Treatment of acute ocular Moraxella bovis infections in calves with a parenterally administered long-acting oxytetracycline formulation

Acute ocular Moraxella bovis infections were induced in the UV-irradiated eyes of 10 calves. Eight calves developed corneal ulcers in at least 1 eye and were used for the treatment experiment. One randomly selected group of 4 calves with corneal ulcers and M bovis infections in 7 eyes was given a long-acting oxytetracycline formulation in 2 IM dosages of 20 mg/kg of body weight each, 72 hours apart. The other 4 calves with corneal ulcers in 6 eyes and M bovis in all 8 eyes served as nontreated controls. Bilateral ocular cultures were obtained and clinical observations were made daily for 20 days after treatment. After administration of the long-acting drug, new ulcers did not develop in the treated calves, whereas 5 new ulcers developed in the control-group calves during this time. The average durations of increased lacrimation/ulcerated eye were 2 and 12 days after treatment in the treatment and control groups, respectively; the average durations of blepharospasm were 3 and 8 days, respectively. Moraxella bovis was not isolated from any of the eyes of the treatment-group calves for the first 6 days after the antibiotic was administered, but was isolated from 1 eye of 1 treated calf on posttreatment day 7 and daily thereafter, for a total of 14 positive cultures of 160 ocular cultures obtained from the treatment-group calves after treatment. The bacterium was isolated from all eyes and from 144 of 160 cultures from the control-group calves during this time.     

Topically applied benzathine cloxacillin for treatment of experimentally induced infectious bovine keratoconjunctivitis

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 groups had smaller ulcer surface area measurements than did controls on week 2. By week 3, calves of the 375-mg group had smaller scores than did controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of tulathromycin for treatment of cattle with acute ocular Moraxella bovis infections

OBJECTIVE: To evaluate the clinical efficacy of a single injection of tulathromycin, compared with saline (0.9% NaCl) solution-treated control calves, for treatment of induced infectious bovine keratoconjunctivitis in calves. DESIGN: Clinical trial. ANIMALS: 30 Holstein bull calves ranging from 5 to 6 months old and 75 to 200 kg (165 to 440 lb) with no history of Moraxella bovis infections, no history of M bovis vaccination, and negative results for M bovis on 3 consecutive ocular bacterial cultures. PROCEDURES: Both eyes of each calf were infected with 1 X 10(10) colony-forming units of piliated M bovis for 3 consecutive days prior to the trial. On day 0, ocular lesion scores were determined for each calf and the calves were weighed and assigned to a treatment (2.5 mg/kg [1.14 mg/lb] of body weight, SC) or control group according to a stratified random allocation based on weight and lesion score. Eyes were stained with fluorescein and photographed daily to record healing. Eyes were evaluated bacteriologically for M bovis on days 0 to 6 and at 3-day intervals thereafter. RESULTS: Median time to ulcer resolution in calves treated with tulathromycin was 9.1 days. More than 50% of control calves still had ulcers at the end of the trial (21 days). Moraxella sp was isolated less often from the eyes of treated calves than from the control calves. By day 10, the treated calves had lower ocular lesion scores than control calves. CONCLUSIONS AND CLINICAL RELEVANCE: A single dose of tulathromycin (SC) was an effective treatment of calves with experimentally induced infectious bovine keratoconjunctivitis. The long serum half-life of tulathromycin, along with the results of this trial, suggests that tulathromycin may be a rational choice as a single-injection treatment for infectious bovine keratoconjunctivitis.     

Mycoplasma bovoculi infection increases ocular colonization by Moraxella ovis in calves

To determine whether infection with Mycoplasma bovoculi increases ocular colonization of cattle eyes with Moraxella bovis and other bacteria, colonization of ocular gram-negative bacteria were measured in eyes of cattle infected with Mycoplasma bovoculi. Strains of Moraxella ovis were chosen because these are among the most commonly isolated species of gram-negative bacteria from cattle eyes. Five strains of M ovis were characterized biochemically and by pilus structure, permitting the recognition of 2 biotypes. All strains were tested in a mouse corneal pathogenicity model. One strain of each biotype was selected for testing in calves. All 5 strains were apathogenic for mice, and the 2 strains tested in cattle did not induce keratitis. Infection of calves with Mycoplasma bovoculi increased the amount and persistence of colonization with the strains of M ovis.     

