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 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.     

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)     

Attachment of Moraxella bovis to calf corneal cells and inhibition by antiserum

An in vitro assay was developed using calf corneal cells to assess the importance of fimbriae in the colonisation of the bovine ocular surface by Moraxella bovis, and the role of fimbrial antibodies in the bovine immune response and resistance to infectious bovine keratoconjunctivitis (IBK). Fimbriae promoted adherence of M. bovis to calf corneal cells in culture; 15 fimbriate isolates, representative of 6 fimbrial serogroups of M. bovis, adhered to the cells whereas 4 non-fimbriate isolates failed to do so. Fimbrial antibodies in hyperimmune rabbit serum inhibited attachment of all fimbriate strains of the homologous fimbrial serogroup but not those of 5 heterologous serogroups. The relevance of these results to the use of a polyvalent fimbrial vaccine in the control of IBK is discussed.     

Genotypic, phenotypic, and biological characteristics of Moraxella bovis

Several isolates of Moraxella bovis obtained from cattle with infectious bovine keratoconjunctivitis killed monocytes and macrophages in vitro and carried 3 to 5 plasmids. Cloned and noncloned M bovis isolates readily induced the disease, killed the phagocytes in vitro, and carried 3 plasmids. Moraxella bovis isolates of low in vivo virulence carried 5 plasmids and did not kill phagocytes to the extent that isolates with 3 plasmids did. The possible implications of these findings in the pathogenesis of infectious bovine keratoconjunctivitis are discussed. Also, a classification of M bovis colonies into rough or smooth varieties according to their staining characteristics with crystal violet is suggested.     

Laboratory model for infectious bovine keratoconjunctivitis: the pathogenicity of different strains of Moraxella bovis, pathology and ultrastructural observations

Further studies were made using C57 mice pretreated with a corticosteroid and inoculated with Moraxella bovis by instillation, as a model for infectious bovine keratoconjunctivitis. Strains of M bovis which had previously been tested in cattle produced a generally similar range of pathogenicity when tested in mice. The pathology in the mouse model closely resembled that in cattle and the value of the model for studies on M bovis was confirmed.     

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.     

Molecular and phenotypic analysis of Moraxella spp. associated with infectious bovine keratoconjunctivitis in Uruguay

Infectious bovine keratoconjunctivitis (IBK) is a common ocular disease of cattle, which is generally thought to be caused by Moraxella bovis. However, a recently characterized Moraxella, M. bovoculi, has been isolated from animals with IBK. The aim of this study was to identify and characterize strains of Moraxella spp. obtained from IBK cases in different geographic locations within Uruguay. Ribosomal gene sequencing indicated that there were two groups of isolates that showed homology with either M. bovis or M. bovoculi. Phylogenetic analysis confirmed the presence of two species as the isolates grouped in different branches of the dendrogram. Conventional biochemical characterization did not distinguish between the species; only 9/25 isolates which had genetic homology with M. bovoculi showed any differences in biochemistry.     

Bovine Infectious Keratoconjunctivitis: Serological Aspects of Moraxella bovis Infection

A modification of a gel diffusion precipitin test (GDPT) was used to detect antibodies for Moraxella bovis (M. bovis) in the sera of cattle affected with bovine infectious keratoconjunctivitis (BIK). The test was also used for the detection of sequential antibody development in cattle vaccinated with cultures of M. bovis. Also, strains of M. bovis isolated from cattle herds affected with BIK were characterized serologically as a part of an identification scheme using the test.

A comparison of the antigenic properties of various strains of M. bovis and M. bovis-like organisms was conducted using the test. The results indicated that there might be antigenic relationships between M. bovisand M. bovis-like organisms such as Moraxella liquefaciens, Moraxella nonliquefaciens, an unidentified hemolytic diplococcus, Mima polymorpha, Mima polymorpha var. oxidans and Herellea vaginicola

The authors suggest that the GDPT can be used for serological studies of BIK, and the identification and antigenic analysis of M. bovis. They indicate, however, that a more definitive study is needed to evaluate the reliability of the test for quantitative work.

