Infectious Bovine Keratoconjunctivitis (IBK)
John A. Angelos
Infectious bovine keratoconjunctivitis (IBK), or “pinkeye,” is the most common ocular disease of cattle and has been identified in cattle populations worldwide.
The clinical signs of IBK include corneal ulceration, corneal edema, photophobia, blepharospasm, and lacrimation (Color Plate 39.23). In less severely affected animals, recovery with or without permanent corneal scarring occurs. In the most severely affected animals, corneal rupture and lens or iris prolapse may occur, causing permanent blindness. IBK is most common in calves, typically affecting one eye, although both eyes may be affected. Estimates of annual incidence of IBK are 5% of all beef cattle, with more than 50% of herds affected.1 Epizootic outbreaks occur, with case attack rates approaching 90% to 100% of yearling cattle.2,3 Cattle of all breeds may be affected; however, a higher incidence is reported in Herefords,4 and a lower incidence is reported in Brahmans and cattle with more pigmentation at the ocular margins.5,6Genetic investigations of cattle affected with IBK have suggested the presence of loci that are associated with IBK.7,8 Hereford-Friesian crossbred calves with full eyelid pigmentation had a lower incidence of IBK versus calves with less than total pigmentation.9 Five single nucleotide polymorphisms (SNPs) on bovine chromosome 20 that are significantly associated with IBK have also been identified.10 A relationship was also reported between a specific SNP in the Toll-like receptor 4 gene and the rate of IBK infection in Angus cattle.11
■ Economic Impact Cattle with IBK have reduced weight gain that results in economic losses to producers. Postweaning 205-day weights of bulls and heifers with IBK showed losses of 36 and 40 pounds (16 and 18 kg), respectively, over unaffected herdmates.12 In 1169 pasture-raised calves over a 4-year period, the average weight reduction was 11 pounds (5 kg) for calves with IBK in one eye and 35 pounds (15.75 kg) for calves with IBK in both eyes.13 A study of more than 45,000 health records of weaned calves demonstrated almost 20-pound (9-kg) lighter weaning weights in calves diagnosed with IBK versus healthy calves.4 A retrospective population-based cohort study of approximately 1800 Angus calves over a 7-year period indicated that yearlings with evidence of IBK at weaning had reduced twelfth rib fat depth, ribeye area, and body weight versus cohorts without IBK.14 The economic losses from reduced weight gain along with treatment-associated expenses accounted for annual losses in the United States that many years ago were estimated to be $150 million.15
■ Etiology and Epidemiology Moraxella bovis is the only organism for which Koch's postulates have been fulfilled with respect to IBK16; however, other viruses and bacteria have been associated with IBK, including infectious bovine rhinotracheitis (IBR) virus17,18 and Mycoplasma spp.19,20 These organisms may increase risk for IBK by enhancing opportunities for corneal injury21-23 and increasing ocular and nasal discharge, which may facilitate transmission of Moraxella bovis.
In an outbreak of IBK in young calves, Mycoplasma bovoculi and Mycoplasma bovis were isolated from affected animals, whereas Moraxella bovis was not isolated.24 In a recent study, IBR exposure as indicated by positive BHV-1 titer was not found to predispose calves to developing IBK in a herd where Moraxella bovis was present.25For many years, a role for hemolytic gram-negative cocci in IBK pathogenesis has been suggested from the observations of researchers in the field. In 1966 hemolytic gram-negative cocci were isolated from calves with severe keratitis and corneal ulceration.26 In Australia, Neisseria species were isolated in 24 of 25 outbreaks of IBK; Moraxella bovis was identified in only two of these outbreaks.27 Neisseria (Branhamella) catarrhalis was reported in almost 45% of IBK cases, whereas Moraxella bovis was isolated from only 28% of IBK cases.28 Moraxella bovis and Neisseria species were also cultured from normal eyes of cattle.29,30 Moraxella ovis and Neisseria ovis were also reported from cattle in Israel with IBK.31,32 Neisseria ovis experimentally inoculated into the eyes of calves did not cause lesions typical of IBK, despite previous corneal irradiation.33 M. ovis was reported in mule deer with keratoconjunctivitis, although experimental inoculation of M. ovis isolates into eyes of mule deer fawns did not cause IBK.34 Recently, hemolytic and cytolytic activity from culture filtrates of M. ovis isolated from cattle with IBK has been described, suggesting a possible role for bacteria other than Moraxella bovis in the pathogenesis of IBK.35
During an IBK drug efficacy trial during summer 2002 in northern California, the majority of bacterial isolates from IBK-affected calves were found to be hemolytic gram-negative cocci.36 Genetic and biochemical characterization of these isolates subsequently identified these organisms as a novel species that was distinct from Moraxella bovis and M.