Experimental production of infectious bovine keratoconjunctivitis

The left eyes of 10 conventional dairy cross calves were inoculated with a pathogenic strain of Moraxella bovis and lesions of infectious bovine keratoconjunctivitis developed in nine of these eyes. M bovis was isolated from all inoculated eyes and lesions developed in five out of 10 eyes which had become naturally infected. The clinical and microbiological findings were similar to those described in field cases.     

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

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

Effectiveness of two commercial infectious bovine keratoconjunctivitis vaccines

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 greater than or equal to 83% of the eyes of calves in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Efficacy of florfenicol in the treatment of experimentally induced infectious bovine keratoconjunctivitis.

OBJECTIVE: To evaluate efficacy of florfenicol in an induced model of infectious bovine keratoconjunctivitis, using a blinded randomized, controlled trial. ANIMALS: 48 male Holstein calves, 2 to 4 months old. PROCEDURE: Moraxella bovis infection was induced in all calves. When corneal ulcers developed, each calf was assigned randomly to 1 of 3 treatment groups, using a block design determined by corneal ulcer size (day 0). Calves were treated with florfenicol (20 mg/kg of body weight, IM) on days 0 and 2 (IM group; n = 16). Calves of a second group received a single dose of florfenicol (40 mg/kg, SC) on day 0 (SC group; n = 16). The third group of calves was not treated (control group; n = 16). Corneal ulcers were photographed, and each calf was assessed for 30 days after treatment for 10 clinical signs of infection. Corneal ulcer surface areas were measured, and clinical scores were calculated. Ocular secretions for microbiologic culture were obtained weekly from each eye. RESULTS: A Cox regression model indicated that, after adjustment for initial ulcer size, healing rates were 6.2 and 4.8 times greater in calves of the IM and SC groups, respectively, compared with the control group. Clinical scores and surface area measurements for treatment groups were significantly smaller than those for controls during posttreatment weeks 1 through 4. From day 8 through day 29, M bovis was isolated from ocular secretions of 14 of 16 control calves and 1 of 32 treated calves. CONCLUSIONS AND CLINICAL RELEVANCE: Parenterally administered florfenicol reduces corneal ulcer healing time, lessens clinical severity, and reduces the amount of bacterial shedding from calves infected with M bovis.     

Enhancement of infectious bovine keratoconjunctivitis by modified-live infectious bovine rhinotracheitis virus vaccine

The effects of a modified-live infectious bovine rhinotracheitis virus vaccine (administered ocularly or intranasally) on experimentally induced infectious bovine keratoconjunctivitis were evaluated. The modified-live infectious bovine rhinotracheitis virus vaccine was administered to 13 male Holstein calves (intranasally in 4 and ocularly in 9; day 0). Five calves were not vaccinated and served as controls. Calves were examined daily and, starting on day 4, Moraxella bovis was administered ocularly to all 18 calves once daily for 4 days. The eyes of all calves were assigned a clinical score, and the ocular secretions were evaluated for presence of infectious bovine rhinotracheitis virus and M bovis daily until day 19. The severity of the ocular lesions was estimated by scoring the lesions clinically and by determining the protein concentration, myeloperoxidase activity, and WBC count in the tears. By day 5, conjunctivitis, chemosis, and epiphora were observed in all of the calves vaccinated ocularly. The calves vaccinated intranasally developed conjunctival plaques, but did not develop chemosis or photophobia. All of the calves developed keratitis after inoculation with M bovis. The median lesion scores were greater in both groups of vaccinated calves than in the controls. Corneal perforations developed exclusively in the vaccinated calves. The frequency of M bovis isolation from ocular secretions was significantly (P less than 0.05) greater in the vaccinated calves than in the controls. The tears from the intranasally vaccinated calves contained the highest myeloperoxidase activity and WBC count. The mean protein concentration in the tears of vaccinated calves was not significantly different from that in tears of controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation of Moraxella bovoculi sp. nov. from other coccoid moraxellae by the use of polymerase chain reaction and restriction endonuclease analysis of amplified DNA.

