Deborah L. Taylor

Experimental infection model for Johne's disease using a lyophilised, pure culture, seedstock of Mycobacterium avium subspecies paratuberculosis

Johnes disease is a severe chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map). Repeatable infections of known duration are required for validation of new diagnostic tests, evaluation of pathogenesis and development of improved vaccines. In the first study of its type, a standardised experimental model for Johnes disease was developed based on a lyophilised, low passage, pure culture, seedstock of Map. Experimental inoculations of sheep with accurately enumerated doses of Map resulted in infection outcomes across multiple trials that were modulated by the interval between inoculation and examination. Compared to an inoculum consisting of an intestinal mucosal homogenate from a naturally affected sheep, clinical signs from the pure culture of Map were manifested later, but other measures of infection were similar. Immunological assays showed that most of the inoculated animals were IFN-gamma positive in the early stages of the infection. Over time, an increasing number of sheep became Map-specific antibody positive, developed typical histopathological lesions and shed Map in their faeces. The repeatability and utility of this experimental infection model will enable study of many aspects of Johnes disease. It is the first study to show that models for Johnes disease can be standardised in relevant species using traditional microbiological approaches to production and storage of seedstock. It is recommended that an international bank of master seedstock be established, containing low passage isolates that are representative of the major strains of Map, S and C.

Toll-like receptor genes are differentially expressed at the sites of infection during the progression of Johne's disease in outbred sheep

Toll-like receptors (TLR) are engaged by ligands on microbial pathogens to initiate innate and adaptive immune responses. Little is known about TLR involvement during infection with Mycobacterium avium subsp. paratuberculosis (M. ptb), the cause of Johnes disease in ruminants, although there is a profound immunopathological response in affected animals. We have analyzed the expression of 10 TLR genes relative to validated reference genes at predilection sites in ileum, jejunum and associated lymph nodes as well as in peripheral blood, to determine if TLR expression is altered in response to infection with M. ptb in outbred sheep. Previously unexposed animals from two flocks and animals from three naturally infected flocks were used with restricted maximum likelihood linear mixed modeling applied to determine significant differences. These were related to the pathologies observed at different stages of infection in exposed sheep, after allowing for other sources of variation. In most cases there were differences in TLR expression between early paucibacillary and multibacillary groups when compared to uninfected sheep, with most TLRs for the paucibacillary group having lower expression levels than the multibacillary group. Increased expression of TLR1-5, and 8 was observed in ileum or jejunum, and TLR1-4, 6, and 8 in mesenteric lymph nodes. There was a trend for increased expression of TLR1, 2, and 6-8 in PBMCs of exposed compared to non-exposed animals. Further study of TLR expression in Johnes disease in ruminants is warranted as these observed differences may help explain pathogenesis and may be useful in the future diagnosis of M. ptb infection.

Growth pattern and partial proteome of Mycobacterium avium subsp. paratuberculosis during the stress response to hypoxia and nutrient starvation.

Mycobacterium avium subsp. paratuberculosis is an important pathogen that causes Johnes disease in animals and has been implicated in Crohns disease in man yet few data exist on its physiological adaptation in either the host or the environment. In this study, the proteomic responses of the two distinct strains of M. a. paratuberculosis, cattle (C) and sheep (S), to hypoxia and starvation were studied in vitro. Nutrient starvation inhibited growth of both strains and was lethal for S strain after 12 weeks. Hypoxia induced a state of very low metabolic activity but rapid resuscitation occurred upon restoration of an aerobic atmosphere, consistent with the dormancy response of other mycobacteria. A total of 55 protein spots differentially expressed in response to starvation and/or hypoxic stress in one or both strains were identified from 2D gels and classified based on biological function. Antioxidant enzymes, oxidoreducatse enzymes and proteins involved in amino acid metabolism, fatty acid metabolism, ATP and purine biosynthesis, proteolysis, cell wall synthesis, protein synthesis, signal recognition and hypothetical proteins with putative functions including dormancy response regulators and universal stress proteins were identified. These proteins are potential screening targets for future diagnosis, prevention and control of M. a. paratuberculosis infection and their identification will assist understanding the pathogenesis of diseases caused by this organism.