ovis; it was named Moraxella bovoculi3 It is likely that Moraxella bovoculi has circulated within cattle populations for many years, and until this gram-negative coccus had been shown to be distinct from M. ovis, it may have been misidentified by diagnostic laboratories as M. ovis, Branhamella ovis, M. ovis-like, or B. ovis-like. A weak causal role for Moraxealla bovoculi (as well as for Moraxella bovis) was reported in a cohort study that looked for Moraxella bovis, M. ovis, and Moraxella bovoculi DNA in swabs from eyes of beef calves.38 However, in an experimental challenge study using a corneal scarification model for infection with the type strain of Moraxella bovoculi, corneal ulceration could not be demonstrated.39 A point prevalence study that evaluated ocular flora in animals from herds in varying stages of IBK outbreaks for the presence of DNA from Moraxella bovis, M. ovis, Moraxella bovoculi, Mycoplasma bovoculi, and Mycoplasma bovis determined that Moraxella bovoculi was the only organism that correlated with clinical signs of IBK.40 In that study, herds with high Mycoplasma bovoculi were more predisposed to IBK outbreaks, and authors postulated that there may be synergistic interactions between Mycoplasma bovoculi and Moraxella spp. in IBK. During 2010 to 2013 Moraxella bovoculi was the most frequently isolated organism from IBK-affected cattle in the central United States.41 When the microbial community in the eyes of a cohort of calves (some of which subsequently developed IBK and some of which did not) were examined using high-throughput sequencing of 16S ribosomal RNA, Moraxella was found to be among the top 10 most abundant genera; however, the difference in abundance between calves that never developed IBK and those that did was not significant.42 Marked genetic differences were determined after whole genome sequence analysis of Moraxella bovoculi from cattle with IBK and from nonocular sites in asymptomatic cattle; these data suggest the presence of genetically distinct strains of Moraxella bovoculi.4 In a study of ocular flora from normal goats, Moraxella bovoculi was the most common single bacterial species isolated.44In addition to bacterial infection, other risk factors for IBK include flies, solar irradiation, and mechanical trauma from plant awns.
Moraxella bovis survives up to 3 days on the external surface45 and 2 days in the gut46 of face flies (Musca autumnalis). IBK was experimentally induced in cattle that were exposed to face flies that had fed on Moraxella bovis cultures.47 Insecticide- impregnated ear tags or back and face rubbers to reduce fly populations have proved effective in reducing IBK in cattle populations.48 An association between solar irradiation and IbK has also been documented.49-51 Corneas of calves exposed to UV irradiation incur corneal epithelial cell degeneration52 that predisposes eyes to establishment of Moraxella bovis and IBK. An experimental model for IBK has been developed in which calves are exposed once daily for 2 or 3 days to UV irradiation using sunlamps held 24 inches (60 cm) from the corneal surface; after the last exposure, animals are infected by instillation of Moraxella bovis into the eye.53-55 Plant awns have been associated with IBK, most likely through direct mechanical damage to the cornea that facilitates infection with Moraxella bovis and subsequent development of IBK.Hemolytic strains of Moraxella bovis are considered pathogenic. Nevertheless, nonhemolytic, nonpathogenic Moraxella bovis can be isolated from normal cattle,2,56-58 from cattle exhibiting conjunctivitis,59 and simultaneously with hemolytic Moraxella bovis from cattle with IBK.58 Outbreaks of IBK typically occur annually during summer months, and such outbreaks were postulated to be caused in part by cattle harboring Moraxella bovis subclinically.58,60
■ Pathophysiology of Moraxella Bovis Moraxella bovis produces many hydrolytic enzymes that may be important in facilitating ocular injury, including C4 esterase, C8 esteraselipase, C14 lipase, phosphoamidase, phosphatase, leucine and valine aminopeptidases, and gelatinase.61 To date, however, only two Moraxella bovis proteins have been linked to pathogenicity: pili and cytotoxin.
