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American Journal of Veterinary Research Sep 2016OBJECTIVE To determine the efficacy of Bdellovibrio bacteriovorus 109J for the treatment of calves with experimentally induced infectious bovine keratoconjunctivitis...
OBJECTIVE To determine the efficacy of Bdellovibrio bacteriovorus 109J for the treatment of calves with experimentally induced infectious bovine keratoconjunctivitis (IBK). ANIMALS 12 healthy dairy calves. PROCEDURES For each calf, a grid keratotomy was performed on both eyes immediately before inoculation with Moraxella bovis hemolytic strain Epp63-300 (n = 11 calves) or nonhemolytic strain 12040577 (1 calf). For each calf inoculated with M bovis Epp63-300, the eyes were randomly assigned to receive an artificial tear solution with (treatment group) or without (control group) lyophilized B bacteriovorus 109J. Six doses of the assigned treatment (0.2 mL/eye, topically, q 48 h) were administered to each eye. On nontreatment days, eyes were assessed and corneal swab specimens and tear samples were collected for bacterial culture. Calves were euthanized 12 days after M bovis inoculation. The eyes were harvested for gross and histologic evaluation and bacterial culture. RESULTS The calf inoculated with M bovis 12040577 did not develop corneal ulcers. Of the 22 eyes inoculated with M bovis Epp63-300, 18 developed corneal ulcers consistent with IBK within 48 hours after inoculation; 4 of those eyes developed secondary corneal ulcers that were not consistent with IBK. Corneal ulcer size and severity and the time required for ulcer healing did not differ between the treatment and control groups. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that B bacteriovorus 109J was not effective for the treatment of IBK; however, the experimental model used produced lesions that did not completely mimic naturally occurring IBK.
Topics: Animals; Bdellovibrio bacteriovorus; Cattle; Cattle Diseases; Conjunctivitis, Bacterial; Cornea; Keratoconjunctivitis; Keratoconjunctivitis, Infectious; Male; Moraxella bovis; Moraxellaceae Infections; Vaccination
PubMed: 27580114
DOI: 10.2460/ajvr.77.9.1017 -
Frontiers in Cellular and Infection... 2021is the bacterium most often cultured from ocular lesions of cattle with infectious bovine keratoconjunctivitis, also known as bovine pinkeye. Some strains of contain...
is the bacterium most often cultured from ocular lesions of cattle with infectious bovine keratoconjunctivitis, also known as bovine pinkeye. Some strains of contain operons encoding for a repeats-in-toxin (RTX) toxin, which is a known virulence factor of multiple veterinary pathogens. We explored the utility of MALDI-TOF MS and biomarker detection models to classify the presence or absence of an RTX phenotype in . Ninety strains that had undergone whole genome sequencing were classified by the presence or absence of complete RTX operons and confirmed with a visual assessment of hemolysis on blood agar. Strains were grown on Tryptic Soy Agar (TSA) with 5% sheep blood, TSA with 5% bovine blood that was supplemented with 10% fetal bovine serum, 10 mmol/LCaCl, or both. The formulations were designed to determine the influence of growth media on toxin production or activity, as calcium ions are required for toxin secretion and activity. Mass spectra were obtained for strains grown on each agar formulation and biomarker models were developed using ClinProTools 3.0 software. The most accurate model was developed using spectra from strains grown on TSA with 5% bovine blood and supplemented with CaCl, which had a sensitivity and specificity of 93.3% and 73.3%, respectively, regarding RTX phenotype classification. The same biomarker model algorithm developed from strains grown on TSA with 5% sheep blood had a substantially lower sensitivity and specificity of 68.0% and 52.0%, respectively. Our results indicate that MALDI-TOF MS biomarker models can accurately classify strains of regarding the presence or absence of RTX toxin operons and that agar media modifications improve the accuracy of these models.
Topics: Agar; Animals; Biomarkers; Calcium Chloride; Cattle; Cattle Diseases; Keratoconjunctivitis, Infectious; Moraxella; Phenotype; Sheep; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 33796479
DOI: 10.3389/fcimb.2021.632647 -
Antimicrobial Agents and Chemotherapy Dec 2015Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti) but not against M. bovis and M. avium. The...
Esters of Pyrazinoic Acid Are Active against Pyrazinamide-Resistant Strains of Mycobacterium tuberculosis and Other Naturally Resistant Mycobacteria In Vitro and Ex Vivo within Macrophages.
Pyrazinamide (PZA) is active against major Mycobacterium tuberculosis species (M. tuberculosis, M. africanum, and M. microti) but not against M. bovis and M. avium. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257-263, 2009, http://dx.doi.org/10.1016/j.ejps.2009.02.012). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy in vitro and ex vivo against several species and strains of Mycobacterium with natural or acquired resistance to PZA, including M. bovis and M. avium. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activity per se, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development.
