The cytotoxic effect of Moraxella bovis 118F on bovine neutrophils was evaluated and characterized by use of a 51Cr release assay. Neutrophils harvested from healthy adult cattle were labeled with 51Cr. The leukocidic activity produced by M bovis 118F, a hemolytic strain of M bovis, was heat-labile. A live culture of strain 118F, at a ratio of 100 bacteria/neutrophil, released 97.7% of the 51Cr from labeled neutrophils. Neither a heat-killed preparation of M bovis 118F nor a live or heat-killed preparation of M bovis IBH63 (a nonhemolytic and nonpathogenic strain) induced significant (P > 0.05) release of 51Cr. Moraxella bovis 118F broth culture filtrates prepared for evaluation of leukocidic activity also were evaluated for hemolytic activity. These 2 toxic activities had several characteristics in common. Both were filterable, heat-labile, produced by a hemolytic strain, and were released during early logarithmic phase growth from broth cultures. Leukocidic and hemolytic activities were protected from degradation by phenylmethyl sulfonyl fluoride, a serine protease inhibitor. Leukocidic and hemolytic activities were dependent on calcium ions. Filtrate resulted in 54.1% 51Cr release from labeled neutrophils and contained 646.7 hemolytic U/ml, respectively, when saline (0.85% NaCl) + 10 mM CaCl2 solution was used as diluent. Neither saline solution nor saline + 10 mM MgCl2 solution supported leukocidic or hemolytic activity. Serum, obtained from several calves 10 to 38 days after M bovis inoculation, substantially neutralized leukocidic and hemolytic activities, compared with paired preinoculation serum samples. In addition, no significant difference (P > 0.05) was detected when the ability of each calf's postinfection serum to neutralize leukocidic activity was compared with the ability of the serum to neutralize hemolytic activity.

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

Plasmid profiles were compared between nonpiliated and piliated forms of Moraxella bovis isolates. The piliated form of M bovis isolate IBH64 contained 1 fewer plasmid than did the nonpiliated form. Piliated and nonpiliated cells of IBH64 contained plasmids having molecular size of 45, 32.8, 4.9, and 4.6 kilobases (kb). Single- and double-restriction endonuclease digestion by Ava I and Nde I indicated that the size of the additional plasmid carried by the nonpiliated form of IBH64 was approximately 43.6 kb. The M bovis isolates, Newport and GRS, contained the same number of plasmids in either their piliated or nonpiliated form.

The cytotoxic effect of Moraxella bovis 118F on bovine neutrophils was evaluated and characterized by use of a 51Cr release assay. Neutrophils harvested from healthy adult cattle were labeled with 51Cr. The leukocidic activity produced by M bovis 118F, a hemolytic strain of M bovis, was heat-labile. A live culture of strain 118F, at a ratio of 100 bacteria/neutrophil, released 97.7% of the 51Cr from labeled neutrophils. Neither a heat-killed preparation of M bovis 118F nor a live or heat-killed preparation of M bovis IBH63 (a nonhemolytic and nonpathogenic strain) induced significant (P > 0.05) release of 51Cr. Moraxella bovis 118F broth culture filtrates prepared for evaluation of leukocidic activity also were evaluated for hemolytic activity. These 2 toxic activities had several characteristics in common. Both were filterable, heat-labile, produced by a hemolytic strain, and were released during early logarithmic phase growth from broth cultures. Leukocidic and hemolytic activities were protected from degradation by phenylmethyl sulfonyl fluoride, a serine protease inhibitor. Leukocidic and hemolytic activities were dependent on calcium ions. Filtrate resulted in 54.1% 51Cr release from labeled neutrophils and contained 646.7 hemolytic U/ml, respectively, when saline (0.85% NaCl) + 10 mM CaCl2 solution was used as diluent. Neither saline solution nor saline + 10 mM MgCl2 solution supported leukocidic or hemolytic activity. Serum, obtained from several calves 10 to 38 days after M bovis inoculation, substantially neutralized leukocidic and hemolytic activities, compared with paired preinoculation serum samples. In addition, no significant difference (P > 0.05) was detected when the ability of each calf's postinfection serum to neutralize leukocidic activity was compared with the ability of the serum to neutralize hemolytic activity.

Two commercially available infectious bovine keratoconjunctivitis (IBK) vaccines were evaluated for their effectiveness in protecting cattle from disease caused by experimental challenge exposure and natural transmission of Moraxella bovis infections. The study was conducted as 2 experiments, using a total of 81 cattle that were culture-negative for M bovis prior to vaccination. In each experiment, young adult cattle were randomly allotted to 4 groups. Each calf in groups 1 and 2 was vaccinated according to the vaccine manufacturer's directions. Groups 3 and 4 were unvaccinated controls. Three weeks after the last vaccination, each calf in groups 1 and 3 was experimentally challenge exposed by dropping a suspension of viable cells of a virulent strain of M bovis directly onto the corneal surface of each eye. Calves in all 4 groups were then commingled in open pastures so that calves in groups 2 and 4 could be naturally exposed to the calves with experimentally induced infections. Each calf was examined for signs of ocular disease on a regular basis by 2 experienced clinicians who scored each eye for severity of disease on the basis of a prearranged scale. Neither clinician was aware of the vaccination or exposure status of the calf nor to which experimental group they belonged. Lacrimal secretions were collected regularly to determine the number of eyes in which the virulent organism became established. Moraxella bovis with bacterial cultural characteristics similar to those of the virulent strain placed in the eyes of groups 1 and 3 was cultured from > 83% of the eyes of calves in all groups. The incidence of clinical IBK was > 50% in each group. There was no significant difference in the ability of vaccinated calves to resist experimental or natural challenge infection. Compared with that in nonvaccinated controls, no decreased incidence or severity of clinical IBK was noticed in vaccinated calves. Though the challenge strain used was not homologous to those used to prepare the vaccine, it was no different than those that may be expected to cause disease under field conditions.

