Abstract
BackgroundEffective diagnosis of Johne's disease (JD), particularly at the stage of early subclinical infection, remains one of the greatest challenges for the control of JD worldwide. The IFN-γ test of cell mediated immunity is currently one of the most suitable diagnostics for subclinical infections, however a major limitation of this test is the lack of a standardised purified protein derivative (PPD) antigen (also referred to as Johnin PPD or PPDj). While attempting to replace PPDj with more specific individual antigens is an attractive proposition, bacterial culture derived PPDj remains the most effective antigen preparation for the diagnosis of subclinical JD. It may be possible to increase the reproducibility and specificity of PPDj preparations by further characterising and standardising the PPDj production.ResultsUsing a standardised protocol, five in-house preparations of PPDj were prepared from cultures of Mycobacterium avium subsp. paratuberculosis (MAP). Compared to PPDs obtained from other institutes/laboratories, these preparations appeared to perform similarly well in the IFN-γ test. Although the broad proteomic composition of all PPDj preparations was remarkably similar, the absolute abundance of individual proteins varied markedly between preparations. All PPDj preparations contained common immunogenic proteins which were also observed in PPD preparations from Mycobacterium avium subsp. avium (PPDa) and Mycobacterium bovis (PPDb). Temporal difference in protein secretion of in vitro cultured MAP was observed between 20 and 34 weeks suggesting that the age of MAP culture used for PPDj preparations may markedly influence PPDj composition.ConclusionsThis study describes a protocol for the production of PPDj and its subsequent proteomic characterisation. The broad proteomic composition of different preparations of PPDj was, for the most part, highly similar. Compositional differences between PPDj preparations were found to be a direct reflection of genetic differences between the MAP strain types used to produce these preparations and the age of MAP cultures they were derived from. A number of conserved immunogenic proteins, such as members of the cutinase-like protein family, were found to be more abundant in PPDj compared to PPDa and should be considered as possible diagnostic antigens for the future.
Highlights
Effective diagnosis of Johne’s disease (JD), at the stage of early subclinical infection, remains one of the greatest challenges for the control of JD worldwide
The ability of the in-house produced AAHL1101 PPDj to generate an interferon gamma (IFN-g) response was demonstrated across 13 Holstein-Friesian cattle and 13 Merino sheep experimentally infected with Mycobacterium avium subsp. paratuberculosis (MAP) (Figure 1A and 1B)
The IFN-g response of infected cattle against AAHL1101 PPDj was not significantly different compared to the CSL or CAN6 PPDj preparations (p > 0.05)
Summary
Effective diagnosis of Johne’s disease (JD), at the stage of early subclinical infection, remains one of the greatest challenges for the control of JD worldwide. A range of diagnostic tests, varying in their sensitivity, specificity and suitability have been trialled for the diagnosis of MAP infections [5,6,7,8,9]. Serological-based tests such as ELISA [9], gel immuno-diffusion [7,10] and complement fixation [5] tests have proven successful for the diagnosis of the clinical stages of disease when a robust antibody response has developed. The early and subclinical stages of infection were thought to be dominated by cell-mediated immunity (CMI) rather than circulating antibodies [11]. The CMI response against MAP is characterised by a Th1-type cellular response leading to the secretion of a number of cytokines, including interferon gamma (IFN-g) [11]
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