     

Evaluation of the clinical efficacy of subconjunctival injection of clindamycin in the treatment of naturally occurring infectious bovine keratoconjunctivitis

OBJECTIVE: To determine the clinical efficacy of subconjunctival injection of clindamycin in the treatment of naturally occurring infectious bovine keratoconjunctivitis (IBK). ANIMALS STUDIED: Clinically, out of 81 animals examined, 46 were found to be suffering from IBK of variable severity. The ocular secretions were collected and cultured for Moraxella bovis. The study included 36 Holstein cattle from which M. Bovis was isolated. These animals ranged between 4 and 28 months of age. PROCEDURES: The severity of the clinical findings were scored as normal, mild, moderate, and severe. Clindamycin was injected subconjunctivally at a total dose of 150 mg (1 mL), once daily for 3 days to the test group (n = 18); isotonic saline solution (1 mL) was administered to the control group. After treatment, all cattle were re-examined and clinical response was evaluated on days 3, 7 and 15 post-treatment. RESULTS: Compared with the control group and prior to treatment, all active lesions such as blepharospasm, epiphora, photophobia, chemosis, corneal edema, and corneal ulceration were generally resolved by day 15 after subconjunctival injection of clindamycin. Severity of IBK lesions increased on days 3 and 7, compared to baseline in the control group administered isotonic saline solution. CONCLUSIONS: The results of this study suggest that subconjunctival injection of clindamycin is effective in the treatment of naturally occurring infectious bovine keratoconjunctivitis.     

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.     

Diagnosis of a mixed mycoplasma infection associated with a severe outbreak of bovine pinkeye in young calves.

Mycoplasma bovoculi and Mycoplasma bovis were both isolated from conjunctival swabs taken from young calves showing symptoms consistent with infectious bovine keratoconjunctivitis (pinkeye). No Moraxella spp. or other nonmycoplasma bacteria were isolated in association with this severe clinical outbreak. Based on laboratory tests and clinical observations, the first phase of the disease was likely pneumonic in nature, possibly caused by bovine respiratory syncytial virus and M. bovis. In the subsequent phase of the disease course, infection with both M. bovoculi and M. bovis resulted in ocular disease. A combination of microbiological, serological, and molecular diagnosticmethods was used to elucidate the etiology of the outbreak.     

Detection of Moraxella bovis antibodies in the SIgA, IgG, and IgM classes of immunoglobulin in bovine lacrimal secretions by an indirect fluorescent antibody test.

The relationship between clinical infectious bovine keratoconjunctivitis (IBK) and Moraxella bovis antibodies was evaluated in a herd of calves during one summer. The detection and the distribution of antibody response in lacrimal secretions of beef calves to natural exposure of M bovis were determined by an indirect fluorescent antibody test. Three classes of immunoglobulins--secretory IgA, IgM, and IgG--were monitored in lacrimal secretions over a 5-month period when IBK was enzootic in the herd. The 3 classes of antibody to M bovis were detected in all but 2 calves at the start of the monitoring, and the highest and most persistent M bovis antibody titers were in the IgG immunoglobulin class, and less so in IgM and secretory IgA classes. The specific antibodies present in the lacrimal secretions did not prevent the development of clinical IBK in the calves.     

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)     

Experimental production of infectious bovine keratoconjunctivitis: comparison of serological and immunological responses using pili fractions of Moraxella bovis.

