Pathogenesis of corneal lesions caused by Moraxella bovis in gnotobiotic calves

Moraxella bovis was instilled into the conjunctival sac of gnotobiotic calves and corneas were sampled serially after infection. Lesions developed in seven of eight infected calves, but were absent in a noninfected control calf. Histologically, M. bovis was first seen in foci of swollen epithelium and within basal epithelial cells adjacent to ulcers. Corneal ulcers were severe in later stages of infection; fibrin deposits, neutrophils, and bacteria were present in the stromas. Examination of early lesions by scanning electron microscopy showed M. bovis in pits on the surfaces of dark epithelial cells, enmeshed in degenerate epithelial cells and within erosions and an ulcer; in later samples, bacteria were rare. Ultrastructurally, M. bovis was seen in surface pits in superficial epithelial cell processes and within swollen epithelial cells. In stroma, M. bovis was frequently seen among collagen fibrils, within neutrophil phagosomes, and associated with cellular debris. This study demonstrates that a virulent strain of M. bovis can invade bovine corneal epithelial cells and can cause keratitis in the absence of injurious ultraviolet irradiation or other known predisposing environmental factors.     

Cytopathic effects of Moraxella bovis on cultured bovine neutrophils and corneal epithelial cells

The effects of Moraxella bovis on the morphologic features of purified bovine neutrophils and bovine corneal epithelial cells were examined, using transmission and scanning electron microscopy and light microscopy. Within 2 minutes after incubation of bovine neutrophils with living M bovis, electron microscopic cellular changes included vacuolation, swelling, and loss of microplicae. Most of the neutrophils were lysed by 10 minutes of incubation. Human neutrophils phagocytosed the M bovis and remained intact, even after 30 minutes of incubation with the bacteria. Living M bovis killed bovine corneal epithelial cells in vitro. Sterile filtrates prepared from 6-hour shaker cultures of M bovis also killed bovine corneal epithelial cells, but the cytotoxic activity was less than that produced by the living bacteria. Cellular changes were first observed in specimens collected 1 hour after corneal cell monolayers were inoculated with sterile culture filtrates. The changes in these cells included pit-like lesions on the cellular surface, cellular separation, and vacuolation.     

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.     

Detection of cell detachment activity induced by Moraxella bovis

OBJECTIVE: To characterize the effect that filtrate obtained from cultures of Moraxella bovis has on cultured corneal epithelial cells and other types of cultured mammalian cells. SAMPLE POPULATION: Cultured hamster corneal epithelial cells, bovine epithelial cells, and several transformed cell lines exposed to culture filtrate from a pathogenic isolate of M bovis. PROCEDURE: Moraxella bovis was cultured, and bacteria were removed by filtration. The resulting bacterial culture filtrate was incubated with various types of cultured cells, and effects of the filtrate on detachment of various mammalian cell types was quantified by the use of neutral red dye. Additionally, bacterial culture filtrate was treated with protease inhibitors as well as trypsin and heat prior to incubation with cultured mammalian cells. RESULTS: Bacterial culture filtrate of M bovis caused all types of cultured cells to detach from each other and from the substrate, with the maximal effect evident 2 hours after initiating incubation. Detached cells were alive, and detachment was reversible. Serine protease inhibitors (phenylmethylsulfonylfluoride and alpha2-macroglobulin) inhibited cell detachment attributable to bacterial culture filtrate. Heating and treatment with trypsin also inhibited cell detachment. CONCLUSIONS AND CLINICAL RELEVANCE: Moraxella bovis produces a soluble factor that causes reversible detachment of cultured cells. This activity may play a role in the pathogenesis of 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.     

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.     

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.     

Growth of Mycoplasma bovis in organ cultures of bovine foetal trachea and comparison with Mycoplasma dispar

Inoculation of tracheal organ cultures from bovine foetuses with Mycoplasma bovis resulted in a loss of cellular structure of the lamina propria, followed 20-22 days later by lifting and detachment of overlying epithelium. The effect was associated with large numbers of M. bovis, identified by immunoperoxidase labelling and electromicroscopy, infiltrating between the epithelial cells and amassing in the lamina propria, especially in the region of the basement membrane of the epithelium. Ciliary activity was undiminished for up to 18 days following inoculation and little or no cytopathic effect on the ciliated epithelium was seen in spite of the close proximity of large numbers of organisms. In contrast, M. dispar was restricted to the margin of the ciliated epithelium where, as previously reported, it caused pyknosis, sloughing and flattening of the epithelium with consequent loss of ciliary activity. The cytopathology observed for each mycoplasma bore a close similarity to the behaviour of the two mycoplasmas in vivo and it is suggested that the organ culture system may be a useful and relevant system to elucidate the pathogenic mechanisms for each mycoplasma.     