Pilin proteins of Moraxella bovis are of the N-methylphenylalanine type (type IV pili62-64) and enable bacteria to adhere to the corneal epithelium and colonize the surface of the cornea.65-67 Moraxella bovis is capable of forming biofilms through formation of type IV pili, and disruption of these pili prevents biofilm formation.68The Moraxella bovis cytotoxin (cytolysin/hemolysin) is a pore-forming protein that is also considered necessary for pathogenesis. Broth supernatants of hemolytic strains of Moraxella bovis will cause lysis of bovine erythrocytes, neutrophils, lymphoma cells, and corneal epithelial cells in vitro.69-72 The lytic activity of Moraxella bovis cytotoxin occurs through calcium-dependent formation of transmembrane pores in target cell membranes.73 Ocular lesions induced by a purified hemolytic and cytolytic fraction of Moraxella bovis are identical to the ocular lesions observed in naturally occurring IBK; extracts from nonhemolytic Moraxella bovis do not cause corneal lesions.74
An association between the Moraxella bovis cytotoxin and the RTX (repeats in the structural toxin) family of bacterial exoproteins followed the discovery that Moraxella bovis cytotoxin induces the formation of pores in target cell membranes.73 It was subsequently shown that an approximately 110-kilodalton protein in concentrated culture supernatants from cytolytic Moraxella bovis cultures could be recognized by a monoclonal antibody to HlyA, an RTX toxin of uropathogenic E. coli7 The presence of RTX toxins has been reported in numerous animal pathogens, including Mannheimia haemolytica, the agent of shipping fever pleuropneumonia in cattle76; Actinobacillus pleuropneumoniae, an agent of swine pleuropneumonia77; Pas- teurella aerogenes, an abortifacient in swine and other mammals78; and Actinobacillus equuli, associated with foal septicemia.79 Enterohemorrhagic E.
coli O157:H7 also harbors a plasmid- encoded RTX toxin.80The best-characterized RTX toxin is HlyA of uropathogenic E. coli. The gene encoding this toxin is assigned the abbreviation hlyA and is contained within a four-gene operon organized 5,-C-A-B-D-3,. The product of the RTX A gene is a structural toxin that must be activated by the RTX C gene product to become hemolytic.81-83 The activation occurs through fatty acylation of conserved lysine residues.84,85 After activation the toxin is secreted by membrane transport proteins encoded by the B and D genes and a third protein, TolC.86,87 The regulation of transcription through the RTX operon in E. coli is a complex process and involves the protein RfaH and JUMPStart DNA sequences.88 As with E. coli hemolysin, the Moraxella bovis cytotoxin gene (mbxA) is contained within an RTX operon (mbx operon) that encodes activation and export proteins; this operon is absent in nonhemolytic Moraxella bovis.89 The mbx operon defines a pathogenicity island, and acquisition or loss of mbx genes may explain the ability of Moraxella bovis to change from the hemolytic to nonhemolytic phenotype.90 Moraxella bovoculi and Moraxella ovis have also been shown to encode RTX toxins that reside within classical RTX operons.91 Near-complete identity between the deduced amino acid sequences of cytotoxin genes and geographically diverse Moraxella bovis isolates has been reported and demonstrates that there is a high degree of conservation in the gene encoding this important pathogenic factor.92
■ Immunity to Moraxella Bovis Secretory IgA is the major immunoglobulin found in normal bovine lacrimal secre- tions.93 During experimentally induced IBK, tear IgGl and IgG2 concentrations increase.94 Early studies suggested that calves with more severe IBK had higher lacrimal IgA titers to crude Moraxella bovis antigen preparations than calves with less severe IBK.95 A subsequent study identified a predominant tear IgG response to a crude, whole-cell Moraxella bovis antigen in calves with naturally occurring IBK and concluded that Moraxella bovis-specific antibodies in lacrimal secretions did not prevent IBK in calves.96 Enzyme-linked immunosorbent assay (ELISA) has been used to quantify nonspecific Moraxella bovis antigens in tears and has revealed that IgA titers are higher than IgG titers.97 A study on a small number of calves then suggested that both lacrimal (secretory IgA) and humoral (IgG) antibodies against Moraxella bovis whole-cell antigen conferred resistance against IBK, versus a humoral IgG antibody response alone.98 It was concluded that serum antibodies against Moraxella bovis may account for a reduction in the length and severity of clinical signs associated with IBK. Unfortunately, none of these studies adequately controlled for total antibody isotypes present in serum or ocular secretions. In addition, crude Moraxella bovis antigen preparations were used in these assays.
