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by M.W. Heuzenroeder, M. D. Barton. T. Vanniasinkam and T. Phumoonna
RIRDC Publication No W05/58 RIRDC Project No IMV-4A
Executive Summary
I Background
R. equi has long
been considered a pathogen in horses principally in foals fewer than 6
months old (particularly 1-3 months old). Infections are often fatal if
untreated. Accurate figures on the incidence of R. equi infection
and fatalities due to infection are hard to find, but it is considered
a major problem for the industry.
Many vaccines have been developed over the years for the prevention of R. equi infection but none has been particularly effective. In the previous studies we determined the DNA sequence of the groEL gene from Rhodococcus equi. The vapA gene and its product have been proven to be essential for the virulence of R. equi.
In this project, a DNA vaccine will be developed using the groEL and vapA gene products as the vaccine candidates. However, the current model of R. equi infection and challenge in mice uses an intravenous route of administration, this model does not mimic the respiratory route of infection that occurs in horse. There is a great need to develop an improved non- equine model of infection using the respiratory route, for vaccine testing prior to moving to horses.
II Specific aims of the project
III Results
Two chimeric groEL2/vapA
vaccine constructs were prepared by the insertion of the immunogenic epitope
NLQKDEPNGRA of VapA into a hydrophilic region of GroEL2
protein. One construct pcDNA3-Re3 was a DNA vaccine and pIMVS-Re4
was a protein vaccine. Both vaccine constructs were shown to produce products
by in vitro tests. This task was successfully completed and satisfies the
first aim.
We then tested the immunogenicity of both vaccines in a Balb/c mouse model employed in previous studies.
The DNA vaccine was shown to produce a cell mediated Th1 immune response and judged by antibody subclassing. This type of immune response is predicted to be necessary to protect against R. equi infection.
Looking for enhanced clearance from the liver and spleen in vaccinated animals after intravenous challenge by virulent R. equi tested efficacy of the vaccines. The vaccines did not enhance clearance of the organism in comparison to control animals. This result was postulated to be a result of the large challenge dose and the route of infection. This task can be judged as successfully completed as the DNA vaccine produced a Th1 biased immune response. The result of the efficacy testing emphasises the need for an animal model that more closely mimics natural R. equi infection in horses.
Both guinea pigs and mice were investigated for aerosol inoculation of R. equi. The guinea pig was specifically chosen because of their sensitivity to respiratory pathogens. Disease in guinea pigs with lesions in the lung similar to those produced by R. equi could be produced by aerosol inoculation, although the animals did not exhibit any obvious symptoms of disease. The DNA vaccine was also shown to be immunogenic in the guinea pig. However a major problem emerged. This was underlying mild pneumonia in the guinea pig that confounded pathological examination of lung tissue. Sourcing of animals from other suppliers did not overcome this problem. The focus of the study then moved to mice where two strains were subjected to aerosol inoculation of R. equi. One strain C57B appeared to be resistant to R. equi infection. A second strain, C3H, could be infected to produce lesions in the lung similar to that ascribed to R. equi. Time did not allow further testing. The preliminary results of this study with C3H mice are encouraging and suggest that with more work this system could be suitable for vaccine testing.
IV Conclusions
A need for a non-equine
model of R. equi infection is still needed. However further work
with larger numbers of animals and differing R. equi dosing strategies
is required to fully validate the C3H mouse model and the efficacy of the
vaccines developed in this study.