Moraxella ovis was historically the only coccoid Moraxella identified in cultures of ocular fluid from cattle with infectious bovine keratoconjunctivitis (IBK) and could be morphologically and biochemically differentiated from Moraxella bovis. Moraxella bovoculi sp. nov. is a recently characterized Moraxella isolated from ulcerated eyes of calves with IBK in northern California in 2002. Like Moraxella ovis, M. bovoculi sp. nov. is a gram-negative coccus/diplococcus. All 18 original isolates of M. bovoculi sp. nov. possessed phenylalanine deaminase (PADase) activity and could therefore be differentiated from M. ovis and M. bovis. During the characterization of 44 additional isolates of hemolytic gram-negative cocci that were cultured from ulcerated eyes of IBK-affected calves, 2 PADase-negative isolates were identified that could not be differentiated biochemically from M. ovis; however, the DNA sequence of the 16S-23S intergenic spacer region (ISR) of the isolates matched the 16S-23S ISR DNA sequence of M. bovoculi sp. nov. To facilitate the identification of PADase-negative moraxellae, a polymerase chain reaction (PCR) coupled with restriction enzyme digestion analysis of amplified DNA was developed. Amplification of the 16S-23S ISR followed by AfaI digestion of amplified DNA could differentiate M. bovoculi sp. nov. from M. ovis and other moraxellae. The DNA sequence analysis of the amplified 16S-23S ISR from the 42 PADase-positive isolates of hemolytic gram-negative cocci indicated that all were M. bovoculi sp. nov. and all possessed an AfaI site. A PCR coupled with restriction analysis of amplified DNA can aid in identifying M. bovoculi sp. nov.     

Effects of aflatoxin ingestion on the development of Moraxella bovis infectious bovine keratoconjunctivitis

A study was conducted to determine whether measured doses of aflatoxin given under different schedules would influence the pathogenesis of Moraxella bovis induced infectious bovine keratoconjunctivitis (IBK). Calves were allotted to 4 groups (groups I-IV) of 9, 9, 9, and 8 calves, respectively. Group I calves were given aflatoxin for 11 consecutive days starting 5 days before their eyes were exposed to M. bovis. Group II calves were given aflatoxin for 5 consecutive days starting 7 days after their eyes were exposed to M. bovis. Group III calves were given aflatoxin for 5 consecutive days starting 21 days after their eyes were exposed to M. bovis. Group IV calves were not given aflatoxin; but their eyes were exposed to M. bovis on the same day as were the eyes of calves in groups I-III; these calves served as controls. Aflatoxin had little if any influence on the pathogenesis of IBK under the conditions of this study. The results did not rule out an exacerbating effect of M bovis infection on aflatoxicosis in calves. Calves with the highest concentration of aflatoxin in their blood had the more severe signs of aflatoxicosis. Possible reasons for the equivocal results are discussed.     

Infectious bovine keratoconjunctivitis: experimental induction of infection in calves with mycoplasmas and Moraxella bovis.

Eyes of 14 calves were exposed by conjunctival instillation to cultures of either Mycoplasma conjunctivae (6 calves) or Acholeplasma laidlawii (8 calves). Calves were observed for clinical signs of infectious bovine keratoconjunctivitis (IBK), and eyes were examined for the test organisms by bacteriologic cultural technique for 60 days. Acholeplasma laidlawii became established in the eyes of 5 of 8 calves; M conjunctivae became established in the eyes of 4 of 6 calves. On day 28, eyes of 9 of the 14 calves were exposed by conjunctival instillation to Moraxella bovis, and all developed IBK. Five calves exposed to Moraxenjunctivae or A laidlawii, but not to Mor bovis, did not develop IBK. Four calves not exposed to M conjunctivae or A laidlawii, but exposed to Mor bovis, developed IBK. Mycoplasmas do not have a major role in IBK, but might produce ancillary effects similar to those of infectious bovine rhinotracheitis virus, wind, ultraviolet radiation, dust, and other irritants.     

