Kumudika de Silva

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.

Candidate gene and genome-wide association studies of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep: A review

Paratuberculosis (Johnes disease), caused by Mycobacterium avium subspecies paratuberculosis, is responsible for significant economic losses in livestock industries worldwide. This organism is also of public health concern due to an unconfirmed link to Crohns disease. Susceptibility to paratuberculosis has been suggested to have a genetic component. In livestock, a number of candidate genes have been studied, selected on their association to susceptibility in other mycobacterial diseases, their known role in disease pathogenesis or links to susceptibility of humans to Crohns disease. These genes include solute carrier family 11 member 1 (SLC11A1, formerly NRAMP1), toll-like receptors, caspase associated recruitment domain 15 (CARD15, formerly NOD2), major histocompatibility complex (MHC) and cytokines (interleukin-10 and interferon-gamma) and their receptors. Genome wide association studies have attempted to confirm associations found and identify new genes involved in pathogenesis and susceptibility. There are a number of limitations and difficulties in these approaches, some peculiar to paratuberculosis but others generally applicable to identification of genetic associations for complex traits. The technical approaches and available information for paratuberculosis have expanded rapidly, particularly relating to sheep and cattle. Here we review the current published evidence for a genetic association with paratuberculosis susceptibility, technological advances that have progressed the field and potential avenues for future research.

High-Throughput Direct Fecal PCR Assay for Detection of Mycobacterium avium subsp. paratuberculosis in Sheep and Cattle

ABSTRACT Johnes disease (JD) is a chronic enteric disease caused by Mycobacterium avium subsp. paratuberculosis that affects ruminants. Transmission occurs by the fecal-oral route. A commonly used antemortem diagnostic test for the detection of M. avium subsp. paratuberculosis in feces is liquid culture; however, a major constraint is the 2- to 3-month incubation period needed for this method. Rapid methods for the detection of M. avium subsp. paratuberculosis based on PCR have been reported, but comprehensive validation data are lacking. We describe here a new test, the high-throughput-Johnes (HT-J), to detect M. avium subsp. paratuberculosis in feces. Its diagnostic accuracy was compared with that of liquid radiometric (Bactec) fecal culture using samples from cattle (1,330 samples from 23 herds) and sheep (596 samples from 16 flocks). The multistage protocol involves the recovery of M. avium subsp. paratuberculosis cells from a fecal suspension, cell rupture by bead beating, extraction of DNA using magnetic beads, and IS900 quantitative PCR. The limit of detection of the assay was 0.0005 pg, and the limit of quantification was 0.005 pg M. avium subsp. paratuberculosis genomic DNA. Only M. avium subsp. paratuberculosis was detected from a panel of 51 mycobacterial isolates, including 10 with IS900-like sequences. Of the 549 culture-negative fecal samples from unexposed herds and flocks, 99% were negative in the HT-J test, while 60% of the bovine- and 84% of the ovine-culture-positive samples were positive in the HT-J test. As similar total numbers of samples from M. avium subsp. paratuberculosis-exposed animals were positive in culture and HT-J tests in both species, and as the results of a McNemars test were not significant, these methods probably have similar sensitivities, but the true diagnostic sensitivities of these tests are unknown. These validation data meet the consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (S. A. Bustin et al., Clin. Chem. 55:611–622, 2009, doi:10.1373/clinchem.2008.112797). The HT-J assay has been approved for use in JD control programs in Australia and New Zealand.

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.

Can early host responses to mycobacterial infection predict eventual disease outcomes

Diagnostic tests used for Johnes disease in sheep either have poor sensitivity and specificity or only detect disease in later stages of infection. Predicting which of the infected sheep are likely to become infectious later in life is currently not feasible and continues to be a major hindrance in disease control. We conducted this longitudinal study to investigate if a suite of diagnostic tests conducted in Mycobacterium avium subspecies paratuberculosis (MAP) exposed lambs at 4 months post infection can accurately predict their clinical status at 12 months post infection. We tracked cellular and humoral responses and quantity of MAP shedding for up to 12 months post challenge in 20 controls and 37 exposed sheep. Infection was defined at necropsy by tissue culture and disease spectrum by lesion type. Data were analysed using univariable and multivariable logistic regression models and a subset of variables from the earliest period post inoculation (4 months) was selected for predicting disease outcomes later on (12 months). Sensitivity and specificity of tests and their combinations in series and parallel were determined. Early elevation in faecal MAP DNA quantity and a lower interferon gamma (IFNγ) response were significantly associated with sheep becoming infectious as well as progressing to severe disease. Conversely, early low faecal MAP DNA and higher interleukin-10 responses were significantly associated with an exposed animal developing protective immunity. Combination of early elevated faecal MAP DNA or lower IFNγ response had the highest sensitivity (75%) and specificity (81%) for identifying sheep that would become infectious. Collectively, these results highlight the potential for combined test interpretation to aid in the early prediction of sheep susceptibility to MAP infection.

