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Infectious bovine keratoconjunctivitis (IBK) is an acute disease caused by Moraxella bovis (Mb). Several factors may predispose animals to an IBK outbreak; one commonly observed is infection with bovine herpes virus type 1 (BHV-1). The aim of this study was to investigate the dynamics of BHV-1 virus infection and its relation with clinical cases of IBK in weaned calves from a beef herd with a high prevalence of lesions caused by Mb. Sampling was carried out in six stages and included conjunctival swabs for isolating Mb as well as blood samples for identifying antibodies specific for BHV-1. A score for IBK lesions after observing each eye was determined. The findings of this study showed a high prevalence of BHV-1 virus infection (100% of animals were infected at the end of the trial); 67% of animals were culture-positive for Mb, but low rates of clinical IBK (19% of calves affected) were detected at the end of the trial. These results suggest that infection with BHV-1 did not predispose these animals to IBK, and that Mb infection produced clinical and subclinical disease in the absence of BHV-1 co-infection.

Background A randomized, masked, 2-arm parallel trial was conducted to assess the efficacy of a Moraxella bovis (M. bovis) autogenous vaccine to prevent naturally occurring infectious bovine keratoconjunctivis (IBK) in beef calves. Hypothesis The null hypothesis was that treatment group was not associated with either risk of IBK or last observed weight. Animals The trial was conducted between May and November 2009 and 2010 on a university-owned farm in Iowa. The vaccine contained 2 randomly selected M. bovis from IBK cases that occurred at the farm in 2008. Calves born between January and May 2009 and 2010 without visible corneal lesions were randomly allocated to receive vaccine (n = 191) or placebo (n = 178). Methods Two SC doses were administered 21–28 days apart. Allocation to treatment was concealed using bottles marked A or B. Staff observing the animals for IBK could not determine the treatment grouping. The herd met the “at-risk” criteria (ie, >15% IBK in unvaccinated calves and M. bovis detection in IBK cases). Analysis was “per-protocol”. Results The risk of IBK was 58/185 (31%) in vaccinated calves and 66/173 (38%) in unvaccinated calves (adjusted risk ratio = 0.78; 95% CI, 0.49–1.24). Average weight before sale did not differ between the vaccinated calves (196.6 kg, SD ± 39.9) and unvaccinated calves (198.1 kg, SD ± 42.7) (P value = .19). No adverse effects were noted. Conclusion and Clinical Importance Combination of the study results with previous studies suggests that autogenous M. bovis vaccines often are ineffective in controlling naturally occurring IBK.

É descrito um surto de ceratoconjuntivite infecciosa bovina (CIB) e hemoncose intercorrente afetando um grupo de 116 bezerros de 1-2 anos de idade na região central do Rio Grande do Sul. Vinte bezerros foram afetados e nove morreram. Os sinais clínicos incluíam perda de peso, marcada palidez da mucosa oral, lacrimejamento, opacidade focal da córnea e avermelhamento da conjuntiva. Sinais mais avançados incluíam lacrimejamento copioso, opacidade da córnea, ceratocone, ceratomalacia e cegueira. O diagnóstico de CIB foi baseado nos achados epidemiológicos e clínico-patológicos e confirmado pela cultura de uma cepa hemolítica de Moraxella bovis dos olhos de dois terneiros afetados. O alto coeficiente de letalidade (45%) neste surto foi atribuído a grave hemoncose intercorrente.

Thirty-seven yaks (Bos grunniens) with keratoconjunctivitis and 22 healthy yaks were used to investigate the role of bovine herpesvirus 1 (BoHV-1) in keratoconjunctivitis in yaks. Nucleic acid sequences of BoHV-1 glycoproteins B and E were detected in conjunctival swabs from all yaks with keratoconjunctivitis using a nested polymerase chain reaction (PCR). In 21 yaks, BoHV-1 sequences were detected along with Moraxella bovis (M. bovis) and Neisseria spp. The amplified BoHV-1 sequences were identical, and no nucleotide variation was observed when compared with a BoHV-1 reference strain using single-strand conformation polymorphism analysis of the amplified DNA sequences. Interestingly, BoHV-1 sequences could not be detected in samples from healthy yaks. However, conjunctival swabs from two healthy yaks (9.09%) yielded M. bovis and Neisseria spp. Samples from 35 yaks with keratoconjunctivitis showed positive reactions in an avidin biotin enzyme-linked immunosorbent assay for BoHV-1 antibodies; all the healthy yaks were seronegative. This is the first report of a possible association of BoHV-1 with keratoconjunctivitis in yaks.

Summary The bacteriological findings in infectious kerato-conjunctivitis of chamois and sheep in Switzerland are described. In addition to a relatively heterogeneous flora (Staph. aureus, Corynebacterium pyogenes, Moraxella ovis, Haemophilus sp., Actinobacillus lignieresii, micrococci and streptococci) we isolated constantly M. conjunctivae, sometimes in mixed infection with M. arginini. In one instance M. conjunctivae was associated with pneumonia. The possible role of M. conjunctivae as an etiological agent in infectious kerato-conjunctivitis is discussed. Zusammenfassung Nachweis von Mycoplasma conjunctivae bei der Keratoconjunctivitis der Gemsen und Schafe Bei der infektiosen Keratoconjunctivitis der Gemsen und Schafe konnte neben einer heterogenen Bakterienflora (Staph. aureus, Corynebacterium pyogenes, Moraxella ovis, Haemophilus sp., Actinobacillus lignieresii, Mikrokokken und Streptokokken) regelmasig M. conjunctivae isoliert werden. Zum Teil lag eine Mischinfektion mit M. arginini vor. In einem weiteren Fall trat M. conjunctivae im Zusammenhang mit einer Pneumonie auf. Die Befunde sprechen fur die aetiologische Bedeutung von M. conjunctivae fur die infektiose Keratoconjunctivitis. Resume Mise en evidence de Mycoplasma conjunctivae dans la Keratoconjonctivite des chamois et des moutons M. conjunctivae a ete regulierement isole dans la Keratoconjonctivite infectieuse des chamois et des moutons a cote d'une flore bacterienne heterogene (Staph. aureus, Corynebacterium pyogenes, Moraxella ovis, Haemophilus sp., Actinobacillus lignieresii, des microcoques et des streptocoques). On a parfois rencontre une infection mixte avec M. arginini. M. conjunctivae est intervenu dans d'autres cas en relation avec une pneumonie. Les resultats parlent en faveur du role etiologique de M. conjunctivae dans la Keratoconjonctivite infectieuse. Resumen Deteccion de Mycoplasma conjunctivae en la queratoconjuntivitis de las gamuzas y ovejas En la queratoconjuntivitis infecciosa de las gamuzas y ovejas se pudo aislar con regularidad Mycoplasma conjunctivae al lado de una flora bacteriana heterogenea (Staph. aureus, Corynebacterium pyogenes, Moraxella ovis, Haemophilus spp., Actinobacillus lignieresii, micrococos y estreptococos). En parte se trataba de una infeccion mixta con M. arginini. En otro caso mas se presento en relacion con una pneumonia. Los hallazgos abogan a favor de la importancia etiologica de M. conjunctivae para la queratoconjuntivitis infecciosa.