The effect of vaccinating cattle and mice on the development of keratoconjunctivitis was studied. Cattle were vaccinated with whole cells, disrupted cells and pili fractions of three strains of Moraxella bovis. Mice were vaccinated with pili fractions of three strains. The resistance of all vaccinated animals was challenged with virulent cultures of M. bovis. In an attempt to correlate the response seen after vaccination and challenge with a pili fraction of M. bovis, vaccinated cattle and mice were grouped on the basis of signs of disease manifested and compared on the basis of serological responses. Serum samples were tested for antibodies by a gel diffusion precipitin test. A greater number of the sera of resistant cattle had antibodies to the homologous pili antigen than those of vaccinated nonresistant cattle. Cattle vaccinated with disrupted cells were not resistant to infectious bovine kerato-conjuctivitis and their sera lacked antibodies against the pili antigens. Vaccinated mice were more resistant to infectious bovine kerato-conjuctivitis and their sera lacked antibodies against the pili antigens. Vaccinated mice were more resistant to challenge exposure by homologous than heterologous cultures. A greater number of the sera of resistant mice had antibodies to pili antigens than nonresistant mice.     

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.     

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 vaccine development

Infectious bovine keratoconjunctivitis is a common and highly contagious ocular disease affecting cattle worldwide. The tremendous economic losses attributable to this disease warrant continued investigation into methods of prevention. Multiple virulence factors have been linked to the primary aetiologic agent, Moraxella bovis. Efforts to develop an efficacious vaccine have primarily focused upon the use of surface pili or cytolysin to stimulate host immunity; however, M. bovis possesses other virulence determinants that include proteases, fibrinolysins, phospholipases and other cell surface components such as outer membrane proteins. These potentially conserved antigens provide additional possibilities for vaccine development. Examination of appropriate antigen presentation is necessary to attain an adequate immune response. Further, the potential for antigenic diversity as well as epitope conversion requires continuous epidemiological surveillance of isolates recovered from outbreaks. Current work targeting conserved immunogens provides hope for efficacious vaccines that when used in tandem with proper management may control, if not prevent, infectious bovine keratoconjunctivitis.     

Assessment of Bdellovibrio bacteriovorus 109J killing of Moraxella bovis in an in vitro model of infectious bovine keratoconjunctivitis

The objective of this study was to determine the potential of Bdellovibrio bacteriovorus 109J as an alternative non-chemotherapeutic treatment of infectious bovine keratoconjunctivitis (IBK). To accomplish this, various parameters of B. bacteriovorus predation of Moraxella bovis were determined in vitro. Initial passage of B. bacteriovorus using M. bovis as prey required 10 d for active cultures to develop compared with 2 d for culture on normal Escherichia coli prey; however by the 5th passage, time to active predatory morphology was reduced to 2 d. This high passage B. bacteriovorus culture [1 × 10(10) plaque forming units (PFU)/mL] killed 76% of M. bovis [1 × 10(7) colony forming units (CFU)/mL] present in suspension broth in a 4 h assay. The minimal level of M. bovis supporting B. bacteriovorus predation was 1 × 10(4) CFU/mL. To assess the ability of B. bacteriovorus to kill M. bovis on an epithelial surface mimicking IBK, an in vitro assay with Madin-Darby bovine kidney (MDBK) cells inoculated with 4 × 10(7) CFU/mL M. bovis was used. Treatment with a B. bacteriovorus suspension (1.6 × 10(11) PFU/mL) decreased adherence of M. bovis to MDBK cells by 6-fold at 12 h of treatment, as well as decreased the number of unattached M. bovis cells by 1.4-fold. This study demonstrates that B. bacteriovorus has potential as an effective biological control of M. bovis at levels likely present in IBK-infected corneal epithelia and ocular secretions.     

Moraxella bovis pathogenicity: an update

Moraxella bovis is the etiologic agent of infectious bovine keratoconjunctivitis, the most important ocular disease affecting cattle worldwide. The severity of the cases varied from eyes that exhibited mild signs to severe clinical cases with profuse lacrimation, conjunctival swelling, corneal opacity, and ulceration. Although the mortality is low, there is a high morbidity and important economic loss in terms of significant reduction in production. This paper examines aspects such as the pathogenesis of the disease and the mechanisms by which this unique bacterium is able to disrupt the corneal epithelium and cause infection.