Topics: Alcohols; Antitubercular Agents; Cell Line; Cell Survival; Drug Resistance, Bacterial; Esters; Humans; Macrophages; Microbial Sensitivity Tests; Moraxella bovis; Mycobacterium; Mycobacterium avium Complex; Mycobacterium tuberculosis; Prodrugs; Pyrazinamide
PubMed: 26438493
DOI: 10.1128/AAC.00936-15 -
PLoS Biology Jun 2024CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new...
CRISPR-Cas12a, often regarded as a precise genome editor, still requires improvements in specificity. In this study, we used a GFP-activation assay to screen 14 new Cas12a nucleases for mammalian genome editing, successfully identifying 9 active ones. Notably, these Cas12a nucleases prefer pyrimidine-rich PAMs. Among these nucleases, we extensively characterized Mb4Cas12a obtained from Moraxella bovis CCUG 2133, which recognizes a YYN PAM (Y = C or T). Our biochemical analysis demonstrates that Mb4Cas12a can cleave double-strand DNA across a wide temperature range. To improve specificity, we constructed a SWISS-MODEL of Mb4Cas12a based on the FnCas12a crystal structure and identified 8 amino acids potentially forming hydrogen bonds at the target DNA-crRNA interface. By replacing these amino acids with alanine to disrupt the hydrogen bond, we tested the influence of each mutation on Mb4Cas12a specificity. Interestingly, the F370A mutation improved specificity with minimal influence on activity. Further study showed that Mb4Cas12a-F370A is capable of discriminating single-nucleotide polymorphisms. These new Cas12a orthologs and high-fidelity variants hold substantial promise for therapeutic applications.
Topics: Gene Editing; CRISPR-Cas Systems; CRISPR-Associated Proteins; Alleles; Humans; Endodeoxyribonucleases; Animals; Protein Engineering; Bacterial Proteins; Polymorphism, Single Nucleotide; Mutation; DNA; HEK293 Cells
PubMed: 38865309
DOI: 10.1371/journal.pbio.3002680 -
PNAS Nexus Apr 2024Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly...
Mammalian hosts combat bacterial infections through the production of defensive cationic antimicrobial peptides (CAPs). These immune factors are capable of directly killing bacterial invaders; however, many pathogens have evolved resistance evasion mechanisms such as cell surface modification, CAP sequestration, degradation, or efflux. We have discovered that several pathogenic and commensal proteobacteria, including the urgent human threat , secrete a protein (lactoferrin-binding protein B, LbpB) that contains a low-complexity anionic domain capable of inhibiting the antimicrobial activity of host CAPs. This study focuses on a cattle pathogen, , that expresses the largest anionic domain of the LbpB homologs. We used an exhaustive biophysical approach employing circular dichroism, biolayer interferometry, cross-linking mass spectrometry, microscopy, size-exclusion chromatography with multi-angle light scattering coupled to small-angle X-ray scattering (SEC-MALS-SAXS), and NMR to understand the mechanisms of LbpB-mediated protection against CAPs. We found that the anionic domain of this LbpB displays an α-helical secondary structure but lacks a rigid tertiary fold. The addition of antimicrobial peptides derived from lactoferrin (i.e. lactoferricin) to the anionic domain of LbpB or full-length LbpB results in the formation of phase-separated droplets of LbpB together with the antimicrobial peptides. The droplets displayed a low rate of diffusion, suggesting that CAPs become trapped inside and are no longer able to kill bacteria. Our data suggest that pathogens, like , leverage anionic intrinsically disordered domains for the broad recognition and neutralization of antimicrobials via the formation of biomolecular condensates.
PubMed: 38633880
DOI: 10.1093/pnasnexus/pgae139 -
Journal of Dairy Science Oct 2016Kelp (Ascophyllum nodosum) is rich in iodine and often fed by organic dairy producers as a mineral supplement to support animal health. A commonly held belief is that...