Summary The clinical features of peste des petits ruminants (PPR) virus infection in goats were studied in two field outbreaks and by contact exposure of susceptible goats. Clinical signs observed included sudden onset of pyrexia, necrosis and erosions of the oral cavity, diarrhoea, and pneumonia, in that chronological order. Erosions of the vulva and prepuce as well as abortion were recorded in a few cases. Bacteriological examination of ante — in and post mortem samples showed that the most significant bacteria associated with PPR — infected goats were Pasteurella haemolytica, Klebsiella sp., Pseudomonas aeruginosa and Staphylococus pyogenes from the lungs, Salmonella sp. and E. coli from the faeces, Moraxella bovis from the eyes and Staphylococcus pyogenes from the oral cavity. The pathological lesions seen in natural PPR included necrosis, erosions and ulcerations of the epithelial layers of the labium, the vulva, and the alimentary tract as well as giant-cell type pneumonia. However, some of the affected goats had fibrinous or purulent bronchopneumonia, most probably caused by secondary bacteria. Zusammenfassung Peste des Petits Ruminants (PPR) bei Ziegen in Nigeria: Klinische, mikrobiologische und pathologische Charakteristika Die klinischen Charkteristika der Peste des Petits Ruminants (PPR)-Virusinfektion bei Ziegen wurden wahrend zwei Feldausbruchen und mittels Kontaktexposition empfanglicher Ziegen untersucht. Die beobachteten klinischen Krankheitszeichen waren plotzlich auftretendes Fieber, Nekrosen und Erosionen in der Maulhohle, Durchfalle und Pneumonien in der aufgefuhrten Reihenfolge. Erosionen an Vulva und Praputium ebenso wie Aborte wurden in wenigen Fallen beobachtet. Bakteriologische Untersuchungen von ante- und post mortem Proben zeigten, daβ die wichtigsten bakteriellen Infektionen in Verbindung mit der PPR verursacht wurden von Pasteurella haemolytica, Klebsiella sp., Pseudomonas aeruginosa und Staphylococcus pyogenes, isoliert aus der Lunge, ferner von Salmonella sp. und E. coli, isoliert aus den Faeces, sowie von Moraxella bovis, isoliert aus den Augen und von Staphylococcus pyogenes isoliert, aus der Maulhohle. Die pathologischen Lasionen, die bei den naturlichen Ausbruchen der PPR beobachtet wurden, waren Nekrosen, Erosionen und Ulcera des Lippen-Epithels, des Epithels der vulva und des Verdauungstrakts und das Auftreten von Pneumonien des Riesenzell-Typs. Obwohl einige der befallenen Ziegen fibrinose oder purulente Bronchopneumonien hatten, durften diese hochstwahrscheinlich durch bakterielle Sekundarinfektionen verursacht sein. Resume Peste des petits ruminants (PFR) chez des chevres au Nigeria: caracteristiques cliniques, microbiologiques et pathologiques Les caracteristiques cliniques de l'infection virale de la peste des petits ruminants (PPR) chez des chevres ont ete examinees durant deux epizooties dans le terrain et au moyen d'une exposition par contact de chevres receptives. Les signes cliniques observes ont ete une fievre brutale, des necroses et des errosions dans la gueule, de la diarrhee et des pneumonies dans l'ordre cite. Des errosions sur la vulve et le prepuce ainsi que des avortements ont ete observes dans quelques cas. Les recherches bacteriologiques d'echantillons ante- est post-mortem ont montre que les infections bacteriennes les plus importantes, liees a PPR etaient dues a Pasteurella haemolytica, Klebsiella sp., Pseudomonas aeruginosa et Staphylococcus pyogenes dans les poumons, a Salmonella sp. et E. coli dans les matieres fecales, a Moraxella bovis dans les yeux et a Staphylococcus pyogenes dans la gueule. Les lesions pathologiques observees lors de PPR dans le terrain furent des necroses, des errosions et des ulceres de l'epithelium des levres, de la vulve et du tube digestif et l'apparition de pneumonies du type «cellules geantes». Quelques chevres atteintes ont presente des bronchopneumonies fibrineuses et purulentes, tres vraisemblablement dues a des infections bacteriennes secondaires. Resumen La peste de los pequenos rumiantes (PPR) en cabras en Nigeria Caracteres distintivos clinicos, microbiologicos y patologicos Se estudiaron las caracteristicas clinicas de la infeccion virosica peste de los pequenos rumiantes (PPR) en cabras durante dos brotes campales y mediante exposicion de contacto de cabras receptibles. Los signos nosologicos observados eran fiebre que aparecia repentinamente, necrosis y erosiones en la cavidad bucal, diarreas y pneumonias en la ringlera citada. En casos contados, se observaron erosiones en la vulva y prepucio, e incluso abortos. Los estudios bacteriologicos de muestras ante y post mortem mostraron que las infecciones bacterianas mas importantes fueron ocasionadas, en combinacion con la PPR, por Pasteurella haemolytica, Klebsiella sp., Pseudomonas aeruginosa y Staphylococcus pyogenes, aisladas del pulmon, ademas por Salmonella sp. y E. coli, aisladas de heces, asi como por Moraxella bovis, aislada en los ojos, y por Staphylococcus pyogenes, aislado de la cavidad bucal. Consistian las lesiones patologicas que se observaron en los brotes naturales de PPR en necrosis, erosiones y ulceras del epitelio labial, del epitelio vulvar y del tracto digestivo y la presentacion de pneumonias del tipo celular gigante. Aunque algunas de las cabras afectadas tenian broncopneumonias fibrinosas o purulentas, estas podrian haber sido ocasionadas con toda posibilidad por infecciones bacterianas secundarias.

A randomized, blinded, controlled field trial was conducted during summer 2006 in a northern California, USA, herd of beef cattle to evaluate the efficacy of a recombinant Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye). A convenience sample comprised of 127 steers were administered a subcutaneous dose of either adjuvant alone (ISCOM matrices; control group) or recombinant M. bovoculi cytotoxin carboxy terminus adjuvanted with ISCOM matrices (MbvA group) and were boostered 21 days later. The steers were examined once weekly for 15 weeks for evidence of IBK. No significant difference in the cumulative proportion of corneal ulcerations was detected between groups. Compared to the control calves, the MbvA vaccinates had significantly higher increases in serum neutralizing titers to M. bovoculi hemolysin between week 0 and week 6. The prevalence of M. bovis isolations was higher from ulcerated eyes of calves vaccinated with MbvA as compared to control calves. Vaccination of calves against the carboxy terminus of M. bovoculi RTX toxin resulted in significant increases in serum hemolysin neutralizing titers and may modulate organism type cultured from ulcerated eyes of calves in herds where both M. bovis and M. bovoculi exist. Use of M. bovoculi antigens alone in vaccines to prevent IBK may not be beneficial in herds where IBK is associated with both M. bovoculi and M. bovis.

