fold changes in serum and tear MbxA-neutralizing antibody titers and concentrations of tear MbxA-specific IgA, tear MbxA-specific IgG, and serum MbxA-specific IgG between days 0 and 14 (D0-D14), days 0 and 28 (D0-D28), days 0 and 42 (D0-D42), and days 0 and 55 (D0-D55). Fold changes in antibody isotype concentrations within individuals were calculated by the use of results obtained from the same sample dilution across all study days. Post hoc analyses of differences between the 3 treatment groups (MbxA-P, MbxA-S, and control) were determined by use of the Wilcoxon signed rank test with Bonferroni adjustment for multiple comparisons and the Dunnett multiple comparison test for nonparametric or repeated-measures ANOVA. A commercial statistical software programbb was used for analyses when applicable. Values of P < 0.05 were considered significant for overall analyses, and values of P < 0.017 (0.05/3) were considered significant for the Bonferroni adjustments.

Results

The mean fold changes in serum MbxA-neutralizing antibody titer for the MbxA-S and MbxA-P groups were greater than those for the control group but did not differ significantly (Figure 1) among the 3 groups. The mean fold changes in tear MbxA-neutralizing antibody titer for the MbxA-P group were significantly (P = 0.014) greater than those for the MbxA-S and control groups, but did not differ significantly between the MbxA-S and control groups. Similarly, the mean fold changes in MbxA-specific IgG concentration in serum (P = 0.007) and tears (P = 0.031) for the MbxA-P group were significantly greater than those for the MbxA-S and control groups, whereas those for the MbxA-S and control groups did not differ significantly (Figure 2). Although the overall fold change in tear MbxA-specific IgA concentration differed significantly (P = 0.001) among the 3 groups, none of the pairwise comparisons between groups were significant (Figure 3).

Figure 1—
Figure 1—

Individual and mean ± SE treatment group fold changes in MbxA-neutralizing antibody titer in serum (A) and tear (B) samples at various intervals for steers that underwent IN administration of 500 μg of partially solubilized (MbxA-S; circles and striped bars; n = 5) or precipitated (MbxA-P; squares and gray bars; 5) recombinant Mbx-A subunit adjuvanted with polyacrylic acid or 2 mL of polyacrylic acid adjuvant (control; triangles and white bars; 3) on days 0 (D0) and 28 (D28). The fold change was calculated as the MbxA-neutralizing antibody titer (or IgA or IgG concentration) on days 14, 28, 42, or 55/MbxA-neutralizing antibody titer (or IgA or IgG concentration) on day 0. The fold change in MbxA-neutralizing antibody titer in serum did not differ among the 3 groups. The fold change in MbxA-neutralizing antibody titer in tears differed significantly (P = 0.014) among the 3 groups; the fold change for the MbxA-P group was significantly greater than that for the MbxA-S and control groups but did not differ significantly between the MbxA-S and control groups.

Citation: American Journal of Veterinary Research 77, 12; 10.2460/ajvr.77.12.1411

Figure 2—
Figure 2—

Individual and mean ± SE treatment group fold changes in MbxA-specific IgG concentration in serum (A) and tear (B) samples at various intervals for the steers of Figure 1. The fold change in MbxA-specific IgG concentration in both serum (P = 0.007) and tear (P = 0.031) samples differed significantly among the 3 groups; in both samples, the fold change for the MbxA-P group was significantly greater than that for the MbxA-S and control groups but did not differ significantly between the MbxA-S and control groups. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 77, 12; 10.2460/ajvr.77.12.1411

Figure 3—
Figure 3—

Individual and mean ± SE treatment group fold changes in MbxA-specific IgA concentration in tear samples at various intervals for the steers of Figure 1. Although the overall fold change in tear MbxA-specific IgA concentration differed significantly (P = 0.001) among the 3 groups, none of the pairwise comparisons between groups were significant. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 77, 12; 10.2460/ajvr.77.12.1411

