The humoral immune response is essential for successful vaccine protection against paratuberculosis in sheep.

BACKGROUND: The role played by the humoral immune response in animals vaccinated against a mycobacterial disease such as paratuberculosis, is not well understood. Sheep vaccinated against Mycobacterium avium subsp. paratuberculosis (MAP) can still become infected and in some cases succumb to clinical disease. The strength and location of the humoral immune response following vaccination could contribute to the ability of sheep to clear MAP infection. We examined the peripheral antibody response along with the localised humoral response at the site of paratuberculosis infection, the ileum, to better understand how this contributes to MAP infection of sheep following vaccination and exposure. RESULTS: Through assessing MAP specific serum IgG1 and IgG levels we show that the timing and strength of the humoral immune response directly relates to prevention of infection following vaccination. Vaccinated sheep that subsequently became infected had significantly reduced levels of MAP specific serum IgG1 early after vaccination. In contrast, vaccinated sheep that did not subsequently become infected had significantly elevated MAP specific serum IgG1 following vaccination. Furthermore, at 12 months post MAP exposure, vaccinated and subsequently uninfected sheep had downregulated expression of genes related to the humoral response in contrast to vaccinated infected sheep where expression levels were upregulated. CONCLUSIONS: The timing and strength of the humoral immune response following vaccination against paratuberculosis in sheep directly relates to subsequent infection status. An initial strong IgG1 response following vaccination was crucial to prevent infection. Additionally, vaccinated uninfected sheep were able to modulate that response following apparent MAP clearance, unlike vaccinated infected animals where there was apparent dysregulation of the humoral response, which is associated with progression to clinical disease.

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays.

UNLABELLED: Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE: Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing.

Evaluation of the limitations and methods to improve rapid phage-based detection of viable Mycobacterium avium subsp. paratuberculosis in the blood of experimentally infected cattle.

BACKGROUND: Disseminated infection and bacteraemia is an underreported and under-researched aspect of Johne's disease. This is mainly due to the time it takes for Mycobacterium avium subsp. paratuberculosis (MAP) to grow and lack of sensitivity of culture. Viable MAP cells can be detected in the blood of cattle suffering from Johne's disease within 48 h using peptide-mediated magnetic separation (PMMS) followed by bacteriophage amplification. The aim of this study was to demonstrate the first detection of MAP in the blood of experimentally exposed cattle using the PMMS-bacteriophage assay and to compare these results with the immune response of the animal based on serum ELISA and shedding of MAP by faecal culture. RESULTS: Using the PMMS-phage assay, seven out of the 19 (37 %) MAP-exposed animals that were tested were positive for viable MAP cells although very low numbers of MAP were detected. Two of these animals were positive by faecal culture and one was positive by serum ELISA. There was no correlation between PMMS-phage assay results and the faecal and serum ELISA results. None of the control animals (10) were positive for MAP using any of the four detection methods. Investigations carried out into the efficiency of the assay; found that the PMMS step was the limiting factor reducing the sensitivity of the phage assay. A modified method using the phage assay directly on isolated peripheral blood mononuclear cells (without PMMS) was found to be superior to the PMMS isolation step. CONCLUSIONS: This proof of concept study has shown that viable MAP cells are present in the blood of MAP-exposed cattle prior to the onset of clinical signs. Although only one time point was tested, the ability to detect viable MAP in the blood of subclinically infected animals by the rapid phage-based method has the potential to increase the understanding of the pathogenesis of Johne's disease progression by warranting further research on the presence of MAP in blood.

Efficient, validated method for detection of mycobacterial growth in liquid culture media by use of bead beating, magnetic-particle-based nucleic acid isolation, and quantitative PCR.

Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 10(4)-fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n=54) and sheep fecal and tissue (n=90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis.

High-throughput direct fecal PCR assay for detection of Mycobacterium avium subsp. paratuberculosis in sheep and cattle.

Johne's 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 McNemar's 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.

Environmental survival of Mycobacterium avium subsp. paratuberculosis in different climatic zones of eastern Australia.