■ Experimental Vaccination Early studies that reported reduced Moraxella bovis infection rates and decreased occurrence of IBK after reexposure to Moraxella bovis indicated that vaccination against IBK might prevent disease.50 Subsequent work showed that calves vaccinated intramuscularly with live Moraxella bovis had less severe IBK after challenge.99 Formalin-killed Moraxella bovis was also reported to be as effective as live cultures in preventing experimentally induced IBK100; under field conditions, however, a formalin-killed autogenous bacterin was not efficacious.101
In an effort to identify other candidate Moraxella bovis vaccine antigens, researchers began to examine the use of component or subunit vaccines to prevent IBK. In early studies, Moraxella bovis pilin antigens were found to protect calves from homologous challenge.102 Purified Moraxella bovis ribosomes were not protective.103 Bacterin-containing pili plus corneal-degrading enzymes were protective in field trials, and protection was correlated with the corneal-degrading enzyme level in the vaccine.104 In that study, however, the exact composition of corneal-degrading enzymes was not reported. In a later study, two commercial Moraxella bovis pilus-based vaccines were not protective for calves in a heterologous challenge model.105 Although pilin is immunogenic, there is marked antigenic diversity between different pilus types because of the presence of two structural pilin genes and variability in the amino acid composition of the pilin molecule caused by inversions within pilin genes.66,67,106,107 Limited antigenic cross-reactivity was reported between heterologous pili,108,109 and emergence of novel pilus types can precipitate IBK outbreaks.110 Such antigenic variability is believed to reduce the overall efficacy of pilus-based vaccines. Nevertheless, more recent work has demonstrated conserved epitopes across different pilus types,111,112 suggesting a possible future role for conserved pilus antigens in IBK vaccines. In Australia, the prevalence of a limited number of pilus antigens in Moraxella bovis suggests that the use of vaccines containing certain broadly represented pilus types may help reduce losses associated with IBK.113
Unlike pili, the Moraxella bovis cytotoxin is more conserved across different Moraxella bovis strains. IBK-affected cattle were shown to develop systemic immune responses to cytotoxin,114-117 and antihemolysin antibodies to one Moraxella bovis strain neutralized hemolysin from 33 different strains of Moraxella bovis11 A partly purified cytotoxin vaccine also protected calves against IBK after challenge with heterologous Moraxella bovis.114 These reports suggest a role for a cytotoxin-based vaccine to prevent IBK from multiple Moraxella bovis strains, which could therefore be superior to traditional pilus-based vaccines.
Methods to partially purify the Moraxella bovis cytotoxin have been published,118 and its efficacy in a vaccine to prevent IBK has been demonstrated.119 Calves vaccinated with the recombinant carboxy terminus of Moraxella bovis cytotoxin had a lower cumulative proportion of ulcerated eyes compared with saline and adjuvant control calves.120 Along with recombinant Moraxella bovis cytotoxin, other recombinant cytotoxin- based vaccines to prevent IBK have been investigated, including recombinant Moraxella bovis pilin plus cytotoxin,121 recombinant Moraxella bovoculi cytotoxin,122 and recombinant Moraxella bovis pilin-cytotoxin plus recombinant Moraxella bovoculi cytotoxin.123 Trends for reduced IBK in calves vaccinated with recombinant Moraxella bovis cytotoxin suggest promise for such recombinant vaccines, but it is likely that additional protective antigens will need to be included in such vaccines to increase their efficacy against IBK.
Although the vast majority of IBK vaccine trials have evaluated vaccines administered by a parenteral route, the use of mucosal routes of vaccine delivery has been investigated. In one study, aerosol vaccination of cattle with uncharacterized Moraxella bovis antigens was reportedly effective against IBK.124 Intranasal Moraxella bovis pilin vaccines stimulated pilin-specific IgA in ocular secretions; the presence of such antibodies, however, could not be correlated with prevention of Moraxella bovis ocular infection or the development of IBK lesions.125 An experimental intranasal recombinant Moraxella bovis cytotoxin-based vaccine has been developed126,127 that appears to reduce severity of corneal ulceration associated with IBK.128 ■ Diagnosis, Treatment, and Prevention Diagnosis of IBK is usually based on clinical signs (see Color Plate 39.23). Ocular swabs submitted for bacterial culture can assist practitioners in identifying a specific bacterial species involved in infection and developing more specific therapeutic recommendations based on antimicrobial sensitivity data. When collecting ocular samples for culture, swabbing the periphery of active corneal ulcers and avoiding the subconjunctival cul- de-sac can help reduce contaminant overgrowth and may enhance recovery of Moraxella spp. (K. Clothier, personal communication). To culture Mycoplasma from ocular swabs, special media and handling may be required, and it is a good idea to consult with your diagnostic laboratory prior to sample collection.