Discussion

Results of the present study indicated that IN administration of a precipitated formulation of a carboxy subunit of recombinant MbxA (MbxA-P) adjuvanted with polyacrylic acid was superior to a partially solubilized formulation of the same cytotoxin subunit (MbxA-S) adjuvanted with polyacrylic acid in stimulating systemic and ocular MbxA-specific IgG and tear MbxA-neutralizing antibody responses. The finding that IN administration of an MbxA vaccine induced a systemic immune response in the present study was consistent with results of other studies41–46 that involved mucosal (IN) administration of various vaccines in different animal models. The steers of the present study that received the MbxA-P vaccine also had the highest mean fold changes in serum MbxA-neutralizing antibody titer, although those fold changes did not differ significantly from those for steers that received the MbxA-S and control (polyacrylic acid adjuvant) vaccines. To our knowledge, the minimum systemic or local MbxA-neutralizing antibody titer required for protection against IBK has not been determined, and it is unknown whether the antibody titers achieved in the steers of this study following vaccination with either the MbxA-P or MbxA-S vaccine would protect against the disease.

In the present study, we expected that steers vaccinated with the MbxA vaccines would have greater fold changes in mucosal (tear) MbxA-specific IgA concentrations than steers vaccinated with the control vaccine; however, a control steer had the greatest fold change in tear MbxA-specific IgA concentration. It is possible that steer developed an immune response to a naturally occurring M bovis infection following study enrollment. Although steers were examined for the presence of previous or active IBK prior to enrollment, ocular swab specimens were not obtained for bacterial culture to determine whether they were harboring M bovis prior to vaccination. Clinical signs of IBK were not observed in any of the steers during the weekly examinations conducted throughout the observation period, but cattle can be inapparent carriers of M bovis.47 Additional studies on the types of systemic and local antibody responses induced by M bovis antigens in carrier cattle are necessary to provide insight into the ocular IgA response that should be expected in inapparent M bovis carriers. In the absence of that data, we recommend that bacterial culture of specimens obtained from ocular and nasal surfaces of cattle be performed prior to enrollment in future studies that require interpretation of the immune response to M bovis vaccine antigens, especially when cattle are housed in environments such as feedlots, where natural exposure to M bovis can occur. Those culture results could provide additional information and aid in the interpretation of study results, particularly in the event that unexpectedly high antibody titers are detected in unvaccinated cattle.

Another possible explanation for the unexpectedly high fold changes in tear IgA concentration observed for the control steers was that the ELISA used detected nonspecific IgA that recognized the recombinant MbxA, the target antigen in the assay. Mannheimia haemolytica leukotoxin and MbxA are both repeats-in-toxin exoproteins with amino acid sequences that are approximately 50% identical48; thus, M haemolytica leukotoxin could induce IgA that might cross-react with MbxA. All steers received an M haemolytica leukotoxin toxoid prior to study enrollment; however, we analyzed fold changes in IgA concentration from the start of the study (day 0), and therefore, preexisting IgA titers were accounted for in the analysis. It is possible that at least 1 control steer was exposed to M haemolytica during the observation period, which induced an anamnestic IgA response that cross-reacted with the ELISA target antigen. The mean fold change in tear MbxA-neutralizing antibody titer remained fairly low in the control group throughout the observation period, whereas that for the MbxA-P group steadily increased from day 0. That finding further supported that the high fold change in tear IgA concentration for at least one of the control steers was the result of the detection of nonspecific IgA by the ELISA. In fact, if the data for the control steer with the greatest fold change in tear MbxA-neutralizing antibody titer were excluded, the magnitude of the difference in the mean fold changes for that titer between the MbxA-P and control groups would have been more substantial. We recommend that functional (antigen neutralizing) antibody assays be performed in addition to ELISA whenever possible when changes in antibody responses to M bovis antigens are evaluated because the information provided may be valuable for the interpretation of unexpected results.

Results of the present study extended the findings from our first study36 that involved the IN administration of an MbxA subunit antigen. In that study,36 the high-dose group received the same vaccine and antigenic dose as that administered to the MbxA-P group in the present study, and although the changes in tear IgG ratios were greatest for that group, they did not differ significantly from those for the unvaccinated control group. In the present study, the mean fold change in MbxA-specific tear IgG for the MbxA-P group was significantly greater than that for the control group. Compared with the previous study,36 the present study had larger study groups and the vaccine was administered through a longer nasal cannula. Also, in the previous study,36 the immune function test results were standardized by tear protein concentration after running the test, whereas in the present study, the protein concentration in tear samples was standardized before the ELISA and MbxA-neutralization tests were performed. Steers were not prescreened to identify those with low preexisting anti-MbxA antibody titers in either study; instead, changes in the immune response relative to day 0 were evaluated to account for differences in prevaccination immunity among study steers.

