RESUMO
Infectious bovine keratoconjunctivitis (IBK) is an ocular disease that affects bovines and has significant economic and health effects worldwide. Gram negative bacteria Moraxella bovis and Moraxella bovoculi are its main etiological agents. Antimicrobial therapy against IBK is often difficult in beef and dairy herds and, although vaccines are commercially available, their efficacy is variable and dependent on local strains. The aim of this study was to analyze for the first time the genomes of Uruguayan clinical isolates of M. bovis and M. bovoculi. The genomes were de novo assembled and annotated; the genetic basis of fimbrial synthesis was analyzed and virulence factors were identified. A 94% coverage in the reference genomes of both species, and more than 80% similarity to the reference genomes were observed. The mechanism of fimbrial phase variation in M. bovis was detected, and the tfpQ orientation of these genes confirmed, in an inversion region of approximately 2.18kb. No phase variation was determined in the fimbrial gene of M. bovoculi. When virulence factors were compared between strains, it was observed that fimbrial genes have 36.2% sequence similarity. In contrast, the TonB-dependent lactoferrin/transferrin receptor exhibited the highest percentage of amino acid similarity (97.7%) between strains, followed by cytotoxins MbxA/MbvA and the ferric uptake regulator. The role of these virulence factors in the pathogenesis of IBK and their potential as vaccine components should be explored.
Assuntos
Doenças dos Bovinos , Genoma Bacteriano , Ceratoconjuntivite Infecciosa , Moraxella bovis , Moraxella , Animais , Moraxella/genética , Moraxella/isolamento & purificação , Bovinos , Moraxella bovis/genética , Ceratoconjuntivite Infecciosa/microbiologia , Doenças dos Bovinos/microbiologia , Infecções por Moraxellaceae/microbiologia , Infecções por Moraxellaceae/veterinária , Uruguai , Fatores de Virulência/genéticaRESUMO
AIMS: This study aimed to verify the formation of biofilms by Moraxella bovis, Moraxella ovis and Moraxella bovoculi isolates from ruminants. In addition, the lysozyme activity against the isolates of M. bovis, M. ovis and M. bovoculi in free form and in biofilms was determined. METHODS AND RESULTS: In this study, 54 isolates of Moraxella sp. obtained from bovine and ovine clinical samples were evaluated in vitro for capacity of biofilm formation and lysozyme susceptibility in planktonic and sessile cells. In addition, biofilms produced by four Moraxella sp. isolates were visualized under scanning electron microscope (SEM). It was possible to demonstrate, for the first time, the ability to form biofilms by M. ovis and M. bovoculi. The isolates of Moraxella sp. have the capacity to form biofilms in different intensities, varying among weak, moderate and strong. It was verified that the lysozyme shows activity on Moraxella sp. in planktonic form. However, on biofilms there was a reduction in the production, but without impairing its formation, and on consolidated biofilms the lysozyme did not have the capacity to eradicate the preformed biofilms. CONCLUSIONS: This work shows the capacity of biofilm formation by Moraxella sp. of veterinary importance. The lysozyme susceptibility of Moraxella sp. in planktonic form shows that this enzyme has bacteriostatic activity on this micro-organism and it reduced the production of biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the results, it is possible to infer that the biofilm formation capacity by Moraxella sp. and the resistance to lysozyme concentrations equal to or greater than the physiological levels of the ruminant tear may be linked not only to the capacity to colonize the conjunctiva, but also to remain in this place even after healing of the lesions, being a reservoir of Moraxella sp. in a herd.
Assuntos
Biofilmes , Moraxella bovis/fisiologia , Moraxella/fisiologia , Muramidase/metabolismo , Animais , Bovinos , Doenças dos Bovinos , Ceratoconjuntivite Infecciosa , Moraxella/isolamento & purificação , Infecções por Moraxellaceae , Ovinos/microbiologiaRESUMO
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.