Identification and characterization of complete RTX operons in Moraxella bovoculi and Moraxella ovis

To determine if Moraxella bovoculi (M. bovoculi), a recently characterized coccoid Moraxella that was isolated from the eyes of calves affected with infectious bovine keratoconjunctivitis (IBK), and Moraxella ovis (M. ovis), originally isolated from sheep with conjunctivitis, possessed genes encoding RTX proteins, genomic DNA was amplified with oligonucleotide primers targeting RTX operon genes of Moraxella bovis (M. bovis). Complete classical RTX operons composed of RTXCABD genes closely linked to a putative secretion accessory protein encoding gene (tolC) were identified in M. bovoculi and M. ovis and were designated mbvCABDtolC and movCABDtolC, respectively. These genes were closely related to M. bovis mbxCABDtolC. Polyclonal rabbit antiserum against the carboxy terminus of M. bovoculi MbvA neutralized hemolytic activity of both M. bovoculi and M. ovis; this antiserum did not neutralize the hemolytic activity of M. bovis. M. bovoculi and M. ovis possess genes that encode proteins related to pathogenic factors of M. bovis.     

Infectious bovine keratoconjunctivitis II. Antibodies in lacrimal secretions of cattle naturally or experimentally infected with Moraxella bovis.

One or both eyes of 20 calves were inoculated one or more time with variou(s combinations of microorganism (live oor killed Moraxella bovis, infectious bovine rhinotracheitis virus, bovine adenovirus, bovine parainfluenza-3 virus and Mycoplasma bovoculi) by conjunctival instillation or direct inoculation of the conjunctivea or cornea. The eyes of all the calves received natural or artificial ultraviolet irradiation. Neither the adenovirus nor parainfluenza-3 virus became established in the eye or produced keratoconjunctivitis. Both M. bovis and infectious bovine rhinotracheitis virus became established in the bovine eye and produced disease. Subconjunctival or intracorneal inoculation of M. bovis caused a severe disease, simulating natural infectious bovine keratoconjunctivitis. Only the intracorneal inoculation of mycoplasma produced severe keratoconjunctivits. Eyes that on initial exposure to M. bovis became severly inflamed were more resistant to a second or third exposure to M. bovis, presumably by enhanced local defence mechanisms.     

Identification of bovine carriers of Moraxella bovis by comparative cultural examinations of ocular and nasal secretions

The carrier state of Moraxella bovis was investigated, using bacteriologic examinations of ocular and nasal secretions from cattle under experimental and natural conditions of exposure and management. Moraxella bovis was isolated throughout the year from the ocular and nasal secretions of cattle naturally affected with infectious bovine keratoconjunctivitis. There was also 1 case of nasal transmission of M bovis without isolation of M bovis from ocular secretions and 1 case of M bovis isolation from the vagina of a calf contracted by contact with a calf affected with infectious bovine keratoconjunctivitis. The frequency of isolations and duration of infections, as determined by examination of ocular and nasal secretions, indicated that these secretions were comparable in the identification of M bovis carriers. The increased cultural isolations of M bovis from nasal secretions after shipment relative to the number of isolations before shipment indicated that shipment may serve as a stress factor causing an increase in the number of carriers.     

A Mycoplasma Isolated from Cattle with Infectious Bovine Keratoconjunctivitis

A mycoplasma has been recovered from the eyes of calves in two naturally-occurring outbreaks of infectious bovine keratoconjunctivitis; also from a third group of calves accidentally exposed to an animal which had ocular exudates from one of the outbreaks instilled into its eyes.

The severity of the ocular lesions in infectious bovine keratoconjunctivis outbreaks may be related to a mixed infection with the mycoplasma and Moraxella bovis.

Preliminary typing studies indicate the mycoplasma is not serologically related to any known bovine mycoplasma.

     

Infectious bovine keratoconjunctivitis: evidence for general immunity

A study was conducted to determine whether acquired immunity to Moraxella bovis during episodes of infectious bovine keratoconjunctivitis (IBK) was local or general. Twelve calves (principals) were exposed to M bovis in the left eye by conjunctival sac instillation on day 1 and in both eyes on day 21. Six calves exposed in both eyes at day 1 and six others exposed at day 21 served as controls. The mean duration of infection in the right eyes of principal calves was significantly less (P less than 0.001) than that in the left eyes of principal calves, as well as those of the control calves. There were no significant differences (P less than 0.2) in the durations of infection between the 2 groups of the control calves or the duration of infection in the left eyes of principal calves. Five of 6 calves (7 of 12 eyes) in the day 1 controls and 4 of 6 calves (7 of 12 eyes) in the day 21 controls developed IBK. Ten of 12 of the principals developed IBK in the left eye when exposed on day 1, but only 1 of 12 principals developed IBK in the right eye when exposed on day 21. It was concluded that the resistance to infection after initial infection is probably due to a generalized immune response, rather than to a localized response.     