The duration of survival of both the S and C strains of Mycobacterium avium subsp. paratuberculosis in feces was quantified in contrasting climatic zones of New South Wales, Australia, and detailed environmental temperature data were collected. Known concentrations of S and C strains in feces placed on soil in polystyrene boxes were exposed to the environment with or without the provision of shade (70%) at Bathurst, Armidale, Condobolin, and Broken Hill, and subsamples taken every 2 weeks were cultured for the presence of M. avium subsp. paratuberculosis. The duration of survival ranged from a minimum of 1 week to a maximum of 16 weeks, and the provision of 70% shade was the most important factor in extending the survival time. The hazard of death for exposed compared to shaded samples was 20 and 9 times higher for the S and C strains, respectively. Site did not affect the survival of the C strain, but for the S strain, the hazard of death was 2.3 times higher at the two arid zone sites (Broken Hill and Condobolin) than at the two temperate zone sites (Bathurst and Armidale). Temperature measurements revealed maximum temperatures exceeding 60°C and large daily temperature ranges at the soil surface, particularly in exposed boxes.

Development and validation of a liquid medium (M7H9C) for routine culture of Mycobacterium avium subsp. paratuberculosis to replace modified Bactec 12B medium.

Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 µl inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained <10 viable bacilli per gram, but these results were relatively uncommon, and the performances of the two media were not significantly different. M7H9C medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period.

IP10 is a predictor of successful vaccine protection against paratuberculosis infection in sheep.

The cell mediated immune response and ability of immune cells to migrate to the site of infection are both key aspects of protection against many pathogens. Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular pathogen and the causative agent of paratuberculosis, a chronic wasting disease of ruminants. Current commercial vaccines for paratuberculosis reduce the occurrence of clinical disease but not all animals are protected from infection. Therefore, there is a need to understand the immune responses triggered by these vaccines at the site of infection, in circulating immune cells and their relationships to vaccine-mediated protection. The magnitude and location of gene expression related to the cell mediated immune response and cellular migration were studied in the ileum of sheep. In addition, longitudinal IP10 (also known as IP10) secretion by circulating immune cells was examined in the same sheep. Animals were grouped based on vaccination status (vaccinated vs non-vaccinated) and MAP exposure (experimentally exposed vs unexposed). Vaccination of unexposed sheep increased the expression of IP10, CCL5 and COR1c. Sheep that were successfully protected by vaccination (uninfected following experimental exposure) had significantly reduced expression of IP10 in the ileum at 12 months post exposure compared to vaccine non-responders (those that became infected) and non-vaccinated infected sheep. Successfully protected sheep also had significantly increased secretion of IP10 in in vitro stimulated immune cells from whole blood compared to vaccine non responders at 4 months post exposure. Therefore, the IP10 recall response has the potential to be used as marker for infection status in vaccinated sheep and could be a biomarker for a DIVA test in sheep.

Evaluating serological tests for foot-and-mouth disease while accounting for different serotypes and uncertain vaccination status.

Controlling foot-and-mouth disease (FMD) by vaccination requires adequate population coverage and high vaccine efficacy under field conditions. To assure veterinary services that animals have acquired sufficient immunity, strategic post-vaccination surveys can be conducted to monitor the coverage and performance of the vaccine. Correct interpretation of these serological data and an ability to derive exact prevalence estimates of antibody responses requires an awareness of the performance of serological tests. Here, we used Bayesian latent class analysis to evaluate the diagnostic sensitivity and specificity of four tests. A non-structural protein (NSP) ELISA determines vaccine independent antibodies from environmental exposure to FMD virus (FMDV), and three assays measuring total antibodies derived from vaccine antigen or environmental exposure to two serotypes (A, O): the virus neutralisation test (VNT), a solid phase competitive ELISA (SPCE), and a liquid phase blocking ELISA (LPBE). Sera (n = 461) were collected by a strategic post-vaccination monitoring survey in two provinces of Southern Lao People's Democratic Republic (PDR) after a vaccination campaign in early 2017. Not all samples were tested by every assay and each serotype: VNT tested for serotype A and O, whereas SPCE and LPBE tested for serotype O, and only NSP-negative samples were tested by VNT, with 90 of them not tested (missing by study design). These data challenges required informed priors (based on expert opinion) for mitigating possible lack of model identifiability. The vaccination status of each animal, its environmental exposure to FMDV, and the indicator of successful vaccination were treated as latent (unobserved) variables. Posterior median for sensitivity and specificity of all tests were in the range of 92-99 %, except for the sensitivity of NSP (∼66%) and the specificity of LPBE (∼71 %). There was strong evidence that SPCE outperformed LPBE. In addition, the proportion of animals recorded as having been vaccinated that showed a serological immune response was estimated to be in the range of 67-86 %. The Bayesian latent class modelling framework can easily and appropriately impute missing data. It is important to use field study data as diagnostic tests are likely to perform differently on field survey samples compared to samples obtained under controlled conditions.

