Moraxella Bovis Research Articles

Evaluation of cytokines as adjuvants of infectious bovine keratoconjunctivitis vaccines

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.

Epidemiological investigation of bovine blindness syndrome in local and improved herds in Mkinga district of Tanzania

Aim: A demand-driven epidemiological field and laboratory investigation of a 'bovine blindness syndrome, BBS' was conducted in Mkinga district of Tanzania during the period of June through August, 2009. Materials and methods: Participatory epidemiological (PE) methods including focus group discussion (FGD), proportional piling and matrix scoring were used to characterize clinical signs, disease impact and risk factors for BBS as perceived by livestock keepers. PE investigation was complimented by microbiological culture of ocular discharges (n = 96 swab samples), clinical examination of animals and entomological investigation of the 'butterfly insects like' that were suspected by livestock keepers to be associated with BBS. Results: Matrix scoring showed moderate to good agreement between informant groups on the clinical signs, risk factors, disease impact and occurrence which coincides with rainy season. Clinical examination of cattle (n=177) from six villages revealed that 72 (40.7%) presented discharges from eyes, of which the majority had serous discharges (n=35; 48.6%) while muco-purulent and dried discharges were recorded in 19.2% and 32.2% of the animals, respectively. Of the 37 cattle that were found to be blind (based on obstacle test), most were affected in the right eye and the prevalence of blindness was significantly higher in indigenous than in cross-bred cattle (26.1% vs 5.4%, p=0.0027). Entomological examination of insect specimens identified the suspected 'butterfly insects' to be moths that belong to the species Arcyophora longivalvis, reported to serve as mechanical carriers for Moraxella bovis, a bacterium that causes Infectious Bovine Keratoconjuctivitis (IBK or Pinkeye) which among others, it causes blindness in cattle. M. bovis was isolated from 83.7% of 96 samples cultured. Conclusion: It is concluded that blindness in cattle in the area under study district was due to M. bovis infection. To the best of our knowledge, this is the first study in East Africa which has associated A. longivalvis moths with transmission of IBK. Appropriate disease prevention and control measures for pinkeye should be adopted in order to safeguard animal health in

A Randomized Clinical Trial Evaluating a Farm‐of‐Origin Autogenous Moraxella bovis Vaccine to Control Infectious Bovine Keratoconjunctivis (Pinkeye) in Beef Cattle

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. The null hypothesis was that treatment group was not associated with either risk of IBK or last observed weight. 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). Two s.c. 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". 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. Combination of the study results with previous studies suggests that autogenous M. bovis vaccines often are ineffective in controlling naturally occurring IBK.

Descriptive epidemiology of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in beef calves with naturally occurring infectious bovine keratoconjunctivitis (Pinkeye)

Problem addressedInfectious bovine keratoconjunctivitis (IBK) is an ocular disease that causes substantial weight loss in beef calves. ObjectiveThe objective of this study was to evaluate the association between Moraxella bovoculi, Moraxella bovis and Moraxella ovis and IBK incidence. Methods and approachA cohort design was used. From 239 calves and 478 eyes, 77 randomly chosen eyes were monitored for M. bovoculi, M. bovis, M. ovis and IBK incidence over 4 months. One hypothesis tested was that IBK hazard in eyes was not associated with detection of M. bovoculi, M. bovis and M. ovis. A secondary hypothesis tested that IBK cases were not associated with increased prevalence of M. bovoculi, M. bovis and M. ovis. Results23% of 77 eyes developed IBK. M. ovis was identified in one IBK-negative eye. The adjusted hazard ratio (HR) for IBK incidence from eyes where M. bovoculi or M. bovis were recovered prior to disease occurrence were not statistically significant (M. bovoculi HR=1.38, 95% CI: 0.54–3.53, p=0.49, M. bovis HR=1.60, 95% CI: 0.48–5.53, p=0.44). The adjusted hazard ratio for M. bovoculi in IBK lesions was 6.45 (95% CI: 3.35–12.44, p<0.001). The adjusted hazard ratio for M. bovis in IBK lesions was 2.33 (95% CI: 1.22–4.45, p=0.01). ConclusionA temporal association between prior exposure to M. bovoculi or M. bovis and subsequent IBK incidence was not demonstrated. However, M. bovoculi and M. bovis are more frequently recovered from eyes with IBK lesions than unaffected eyes and this provides weak evidence for a causal role.