Given the possibility that local ocular immune responses to M bovis antigens may exacerbate ocular injury in cattle with IBK,31 it seemed logical to investigate mucosal vaccination as a method to augment local IgA titers against MbxA. Unfortunately, the minimum systemic or mucosal antibody titer against M bovis required to protect cattle against IBK has not been determined. Consequently, we were unable to speculate whether the immune responses observed in the present study would protect cattle against IBK. It is also unclear whether the high tear MbxA-specific IgG concentrations observed in the steers of the MbxA-P group might be detrimental to ocular health; however, the tear MbxA-specific IgA concentrations detected in those steers might protect the ocular surface despite the presence of IgG.

In the present study, the greatest change in the mean immune response for steers in the MbxA-P group was observed at 42 days after vaccination, and the immune responses were generally declining by 55 days after vaccination. It is unknown whether the immune response observed on day 55 after vaccination would be adequate to protect cattle against naturally occurring IBK. The duration of the immune response observed in this study might be adequate to protect calves against IBK during a typical summer when the risk for IBK is highest; however, additional studies are necessary to fully evaluate whether the MbxA-P vaccine is protective against IBK and determine how the kinetics of the immune response change with and without booster vaccination. Another issue that needs to be investigated is the comparison of the immune response following administration of the vaccine in 1 nostril with that following administration of the vaccine in both nostrils. We divided the vaccine dose equally and administered it in both nostrils (1 mL/nostril) in the present study. It is possible that administration of the entire 2-mL dose in 1 nostril might result in a greater amount of the vaccine antigen reaching the nasal-associated lymphoid tissues of the pharyngeal lymphoid ring than splitting the dose between the 2 nostrils.

Some investigators may prefer randomization into study groups that takes into consideration rankings by weight; however, because we considered the steers in this study to be fairly uniform in age and body weight at the time of study enrollment, weight rankings were not used in our randomization scheme. We subsequently performed a Kruskal-Wallis test to compare steer weights among the 3 treatment groups, and results indicated that body weight did not differ significantly (P = 0.480) among the 3 treatment groups. Therefore, we feel that the randomization method used in this study was adequate.

Mixed results have been reported by other researchers9,49–51 who have investigated mucosal vaccination with M bovis antigens. Cattle were protected against IBK following aerosol administration of an M bovis bacterin.9,49 Intranasal administration of native M bovis pili to calves resulted in the stimulation of high anti-pili IgA responses, but those responses could not be correlated with protection against IBK.50 It is possible that antigenic variation associated with the various pilus types of naturally occurring M bovis strains accounted for the apparent lack of vaccine efficacy in that study.50 However, some protective benefits were observed in calves following topical administration of an M bovis bacterin adjuvanted with interleukin-2 and interferon-α.51

In the present study, systemic and ocular immune responses induced by IN administration of the MbxA-P vaccine were superior to those induced by the MbxA-S vaccine. Further research is necessary to determine whether the MbxA-P vaccine is protective against naturally occurring IBK and whether it has better efficacy than a parenterally administered formulation of recombinant MbxA.

Acknowledgments

Supported by USDA Formula Funds administered through the University of California-Davis Center for Food Animal Health (Project #CALV-AH-322).

Presented in abstract form at the Conference of Research Workers in Animal Diseases Meeting, Chicago, December 2014.

The authors thank James Moller for technical assistance.

ABBREVIATIONS

IBK

Infectious bovine keratoconjunctivitis

IN

Intranasal

MbxA

Moraxella bovis cytotoxin

Footnotes

a.

Se 365 bolus selenium supplement, Pacific Trace Minerals Inc, Sacramento, Calif.

b.

Inforce 3, Zoetis Inc, Florham Park, NJ.

c.

Ultrabac 7/Somubac, Zoetis Inc, Florham Park, NJ.

d.

One Shot, Zoetis Inc, Florham Park, NJ.

e.

Dectomax Pour-On, Zoetis Inc, Florham Park, NJ.

f.

BCA Kit, Thermo Fisher Scientific Inc, Waltham, Mass.

g.