The contribution of a type II restriction-modification system (R-M system) to genome integrity and cell viability was investigated. We established experimental conditions which enabled the achievement of hemimethylated and unmethylated states for the specific bases of the recognition sequences of the host's DNA. To achieve this, we constructed the MboII R-M system containing only one (i.e. M2.MboII) out of two functional MboII methyltransferases found in Moraxella bovis. Using the incomplete R-M system we were able to perturb the balance between methylation and restriction in an inducible manner. We demonstrate that upon the SOS-induced DNA repair in the mitomycin C treated cells, restriction significantly reduces cell viability. Similar results for the well-studied wild type EcoRI R-M system, expressed constitutively in Escherichia coli, were obtained. Our data provide further insights into the benefits and disadvantages of maintaining of a type II R-M system, highlighting its impact on host cell fitness.

Morphological variants of four Moraxella bovis strains were studied, using different cultural conditions. On bovine blood agar medium two colony types could be distinguished: 1) large, flat, rough and agar-corroding, and 2) most often smaller, convex, smooth and non-corroding forms. When transferred to human blood agar, the flat type formed convex, spreading-corroding colonies (SC type) and the other type remained convex, non-spreading and non-corroding (N type). Some cell lines lost the original flat colony appearance on bovine blood agar after passages for a longer period on human blood agar; on bovine blood agar these SC cells now formed convex, but still corroding colonies. By electron microscopy it was observed that cells from corroding colonies on either medium were strongly fimbriated, whereas preparations from non-corroding populations had non-fimbriated cells or contained only an occasional fimbria. Conjunctival infection experiments in calves revealed that only fimbriated variants of each strain were able to colonize the conjunctival mucosa. Ultraviolet irradiation of the eye prior to inoculation facilitated the bacterial colonization, with respect to initiation and duration. Non-fimbriated M. bovis could not be reisolated even after irradiation. Keratitis and conjunctivitis only occurred in irradiated eyes inoculated with cell lines yielding typically flat, corroding colonies on bovine blood agar. The possible function of fimbriae for attachment of M. bovis to the mucous membranes of the eye is discussed.

Objective To conduct a serologic survey and define pili antigenic variability via the serologic cross-reactivity of Moraxella bovis isolates from naturally occurring infectious bovine keratoconjunctivitis (IBK) outbreaks in Australia. This project applies to the development of an M bovis pili-based vaccine targeting Australian strains originating from intensive cattle producing regions. Procedure Ocular swabs were collected from cattle affected with clinical signs of IBK from 25 veterinary practices. Standard criteria were used to identify 70 M bovis. Pure, piliated isolates were evaluated with a modified competitive enzyme-linked immunosorbent assay (ELISA) for cell-bound M bovis pili to determine their serologic cross-reactivity with pili of vaccinal bacterin strains EPP63, FLA64, and SAH 38. Results Sixty-four percent (45/70) of M bovis isolates demonstrated homologous pili antigens to a vaccinal strain. M bovis isolates homologous to one of the three vaccinal strains were obtained in 77% (34/44) of IBK outbreaks sampled. No IBK outbreak had isolates homologous to more than one vaccinal strain; however, 29% (10/34) of outbreaks with a cross-reacting strain had non-cross-reacting strains as well. Conclusion The similar prevalence of pilus antigen homology to strain FLA64 was observed with isolates derived from NSW, Tasmania, and Victoria, compared with results of prior smaller serologic studies, suggests that the common pilus antigens in M bovis within Australia have been relatively stable over the last 20 years. The prevalence of a limited number of pilus antigens in M bovis suggest that the application of a vaccine containing the bacterial strains EPP63, FLA64, and SAH38 may provide a useful management tool for reducing production losses associated with IBK in Australia.

A review was conducted of all identified literature evaluating Moraxella bovis vaccines efficacy in preventing pinkeye in beef calves. From 292 publications identified by the search, data on 123 unique vaccine-to-control comparisons were extracted from 38 studies published in English from 1960 to 2005. Descriptive analysis was performed and an analysis of sources of variation evaluated. Use of methods to control bias such as randomization and blinding were associated with decreased vaccine efficacy. Only 15 trials reported using randomization and blinding. The authors conclude that when designing and reporting veterinary vaccination studies, researchers must include methodological quality information necessary to judge the evidence produced from the study.

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.

To determine whether amino acid sequence variation exists in the Moraxella bovis ( M. bovis ) cytotoxin (MbxA) from geographically diverse M. bovis isolated in the United States, mbxA was amplified and sequenced. The MbxA deduced amino acid sequence from M. bovis originally isolated in California, Washington, North Carolina, and Georgia, as well as reference strains of M. bovis isolated at the National Animal Disease Laboratory, Ames, IA, USA, all encoded a nearly identical 927 amino acid protein. MbxA from two of the four California isolates (SFS 9a and SFS 100a) differed from all other isolates at two sites at which the polar amino acids glutamine (position 666) and asparagine (position 823) were replaced by ionized amino acids glutamic acid and aspartic acid, respectively. Rabbit antiserum to the expressed carboxy terminus (amino acids 590–927) of MbxA from M. bovis (Tifton I) neutralized the hemolytic activity of SFS 9a and SFS 100a. The M. bovis cytotoxin appears to be conserved amongst geographically diverse isolates of M. bovis from the USA. Antiserum against the carboxy terminus of MbxA common to the majority of isolates neutralized the hemolytic activity of two strains with a divergent MbxA deduced amino acid sequence. Vaccines against IBK that incorporate MbxA as antigen may offer protection against geographically diverse strains of M. bovis .

Eighteen isolates of a Gram-negative coccus (strain 237T) were cultured from the eyes of dairy and beef calves affected with infectious bovine keratoconjunctivitis (IBK; ‘pinkeye’) in northern California, USA, during summer 2002. These isolates had near full-length (1397 bp) 16S rRNA gene sequences that clustered into three groups with 99.9 % sequence similarity. On the basis of 16S rRNA gene sequence, the isolates were most closely associated with Moraxella bovis and Moraxella ovis in clade I of the classical moraxellae. Biochemically, the novel isolates could be distinguished from the other members of the genus Moraxella isolated from animals on the basis of phenylalanine deaminase activity. The results of partial sequence analysis of six housekeeping genes, the 16S–23S rRNA gene interspacer region and partial 23S rRNA gene provide strong support for the inclusion of these isolates in a novel taxon, for which the name Moraxella bovoculi sp. nov. is proposed. The type strain is strain 237T (=ATCC BAA-1259T=CCUG 52049T).

The purpose of the present study was to identify Moraxella ( M. )—like organisms recovered from calves suffering from respiratory disease down to species level by means of tDNA-intergenic spacer length polymorphism analysis (tDNA-PCR), and to perform antimicrobial susceptibility testing of these isolates using an agar dilution technique. A total of 16 isolates originating from 12 unrelated occasions were identified as Moraxella ovis , and tDNA fingerprinting showed clear delineation from other Moraxella species. The minimal inhibitory concentrations (in μg/mL) for 90% of the investigated isolates were ≤0.03 for ampicillin; 0.25 for ceftiofur; 0.5 for oxytetracycline; 8 for gentamicin; 64 for spectinomycin; 0.5/9.5 for the combination trimethoprim-sulfonamides; 4 for erythromycin; 8 for tilmicosin; 1 for florfenicol and 0.125 for enrofloxacin.