Correlates of vaccine protection against Mycobacterium avium sub-species paratuberculosis infection revealed in a transcriptomic study of responses in Gudair® vaccinated sheep.

A critical hindrance in the development of effective vaccine strategies to combat infectious disease is lack of knowledge about correlates of protection and of the host responses necessary for successful adaptive immunity. Often vaccine formulations are developed by stepwise experimentation, with incomplete investigation of the fundamental mechanisms of protection. Gudair® is a commercially available vaccine registered for use in sheep and goats for controlling spread of Mycobacterium avium sub-species paratuberculosis (MAP) infections and reduces mortality by up to 90%. Here, using an experimental infection model in sheep, we have utilized a transcriptomics approach to identify white blood cell gene expression changes in vaccinated, MAP-exposed Merino sheep with a protective response in comparison to those vaccinated animals that failed to develop immunity to MAP infection. This methodology facilitated an overview of gene-associated functional pathway adaptations using an in-silico analysis approach. We identified a group of genes that were activated in the vaccine-protected animals and confirmed stability of expression in samples obtained from naturally exposed commercially maintained sheep. We propose these genes as correlates of vaccine induced protection.

Sheep vaccinated against paratuberculosis have increased levels of B cells infiltrating the intestinal tissue.

Systemic immunisation delivered subcutaneously is currently used to control paratuberculosis, a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). These vaccines do not provide complete protection and a small cohort of animals still succumb to clinical disease. The aim of this study was to assess mycobacterial infection site-specific variations in immune cells in vaccinated sheep that did or did not develop the disease following controlled exposure to MAP. Immunohistochemical staining of terminal ileum demonstrated that vaccination increased infiltration of CD4 + T cells and B cells. Infiltration of large numbers of CD4 + T and B cells was also seen in sheep that successfully cleared infection. Vaccination promoted the polarisation of macrophages to an M1 activation state. The presence of certain cells at the site of infection, especially CD4 + T cells, is likely to contribute to vaccine success by increasing the speed and potency of the local immune response. Systemic immunisation against MAP can alter the composition of innate and adaptive immune cell populations at the predilection site for MAP infection in the ileum one year after vaccination. This informs understanding of the impact of vaccination at the site of infection and also the duration of vaccine-elicited changes. This information may assist vaccine development and allow targeting of protective immune responses in the gut of ruminants.

Indoleamine 2,3-dioxygenase, tryptophan catabolism, and Mycobacterium avium subsp. paratuberculosis: a model for chronic mycobacterial infections.

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 Johne's 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.

Engineering Synthetic Lipopeptide Antigen for Specific Detection of Mycobacterium avium subsp. paratuberculosis Infection.

Unlike other MAC members, Mycobacterium avium subsp. paratuberculosis (MAP) does not produce glycopeptidolipids (GPL) on the surface of the cell wall but a lipopentapeptide called L5P (also termed Lipopeptide-I or Para-LP-01) characterized in C-type (bovine) strains. This lipopeptide antigen contains a pentapeptide core, D-Phenylalanine-N-methyl-L-Valine-L-Isoleucine-L-Phenylalanine-L-Alanine, in which the N-terminal D-Phenylalanine is amido-linked with a fatty acid (C18-C20). The molecular and genetic characterization of this antigen demonstrated that L5P is unique to MAP. Knowledge of the structure of L5P enabled synthetic production of this lipopeptide in large quantities for immunological evaluation. Various studies described the immune response directed against L5P and confirmed its capability for detection of MAP infection. However, the hydrophobic nature of lipopeptide antigens make their handling and use in organic solvents unsuitable for industrial processes. The objectives of this study were to produce, by chemical synthesis, a water-soluble variant of L5P and to evaluate these compounds for the serological diagnosis of MAP using well-defined serum banks. The native L5P antigen and its hydrosoluble analog were synthesized on solid phase. The pure compounds were evaluated on collections of extensively characterized sera from infected and non-infected cattle. ROC analysis showed that L5P and also its water-soluble derivative are suitable for the development of a serological test for Johne's disease at a population level. However, these compounds used alone in ELISA have lower sensitivity (Se 82% for L5P and Se 62% for the water-soluble variant of L5P) compared to the Se 98% of a commercial test. Advantageously, these pure synthetic MAP specific antigens can be easily produced in non-limiting quantities at low cost and in standardized batches for robust studies. The fact that L5P has not been validated in the context of ovine paratuberculosis highlights the need to better characterize the antigens expressed from the different genetic lineages of MAP to discover new diagnostic antigens. In the context of infections due to other mycobacteria such as M. bovis or the more closely related species M. avium subsp. hominissuis, the L5P did not cross react and therefore may be a valuable antigen to solve ambiguous results in other tests.