The early lymphocyte proliferation response in sheep exposed to Mycobacterium avium subsp. paratuberculosis compared to infection status.

Johnes disease (JD) is an enteric mycobacterial infection of ruminants that has significant global economic impact. Not all individuals exposed to pathogenic mycobacteria succumb to disease. Therefore although early detection of infectious individuals is vital, it is equally important to distinguish how the host response differs in those that are able to successfully clear or control mycobacterial infection and those that are unable to do so. To further our understanding of this issue we studied the antigen-specific proliferation of lymphocytes, including lymphocyte subsets, during the course of early experimental ovine paratuberculosis and assessed differences in this response between animals that eventually succumbed to disease and those that remained disease-free. While proliferation of blood lymphocytes was significantly higher in sheep exposed to Mptb compared to unchallenged controls as early as 4 months post inoculation (p.i.), there was no difference in PBMC proliferation between sheep that had been exposed to Mptb and became infected and those that were disease-free at the termination of the study. However, jejunal lymph node cells of Mptb-exposed infected animals had a significantly lower antigen-specific proliferative response compared to exposed uninfected animals. This may be useful when assessing JD status in animals of high commercial value. Multibacillary sheep had a lower proliferative response in peripheral blood compared to paucibacillary animals as early as 8 months p.i. and this was significantly different at 13 months p.i.

Enzyme-Linked Immunospot: An Alternative Method for the Detection of Interferon Gamma in Johne's Disease:

To date, the sensitivity of the interferon gamma (IFN-γ) enzyme-linked immunosorbent assay (ELISA) to detect Johnes disease (JD) has been poor, especially in the early stages of disease. To improve the sensitivity of IFN-γ detection in the early stages of infection, an alternate assay needs to be developed. The enzyme-linked immunospot (ELISPOT) assay is a highly sensitive technique for the detection of cytokines and has the potential to improve the diagnosis of JD. Of the variables examined, choice of capture antibody and the method by which the peripheral blood mononuclear cells were isolated significantly affected the ability to enumerate IFN-γ–secreting cells. The ELISPOT assay was as sensitive as or better than the IFN-γ ELISA at detecting ovine JD and could also detect disease at early time points postinoculation. The IFN-γ ELISPOT could distinguish infected from unexposed animals; however, neither the IFN-γ ELISA nor the ELISPOT assay could distinguish between sheep experimentally infected with Mycobacterium avium subspecies paratuberculosis and those exposed to the bacterium but diagnosed as uninfected at necropsy.

Evaluation of the immunogenicity of recombinant stress-associated proteins during Mycobacterium avium subsp. paratuberculosis infection: Implications for pathogenesis and diagnosis.

The aim of this study was to assess the immunogenicity of recombinant stress-associated proteins of Mycobacterium avium subsp. paratuberculosis in sheep infected with the organism compared to control sheep. Five proteins - MAP2411, ClpP, Ppa, MAP0593c and GreA - which were identified previously in in vitro stress or dormancy responses of M. paratuberculosis to hypoxia, nutrient starvation and heat, were cloned, expressed and purified as His-tag recombinant proteins from the pET-15b vector in a BL21(DE3)pLysS strain of E. coli. The immunogenicity of MAP2411 did not differ between infected and control sheep. However, the serological reactivity of the other recombinant antigens, and combinations of them, varied according to the histological stage of paratuberculosis. Interestingly, the sera from some animals with paucibacillary lesions, which were not immunoreactive in a commercial paratuberculosis ELISA that was based on non-defined native antigens, recognised the recombinant antigens. We infer from their differential immunogenicity in infected and control sheep that four of the stress-associated proteins were expressed by M. paratuberculosis in vivo. These data provide fundamental information on host-mycobacterial interactions and have conceptual implications for the development of future diagnostic tests for early immune responses in animals infected with mycobacteria.