Moraxella bovis is a Gram-negative gammaproteobacterium and is one of the causative agents of infectious bovine keratoconjunctivitis. The structure of lipooligosaccharide (LOS) from strain Epp63 was recently elucidated. In the present study a genetic locus of seven encoding genes with high similarity to glycosyltransferases has been identified. Mutation of these putative glycosyltransferase genes resulted in M. bovis mutant bacteria that expressed truncated LOS structures. The structures of the oligosaccharide (OS) expressed by the mutant strains were elucidated and demonstrated the role of the glycosyltransferase enzymes in the LOS biosynthesis of M. bovis. The glycosyltransferase genes designated lgt1, lgt3, and lgt6 are highly similar to the genes in the related bacterium M. catarrhalis. In addition, there are syntenic similarities with the corresponding LOS biosynthesis locus in M. catarrhalis and other members of Moraxellaceae.

Copper (Cu) deficiency increases occurrence of certain infectious diseases in animals, including infectious keratoconjunctivitis in bovines, a bacterial ocular inflammation caused by Moraxella bovis. Neutrophil leukocytes constitute the first phagocytic cells to arrive at infection sites for bacterial neutralization. The objective of this work was to evaluate whether the functionality of neutrophils against M. bovis is impaired in experimentally induced Cu deficiency in bovines using high molybdenum and sulfur levels in the diet. The Cu tissue values and the periocular achromotrichia observed in +Mo animals showed that the clinic phase of Cu deficiency was reached in this group. Instead, +Cu animals have not evidenced clinical signs or biochemical parameters of hypocuprosis. On the basis of our observations, we concluded that Cu deficiency has no effect on phagocytic and bactericidal activities of neutrophils against M. bovis. However, superoxide dismutase activity and peroxide hydrogen generation were significantly different between groups. Therefore, additional research to explain these results is merited to fully characterize the consequences of Cu status on the risk for infections under field conditions.

The aim of this study was to determine the differences in the antimicrobial susceptibility profiles of Moraxella bovis, M. bovoculi and M. ovis. Thirty-two strains of Moraxella spp. isolated from cattle and sheep with infectious keratoconjunctivitis were tested via broth microdilution method to determine their susceptibility to ampicillin, cefoperazone, ceftiofur, cloxacillin, enrofloxacin, florfenicol, gentamicin, neomycin, oxytetracycline and penicillin. The results demonstrated that Moraxella spp. strains could be considered sensitive for most of the antimicrobials tested in this study, but differences between the antimicrobial susceptibility profiles of these three Moraxella species were found. M. bovis might differ from other species due to the higher MIC and MBC values it presented.

Copyright © 2015 Elsevier Inc. All rights reserved. As is the case for controlling other infectious livestock diseases, the most successful efforts to control infectious bovine keratoconjunctivitis (IBK) will include consideration of the host, the environment, herd management, and ongoing surveillance even after the immediate crisis has passed. Research over many years has led to the discovery of a variety of antibiotic treatments and antibiotic regimens that can be effective against IBK. The discoveries of Mor bovoculi and reports of IBK associated with Mycoplasma spp without concurrent Mor bovis or Mor bovoculi have raised new questions into the roles that other organisms may play in IBK pathogenesis.

The cytotoxin A (MbxA) is one of the main virulence factors of Moraxella bovis involved in the pathogenesis of infectious bovine keratoconjunctivitis (IBK). Moraxella ovis and Moraxella bovoculi, suspected to be associated with infectious keratitis in sheep and cattle respectively, also have a gene that encodes the cytotoxin A (movA and mbvA, respectively). The aim of this study was to determine the molecular sequence of the 3' region of the cytotoxin gene of Moraxella spp. strains isolated from clinical cases to establish phylogenetic and evolutionary comparisons. PCR amplification, nucleotide sequencing (nt) and amino acid (aa) sequence prediction were performed, followed by the sequences comparison, identity level calculation and selective pressure analysis. The phylogenetic reconstruction based on nt and aa sequences clearly differentiate M. bovis (n=15), M. bovoculi (n=11) and M. ovis (n=7) and their respective reference strains. An alignment of 843nt revealed high similarity within bacterial species (MbxA=99.9% nt and aa; MbvA=99.3% nt and 98.8% aa; MovA=99.5% nt and 99.3% aa). The similarity of partial sequences (nt 1807-2649) of MbxA in relation to MbvA and MovA ranged from 76.3 to 78.5%; similarity between MbvA and MovA ranged from 95.7 to 97.5%. A negative selection on mbvA and movA sequences was revealed by the molecular evolution analysis. The phylogenetic analysis of movA and mbvA allowed different strains of Moraxella spp. to be grouped according to the period of isolation. Sequence analysis of cytotoxin may provide insights into genetic and evolutionary relationships and into the genetic/molecular basis of Moraxella spp.

Dissertação de Mestrado Integrado em Medicina Veterinária A Queratoconjuntivite Infeciosa Bovina (QIB) é a patologia ocular mais comum em bovinos a nível mundial. O agente responsável por esta infeção, referenciada pela primeira vez em 1889, é uma bactéria coco Gram-negativa denominada Moraxella bovis (M. bovis) com características patogénicas específicas, o que suscita cada vez mais interesse no seu estudo tendo em vista o desenvolvimento de novos e melhores protocolos de tratamento e prevenção. A incidência desta patologia ocular é mais elevada no Verão e no Outono tendo sido, precisamente nesses dois períodos, observada em muitas explorações da região do Alto Alentejo durante um estágio de 4 meses de duração, no ano de 2014. O impacto da Queratoconjuntivite Infeciosa Bovina na produção leiteira e de carne é de grande importância económica, pois há uma diminuição do peso dos animais ao desmame, as vacas aleitantes produzem menos leite devido à dor provocada pela irritação ocular, aumentam os custos de tratamento médico-veterinário dos animais afetados e o seu valor no mercado diminui, pela desfiguração provocada pela infeção conjuntamente com cegueira, por vezes associada. O principal objetivo deste trabalho foi realizar uma revisão bibliográfica aprofundada sobre o tema e comparar com o observado no trabalho de campo durante o estágio, principalmente no que concerne ao diagnóstico e tratamento. Foram recolhidas amostras para o isolamento da bactéria tendo como objetivo a instituição de um tratamento eficaz. Os erros de maneio que propiciavam o aparecimento e desenvolvimento da bactéria foram alvo de particular análise. O resultado das amostras das secreções da conjuntiva ocular efetuadas na exploração com maior prevalência da doença, foi positivo para M. bovis. As principais falhas observadas no maneio dos animais foram a ausência de dispositivos de controlo de vetores da bactéria e erros relacionados com as infra-estruturas da exploração tendo em conta o efetivo. The infectious bovine keratoconjunctivitis (IBK) is the most common eye disease in cattle worldwide. The agent responsible for this infection, referenced in 1889 for the first time, is a Gram-negative coccobacilli bacterium called Moraxella bovis (M. bovis) with specific pathogenic features, which raises a special interest in its study with the objective of developing new and better protocols for treatment and prevention. The incidence of ocular pathology is higher in summer and in autumn and, precisely during these two periods, it was observed in many farms of the Alto Alentejo region during a 4-month-long externship in 2014. The impact of infectious bovine keratoconjunctivitis in milk and meat production is of great economic importance, as there is a decreased animals weight at weaning, the suckler cows produce less milk due to the pain caused by eye irritation, increased veterinary medical treatment costs of affected animals and a decreased market value of this animals due to the disfigurement caused by the infection and to the blindness sometimes associated. The main objective of this study was to conduct a review of the literature on the topic and compare the observed field work, especially regarding the diagnosis and the treatment. Samples were collected for bacteria isolation with the objective of establishing an effective treatment. The management errors that gave rise to the appearance and development of the bacteria were subject to particular analysis The results from the swabs collected from the ocular conjuntiva in the farm with the highest prevalence of this disease were positive for M. bovis. The main flaws observed in the animal handling were the absence of devices to control the bacteria vectors and errors related to the exploitation infrastructure, taking into account the animal population.