Development and evaluation of a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in pure culture isolates and lacrimal swabs collected from conventionally raised cattle

To develop a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis (M. bovis), M. bovoculi and M. ovis. The multiplex real-time PCR assay was validated on three reference strains, 57 pure culture isolates and 45 lacrimal swab samples. All reference strains were identified correctly with no cross-reactions between species. Sequencing of 53 of the 57 culture isolates confirmed the results obtained with the multiplex real-time PCR, and the assay had 96·5% (55/57) concordance with a Moraxella spp. multiplex conventional PCR assay on the isolates. Among the lacrimal swab samples, the concordance between the multiplex real-time PCR and culture was 86·7% (39/45) for M. bovoculi and 75·6% (34/45) for M. bovis. The multiplex real-time PCR assay is specific and sensitive and can be used directly on lacrimal swab samples. The lack of a rapid, specific and sensitive detection method is a barrier for determining the roles of M. bovis, M. bovoculi and M. ovis in infectious bovine keratoconjunctivitis cases, and the developed PCR assay will contribute to improved understanding of the epidemiology and pathogenesis of these three Moraxella species.

Isolation and identification of conjunctival bacteria in cattle in Mosul

This study included examination of (120) eye swabs, from cows from different ages and regions in Mosul city. This study extended from September – December 2009. The samples were collected from clinically healthy and infected eyes of animals. 11 bacterial species were isolated they included Staphylococcus spp. 22.4%, Bacillus spp. 18.2%, Corynebacterium 17.6%, Streptococcus spp. 7%, Staphylococcus aureus 8.2%, Moraxella bovis 10.6%, E. coli 5.9%, Pseudomonas spp. 4.7%, Klebsiella spp. 2.4%, Micrococcus spp. 1.8%, Proteus spp. 1.2%. The isolated bacteria were 170, Gram positive bacteria were 128/170 while Gram negative bacteria 42/170 (24.7%). Many types of bacteria isolated from healthy swabs 110 (64.7%) and 60 (35.3%) from infected swabs.

Evaluation of anti-Moraxella bovis pili immunoglobulin-A in tears following intranasal vaccination of cattle

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.

Minimum inhibitory concentrations of selected antimicrobial agents for Moraxella bovoculi associated with infectious bovine keratoconjunctivitis

Infectious bovine keratoconjunctivitis (IBK) has been associated with ocular infections by Moraxella bovis, the established etiologic agent of IBK, and more recently, Moraxella bovoculi, a recently described species of Moraxella. To assist in designing rational treatment regimens for M. bovoculi infections associated with IBK, the in vitro susceptibilities of 57 M. bovoculi field isolates cultured from eyes of cattle with IBK in California from 2002 through 2007 were determined. The minimum inhibitory concentration required to inhibit the growth of 90% of organisms (MIC(90)) of the following 18 antibiotics tested in the present study were: danofloxacin and enrofloxacin: ≤0.12 µg/ml; ampicillin and ceftiofur: ≤0.25 µg/ml; penicillin: 0.25 µg/ml; gentamicin: ≤1 µg/ml; chlortetracycline, oxytetracycline, and tiamulin: 1 µg/ml; florfenicol: 0.5 µg/ml; trimethoprim-sulfamethoxazole: ≤2/38 µg/ml; clindamycin: 2 µg/ml; neomycin and tilmicosin: ≤4 µg/ml; tulathromycin: 4 µg/ml; spectinomycin and tylosin: 16 µg/ml; and sulfadimethoxine: >256 µg/ml. The low MIC(90) of these M. bovoculi isolates suggests that commonly used antibiotics for treatment of IBK associated with M. bovis should also be effective against M. bovoculi.

