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Rural Industries Research & Development Corporation
Published on the web in August 2003
RIRDC Publication No 03/096 RIRDC Project No IMV-2A
General Background
Rhodococcus equi has
long been considered a pathogen in horses principally in foals less than
6 months old (particularly 1-3 months old). This organism causes a pyogranulomatous
pneumonia, often accompanied by extra-pulmonary manifestations. Infections
are often fatal if untreated. R.
equi is known to also cause infections in cattle, pigs and goats. It is also known to cause severe pulmonary and disseminated disease in immuno-compromised humans, particularly AIDS patients.
In Australia most equine R. equi infections occur in summer (December to February) when the age of the foals (most foals born the previous year would be under 6 months old) and warm, dry environmental conditions make the animals more susceptible to infection.
An effective vaccine against R. equi is a long-term aim of the project. Vaccines that have been employed in the past have been based upon whole R. equi. These often result in unsightly ulceration of the animal, which may reduce its value. The DNA and protein vaccines we propose to develop only use components of the bacterium that produce antibody that protects against subsequent infection. DNA vaccines using GroEL protein have been shown effective in larger animals. The demonstration in a mouse model of the effectiveness of this approach would justify testing of these vaccines in a controlled study with horses
Further refinement of
the diagnostic test for R. equi infection
Linear epitopes of the R.
equi virulence-associated protein (VapA) were mapped using a synthetic
peptide bank in this study. The peptides were screened in an ELISA with
a total of 70 sera from foals with current R. equi disease (51 sera),
as well as from foals that had either recovered from R. equi infection
ten months previously (3 sera) or had no known history of R. equi disease
(16 sera). An epitope with sequence NLQKDEPNGRA was identified and was
universally recognised by all 51 sera from foals with R. equi disease
and was not recognised by any of the other sera. There was poor reactivity
between all sera and peptides relating to other areas of the VapA protein.
It is proposed that an ELISA based upon a defined peptide epitope, may
be used in an improved serological diagnostic test for R. equi infection
in foals.
Cloning and characterisation
of the R. equi groEL gene
The R. equi gene
encoding the heat shock protein GroEL was cloned and sequenced. The promoter
region of the gene contained two copies of Controlling Inverted Repeat
of Chaperone Expression (CIRCE) motifs, which are well- recognised regulators
of bacterial heat shock proteins. The deduced amino acid sequence of GroEL2
was 90% identical to published GroEL2 sequences of organisms such as Mycobacteria
spp. The R. equi GroEL2 was expressed in E. coli and
its identity to the predicted R. equi GroEL protein sequence was
confirmed.
Development of groEL
and vapA based DNA vaccines against R. equi
There is currently no widely
available commercial vaccine for the prevention of R. equi–induced
disease in foals. In this study, a groEL and vapA based DNA
vaccines were developed and tested in the mouse model. Mice immunised with
the vaccine showed a strong DTH response and high R.
equi GroEL specific IgG2a and a low IgG1 response. Indicating a relatively strong Th1 cell mediated immune response, in contrast to a vaccine based purified GroEL protein. The VapA based DNA vaccine gave a weaker Th1 response than the GroEL based DNA vaccine. The purified VapA protein yielded a stronger Th1 response than its corresponding DNA vaccine. Challenge of immunised mice with R. equi indicated that while the GroEL vaccine elicits a significant cellmediated immune response it did not enhance R. equi clearance in the mouse model. It is also still unclear whether antibodies against VapA are protective, and indeed whether this protein is a bonafide virulence factor.
Discussion
Although all aims were carried
out and in the main part were successful, particularly the improvement
of the ELISA test and the vaccine construction. It was, nevertheless, disappointing
that despite evidence of a relatively strong Th1 immune response that is
postulated to be necessary to afford protection against R. equi infection
no enhanced clearance of bacteria was observed in the mouse model. This
result may be explained by the inherent deficiencies of the mouse model
of infection, ie delivery of very large numbers of organisms via the intravenous
route. It bears little resemblance to the probable route of infection in
horses, which is the respiratory route via aerosol.
The very nature of aerosol inoculation results in the deposition of much smaller numbers of organisms in the lungs. It may be that the vaccines developed in this study may produce a sufficiently high immune response to protect against an aerosol challenge since the intravenous route delivers up to a thousand fold more bacteria that subsequently overwhelms the immune response.
Implications
An accurate serological
test to test for early R. equi infection has been further developed.
A number of promising vaccine candidates have been constructed, however,
it is likely that the murine model of R. equi infection may not
accurately predict the immune response in horses and has serious deficiencies
in the measurement of vaccine efficacy.
Recommendations
That an improved non-equine
model of R. equi infection by the aerosol route be researched and
developed The vaccines developed in this study be further refined and tested
in an improved non-equine model as a prelude for field testing in horses.