Kelp (Ascophyllum nodosum) is rich in iodine and often fed by organic dairy producers as a mineral supplement to support animal health. A commonly held belief is that kelp supplementation decreases susceptibility to infectious bovine keratoconjunctivitis due to increased iodine concentrations in tears. Whereas serum and milk iodine concentrations are positively correlated and modulated by oral iodine supplementation, nothing is known about the iodine concentration of tears. Therefore, the 3 objectives of this pilot study were to determine (1) the iodine content of tears, milk, and serum of cows after being fed kelp for 30d; (2) the trace mineral and thyroid status of cows before (d 0) and after being fed kelp for 30d; and (3) the in vitro growth rate of bacteria in tears (Moraxella bovis) or milk (Staphylococcus aureus, Escherichia coli, Streptococcus uberis) collected from cows fed no kelp (d 0) or kelp (d 30). Cows (n=3/treatment) were individually fed 56g of kelp per day (n=3/treatment) or not (n=3/no treatment) for 30 d. Daily feed intake of the TMR was recorded and weekly TMR, kelp, milk, blood and tear samples were collected and analyzed for iodine. The feed samples were pooled and further analyzed for other minerals. On d 0 and 30, liver biopsies and blood samples were collected and analyzed for mineral content and thyroid hormone concentrations, respectively. An inhibition test used milk and tear-soaked plates from kelp-fed cows (d 0 and 30) as well as 1 and 7.5% iodine as positive and distilled water as negative control. As expected, serum iodine concentrations were positively correlated with milk and tear iodine concentrations. Whereas the iodine concentrations in serum increased significantly in the kelp-fed cows during the 30-d study, milk and tear iodine concentrations increased only numerically in these cows compared with the control group. Liver mineral profiles were comparable between groups and generally did not change over the course of the study. Thyroid hormones remained overall within the reference range throughout the trial. Neither milk nor tears from kelp-fed cows inhibited in vitro growth of any of the plated bacteria. In summary, serum iodine concentration was correlated with the iodine concentration in milk and tears and feeding kelp increased only the serum iodine levels of cows in this trial. Bacterial growth was not inhibited in milk and tears of kelp-fed cattle in vitro, and prevention of infectious bovine keratoconjunctivitis would not be based solely on increased iodine concentrations in tears.
Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Ascophyllum; Cattle; Diet; Escherichia coli; Female; Iodine; Milk; Moraxella bovis; Pilot Projects; Staphylococcus aureus; Streptococcus; Tears
PubMed: 27448858
DOI: 10.3168/jds.2015-10810 -
Nucleic Acids Research Apr 2015DNA-dependent T7 RNA polymerase (T7 RNAP) is the most powerful tool for both gene expression and in vitro transcription. By using a Next Generation Sequencing (NGS)...
DNA-dependent T7 RNA polymerase (T7 RNAP) is the most powerful tool for both gene expression and in vitro transcription. By using a Next Generation Sequencing (NGS) approach we have analyzed the polymorphism of a T7 RNAP-generated mRNA pool of the mboIIM2 gene. We find that the enzyme displays a relatively high level of template-dependent transcriptional infidelity. The nucleotide misincorporations and multiple insertions in A/T-rich tracts of homopolymers in mRNA (0.20 and 0.089%, respectively) cause epigenetic effects with significant impact on gene expression that is disproportionally high to their frequency of appearance. The sequence-dependent rescue of single and even double InDel frameshifting mutants and wild-type phenotype recovery is observed as a result. As a consequence, a heterogeneous pool of functional and non-functional proteins of almost the same molecular mass is produced where the proteins are indistinguishable from each other upon ordinary analysis. We suggest that transcriptional infidelity as a general feature of the most effective RNAPs may serve to repair and/or modify a protein function, thus increasing the repertoire of phenotypic variants, which in turn has a high evolutionary potential.
Topics: Amino Acids; DNA Repair; DNA-Directed RNA Polymerases; Epigenesis, Genetic; Frameshifting, Ribosomal; INDEL Mutation; Methyltransferases; Moraxella bovis; Phenotype; RNA Editing; RNA, Messenger; Viral Proteins
PubMed: 25824942
DOI: 10.1093/nar/gkv269 -
Acta Crystallographica. Section F,... Oct 2014Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the genera Neisseria...
Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the genera Neisseria and Moraxella have evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogen Neisseria meningitidis is reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogen Moraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin.
Topics: Animals; Bacterial Proteins; Carrier Proteins; Cattle; Crystallography, X-Ray; Host-Pathogen Interactions; Humans; Hydrogen Bonding; Iron; Lactoferrin; Models, Molecular; Neisseria meningitidis; Protein Binding; Protein Interaction Domains and Motifs; Protein Structure, Quaternary; Protein Structure, Secondary
PubMed: 25286931
DOI: 10.1107/S2053230X14019372 -
Microbiology Resource Announcements Aug 2018We report here the complete closed genome sequence of Moraxella bovis strain Epp-63 (300) (Epp63). This strain was isolated from an infectious bovine...
We report here the complete closed genome sequence of Moraxella bovis strain Epp-63 (300) (Epp63). This strain was isolated from an infectious bovine keratoconjunctivitis (IBK) case in 1963. Since then, Epp63 has been used extensively for IBK research. Consequently, the genome sequence of Epp63 should help elucidate IBK host-pathogen interactions.
PubMed: 30533917
DOI: 10.1128/MRA.01004-18