Biomarker discovery for ovine paratuberculosis (Johne's disease) by proteomic serum profiling.

Paratuberculosis (Johnes disease) is a chronic granulomatous enteritis affecting ruminants and other species. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP). In this study, surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI TOF-MS) was used as a platform to identify candidate biomarkers from sheep serum. Multivariate biomarker models which aimed to differentiate sheep with paratuberculosis and vaccinated-exposed sheep from unexposed animals were proposed based on classification and regression tree (CART) and linear discriminant analysis (LDA) algorithms from two array types. The accuracy of classification of sheep into unexposed or exposed groups ranged from 75 to 100% among models. SELDI was used to monitor protein profile changes over time during an experimental infection trial by examining sera collected at 4-, 8- and 13-months post infection. Although three different SELDI instruments were used, nine consistent proteomic features were observed associated with exposure to MAP. Two of the putative serum biomarkers were purified from serum using chromatographic methods and were identified as transthyretin and alpha haemoglobin by tandem mass spectrometry. They belong to highly abundant, acute phase reactants in the serum proteome and have also been discovered as serum biomarkers in human inflammatory conditions and cancer. Their relationship to the pathogenesis of Johnes disease remains to be elucidated.

Proteomic profiling of ovine serum by SELDI-TOF MS: Optimisation, reproducibility and feasibility of biomarker discovery using routinely collected samples

The diagnosis of infectious diseases in animals may be enhanced by study of the serum proteome in which myriad components are influenced by physiological and pathological processes. Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) has the capacity to detect known and unknown immunologically relevant molecules in the serum proteome. Optimum combinations of ProteinChip array surfaces, energy absorbing molecules, sample dilutions and instrument settings were determined for spectral generation from whole ovine sera. The coefficient of variation for within and between chip mass/charge and peak intensity were <0.03% and <23%, respectively. There were minor alterations in spectra associated with storage of chips or machine drift. Clotting times of 30 min to 3h did not greatly alter protein spectra although storage of sera at -20 degrees C led to alterations. However, routinely collected serum samples stored at -20 degrees C were useful for identification of biomarkers associated with vaccination with a bacterial antigen. This information will inform future studies on serum proteome profiling in livestock, but independent assessments are recommended for each species.

Identification of differentially expressed genes in ileum, intestinal lymph node and peripheral blood mononuclear cells of sheep infected with Mycobacterium avium subsp. paratuberculosis using differential display polymerase chain reaction.

Discovery of differentially expressed genes aids in understanding molecular mechanisms underpinning normal and pathological states. When studying animals such as sheep where the entire genome has not been characterized, techniques that do not require knowledge of gene sequences are particularly advantageous. We used one such technique, differential display polymerase chain reaction (DD-PCR), to identify genes that had different degrees of expression in response to Mycobacterium avium subsp. paratuberculosis (M. ptb), the organism that causes Johnes disease in ruminants. Differentially expressed genes were validated by quantitative PCR using especially selected reference genes established in this study. Sheep (n=47) were classified according to history of exposure to M. ptb and infection status by histology and faecal and tissue culture. Differences in levels of gene expression were analyzed using restricted maximum likelihood (REML) in a linear mixed model. Five genes from the ileum and 17 genes from lymph node were differentially expressed in ovine Johnes disease. Expression of seven of these genes was also significantly different in peripheral blood mononuclear cells. Genes identified in association with M. ptb infection had a wide range of functions in pathways including: antigen presentation, signal transduction and cell differentiation, TLR signaling, immune cell activation and chemokine functions, granulomatous inflammation, Th1 suppression and apoptosis.

Corrigendum to “Experimental infection model for Johne's disease using a lyophilised, pure culture, seedstock of Mycobacterium avium subspecies paratuberculosis” [Veterinary Microbiology 141 (2010) 301-311]

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