The complete nucleotide sequence of a plasmid, pMBO-1, from Moraxella bovis strain Epp63 was determined. We identified 30 open reading frames (ORFs) encoded by the 44,215 bp molecule. Two large ORFs, flpA and flpB, encoding proteins with similarity to Bordetella pertussis filamentous haemagglutinin (FHA), were identified on the same plasmid. The gene for a specific accessory protein (Fap), which may play a role in the secretion of Flp protein, was also identified. Reverse transcriptase PCR analysis of total RNA isolated from M. bovis Epp63 indicated that the flpA, flpB, and fap genes are all transcribed. Southern blot analysis indicated that the flp and fap genes are present in other clinical isolates of geographically diverse M. bovis.

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 1010 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.

To characterize flanking regions of thembxoperon inMoraxella bovis, DNA surroundingmbxCABDtolCwas sequenced in haemolytic and nonhaemolytic strains ofM. bovis. In two haemolytic strains ofM. bovis, thembxoperon, including the adjacentM. bovis tolCorthologue, was flanked by approximately 700 bp imperfect repeats. Nonhaemolytic strains ofM. bovishad only one or no such repeats, as well as ORFs identical to those flanking the repeats from haemolyticM. bovis. Two nonhaemolytic strains also contained ORFs with deduced amino acid sequence similarity to bacterialaraJgenes. The G+C content of thembxCABDtolCgene region was lower than the flanking regions. The genetic organization and G+C content ofmbxCABDtolCgenes, and flanking repeats in haemolyticM. bovis, as well as the presence or absence of flanking repeats in nonhaemolyticM. bovis, suggests that this RTX operon is located on a mobile genetic element, and supports the designation of this region as a pathogenicity island, which is believed to be the first such element demonstrated inM. bovis.

The bacterium Moraxella bovis is the causative agent of an economically important disease of cattle: Infectious Bovine Keratoconjunctivitis (IBK), otherwise known as pinkeye. Little is known regarding the structure of the carbohydrates produced by M. bovis. The structure of a capsular polysaccharide from M. bovis (strain Mb25) has been determined using NMR and MS analysis. From these data it is concluded that the polysaccharide is composed of the unmodified chondroitin disaccharide repeat unit.

Objective—To determine the immunogenicity of a Moraxella bovis cytolysin-enriched vaccine for prevention of infectious bovine keratoconjunctivitis (IBK). Animals—104 mixed-breed beef calves ranging between 4 and 8 months of age. Procedure—Vaccines were prepared by the diafiltration of broth culture supernatant from hemolytic M bovisor or sterile media. The diafiltered retentate was combined with Quil A adjuvant. Calves were randomly assigned to receive either the cytolysin vaccine (n = 35) or, as controls, adjuvant (35) or saline (0.9% NaCl) solution (34). Eyes of all calves were examined weekly for signs of IBK for 15 weeks. Calves that developed severe IBK were treated SC with florfenicol. Results—Cytolysin vaccine contained 4 proteins with molecular masses ranging between 65 and 90 kd. Cytolysin-vaccinated calves had fewer instances of IBK than control calves. The time of onset of corneal lesions in cytolysin-vaccinated calves that developed IBK was delayed, compared with that of calves in either control group. The cytolysin-Quil A vaccine contained endotoxin, but calves did not have clinical signs of illness after vaccination. Conclusions and Clinical Relevance—Calves that were vaccinated with a cytolysin-enriched vaccine had some resistance to IBK. Vaccines containing concentrated diafiltered M bovis cytolysin could protect beef calves against IBK. (Am J Vet Res 2005;66:136–142)

The efficacy of a recombinant Moraxella bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK) was evaluated in a randomized, blinded, controlled field trial. Ninety-three cross bred beef calves were vaccinated with either saline, ISCOM matrix (adjuvant control), or a recombinant M. bovis cytotoxin carboxy terminus peptide plus ISCOM matrix and boostered 21 days later. Ocular examinations were performed once weekly for 20 weeks. At week 12, the cumulative proportion of calves with ulcerated eyes in the recombinant vaccine group was significantly lower than in the saline control group. Throughout the 20 week trial, the cumulative proportion of ulcerated calves remained lowest in the recombinant vaccine group. By week 7, nonulcerated calves in the recombinant vaccine group had significantly higher changes in serum neutralizing titers and cytotoxin specific to total IgG ratios in serum and tears as compared to calves in the control groups. The trend for a reduced cumulative proportion of IBK in the vaccinated calves over the 20 week trial suggests that a recombinant M. bovis cytotoxin vaccine may be beneficial in helping to prevent naturally occurring IBK.

The molecular profile of 30 Moraxella bovis strains, recovered from outbreaks of infectious bovine keratoconjunctivitis in Argentina, Brazil and Uruguay between 1974 and 2001, was determined through randomly applied polymorphic DNA (RAPD) analysis. Molecular profiles of nine strains recovered after 1990 varied from those recovered before 1990. The profiles of 13 strains (48%) differed from those of three vaccinal strains extensively used since 1984 in Argentina and Uruguay. Eight Argentinean strains, one from Brazil and two from Uruguay had identical RAPD profiles. Strains belonging to different serogroups had identical RAPD profiles, demonstrating that this technique was not able to discriminate among strains with low cross-reactivity indices. RAPD may be helpful in the primary characterization of M. bovis strains, but it does not replace serological characterization.

Moraxella bovis expresses an iron-repressible 79-kDa outer-membrane protein, IrpA. DNA and N-terminal amino acid sequence analysis indicate that IrpA is closely related to FrpB of Neisseria meningitidis, FetA of Neisseria gonorrhoeae and CopB of Moraxella catarrhalis. The results of manganese mutagenesis and a gel-shift assay suggested that the transcription of irpA is negatively regulated by the ferric uptake regulator. The insertion of an antibiotic resistance cassette into the irpA gene affected the strain's ability to utilize bovine transferrin and lactoferrin. IrpA was detected in geographically diverse clinical isolates, and the antigenicity of IrpA was conserved in all the isolates tested. Therefore, IrpA may have potential as a candidate vaccine.