Assuntos
Antibacterianos/farmacologia , Cobre/deficiência , Cobre/metabolismo , Peróxido de Hidrogênio/farmacologia , Moraxella bovis/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Animais , Antibacterianos/análise , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Peróxido de Hidrogênio/análise , Neutrófilos/microbiologia , Fagocitose/efeitos dos fármacosRESUMO
The difficulties in preventing and treating infectious bovine keratoconjunctivitis (IBK) and the consequent impact on the cattle industry worldwide emphasize the need to better understand this infectious process along with the biology of Moraxella bovis, its primary causative agent. Although there is increasing evidence that bacterial biofilms participate in a variety of ocular infections by direct biofilm formation on the surfaces of the eye, IBK has not been considered as a biofilm-based disease so far, and even more, no information is currently available regarding the ability of M. bovis to adopt a biofilm lifestyle. In the present research, we demonstrated the capacity of M. bovis clinical isolates and reference strains to form biofilms on different abiotic surfaces and culture conditions, and provided qualitative and quantitative information on the biofilm growth and architecture of mature biofilms. In addition, our data indicated that the type IV pili play a critical role in the biofilm formation in vitro. Most significantly, we proved that through exposure to MgCl2 type IV pili are removed from the cell surface, not only preventing M. bovis biofilm formation but also disassembling preformed biofilms. These results could constitute a new approach in the understanding of M. bovis colonization process in cattle eye and/or nasal cavity, and may aid in the development of future antimicrobial strategies for the control of IBK.
Assuntos
Biofilmes , Doenças dos Bovinos/microbiologia , Ceratoconjuntivite Infecciosa/microbiologia , Moraxella bovis/fisiologia , Infecções por Moraxellaceae/veterinária , Animais , Bovinos , Moraxella bovis/isolamento & purificação , Infecções por Moraxellaceae/microbiologiaRESUMO
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.
Assuntos
Doenças dos Bovinos/microbiologia , DNA Bacteriano/genética , Ceratoconjuntivite Infecciosa/microbiologia , Moraxella/genética , Infecções por Moraxellaceae/veterinária , RNA Ribossômico 16S/genética , Animais , Bovinos , Moraxella/classificação , Moraxella/isolamento & purificação , Moraxella bovis/classificação , Moraxella bovis/genética , Moraxella bovis/isolamento & purificação , Infecções por Moraxellaceae/microbiologia , Filogenia , Análise de Sequência de DNA/veterinária , Especificidade da Espécie , UruguaiRESUMO
In this work two cytokines were used in combination with inactivated bacteria (bacterin) to test the bovine conjuctival immune response to the pathogen Moraxella bovis, the causative agent of Infectious bovine keratoconjunctivitis (IBK). Treatments using the bacterin vaccine combined with interleukin-2 and interferon-α as adjuvants (Group A), the bacterin vaccine only (Group B), and controls without treatment (Group C), were applied by ocular spraying to evaluate the local immune response in the corneal structure of cattle experimentally infected with M. bovis. Six weeks after infection, 14 out of a total of 34 animals presented different corneal lesions; 9 corresponding to the control group C, 4 to the group B and only one to the group A. According to the clinical manifestations, a numeric score was calculated. Control animals presented the highest score value (12 points), followed by group B (7.5 points) and group A (1 point). These results suggest that the addition of cytokines to M. bovis treatments can reduce not only eye injuries caused by IBK but also the number of diseased animals.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacinas Bacterianas/uso terapêutico , Doenças dos Bovinos/prevenção & controle , Citocinas/uso terapêutico , Ceratoconjuntivite Infecciosa/prevenção & controle , Moraxella bovis , Infecções por Moraxellaceae/veterinária , Animais , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Interferon-alfa/uso terapêutico , Interleucina-2/uso terapêutico , Ceratoconjuntivite Infecciosa/imunologia , Ceratoconjuntivite Infecciosa/microbiologia , Moraxella bovis/imunologia , Infecções por Moraxellaceae/imunologia , Infecções por Moraxellaceae/prevenção & controleRESUMO
Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease of cattle caused by Moraxella bovis (Mb). Parenterally administered immunogens used to prevent the disease do not offer complete protection possibly because they stimulate a poor ocular mucosal secretory response, in which locally secreted immunoglobulin-A (sIgA) is one of the main components. The principal aim of this study was to evaluate by an indirect enzyme linked immunosorbent assay (ELISA), the local ocular mucosal sIgA response against Mb purified pili, produced after intranasal inoculation of experimental vaccines. Pili were adjuvanted by several different adjuvants (QuilA, Marcol Arlacel, Marcol Span, microencapsulated pili with PLGA polymers). Results were compared to sIgA response produced by adjuvant placebo inoculations and by IBK natural infection. Significantly higher anti-pili IgA response (p<0.05) was detected in calves vaccinated intranasally with pili QuilA and pili Marcol Span compared to control calves, although this specific immune response did not seem to be related to protection against Mb infection or typical IBK lesion development.
