Progress 08/01/10 to 07/31/12
Outputs
OUTPUTS: The goals of this project were to identify and develop potential molecular epidemiological markers and genetic tools for Moraxella bovoculi, an organism that is increasingly being recovered from cases of Infectious Bovine Keratoconjunctivitis in the US. Optimization of PCR assays has been performed that allow facile and robust amplification of 8 genetic targets of M. bovoculi (and several also for the related Moraxella ovis), using boiled lysates of colonies as template. Sequencing of amplicons has enabled multiple alleles for most target genes to be identified, which will form the basis of a multi-locus sequence typing scheme. In addition, a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) locus was identified and found to be strain variable in composition. As an alternate method to explore genome variation and compare discriminatory power of different techniques, effort was expended towards DNA profiling using Pulsed Field Gel Electrophoresis. No usable DNA fingerprints were obtained despite considerable efforts to optimize this technique including different isolates, restriction enzymes, cell densities and growth conditions. To generate a macrorestriction map of the Type strain genome, optical mapping with a single restriction enzyme was used (conducted by Opgen). This profile will facilitate subsequent comparative analysis of genomes of other strains. Having confirmed the circular configuration of two previously unrecognized plasmid-like sequences from M. bovoculi, potential shuttle vectors containing E.coli replicons as chimaeras with either M. bovoculi or M. bovis plasmids were constructed. These vectors were used in pilot efforts to genetically transform M. bovoculi. However, despite repeated attempts it has not been possible to demonstrate succesful transformation with plasmids by electroporation. Different growth conditions have been used and six strains of M. bovoculi have been tested. Preliminary attempts to perform mutagenesis by homologous recombination have also been succesful. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The sequence data of the MLST amplicons, the CRISPR analysis, plasmid sequences and optical map for M. bovoculi are all new pieces of knowlede to the field. The MLST data are likley to be of use for epidemiological studies and potentially will have use in vaccine studies as they could allow discrimination between isolates. The rather limited sequence variation in the putative pilin gene raises the possibility that there may be less antigenic variation between isolates than has been reported for M. bovis. The confirmation of the plasmid sequences may also allow the research community to develop vectors for manipulation of Moraxellae. As the cost of optical mapping continue to decrease, the availability of an optical map for the Type strain should allow maps to be compared to rapidly identify major regions of difference between the genomes of M. bovoculi isolates.
Publications
- No publications reported this period
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: During this project period, an expanded set of Moraxella bovoculi strains have been tested for presence of target genes for Multi-Locus Sequence Typing (MLST) analysis and an additional gene (encoding a putative phospholipase B) has been added to the target list. Several of these targets have also been amplified from the Type strain of Moraxella ovis, for use in planned phylogenetic analysis. Antimicrobial susceptibility testing has been performed for a panel of antimicrobial compounds on selected M. bovoculi strains. Genetic constructs for specific gene disruption of multiple genes have been generated and electroporation of this organism has been carried out. The presence of plasmids related to those present in the M. bovoculi Type strain has been assessed for multiple strains. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The ongoing MLST analysis has revealed additional alleles of the target genes. Further bioinformatic analysis has identified an additional candidate virulence factor, an autotransporter protein that is predicted to encode a phospholipase. This gene has been added to the MLST panel of target genes. Initial attempts to transform Moraxella bovoculi using mutagenic PCR constructs have not been successful. As a complementary approach, fragments of genes have been cloned into an Escherichia coli plasmid, to test whether these can be used to knock out genes by homologous recombination following transformation. Chimaeric replicons comprised of an E. coli replicon and a M. bovoculi plasmid have been generated and will be evaluated as shuttle vectors.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: During the first 6 months of this project, the main activity has focused on design/optimization of PCR and DNA Sequencing of 8 target genes for a Multi Locus Sequence Typing Scheme for Moraxella bovoculi. The initial findings are reported under "Outcomes" and have been presented by Neal Martin, a Veterinary Research Scholar Program student at the Merial Conference in Athens Georgia and at the Phi Zeta Research Day at the University of Missouri. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Successful amplification, DNA sequencing and bioinformatic analyses have identified multiple alleles of each of the 8 initially targeted genes for the MLST Typing scheme that is proposed for Moraxella bovoculi. From a pilot analysis of these genes from 7 field isolates and the Type strain, 6 different "Sequence Types" have been delineated based on the newly identified polymorphisms. In addition, the two putative plasmid sequences have been shown to indeed represent two hitherto unrecognized replicons. Although not initially proposed for study in the grant application, a CRISPR sequence (Clustered Regularly Interspaced Short Palindromic Repeats) was identified. As these regions are also considered useful molecular epidemiological tools, these regions are under analysis from additional field strains.
Publications
- No publications reported this period
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