Slide-A-Lyzer dialysis cassette (extra strength), 10,000-kDa molecular-weight cutoff, Thermo Fisher Scientific Inc, Waltham, Mass.

h.

SuperScript II, Invitrogen, Carlsbad, Calif.

i.

Invitrogen, Carlsbad, Calif.

j.

Topo TA cloning kit, Invitrogen, Carlsbad, Calif.

k.

Davis Sequencing, Davis, Calif.

l.

BL21-AI, Invitrogen, Carlsbad, Calif.

m.

Sorvall SS-34 rotor, Sorvall Inc, Norwalk, Conn.

n.

Superdex 200, GE Healthcare, Pittsburgh, Pa.

o.

ImageJ analysis software, version 1.46, National Institutes of Health, Bethesda, Md. Available at: rsbweb.nih.gov/ij/index.html. Accessed Mar 18, 2013.

p.

MicroCentrifuge HP, Thermo Electron Corp, Milford, Mass.

q.

Carbigen, MVP Technologies, Omaha, Neb.

r.

pH 5–10 strips, EMD Chemicals Inc, Billerica, Mass.

s.

Equine Nasal Applicator, Boehringer Ingelheim Pharmaceuticals Inc, Ridgefield, Conn.

t.

Tyco Healthcare Group LP, Mansfield, Mass.

u.

Patterson Dental Supply Inc, Saint Paul, Minn.

v.

Immulon 4HBX Ultra-high Binding Polystyrene Microtiter Plates, Thermo Fisher Scientific Inc, Rochester, NY.

w.

Bethyl Laboratories, Montgomery, Tex.

x.

Teleostean gelatin, Sigma Life Science, St Louis, Mo.

y.

Tetramethylbenzine peroxidase substrate ELISA, Moss Inc, Pasadena, Md.

z.

SpectraMax 250, Molecular Devices Corp, Sunnyvale, Calif.

aa.

MasterPlex ReaderFit, version 2.0.0.68, Hitachi Solutions America Ltd, MiraiBio Group, Alameda, Calif.

bb.

SAS, version 9.3, SAS Institute Inc, Cary, NC.

References

  • 1. Henson JB, Grumbles LC. Infectious bovine keratoconjunctivitis. I. Etiology. Am J Vet Res 1960; 21: 761766.

  • 2. Iwasa M, Kawada A, Yorita M, et al. Prolonged occurrence of Moraxella bovis infection on a restricted heifer farm. J Vet Med Sci 1994; 56: 429432.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Conceição FR, Paolicchi F, Cobo AL, et al. Antigenic relationships of Moraxella bovis isolates recovered from outbreaks of infectious bovine keratoconjunctivitis in Argentina, Brazil, and Uruguay between 1983 and 2000. Can J Vet Res 2003; 67: 315318.

    • Search Google Scholar
    • Export Citation
  • 4. Alexander D. Infectious bovine keratoconjunctivitis: a review of cases in clinical practice. Vet Clin North Am Food Anim Pract 2010; 26: 487503.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Schnee C, Heller M, Schubert E, et al. Point prevalence of infection with Mycoplasma bovoculi and Moraxella spp. in cattle at different stages of infectious bovine keratoconjunctivitis. Vet J 2015; 203: 9296.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 6. Nayar PS, Saunders JR. Infectious bovine keratoconjunctivitis I. Experimental production. Can J Comp Med 1975; 39: 2231.

  • 7. Yeruham I, Perl S, Elad D. Infectious bovine keratoconjunctivitis and lymphofollicular hyperplasia of the third eyelid in heifers. J Vet Med B Infect Dis Vet Public Health 2001; 48: 137141.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8. Barner RD. A study of Moraxella bovis and its relation to bovine keratitis. Am J Vet Res 1952; 13: 132144.

  • 9. Misiura M. Keratoconjunctivitis infectiosa in calves—attempt at elimination by active immunization. Arch Vet Pol 1994; 34: 187194.

    • Search Google Scholar
    • Export Citation
  • 10. Angelos JA, Dueger EL, George LW, et al. Efficacy of florfenicol for treatment of naturally occurring infectious bovine keratoconjunctivitis. J Am Vet Med Assoc 2000; 216: 6264.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11. Dueger EL, Angelos JA, Cosgrove S, et al. Efficacy of florfenicol in the treatment of experimentally induced infectious bovine keratoconjunctivitis. Am J Vet Res 1999; 60: 960964.