Assuntos
Anticorpos Antibacterianos/análise , Vacinas Bacterianas/imunologia , Fímbrias Bacterianas/imunologia , Imunoglobulina A/análise , Moraxella bovis/imunologia , Infecções por Moraxellaceae/veterinária , Lágrimas/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/uso terapêutico , Bovinos , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática/veterinária , Imunoglobulina A/imunologia , Infecções por Moraxellaceae/imunologia , Infecções por Moraxellaceae/prevenção & controle , Vacinação/métodos , Vacinação/veterináriaRESUMO
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.
Assuntos
Surtos de Doenças/veterinária , Herpesvirus Bovino 1/imunologia , Ceratoconjuntivite Infecciosa/microbiologia , Moraxella bovis , Infecções por Moraxellaceae/veterinária , Animais , Anticorpos Antivirais/sangue , Bovinos , Imunidade Humoral , Ceratoconjuntivite Infecciosa/complicações , Ceratoconjuntivite Infecciosa/imunologia , Ceratoconjuntivite Infecciosa/patologia , Infecções por Moraxellaceae/imunologia , Infecções por Moraxellaceae/virologiaRESUMO
Type-IV pili are cell surface organelles found in a wide variety of Gram-negative bacteria. They have traditionally been detected by electron microscopy and ELISA techniques. However, these methodologies are not appropriate for the rapid discrimination and quantification of piliated and nonpiliated cells in industrial or field conditions. Here, the analysis of FT-IR spectra of piliated, nonpiliated and sheared Moraxella bovis cells, together with purified pili suspensions spectra, allowed the identification of 3 IR regions associated to spectroscopic markers of Type-IV pili: 1750-1600, 1450-1350 and 1280-950 cm(-1). Such IR-specific markers were found for piliated cells grown in different culture systems (liquid or solid media), independently of the strain or pili serotype. They were also sensitive to pili expression levels. Therefore, on the bases of these specific spectral features, an FT-IR ANN-based model was developed to classify piliation levels in 5 distinct groups. An overall classification rate of almost 90% demonstrates the strong potential of the ANN system developed to monitor M. bovis cultures in vaccine production.
Assuntos
Algoritmos , Técnicas de Tipagem Bacteriana/métodos , Fímbrias Bacterianas/classificação , Moraxella bovis/classificação , Moraxella bovis/ultraestrutura , Redes Neurais de Computação , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Biomarcadores/análise , Fímbrias Bacterianas/ultraestrutura , Reconhecimento Automatizado de Padrão/métodosRESUMO
Pili are the principal antigens and virulence factors of Moraxella bovis, the etiological agent of infectious bovine keratoconjunctivitis (IBK). Although it has been reported that the low efficacy of whole cell vaccines against IBK is mainly due to the difficulties in keeping the cellular piliation level of M. bovis during the growth of bacteria in stirred bioreactors, the problem has not yet been overcome because the mechanisms involved in the loss of piliation are still not fully clarified. In this work we found that during the culture of M. bovis in liquid media, around 15% of the cells changed from piliated to non-piliated phenotypes at the end of the growth. Nevertheless, we demonstrated that the main cause of cellular piliation loss in M. bovis growing in stirred and/or sparged bioreactors is due to shear forces, which are a function of the volumetric gassed power drawn (P(g)V(-1)). Therefore, we tested here the use of bubble column bioreactors to protect M. bovis cell-bound pili from mechanical agitation damage effects. These bioreactors operated at a superficial air velocity of 0.0065 m s(-1) yielded a cellular piliation level of 25%, in contrast to 1% obtained for stirred bioreactors. The addition of carboxymethylcellulose (CMC) at 0.10% (w v(-1)) to culture medium proved to be suitable to improve the final piliation level (65%). We demonstrated by FT-IR spectroscopy and ELISA technique, that this chemical additive has a pili protective role interacting with the cells but without affecting pili antigenic properties.