Surto de ceratoconjuntivite infecciosa bovina e hemoncose causando mortalidade em bezerros

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.

A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite

The measurement of elemental selenium (Se0) is needed to assess the rate and magnitude of bacteria reduction of selenite or selenate. We have developed a spectrophotometric method for the measurement Se0 that is rapid and can be employed to measure the quantity of Se0 produced by bacterial cultures. This method employs the use of 1M Na2S to convert the insoluble elemental selenium to a red-brown solution and with this method there is a direct correlation between concentration of elemental selenium and the absorption at 500nm. To demonstrate the utility of this assay, we have followed the reduction of selenite to Se0 by Moraxella bovis, and by bacterial consortia in soil and water samples.

Dynamics ofMoraxella bovisinfection and humoral immune response to bovine herpes virus type 1 during a natural outbreak of infectious bovine keratoconjunctivitis in beef calves

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.

The first report described as an important study: The association of mannose-binding lectin gene 2 polymorphisms in children with down syndrome

BACKGROUND:Mannose-binding lectin gene 2 (MBL2) plays a very important role in the first line of host immune response in Down syndrome (DS). The importance of MBL2 gene polymorphisms in children with DS is unclear, and no research has addressed MBL2 gene polymorphisms in patients with DS. This is the first report describing an important association between MBL2 gene polymorphisms and infections in children with DS.MATERIALS AND METHODS:We compared the frequency of single-nucleotide polymorphisms (SNPs) at two codons of the MBL2 gene in a cross sectional cohort of 166 children with DS and 229 controls. Polymorphisms at codons 54 (GGC→GAC) and 57 (GGA→GAA) in exon 1 of the MBL2 gene were typed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique using the restriction enzymes BshN1 (derivated from Bacillus sphaericus) and MboII (derivated from Moraxella bovis), respectively.RESULTS:MBL2 codon 54 GA genotype frequency was found to be lower in patients with DS (22.9%) than those of healthy controls (35.8%), differences were statistically significant (OR = 0.532, 95% CI = 0.339-0.836, P = 0.008). On the other hand, codon 57 polymorphism in the MBL2 gene was detected in none of the DS patients, but only one person in the control group showed codon 57 GA genotype (OR = 1.004, 95% CI = 0.996-1.013, P = 1.000).CONCLUSION:Our data provides an evidence for the first time that a homozygote or heterozygote for the variant, MBL2 alleles, is not associated with infections in patients with DS, and do not influence the incidence of infections.

Detection of bovine herpesvirus 1sequences in yaks (Bos grunniens) with keratoconjunctivitis, using a highly sensitive nested polymerase chain reaction

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.

Type‐IV pili spectroscopic markers: Applications in the quantification of piliation levels inMoraxella boviscells by a FT‐IR ANN‐based model

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.

Unbalanced restriction impairs SOS-induced DNA repair effects

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.

USO INTRAPALPEBRAL DE FLORFENICOL NO TRATAMENTO DA CERATOCONJUNTIVITE INFECCIOSA BOVINA

The primary etiologic agent of infectious keratoconjunctivitis (IBK) is Moraxella bovis, which causes the majority of ocular inflammations in cattle from central Brazil. It may lead to significant production losses and laborious treatment protocols. Therefore, this study assesses an alternative procedure for treating infectious keratoconjunctivitis that consists of a single intrapalpebral application of florfenicol (600 mg in each affected eye). Our results show both clinical and economic advantages of this technique as well as its prospects for wide use.

Vaccine against infectious bovine keratoconjunctivitis: A new approach to optimize the production of highly piliated Moraxella bovis cells

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.

Isolation of Moraxella bovis from frozen bovine semen and determination of microbial load.

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.

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., α-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.

Moraxella bovis pathogenicity: An update

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