Expression of genes associated with the antigen presentation and processing pathway are consistently regulated in early Mycobacterium avium subsp. paratuberculosis infection

The primary objective of this study was to evaluate early gene expression profiles associated with paratuberculosis in cattle exposed to known infectious doses of Mycobacterium avium sub-species paratuberculosis (MAP). A Johnes disease experimental infection field trial was conducted on a mixed population of Holstein and Holstein Red cattle. Blood samples from four MAP exposed and four unexposed cattle, selected based on IFNγ expressions were taken at 9, 13 and 21 weeks and RNA processed to Affymetrix GeneChip™ Bovine Genome arrays. Ontological analysis revealed consistent differences in gene expression between MAP exposed and control animals. A stark variation was observed in expression of a number of genes along antigen presentation pathways, suggesting that MAP exposure potentially results in the host immune response switching to a CD8(+) biased antigen presentation profile. This requires further in-depth analysis since it exposes a hitherto unconfirmed association between MAP exposure and in vivo MHC gene modulation.

The interleukin 10 response in ovine Johne's disease.

Johnes disease is an enteric mycobacterial infection of ruminants that has significant global economic impact. The classic host reaction is one of an early T-cell mediate immune response, with predominant interferon gamma (IFNγ) activity; there is subsequent lowering of this response as animals reach the terminal stages of disease. Interleukin (IL)-10, which can suppress Th1-type and enhance Th2-type cytokine production, is considered to play a role in the later stages of Johnes disease. To determine the role of IL-10 throughout the course of Johnes disease we studied groups of sheep with either no Johnes disease (n=10), natural infection (n=30) or experimental infection (n=58). Disease status of the animals was comprehensively assessed by culture of Mycobacterium avium subsp. paratuberculosis (Mptb), histopathology and serology. Antigen-specific IL-10 secretion in peripheral blood of sheep exposed to Mptb was significantly higher than in control animals (P<0.001) as early as 4 months post-inoculation, and increased progressively. In ileal and jejunal lymph node cells, IL-10 secretion was also significantly higher in animals that were exposed to Mptb compared to controls (P<0.05). The early IL-10 response seen in peripheral blood cells may be a reflection of early responses at sites of Mptb infection. IL-10 secretion from ileal and jejunal lymph node cells was significantly higher in exposed animals with no lesions or with paucibacillary lesions when compared to animals with multibacillary lesions. These novel findings demonstrate that increased IL-10 activity commences soon after exposure to the causative mycobacterium and may play a role in determining disease outcome.

Indoleamine 2,3-Dioxygenase, Tryptophan Catabolism, and Mycobacterium avium subsp. paratuberculosis: a Model for Chronic Mycobacterial Infections†

ABSTRACT Virulent mycobacterial infections progress slowly, with a latent period that leads to clinical disease in a proportion of cases. Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen that causes paratuberculosis or Johnes disease (JD), a chronic intestinal disease of ruminants. Indoleamine 2,3-dioxygenase (IDO), an enzyme that regulates tryptophan metabolism, was originally reported to have a role in intracellular pathogen killing and has since been shown to have an important immunoregulatory role in chronic immune diseases. Here we demonstrate an association between increased IDO levels and progression to clinical mycobacterial disease in a natural host, characterizing gene expression, protein localization, and functional effects. IDO mRNA levels were significantly increased in M. avium subsp. paratuberculosis-infected monocytic cells. Levels of both IDO gene and protein expression were significantly upregulated within the affected tissues of sheep with JD, particularly at the site of primary infection, the ileum, of animals with severe multibacillary disease. Lesion severity was correlated with the level of IDO gene expression. IDO gene expression was also increased in the peripheral blood cells of M. avium subsp. paratuberculosis-exposed sheep and cattle. IDO breaks down tryptophan, and systemic increases were functional, as shown by decreased plasma tryptophan levels, which correlated with the onset of clinical signs, a stage well known to be associated with Th1 immunosuppression. IDO may be involved in downregulating immune responses to M. avium subsp. paratuberculosis and other virulent mycobacteria, which may be an example of the pathogen harnessing host immunoregulatory pathways to aid survival. These findings raise new questions about the host-mycobacterium interactions in the progression from latent to clinical disease.

Toll-like receptor (TLR)6 and TLR1 differentiation in gene expression studies of Johne's disease

Johnes disease (JD) is a mycobacterial infection of the gut affecting ruminants and other species caused by Mycobacterium avium subspecies paratuberculosis (MAP). The role of toll-like receptors (TLR) in the pathogenesis of JD has been previously identified at the level of gene expression. Gene expression studies using reverse transcriptase (RT)-PCR are widely used and powerful, but the results obtained from such studies are dependent on the specificity of the assay. Here we describe an assay designed to detect ovine TLR6 in blood and tissues from sheep. Discrimination between TLR1 and TLR6 at the level of gene expression was challenging due to extensive tracts of homology and identity within the two sequences. Both TLR1 and 6 can form heterodimers with TLR2 in order to bind the ligands of microbial pathogens. The expression of TLR6 was increased in the ileum and jejunum of sheep infected with MAP, with a trend towards TLR6 upregulation in peripheral blood cells in response to exposure to MAP. A likely role for TLR6/TLR2 heterodimers in the pathogenesis of JD was identified. TLR6 may be a potential marker of exposure and could aid in the development of a gene signature for sheep resistant to MAP infection.