    • Search Google Scholar
    • Export Citation
  • 12. Dueger EL, George LW, Angelos JA, et al. Efficacy of a long-acting formulation of ceftiofur crystalline-free acid for the treatment of naturally occurring infectious bovine keratoconjunctivitis. Am J Vet Res 2004; 65: 11851188.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. Moore LJ, Rutter JM. Attachment of Moraxella bovis to calf corneal cells and inhibition by antiserum. Aust Vet J 1989; 66: 3942.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14. Ruehl WW, Marrs C, Beard MK, et al. Q pili enhance the attachment of Moraxella bovis to bovine corneas in vitro. Mol Microbiol 1993; 7: 285288.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15. Annuar BO, Wilcox GE. Adherence of Moraxella bovis to cell cultures of bovine origin. Res Vet Sci 1985; 39: 241246.

  • 16. Beard MK, Moore LJ. Reproduction of bovine keratoconjunctivitis with a purified haemolytic and cytotoxic fraction of Moraxella bovis. Vet Microbiol 1994; 42: 1533.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17. Gray JT, Fedorka-Cray PJ, Rogers DG. Partial characterization of a Moraxella bovis cytolysin. Vet Microbiol 1995; 43: 183196.

  • 18. Kagonyera GM, George LW, Munn R. Cytopathic effects of Moraxella bovis on cultured bovine neutrophils and corneal epithelial cells. Am J Vet Res 1989; 50: 1017.

    • Search Google Scholar
    • Export Citation
  • 19. Lehr C, Jayappa HG, Goodnow RA. Serologic and protective characterization of Moraxella bovis pili. Cornell Vet 1985; 75: 484492.

  • 20. Jayappa HG, Lehr C. Pathogenicity and immunogenicity of piliated and nonpiliated phases of Moraxella bovis in calves. Am J Vet Res 1986; 47: 22172221.

    • Search Google Scholar
    • Export Citation
  • 21. Lepper AW, Atwell JL, Lehrbach 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: 129138.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 22. Lepper AW, Elleman TC, Hoyne PA, et al. A Moraxella bovis pili vaccine produced by recombinant DNA technology for the prevention of infectious bovine keratoconjunctivitis. Vet Microbiol 1993; 36: 175183.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 23. 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: 310316.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24. Moore LJ, Lepper AW. A unified serotyping scheme for Moraxella bovis. Vet Microbiol 1991; 29: 7583.

  • 25. Marrs CF, Ruehl WW, Schoolnik GK, et al. Pilin-gene phase variation of Moraxella bovis is caused by an inversion of the pilin genes. J Bacteriol 1988; 170: 30323039.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26. Angelos JA, Ball LM. Relatedness of cytotoxins from geographically diverse isolates of Moraxella bovis. Vet Microbiol 2007; 124: 382386.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27. Ostle AG, Rosenbusch RF. Immunogenicity of Moraxella bovis hemolysin. Am J Vet Res 1985; 46: 10111014.

  • 28. Billson FM, Hodgson JL, Egerton JR, et al. A haemolytic cell-free preparation of Moraxella bovis confers protection against infectious bovine keratoconjunctivitis. FEMS Microbiol Lett 1994; 124: 6973.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29. George LW, Borrowman AJ, Angelos JA. Effectiveness of a cytolysin-enriched vaccine for protection of cattle against infectious bovine keratoconjunctivitis. Am J Vet Res 2005; 66: 136142.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30. Angelos JA, Hess JF, George LW. Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine. Vaccine 2004; 23: 537545.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31. Kagonyera GM, George LW, Munn R. Light and electron microscopic changes in corneas of healthy and immunomodulated calves infected with Moraxella bovis. Am J Vet Res 1988; 49: 386395.

    • Search Google Scholar
    • Export Citation
  • 32. Pedersen KB. The origin of immunoglobulin-G in bovine tears. Acta Pathol Microbiol Scand B Microbiol Immunol 1973; 81: 245252.