Restriction endonuclease digestions were performed on plasmids purified from Moraxella bovis isolates GRS, Newport, and IBH64. It was determined from single and double digestions of plasmid DNA that GRS and Newport isolates carried 3 large plasmids having molecular sizes of 43.8, 41.3, and 32.8 kilobases (kb). Digestion of the 3 large plasmids and restriction endonucleases Hae III, HindIII, Nde I, and Ava I strongly indicated that these isolates shared structurally identical large plasmids. Timed single digestions with Ava I revealed that the IBH64 isolate carried 2 large plasmids having molecular sizes of 45 and 32.8 kb. The 32.8-kb plasmid was the only large plasmid that appeared to be shared by all 3 M bovis isolates. Two isolates, Newport and IBH64, carried small plasmids in addition to the large plasmids. Restriction maps were constructed for the 43.8-, 41.3-, and 32.8-kb plasmids.

A field study was performed to determine the effectiveness of benzathine cloxacillin for the treatment of infectious bovine keratoconjunctivits in cattle from 2 farms located in northern California. The study was performed between June and September of 1987. Affected calves ranging from 2 to 9 months of age were selected from the main herd when signs of corneal ulceration were observed. The study was conducted in 2 phases. For phase I, the affected calves of herd 1 (n = 21; Holsteins) and herd 2 (n = 43 Angus crossbred), were randomly assigned to 1 of 3 groups, and were either treated with 250 (n = 23) or 375 mg (n = 21) of benzathine cloxacillin, or mineral oil (n = 20) on days 1 and 4. For phase II, affected calves (n = 16; Angus crossbred, 3 to 9 months of age) from herd 2 were treated with benzathine cloxacillin (250 mg). Eight of these calves were retreated on day 4. After treatment, all calves were examined every 72 hours for 16 days. For examinations, a clinical score was assigned to each eye, and the surface areas of photographed corneal ulcers were measured. The ocular secretions were collected and examined culturally for Moraxella bovis. On days 7, 10, and 13, the calves treated with benzathine cloxacillin had significantly (P less than 0.05) lower lesion scores, compared with the controls. The percentage of day-1 area measurements on posttreatment days 4, 7, and 10 were significantly larger in the controls than in the treated calves. The mean healing times of corneal lesions in the 2 antibiotic treatment groups were significantly (P less than 0.05) shorter than in the controls. In the treated calves, the healing times of corneal ulcers less than or equal to 0.05 cm in diameter on day 1 were significantly (P less than 0.05) shorter than the healing times of larger lesions. The healing times of the corneal ulcers in the Angus crossbred calves were significantly (P less than 0.05) longer than the healing times of the Holsteins. The controls had the greatest number of nonhealed corneal ulcers on day 16. The number of calves that remained infected with M bovis on days 4, 7, 10, and 13 was significantly less in both groups of treated calves than in the controls. The scores, surface area measurements, healing times, and the M bovis isolation frequency in the calves of the 250-mg and 375-mg and 1- and 2-dose treatment groups were not significantly different. The minimal inhibitory concentrations (MIC) of penicillin, cloxacillin, and ampicillin in the day-16 isolates from benzathine cloxacillin-treated herd-2 calves were greater than in isolates from corresponding calves on observation day 1; however, twofold increases of the respective MIC of pretreatment and day-16 specimens were not observed. The highest MIC of ampicillin, cloxacillin, gentamicin, oxytetracycline, and penicillin were 0.5, 8.0, 0.5, 2, and 1 migrogram/ml, respectively, for isolates collected during the final study week.

The efficacy of an ophthalmic ointment containing benzathine cloxacillin for treatment of infectious bovine keratoconjunctivitis was determined in 2 experiments. In the first experiment, Holstein calves (n = 6/group) were inoculated with Moraxella bovis and treated on postinoculation days 3 and 6 with either topically applied benzathine cloxacillin (250 mg/eye) or long-acting oxytetracycline formulation (20 mg/kg of body weight, IM). A third group of inoculated calves remained untreated as controls. For the second experiment, 4 groups of calves (n = 6/group) were inoculated and treated on postinoculation days 3 and 6 with 50, 125, 250, or 375 mg of benzathine cloxacillin; a fifth untreated group served as controls. Ocular specimens were obtained for microbiologic culture, and eyes were observed and assigned a clinical score daily. Eyes were photographed on alternate days. Ulcer surface area was measured, using a planimeter. In experiment 1, the week-2 ulcer surface area measurements for both groups of treated calves were smaller than those for controls. There was a greater frequency of M bovis isolation from the ocular secretions of controls than from those of benzathine cloxacillin-treated calves during postinoculation weeks 2 and 3. The number of M bovis isolations from the benzathine cloxacillin- and oxytetracycline-treated calves was not significantly different at any sample collection interval. On week 3, the scores of the benzathine cloxacillin-treated calves were smaller than those of controls. In experiment 2, calves of the 250- and 375-mg group had smaller scores than did controls. During postinoculation weeks 1 through 3, M bovis was isolated less frequently from the ocular secretions of calves of the 250- and 375-mg groups than from those of the control calves.

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