Construction of a stable non-mucoid deletion mutant of the Streptococcus equi Pinnacle vaccine strain.

The research study focuses on modifying the existing Streptococcus equi Pinnacle vaccine strain, which is currently used to prevent equine strangles, to make it more identifiable and prevent the capsule’s production. The scientists were successful in creating a stable, mutation-identified mutation, which cannot revert to its original form and can be easily recognized from other strains.

Background

• Streptococcus equi is a bacterium that causes a disease called equine strangles, a purulent lymphadenopathy affecting a horse’s head and neck.
• In North America, an avirulent, non-encapsulated strain of this bacterium, known as Pinnacle, has been commonly used as an intranasal vaccine. This vaccine strain has been effective, but has the limitation of potentially reverting to a mucoid phenotype, which could make the disease recurring.
• The aim of this study was to create a specific and permanent mutation in the hasA gene of the Pinnacle strain. This mutation would abolish the production of a capsule, preventing the strain from returning to a harmful form, and also serve as an easily recognisable genetic marker.

Methodology

• The researchers created an internal fragment of the hasA gene using a technique called Polymerase Chain Reaction (PCR).
• This fragment was then integrated into a plasmid (a type of DNA molecule) known as pTW100.
• An encapsulated revertant of the Pinnacle strain was transformed with this recombinant plasmid using a process called electroporation, which involves using an electric field to increase the permeability of the cell membrane, allowing components such as DNA to enter the cell.
• The transformed bacteria were then cultured under specific conditions that promoted homologous recombination, a process where a segment of DNA is swapped with another segment having similar sequence, to incorporate the modified gene into the bacterial genome.

Results and Findings

• Among 90 observed spectinomycin resistant transformants, one non-mucoid (non-encapsulated) spectinomycin resistant colony was detected, indicating successful gene modification.
• The presence of the plasmid sequence within the hasA gene was confirmed by PCR, further validating the success of the modification.
• After growing the bacteria for six generations in a medium without antibiotics, four non-mucoid and spectinomycin-sensitive colonies were found. This display of sensitivity to antibiotics suggested that the plasmid had been lost as desired, and the resultant strain was now stable.
• Sequence analysis of one of these clones, named Pinnacle HasNeg, revealed the absence of parts of the hasA and hasB genes, confirming the presence of the desired gene deletion.
• Their results demonstrated the successful creation of a stable deletion mutant that could not revert back to a mucoid phenotype, providing a new improved vaccine strain with distinguishable genetic encoding.

Conclusion

• This deletion mutant may serve as a worthy replacement for the current Pinnacle vaccine strain. It offers an advantage as it does not have the potential to revert to a mucoid phenotype, thereby reducing the risk of disease recurrence post-vaccination.
• Additionally, this new strain is genetically distinguishable from wild type strains, simplifying identification and monitoring during implementation and future research.