  • 33. Nielsen K, Duncan JR. Bovine IgM: does it fix guinea pig complement in the absence of bovine complement components? Vet Immunol Immunopathol 1987; 14: 335343.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 34. Vaerman JP, Heremans JF. Effect of neuraminidase and acidification on complement-fixing properties of human IgA and IgG. Int Arch Allergy Appl Immunol 1968; 34: 4952.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 35. Marnila P, Korhonen H. Milk proteins: immunoglobulins. In: Fuquay JW, Fox PF, McSweeney PLH, eds. Encyclopedia of dairy sciences. 2nd ed. Cambridge, Mass: Academic Press, 2011; 807815.

    • Search Google Scholar
    • Export Citation
  • 36. Angelos JA, Edman JM, Chigerwe M. Ocular immune responses in steers following intranasal vaccination with recombinant Moraxella bovis cytotoxin adjuvanted with polyacrylic acid. Clin Vaccine Immunol 2014; 21: 181187.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 37. Horwitz J. Alpha-crystallin can function as a molecular chaperone. Proc Natl Acad Sci U S A 1992; 89: 1044910453.

  • 38. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156159.

    • Search Google Scholar
    • Export Citation
  • 39. Studier FW, Rosenberg AH, Dunn JJ, et al. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 1990; 185: 6089.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 40. Nagai K, Thøgersen HC. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol 1987; 153: 461481.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 41. Wang G, Pan L, Zhang Y, et al. Intranasal delivery of cationic PLGA nano/microparticles-loaded FMDV DNA vaccine encoding IL-6 elicited protective immunity against FMDV challenge. PLoS ONE 2011; 6: e27605.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 42. Boland KG, Hayles AN, Miller CB, et al. Regional immune response to immunization with Escherichia coli O157:H7-derived intimin in cattle. Clin Vaccine Immunol 2013; 20: 562571.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 43. Stanley AC, Buxton D, Innes EA, et al. Intranasal immunisation with Toxoplasma gondii tachyzoite antigen encapsulated into PLG microspheres induces humoral and cell-mediated immunity in sheep. Vaccine 2004; 22: 39293941.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 44. Shen X, Lagergård T, Yang Y, et al. Systemic and mucosal immune responses in mice after mucosal immunization with group B streptococcus type III capsular polysaccharide-cholera toxin B subunit conjugate vaccine. Infect Immun 2000; 68: 57495755.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 45. Mapletoft JW, Latimer L, Babiuk LA, et al. Intranasal immunization of mice with a bovine respiratory syncytial virus vaccine induces superior immunity and protection compared to those by subcutaneous delivery or combinations of intranasal and subcutaneous prime-boost strategies. Clin Vaccine Immunol 2010; 17: 2335.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 46. Liu Z, Yuan F, Yang Y, et al. Partial protective immunity against toxoplasmosis in mice elicited by recombinant Toxoplasma gondii malate dehydrogenase. Vaccine 2016; 34: 989994.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 47. Pugh GW Jr, Hughes DE. Bovine infectious keratoconjunctivitis: carrier state of Moraxella bovis and the development of preventive measures against disease. J Am Vet Med Assoc 1975; 167: 310313.

    • Search Google Scholar
    • Export Citation
  • 48. Angelos JA, Hess JF, George LW. An RTX operon in hemolytic Moraxella bovis is absent from nonhemolytic strains. Vet Microbiol 2003; 92: 363377.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 49. Misiura M. Estimation of fimbrial vaccine effectiveness in protection against keratoconjunctivitis infectiosa in calves considering different routes of introducing vaccine antigene. Arch Vet Pol 1994; 34: 177186.

    • Search Google Scholar
    • Export Citation
  • 50. Zbrun MV, Zielinski GC, Piscitelli HC, et al. Evaluation of anti-Moraxella bovis pili immunoglobulin-A in tears following intranasal vaccination of cattle. Res Vet Sci 2012; 93: 183189.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 51. di Girolamo FA, Sabatini DJ, Fasan RA, et al. Evaluation of cytokines as adjuvants of infectious bovine keratoconjunctivitis vaccines. Vet Immunol Immunopathol 2012; 145: 563566.

    • Crossref
    • Search Google Scholar
    • Export Citation

Contributor Notes

Address correspondence to Dr. Angelos (jaangelos@ucdavis.edu).