A homologue of the ferric uptake regulator gene (fur) was isolated from Moraxella bovis by degenerate polymerase chain reaction and cloning. Fur protein of M. bovis exhibited 72.1% amino acid identity with Acinetobacter calcoaceticus Fur. Western blot analysis showed a decrease of Fur expression in response to sufficient-iron conditions compared with deficient-iron conditions. An electrophoretic mobility-shift assay indicated that Fur protein binds to DNA fragments containing a putative Fur-box derived from the upstream region of the M. bovis fur gene. Fur of M. bovis may regulate the expression of iron transport systems in response to iron limitation in the environment.

Pathogenic isolates of Moraxella bovis express a calcium-dependent transmembrane pore forming cytotoxin that is an RTX toxin encoded by mbxA. The DNA flanking mbxA was cloned and sequenced to determine if M. bovis contained a classical RTX operon. Open reading frames (ORFs) with deduced amino acid sequence homology to putative activation (RTX C) and transport (RTX B and D) proteins were identified and have been designated MbxC, MbxB, and MbxD, respectively. Thus, hemolytic M. bovis contains a typical RTX operon comprised of four genes arranged (5'-3') mbxCABD. In addition, the deduced amino acid sequences of DNA flanking mbxCABD revealed ORFs with amino acid sequence similarity to transposases (5'). At the 3' end of the mbx gene cluster, an ORF with homology to bacterial tolC genes was identified. Thus, as with the cya RTX operon of Bordetella pertussis, M. bovis appears to have a secretion accessory protein linked to RTX genes. Analysis of genomic DNA isolated from 5 nonhemolytic M. bovis strains by PCR and Southern blotting revealed the absence of mbxCABD. These strains did, however, amplify with primers specific for the 5' region flanking mbxC. M. bovis harbors a classical RTX operon that is absent in nonhemolytic strains.

Moraxella bovis is the causative agent of infectious conjunctivitis in cattle. Moraxella bovis isolates were shown to specifically bind bovine lactoferrin (bLf) and bovine transferrin (bTf) and to use these proteins as a source of iron to support the growth of iron-limited cells. Affinity isolation experiments with immobilized bTf yielded two proteins readily resolved by SDS-PAGE analysis, whereas only a single band of approximately 100 kDa was detected when immobilized bLf was used as the affinity ligand. Using a novel cloning strategy, regions containing the genes encoding the lactoferrin (Lf) and transferrin (Tf) receptor proteins were isolated and sequenced, demonstrating that they both consisted of two genes, with the tbpB or lbpB gene preceding the tbpA or lbpA gene. The cloned lbp genes were used to generate isogenic mutants deficient in lactoferrin binding protein A and (or) B, and the resulting strains were tested in growth and binding assays. The isogenic mutants were deficient in their use of bLf for growth and had substantially diminished bLf binding capability. The predicted amino acid sequence from the segment encoding Lf binding protein B revealed an internal amino acid homology suggesting it is a bi-lobed protein, with a C-lobe enriched in acidic amino acids, but without the evident clustering observed in Lf-binding proteins from other species.Key words: outer membrane protein, iron-binding protein, lactoferrin, receptor, iron, transport, specificity.

Infectious bovine keratoconjunctivitis (IBK), also known as pinkeye, is the most costly eye disease of cattle. The principal etiologic agent of IBK is the Gram-negative bacterium Moraxella bovis. However, there have been reports of IBK outbreaks associated with Moraxella bovoculi. A retrospective study of IBK diagnostic cases submitted from July 1, 2010 through October 31, 2013 was conducted. Included in the study were 1,042 Moraxella isolates from 1,538 swabs of lacrimal secretions collected from 282 herds from 30 U.S. states. Moraxella isolates were identified to the species level and were composed of M. bovoculi (701 isolates), M. bovis (295 isolates), Moraxella ovis (5 isolates), and other Moraxella spp. (41). Minimum inhibitory concentrations required for 90% growth inhibition (MIC90) was calculated for representative isolates. The MIC90 values for both M. bovis and M. bovoculi were as follows: ampicillin and ceftiofur: ≤0.25 µg/ml; clindamycin: 2 µg/ml; danofloxacin and enrofloxacin: ≤0.12 µg/ml; florfenicol: 0.5 µg/ml; gentamicin: 1 µg/ml; neomycin: 4 µg/ml; tulathromycin: 2 µg/ml; and tylosin: 8 µg/ml. The MIC90 values for M. bovoculi included the following: chlortetracycline: ≤0.5 µg/ml; oxytetracycline: 4 µg/ml; penicillin: 0.25 µg/ml; spectinomycin: 32 µg/ml; sulfadimethoxine: >256 µg/ml; tiamulin: 1 µg/ml; and trimethoprim–sulfamethoxazole: 4 µg/ml. For M. bovis, MIC90 values included the following: chlortetracycline and oxytetracycline: 1 µg/ml; penicillin: ≤0.12 µg/ml; spectinomycin: 16 µg/ml; sulfadimethoxine: ≤256 µg/ml; tiamulin: ≤0.5 µg/ml; and trimethoprim–sulfamethoxazole: ≤2 µg/ml. The current work describes the frequency of isolation and differences in antimicrobial sensitivity observed among Moraxella isolates from case submissions.

The nature of association betweenDemodexmites and bacteria involved in bovine demodectic mange lesions and the normal flora inhabiting the skin of noninfected animals was investigated.Demodex bovisandD. ghanensismites were isolated from the infected purulent material extracted from skin and meibomian gland lesions, respectively. The mites could not be demonstrated in skin brushings or impression smears from the eyes of noninfected cattle. Pathogenic bacteria (Staphylococcus aureusandStreptococcus pyogenes(Group A)) and opportunistic organisms (Proteus vulgaris,Pseudomonas aeruginosa,Staphylococcus epidermidis, andTrueperella pyogenes) were isolated from skin lesions of demodectic mange, andMoraxella bovisandStaphylococcus aureuswere isolated from meibomian gland lesions.Bacillus subtilis,Escherichia coli,Proteus vulgaris,Staphylococcus aureus,Staphylococcus epidermidis, andStreptococcus pyogenes(Group A) were isolated from skin brushings from noninfected cattle. The nature of association betweenDemodexmites and bacteria in demodectic mange lesions is synergistic and of equal significance. Pathogenic and opportunistic bacteria facilitated the establishment ofDemodexmites in the lesions produced and provided an excellent microclimate for the mites to propagate and reproduce, resulting in severe and progressive disease. The “high-turnover” granulomatous reaction which characterized the histopathological changes proved thatDemodexmites and associated bacteria were persistent and immunogenic.

This chapter reviews studies on type 4 pili from the species Moraxella bovis, Moraxella lacunata, and Moraxella nonliquefaciens of the family Moraxellaceae and Eikenella corrodens, Kingella denitrificans, and Kingella kingae of the family Neisseriaceae. K. kingae was originally classified as Moraxella kingii and was then transferred to the new genus Kingella within the family Neisseriaceae in 1976. Further studies with M. nonliquefaciens, M. bovis, and M. kingii confirmed the association between the "spreading-corroding" (SC) colony type and piliation. Researchers studied thin sections of M. bovisin the electron microscope and concluded that the pili were distributed peritrichously around the cell. When M. bovis Epp63 was propagated on 5% sheep blood agar, one N-type and six distinct piliated colony types were observed. Thus, pellicle formation can be used as an enrichment procedure to isolate the often rare variants switching from the nonpiliated to the piliated state. The competence for DNA transformation in piliated E. corrodens has also been demonstrated. Further studies on the function and genetics of type 4 pili may lead to a better understanding of the roles that they play in so many bacterial species.

