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.     

Isolation of Mycoplasma bovoculi from cases of infectious bovine keratoconjunctivitis.

Five outbreaks of infectious bovine keratoconjunctivitis were examined for bacteria and mycoplasmas. Mycoplasma bovoculi was demonstrated in four of the five outbreaks. Other mycoplasmatales were represented by Ureaplasma in one sample. Moraxella bovis and Neisseria ovis were found in all the outbreaks, the former being present in the vast majority of the animals. Transmission experiments with Mycoplasma bovoculi and Moraxella bovis in combination were carried out on four young, colostrumdeprived calves. Mycoplasma bovoculi appeared to have an enhancing effect on the pathogenicity of Moraxella bovis.     

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.     

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.     

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.     

Vaccination of cattle with Mycoplasma bovoculi antigens: evidence for field immunity

Cattle previously exposed to Mycoplasma bovoculi developed a high serum IgG and cellular responses detected by ELISA and lymphocyte blastogenesis respectively, when immunized with three M. bovoculi antigen preparations (two non-ionic detergent extracts and a killed whole organism). Similar responses to the detergent immunogen were observed in colostrum-deprived calves free of M. bovoculi. Following ocular challenge with live M. bovoculi all animals including the control group cleared the organism. However the organism colonized the eyes of the colostrum-deprived calves. These observations indicate that previous exposure to M. bovoculi confers immunity to reinfection and that none of the vaccines provided a protective activity against the organism.     

Microbial flora of the eyes of cattle

The bacteria and mycoplasma occurring in the eyes of normal healthy calves were monitored in three groups of animals from purchase at about one week old to slaughter at about 15 months old. Non-haemolytic Moraxella bovis, Branhamella catarrhalis and Mycoplasma bovoculi were all isolated regularly, though their seasonal occurrence varied. The significance of these findings with respect to infectious bovine keratoconjunctivitis is discussed.     

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.     

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.     

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)     

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.     

Infectious bovine keratoconjunctivitis and lymphofollicular hyperplasia of the third eyelid in heifers

On a dairy cattle farm, infectious bovine keratoconjunctivitis was diagnosed in 29 (24%) calves and heifers aged from 2 weeks to 1 year old. The highest infection rate (18%) occurred in animals aged 3-6 months. The bacteriological examination of swabs from the affected animals yielded several species of bacteria: Moraxella bovis, Neisseria ovis, N. cuniculi, plasma coagulase-negative Staphylococcus spp., alpha-haemolytic Streptococcus spp., Arcanobacterium pyogenes and Escherichia coli. Moraxella bovis and N. ovis were the most common isolates. Hyperplasia of the lymphatic tissue of the third eyelid in the form of nodules 7-8 mm in diameter was diagnosed in two heifers aged 8 and 10 months.     

Interactions of Mycoplasma bovoculi with erythrocytes: role of p94 surface protein.

The attachment of two strains of Mycoplasma bovoculi to erythrocytes was measured using 35S-methionine-labelled organisms. Receptor sites of M. bovoculi involved in this attachment are trypsin-sensitive, since mild trypsin treatment of the intact organisms abolished this process completely. Pretreatment of erythrocytes with trypsin or increasing concentrations of neuraminidase resulted in no measurable effect. Monoclonal antibody MA25.5 directed against a M. bovoculi surface antigen of 94 kDa termed p94 blocked 40% of the attachment, while MA18.13 directed against a 57 kDa protein band of M. bovoculi had no effect on the attachment process. Other properties of M. bovoculi were tested using six strains of the mycoplasma and erythrocytes from several animal species. None of the strains showed haemagglutinating or haemadsorbing activities.     