Preliminary observations of interaction between bacteriophages and Streptococcus bovis bacteria on ruminal epithelium primoculture.

Five Streptococcus bovis strains (47/3, 59/2, 4/1, 46/2 and 44/9) isolated from calf ruminal fluid samples were examined for the adherence to cultured ruminal epithelium cells. Four strains (47/3, 59/2, 4/1 and 46/2) were able to attach to the cultured epithelial cells. However, S. bovis 47/3 strain attached to the target cells in significantly greater numbers than the other strains. Strain 44/9 did not adhere to cells of ruminal epithelium. The adherent bacteria were observed on the surface of differentiated (mainly keratinized) cells of ruminal epithelium primoculture only. The different effect of F4, F5 and F6 bacteriophages was ascertained on S. bovis bacteria adhering to rumen epithelial primoculture. A significant decrease in the number of adherent bacteria was shown after cultivation of strains 47/3 and 4/1 with F6 bacteriophage and of 47/3 strain with F4 phage. The F5 bacteriophage had no significant effect on these bacteria.     

Adherence of Moraxella bovis to cell cultures of bovine origin

The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.     

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.     

Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: Characterization of lung pathology,persistence of variable surface protein antigens and local immune response

Background

Mycoplasma bovis is associated with pneumonia in calves characterized by the development of chronic caseonecrotic lesions with the agent persisting within the lesion. The purposes of this study were to characterize the morphology of lung lesions, examine the presence of M. bovis variable surface protein (Vsp) antigens and study the local immune responses in calves after infection with M. bovis strain 1067.

Methods

Lung tissue samples from eight calves euthanased three weeks after experimental infection with M. bovis were examined by bacteriology and pathology. Lung lesions were evaluated by immunohistochemical (IHC) staining for wide spectrum cytokeratin and for M. bovis Vsp antigens and pMB67 antigen. IHC identification and quantitative evaluation of CD4⁺ and CD8⁺ T lymphocytes and immunoglobulin (IgG1, IgG2, IgM, IgA)-containing plasma cells was performed. Additionally, expression of major histocompatibility complex class II (MHC class II) was studied by IHC.

Results

Suppurative pneumonic lesions were found in all calves. In two calves with caseonecrotic pneumonia, necrotic foci were surrounded by epithelial cells resembling bronchial or bronchiolar epithelium. In all calves, M. bovis Vsp antigens were constantly present in the cytoplasm of macrophages and were also present extracellularly at the periphery of necrotic foci. There was a considerable increase in numbers of IgG1- and IgG2-positive plasma cells among which IgG1-containing plasma cells clearly predominated. Statistical evaluation of the numbers of CD4⁺ and CD8⁺ T cells, however, did not reveal statistically significant differences between inoculated and control calves. In M. bovis infected calves, hyperplasia of bronchus-associated lymphoid tissue (BALT) was characterized by strong MHC class II expression of lymphoid cells, but only few of the macrophages demarcating the caseonecrotic foci were positive for MHC class II.

Conclusions

The results from this study show that infection of calves with M. bovis results in various lung lesions including caseonecrotic pneumonia originating from bronchioli and bronchi. There is long-term persistence of M. bovis as demonstrated by bacteriology and immunohistochemistry for M. bovis antigens, i.e. Vsp antigens and pMB67. The persistence of the pathogen and its ability to evade the specific immune response may in part result from local downregulation of antigen presenting mechanisms and an ineffective humoral immune response with prevalence of IgG1 antibodies that, compared to IgG2 antibodies, are poor opsonins.     