In a recent systematic review of published studies of IBK treatment with antibiotics, most treatments were considered to be effective in IBK, especially during posttreatment days 7 to 14.129 Moraxella bovis is susceptible to penicillin administered subconjunctivally130 parenteral oxytetracycline,131,132 florfenicol administered intramuscularly (two 20-mg/kg injections 48 hours apart) or subcutaneously (40 mg/kg once),133,134 ceftiofur crystalline-free acid administered subcutaneously (6.6 mg ceftiofur equivalents/kg once) in the posterior aspect of the pinna,36 and tulathromycin (2.5 mg/kg) administered subcutane- ously.135 The antibiotic sensitivities of 57 California Moraxella bovoculi field isolates have been reported and suggest that commonly used antibiotics for the treatment of IBK associated with Moraxella bovis should also be effective against Moraxella bovoculi)16 Regional differences in antibiotic susceptibility among Moraxella bovoculi and Moraxella bovis exist41,136,137 and underscore the importance of making informed treatment decisions based on antimicrobial susceptibility data. Nonantibiotic treatments of IBK have become increasingly important for purveyors of antibiotic-free cattle. A recent study found that hypochlorous acid spray can reduce pain, infection, and healing time of corneal lesions in calves experimentally infected with Moraxella bovis)19
Use of an NSAID, such as flunixin meglumine, can help relieve ocular pain and inflammation in severe cases. For individual animals in hospital settings, autologous patient serum applied topically may also assist in healing. Use of denim patches to cover an affected eye is controversial, but it is likely that such patches can relieve ocular pain associated with exposure to bright sunlight and may reduce vector spread of infectious ocular secretions in a herd setting. When applying patches, it is important to allow for ventral drainage; producers should also be advised to check under patches at least once or twice weekly to make sure an affected eye is not deteriorating. Other surgical ocular procedures such as third eyelid flaps may also
139 be considered in severe cases.139
Vaccination against Moraxella bovis has traditionally been considered to be the foundation of a successful IBK prevention program. As with other multifactorial diseases, however, it is likely that the most effective IBK prevention program will require consideration of a variety of factors, including timing of vaccination (ideally within 4 to 6 weeks of anticipated cases); reducing risk factors (such as plant awns, foxtails, flies, and dust); supplementing trace minerals in deficient areas (e.g., copper and selenium); and reducing iatrogenic spread of potentially infectious material between animals (e.g., contaminated instruments, halters, and hands). To reduce fly populations, insecticide-impregnated ear tags placed on calves and topical insecticides with back and face rubbers are recommended. Clipping mature grasses before cattle are turned out may help minimize risks associated with direct mechanical corneal injury from plant awns. In areas where trace mineral deficiencies, especially of copper and selenium, are prevalent, attention to supplementation of these trace minerals is considered an important aspect of overall herd control and prevention. Neutrophils from copper-deficient calves had lower superoxide dismutase activity versus neutrophils from copper-supplemented calves, but no difference was observed in phagocytic and bactericidal activities of neutrophils from these groups.140
For vaccination against IBK, commercially available Moraxella bovis bacterins are available, but these are not universally effective. In 2017 a conditionally licensed Moraxella bovoculi vaccine was also released.141 With the increase in reporting of Moraxella bovoculi from clinical samples following the initial report of that organism in 2007, producers and veterinarians have also sought to improve vaccination coverage by using autogenous Moraxella bovoculi (and/or Moraxella bovis) vaccines in prevention programs. While anecdotal reports that support or refute the use of Moraxella bovis and/or Moraxella bovoculi bacterins are easy to come across on discussion boards of cattle veterinarians, recently published studies investigating the efficacy of autogenous or commercial Moraxella bovis or autogenous Moraxella bovoculi vaccines reported these vaccines to be ineffective.142-144 Some authors have also recommended the use of multivalent Moraxella bovis, Moraxella bovoculi, and M. ovis vaccines to prevent IBK.145