Moraxella bovis is a Gram-negative microorganism that causes Infectious Bovine Keratoconjunctivitis (IBK), colloquially known as 'Pink eye' in cattle worldwide. Lipopolysaccharides/lipooligosaccharides are the predominant glycans on the surface of Gram-negative microorganisms. Structural elucidation of the oligosaccharide structure of the rough phenotype of Moraxella bovis strain Epp63 was determined using GC-MS, methylation analysis, and NMR spectroscopy. The oligosaccharide is a branched structure that comprises 10 sugars in addition to KDO. The unusual features of this oligosaccharide include the fact that the oligosaccharide is devoid of heptose. The KDO residue is directly attached to a (→4,6)-branched glucose and additionally contains a terminal open chain acetal-linked N-acetylgalactosamine, (1S)-GalaNAc residue →4,6-linked to a sub-terminal galactose residue.

A candidate for a vaccine against infectious bovine keratoconjunctivitis (IBK) has been cloned and characterized from Moraxella bovis. The plb gene encodes a protein of 616 amino acids (molecular mass of ∼65.8 kDa) that expresses phospholipase B activity. Amino acid sequence analysis revealed that PLB is a new member of the GDSL (Gly-Asp-Ser-Leu) family of lipolytic enzymes.

Objective—To identify the Moraxella bovis cytotoxin gene. Procedure—Hemolytic and nonhemolytic strains of M bovis were compared by use of western blotting to identify proteins unique to hemolytic strains. Oligonucleotide primers, designed on the basis of amino acid sequences of 2 tryptic peptides derived from 1 such protein and conserved regions of the C and B genes from members of the repeats in the structural toxin (RTX) family of bacterial toxins, were used to amplify cytotoxin-specific genes from M bovis genomic DNA. Recombinant proteins were expressed, and antisera against these proteins were produced in rabbits. Results—Several proteins ranging in molecular mass from 55 to 75 kd were unique to the hemolytic strain. An open reading frame encoding a 927-amino acid protein with a predicted molecular mass of 98.8 kd was amplified from M bovis genomic DNA. The deduced amino acid sequence encoded by this open reading frame was homologous to RTX toxins. Antisera against the recombinant carboxy terminus encoded by this open reading frame neutralized hemolytic and cytolytic activities of native M bovis cytotoxin. Conclusions and Clinical Relevance—A gene was identified in M bovis that encodes a protein with sequence homology to other RTX toxins. Results of cytotoxin neutralization assays support the hypothesis that M bovis cytotoxin is encoded by this gene and belongs in the RTX family of bacterial exoproteins. Identification of this gene and expression of recombinant cytotoxin could facilitate the development of improved vaccines against infectious bovine keratoconjunctivitis. (Am J Vet Res 2001;62:1222–1228)

Swabs of conjunctiva were collected from 44 live and 226 hunter-harvested mule deer (Odocoileus hemionus) from Wyoming and Utah (USA). We identified 29 gram negative and 22 gram positive bacterial taxonomic categories, but many isolates from hunter-harvested animals were environmental contaminants. Staphylococcus spp. and Micrococcus spp. were the most common gram positive bacteria isolated, and Enterobacter spp., Escherichia coli, and Pseudomonas spp. were common gram negative bacteria isolated. Thelazia californiensis were found in 15% of hunter-harvested deer in Utah in 1994 and in 8% in 1995. Nematodes were found in 40% of live deer in 1995 and 66% in 1996. Three live animals showed clinical signs of infectious keratoconjunctivitis (IKC) in 1996, but pathogenic bacteria were not isolated from these individuals. Hemolytic, non-piliated Moraxella ovis was isolated from two clinically normal live deer in 1996 and isolates were similar to those cultured from IKC cases from Wyoming and Utah.

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 α2-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. (Am J Vet Res 2000;61:1145–1149)

Moraxella bovis is a gram-negative bacterium implicated in the pathogenesis of infectious bovine keratoconjunctivitis (IBK), a disease causing significant economic losses in cattle industries worldwide (10, 35, 37). Strains of M. bovis must be fimbriated and hemolytic in cell culture to cause clinical disease, suggesting that these characteristics are important virulence traits. Fimbriae mediate attachment to corneal epithelium (28), while toxin production may cause corneal epithelial cell damage (16, 34). The role of these virulence traits in disease implies the potential of these proteins as vaccine candidates. To date, most investigations have used fimbrial proteins in vaccine preparations, and both native and recombinant M. bovis fimbriae induce serogroup-specific protection (22, 23). However, there is increasing evidence that inclusion of fimbriae from all seven identified serogroups is necessary for protection, which may lead to antigenic competition (33). Therefore, the aim of this investigation was to characterize the hemolysin of M. bovis and assess its potential as an alternative vaccine candidate, as described for other bacterial hemolysins (30). There has been limited characterization of the hemolysin of M. bovis with previous investigations associating hemolytic activity in cell-free preparations to fractions with molecular mass greater than 300 kDa (6). This has been attributed to its release in membrane-bound vesicles and association with cell-wall proteins and has foiled efforts to obtain purified toxin (3). However, several similarities have been observed between the activity of M. bovis hemolysin and the activity of recently documented family of pore-forming bacterial toxins, the RTX exotoxins (1, 4, 11, 15). These activities include pore formation in target-cell membranes and a role for Ca2+ in the lytic activity of M. bovis hemolysin (11). Members of the RTX toxin family include the secreted toxins of Escherichia coli, Pasteurella hemolytica, Actinobacillus pleuropneumoniae, and Actinobacillus actinomycetemcomitans (36, 39). Speculation that the hemolysin of M. bovis may be related to this toxin family has been supported by preliminary findings that a monoclonal antibody (MAb) recognizing E. coli hemolysin also recognizes a 100-kDa protein in hemolytic M. bovis cultures (16). However, the inclusion of the hemolysin of M. bovis in this toxin family has not been confirmed (21). In addition to lytic action on erythrocytes, in vitro exposure of bovine corneal epithelial cells to culture supernatants of hemolytic M. bovis strains results in cell lysis (16, 19, 20). Although nonhemolytic M. bovis strains do not cause epithelial cell lysis, it is not known whether the loss of hemolytic phenotype alone is responsible for the observed loss of cytotoxicity. Consequently, a possible role for separate hemolytic and cytotoxic proteins in the pathogenesis of IBK has been debated (16, 19, 20). Here, we report the generation of a MAb which neutralizes both the hemolytic and epithelial cytotoxic potential of M. bovis culture supernatants. This MAb was used to identify the putative hemolysin in cell-free extracts of hemolytic M. bovis strains by Western blot analysis and to investigate a role for Ca2+ in lytic activity of the hemolysin of M. bovis following exposure to erythrocytes.