Cultivation of corneal epithelial cell sheets on canine amniotic membrane

Objective To develop and assess canine corneal epithelial cell sheets cultivated from limbal stem cells on amniotic membrane. Procedures Canine corneal limbal segments were obtained from six beagle dogs. Cryopreserved denuded amniotic membranes (obtained from Miniature Dachshund and Cavalier King Charles Spaniel breeds) from which the epithelial cells were removed were used as scaffolds. The limbal segments were cultured on these amniotic membranes with 3T3 feeder cells for 2 weeks. The harvested corneal epithelial cell sheets were stained with H&E for histologic analysis. The harvested sheets were analyzed immunohistochemically using a corneal epithelium‐specific marker keratin 3(K3) and putative stem cell markers ABCG2, p63, and vimentin. Results Cultivated cells from the corneal limbal tissues reached confluency in 7–8 days. The cultivated cells adhered to the denuded amniotic membrane and formed a sheet. The cultivated cell sheet was transparent and consisted of five to eight layers. K3 was observed in all layers and ABCG2, p63, and vimentin were notably present in the basal layer of the cultivated canine epithelium by immunofluorescence. Conclusions Canine corneal epithelial cells were successfully cultivated on the canine amniotic membrane. The cultivated epithelial sheets contained putative stem cells in the basal layer and had a stratified epithelium.     

Anterior corneal dystrophy of American Dutch belted rabbits: biomicroscopic and histopathologic findings

Spontaneously occurring anterior corneal opacities were present in related, juvenile American Dutch belted rabbits. Slit lamp biomicroscopy revealed focal opacities of epithelium, basement membrane, and subepithelial corneal stroma. Lesions were characterized histologically by thin and disorganized surface epithelium, thickened and intensely staining epithelial basement membrane, fimbriated and irregular basement membrane-stromal juncture, and disorganized subepithelial stroma. Biomicroscopic and histopathologic features of anterior corneal dystrophy of American Dutch belted rabbits appear similar to those of human anterior corneal dystrophies.     

Diagnosis and management of chronic corneal epithelial defects (indolent corneal ulcerations)

Chronic corneal epithelial defects (CCEDs; indolent corneal ulcerations) are the most common refractory ulcerations in veterinary medicine and are diagnosed by their classic appearance. CCEDs are superficial ulcerations without stromal involvement and have a nonadherent epithelial border (lip). Fluorescein stain adheres to the exposed stroma and extends below the epithelial border, outlining the epithelial lip. CCEDs occur secondary to adnexal disease, keratoconjunctivitis sicca, exposure keratitis, neurotrophic keratitis, and primary corneal disease. In cats, herpes keratitis is associated with the development of CCEDs. Bacterial infections are not responsible for the refractory nature of CCEDs. Because of the refractory nature of CCEDs, treatment can be frustrating for both owner and veterinarian. Current treatment recommendations consist of identifying and treating the underlying cause and performing procedures that stimulate epithelialization and adhesion of the corneal epithelium. Initial treatment of CCEDs includes ulcer debridement and grid keratotomy. Superficial keratectomy is indicated in refractory cases.     

Cyclooxygenase-2 expression in the cornea of dogs with keratitis

Cyclooxygenase-2 (COX-2) can be overexpressed at inflammatory sites, leading to the generation of proinflammatory prostanoids. Selective inhibitors of COX-2 have potential use in treating inflammatory conditions including ophthalmic diseases in veterinary medicine. Keratitis is considered the most common inflammatory eye disease in dogs. In this study we evaluated the expression of COX-2 in normal dog eyes and in dog eyes with keratitis by immunohistochemistry using isoform-specific antibodies. In the normal eye (n = 4), no COX-2 immunoreactivity was observed in the cornea. In keratitis, COX-2 (n = 12) expression was observed in all corneal layers (epithelium, stromal cells, and endothelium). COX-2 immunoreactivity was also noted in the stromal and epithelial cells of the iris and the stromal cells of the trabecular meshwork. These data indicate that COX-2 may play a pathophysiologic role in keratitis and suggest potential therapeutic implications of prostaglandin modulation in inflammatory eye diseases.     

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.     

Keratoconus in a cynomolgus monkey

In a seven-year-old male cynomolgus monkey, erythema of the upper eyelid and forehead and corneal opacity, edema and conical protrusion in the eye were observed. At necropsy, ophthalmological and serological examinations revealed binocular corneal opacity and conical protrusion and a high IgE level, respectively. Thinning of the epithelium and stroma of the cornea were noted histopathologically. At the center of the corneal epithelium, the number of epithelial cells was reduced, their cytoplasm was poorer and the basal cells were flatter than at the periphery. Bowman's membrane was folded with partial loss or breakage. Collagen fibers were compacted or disarranged, and the keratocytes were increased in the stroma, with focal pyknosis or loss of the endothelium and folding of Descemet's membrane. Electron microscopical examination revealed atrophy of the corneal epithelial basal cells. This is the first report of a case of keratoconus in a cynomolgus monkey.