Antibody response in calves experimentally or naturally exposed to Mycoplasma bovoculi

An antiglobulin-ELISA has been developed to detect antibody activity to Mycoplasma bovoculi in sera, nasal fluids and lacrimal fluids of field and experimentally exposed calves. Low IgG activity with no IgM or IgA was detected in sera of experimental calves. In nasal and lacrimal fluids, IgA appeared as early as the first week following exposure to M. bovoculi and predominated in both of these fluids throughout the 9 wk observation period. Sera from field-exposed animals showed high IgG and IgM activities. The metabolic-inhibition (MI) test was applied to detect growth inhibition of M. bovoculi in those fluids. This property was found only in sera of exposed animals and thus could be used to test for M. bovoculi infection. The ELISA test and the MI test were considered reliable tests for the detection of antibodies to M. bovoculi infection. The implications of finding no growth-inhibiting activity in nasal and lacrimal fluids concurrent with a high IgA activity are discussed.     

Isolation of Mycoplasmas from nasal swabs of calves affected with respiratory diseases and antimicrobial susceptibility of their isolates

Nasal swabs of 293 calves were examined for Mycoplasma. The samples were collected from calves affected with respiratory diseases on 71 farms in various parts of Japan between 1996 and 1997. Mycoplasma bovirhinis was isolated from 47 of 293 calves (16.0%). Mycoplasma alkalescens, M. bovis, M. arginini, M. bovigenitalium and Acholeplasma spp. were isolated from 19 (6.5%), seven (2.4%), four (1.4%), four (1.4%) and 18 (6.1%) calves, respectively. Pasteurella multocida and P. haemolytica were isolated from 60% of Mycoplasma-positive calves. However, other bacteria were not isolated from calves. To evaluate the antimicrobial susceptibility of their isolates, 68 M. bovirhinis, 21 M. alkalescens and 10 M. bovis strains were examined for 12 antimicrobial agents. All isolates showed higher susceptibility to tiamulin than to the other drugs used in the study. However, erythromycin had no effect on any of the Mycoplasma strains studied. The field isolates were less susceptible than the type strains to some drugs, such as spiramycin, oxytetracycline and tylosin.     

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.     

Serologic response of vaccinated cattle to strains of Moraxella bovis isolated during epizootics of keratoconjunctivitis.

In studies to determine whether there were antigenic differences between strains (isolates) of Moraxella bovis, the sera from vaccinated calves were tested with isolates of M bovis while the calves were experiencing epizootics of infectious bovine keratoconjunctivitis (IBK). Before the epizootics of IBK, the calves were intramuscularly vaccinated with a formalin-killed autogenous M bovis bacterin. During the epizootics, the eyes were examined by cultural technique, and isolates which were obtained were categorized by catalase activity, source (diseased or nondiseased eyes), and reactivity with the various sera. The serum reactivity of the isolates was compared with that of the vaccinal strain. The vaccinal strain and 8 of the 1 5 selected isolates obtained during the 1974 epizootic were catalase negative. Seven of the 15 isolates from the 1974 epizootic and all of the selected isolates from the 1975 epizootic were catalase positive. A significantly higher (P less than 0.01) percentage of calf sera were serologically reactive with the vaccinal strain and other catalase-negative isolates (45.0%) than with catalase-positive isolates (34.8%). The results, although not definitive, suggest that there may be antigenic differences among strains of M bovis. These differences should be considered when cattle are vaccinated against IBK under natural conditions of exposure.     

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.     

Ovine keratoconjunctivitis experimentally induced by instillation of Mycoplasma conjunctivae

Five sheep, free from Mycoplasma conjunctivae and ocular Chlamydia infection, were experimentally inoculated with M. conjunctivae and five more sheep were exposed to the infection by contact. Keratoconjunctivitis developed in all ten sheep. As in natural outbreaks of infectious keratoconjunctivitis (IKC), clinical signs were generally moderate and transient, and recurred in some sheep. M. conjunctivae was detected throughout the 53-day observation period. Clinical diagnosis was confirmed by histopathologic examination of three sheep. Moraxella ovis, found in six of the ten sheep before the start of the experiment, appeared to play no etiologic role in the development of IKC.