In the present study, isolation of Moraxella bovis was done from the microbiological examination of frozen semen straws from clinically healthy twelve Jersey and one Jersey-cross bull supplied by a semen laboratory. The organism was identified on the basis of colonial morphology and biochemical characteristics. Further, viable cell count of the bacterium was studied in detail in the semen sample.

Moraxella oris 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.

The economic impact of infectious bovine keratoconjunctivitis (IBK) warrants continued investigation of the mechanisms by which Moraxella bovis survives on and colonizes the corneal surface. Virulent strains of M bovis produce hemolysin and exhibit different plasmid profiles than nonvirulent strains. Interactions among host, environment, vector, season, and concurrent infection influence the prevalence of IBK. Mycoplasma sp. or infectious bovine rhinotracheitis virus may enhance or hasten the disease process. The manifestations of IBK may range from mild conjunctivitis to severe ulceration, corneal perforation, and blindness. Treatment of IBK is dictated by economic considerations, intended animal use, and feasibility of administration. Antibiotic therapy is aimed at achieving drug concentrations in tears to meet or exceed the minimum inhibitory concentration for prolonged periods. At present, IBK is not a preventable disease. Affected animals must be separated from the herd and vector control vigorously instituted. Carrier animals must be identified and removed from the herd. Vaccination trials have been unsuccessful because of pili antigen cross-reactivity, variable strains, and uncontrolled environmental factors. Recent investigations have determined that M bovis may utilize host iron sources via iron-repressible outer membrane proteins and siderophores for growth. Elucidation of normal defense mechanisms of the bovine eye may lead to new strategies to enhance the immune response against M bovis.

Pathogenic members of the family Neisseriaceae produce specific receptors facilitating iron acquisition from transferrin (Tf) and lactoferrin (Lf) of their mammalian host. Tf receptors are composed of two outer membrane proteins, Tf-binding proteins A and B (TbpA and TbpB; formerly designated Tbp1 and Tbp2, respectively). Although only a single Lf-binding protein, LbpA (formerly designated Lbp1), had previously been recognized, we recently identified additional bacterial Lf-binding proteins in the human pathogens Neisseria meningitidis and Moraxella catarrhalis and the bovine pathogen Moraxella bovis by a modified affinity isolation technique (R. A. Bonnah, R.-H. Yu, and A. B. Schryvers, Microb. Pathog. 19:285‐297, 1995). In this report, we characterize an open reading frame (ORF) located immediately upstream of the N. meningitidis B16B6 lbpA gene. Amino acid sequence comparisons of various TbpBs with the product of the translated DNA sequence from the upstream ORF suggests that the region encodes the Lf-binding protein B homolog (LbpB). The LbpB from strain B16B6 has two large stretches of negatively charged amino acids that are not present in the various transferrin receptor homologs (TbpBs). Expression of the recombinant LbpB protein as a fusion with maltose binding protein demonstrated functional Lf-binding activity. Studies with N. meningitidis isogenic mutants in which the lbpA gene and the ORF immediately upstream of lbpA (putative lbpB gene) were insertionally inactivated demonstrated that LbpA, but not LbpB, is essential for iron acquisition from Lf in vitro. Elemental iron is essential for the sustained growth of nearly all living organisms. When infecting their mammalian host, invading microbes are confronted with an environment with iron levels far too low to permit their proliferation. The iron stores of the host are primarily intracellular, and extracellular iron in serum and cerebrospinal fluid is bound by the glycoprotein transferrin (Tf), whereas the glycoprotein lactoferrin (Lf) serves to sequester iron on mucosal surfaces. To attain iron, many microbes produce and secrete siderophores, small molecules which are able to chelate both soluble and insoluble environmental iron. The resulting iron-siderophore complex is subsequently bound at the bacterial cell surface by a specific receptor and then transported to the periplasmic region of the cells, where it can be shuttled to the cytoplasm for storage and utilization (15, 32). Some bacteria do not produce siderophores but acquire iron by alternative mechanisms that are seemingly advantageous in their ecological niche. Pathogenic organisms such as the strict human pathogens Neisseria meningitidis (45, 47), Neisseria gonorrhoeae (28), Moraxella (Branhamella) catarrhalis (10‐12, 45), and Moraxella lacunata (11, 34) and the bovine pathogen Moraxella bovis (10) have been shown to possess host Tf- and Lf-specific receptors to circumvent the iron sequestration of the host (see reference 21 for a review of this mechanism of iron acquisition). Thus, capture of iron from these host ironbinding glycoproteins permits proliferation of these organisms in an otherwise iron-restricted environment. There are two outer membrane components of the receptormediated mechanism of iron acquisition: Tf-binding proteins A and B (TbpA and TbpB). Although only a TbpA functional homolog was described for the Lf receptor (LbpA) (37, 39), recent genetic (36, 38) and biochemical (10, 12) evidence implies that bacterial Lf receptors have a second constituent (LbpB) with attributes comparable to those of TbpB. The objective of this study was to examine the role of N. meningitidis LbpA in iron acquisition from Lf and characterize an open reading frame (ORF) located immediately upstream of the lbpA structural gene which has significant sequence homology to the B component of the bacterial Tf receptor (putative lbpB gene). For this purpose, we have prepared a series of isogenic mutants which lack either or both Lf receptor proteins. Given that the Tf receptor proteins characterized to date appear to have an operonic organization (21), we also address the possibility that LbpA and LbpB are cotranscribed and perform preliminary biochemical characterization of the proteins encoded by both ORFs.

Thirty-three strains previously classified into 11 species in the bacterial family Moraxellaceae were subjected to phylogenetic analysis based on 16S rRNA sequences. The family Moraxellaceae formed a distinct clade consisting of four phylogenetic groups as judged from branch lengths, bootstrap values and signature nucleotides. Group I contained the classical moraxellae and strains of the coccal moraxellae, previously known as Branhamella, with 16S rRNA similarity ofor = 95%. A further division of group I into five tentative clusters is discussed. Group II consisted of two strains representing Moraxella atlantae and Moraxella osloensis. These strains were only distantly related to each other (93.4%) and also to the other members of the Moraxellaceae (or = 93%). Therefore, reasons for reclassification of these species into separate and new genera are discussed. Group III harboured strains of the genus Psychrobacter and strain 752/52 of [Moraxella] phenylpyruvica. This strain of [M.] phenylpyruvica formed an early branch from the group III line of descent. Interestingly, a distant relationship was found between Psychrobacter phenylpyruvicus strain ATCC 23333T (formerly classified as [M.] phenylpyruvica) and [M.] phenylpyruvica strain 752/52, exhibiting less than 96% nucleotide similarity between their 16S rRNA sequences. The establishment of a new genus for [M.] phenylpyruvica strain 752/52 is therefore suggested. Group IV contained only two strains of the genus Acinetobacter. Strategies for the development of diagnostic probes and distinctive sequences for 16S rRNA-based species-specific assays within group I are suggested. Although these findings add to the classificatory placements within the Moraxellaceae, analysis of a more comprehensive selection of strains is still needed to obtain a complete classification system within this family.