Infectious bovine keratoconjunctivitis: contact transmission

The transmission of Moraxella bovis was studied in calves in the absence of the face fly (Musca autumnalis) or environmental conditions that might insult the eye. Thirty calves were placed in 10 groups of 1 experimentally infected calf and 2 contact calves each. Over 40 days, only 1 eye in 1 contact calf developed clinical infectious bovine keratoconjunctivitis. The organism was recovered in only 8 of 20 contact calves, whereas infection and disease occurred in all experimentally infected calves. Transmission of M bovis occurring in the absence of some other intervening factor was probably of minimal importance. Seemingly, herd preventive treatment would be most effective when flies and environmental factors are at a minimum.     

Immunogenicity of a Moraxella bovis bacterin containing attachment and cornea-degrading enzyme antigens

An adjuvanted Moraxella bovis bacterin containing attachment antigens and cornea-degrading enzyme antigens protected cattle from infectious bovine keratoconjunctivitis (IBK) when experimentally challenged with homologous and heterologous challenge cultures of M. bovis. This bacterin also protected cattle against field exposure to M. bovis. Transmission electron microscopy and fluorescein labeled anti-M. bovis pili antiserum showed pili on the M. bovis bacterin strain. Scanning electron microscopy demonstrated a fibrillar glycocalyx. The bacterin strain of M. bovis, but not all strains of M. bovis, destroyed bovine corneal cell monolayers in vitro. Bovine corneal cells began to separate from each other within 5 min after M. bovis organisms were added and adhered to the cell monolayers. Moraxella bovis organisms remained attached to the disintegrating cells as the cell membrane separated and was digested. Vaccination stimulated bacterial agglutination antibodies. However, protection against experimental challenge was more closely related to the cornea-degrading enzyme content of the experimental bacterins. Twenty-two of 29 cattle (76%) vaccinated with bacterins containing a relative enzyme activity (REA) greater than 0.4 were protected in a rigorous challenge of immunity test. Only 1 of 21 non-vaccinated calves (5%) was free of IBK. Ninety-two percent (24/26) of calves vaccinated with a bacterin containing a REA greater than 0.29 remained free of IBK following field exposure, whereas 47% (8/17) non-vaccinated calves developed IBK. Only 8 of 12 calves (67%) vaccinated with a bacterin containing a REA of 0.09 remained free of IBK. In a larger field efficacy test consisting of 32 herds in six states, the incidence of IBK in individual herds ranged from 0% to 55%. The overall rate of infection was 11.2%. Vaccination of calves with an M. bovis bacterin that contained a REA of 0.63 reduced the incidence of IBK from 11.2% (217/1931) in the non-vaccinated controls to 4.3% (66/1520) in cattle vaccinated once and to 3.1% (48/1536) in cattle vaccinated twice.     

Conjunctival lesions caused by Moraxella bovis in gnotobiotic calves

Hemolytic Moraxella bovis was instilled into the conjunctival sac of gnotobiotic calves, and conjunctivae were sampled serially after infection. Bilateral lesions developed in seven of eight infected calves. Histologically, M. bovis was first seen within swollen epithelial cells near the lid margins and occasionally within superficial epithelium in other areas. Conjunctival erosions and ulcers were seen in later stages. Scanning electron microscopy showed M. bovis in pits on surfaces of epithelial cells and in erosions on palpebral conjunctivae; lesions were prominent near lid margins. By transmission electron microscopy, M. bovis was seen within swollen epithelial cells near lid margins; many epithelial cells had undergone cytolysis. This study demonstrates that virulent M. bovis can invade bovine conjunctival epithelial cells and cause conjunctivitis in the absence of injurious ultraviolet irradiation or other predisposing environmental factors.