Optimization of a whole blood gamma interferon assay for the detection of sheep infected with Mycobacterium avium subspecies paratuberculosis.

The capacity of a commercially available gamma interferon (IFNgamma) assay to detect infected sheep early in the pathogenesis of Johne's disease enables the removal of such animals from the flock before bacterial shedding and pasture contamination. However, nonspecific IFNgamma responses in the assay have meant that to achieve high-test specificity, there has been a reduction in sensitivity. Although the optimal conditions for the use of the assay in cattle have been well documented, there have been few studies optimizing the assay for use in sheep. The current study details the effect of anticoagulant, duration of incubation, cell concentration, blood storage temperature, time of stimulation of cells with antigen relative to time of sample collection, and temperatures during transit on IFNgamma synthesis. Maximal IFNgamma synthesis occurred with incubation periods of 48 hr in blood collected into heparinized tubes. Decreasing the leukocyte population by diluting the total peripheral blood leukocyte concentration was associated with a decreasing IFNgamma response. Conversely, concentrating the peripheral blood concentration 2-fold resulted in an increase in the IFNgamma production. In field studies, immediate incubation of blood samples with antigen at 37 degrees C resulted in larger IFNgamma responses; however, significantly lower IFNgamma values were obtained if the samples were transported at ambient temperature. The results of this study indicate that optimization of the IFNgamma assay may enable increased synthesis of IFNgamma during the stimulation phase of the assay and that future work may determine whether this translates to increased sensitivity of the assay in detecting early infections in sheep.

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-gamma) enzyme-linked immunosorbent assay (ELISA) to detect Johne's disease (JD) has been poor, especially in the early stages of disease. To improve the sensitivity of IFN-gamma 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-gamma-secreting cells. The ELISPOT assay was as sensitive as or better than the IFN-gamma ELISA at detecting ovine JD and could also detect disease at early time points postinoculation. The IFN-gamma ELISPOT could distinguish infected from unexposed animals; however, neither the IFN-gamma 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.

Gene expression profiles during subclinical Mycobacterium avium subspecies paratuberculosis infection in sheep can predict disease outcome.

Paratuberculosis in ruminants is caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP) however exposure does not predetermine progression to clinical disease. The pathogenesis incorporates a subclinical phase during which MAP is capable of evading host immune responses through adaptation of host cellular immune mechanisms. Presented are results of transcriptomic analysis of Merino sheep experimentally exposed to MAP and repeatedly sampled over the subclinical phase, identifying genes consistently changed over time in comparison to unexposed controls and associated with different disease outcomes. MAP exposed sheep were classified as diseased 45% (n = 9) or resilient 55% (n = 11). Significant gene expression changes were identified in the white blood cells of paucibacillary (n = 116), multibacillary (n = 98) and resilient cohorts (n = 53) compared to controls. Members of several gene families were differentially regulated, including S100 calcium binding, lysozyme function, MHC class I and class II, T cell receptor and transcription factors. The microarray findings were validated by qPCR. These differentially regulated genes are presented as putative biomarkers of MAP exposure, or of the specified disease or resilience outcomes. Further, in silico functional analysis of genes suggests that experimental MAP exposure in Merino sheep results in adaptations to cellular growth, proliferation and lipid metabolism.

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 Johne's 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 Johne's 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.

Experimental animal infection models for Johne's disease, an infectious enteropathy caused by Mycobacterium avium subsp. paratuberculosis.

A critical literature review of experimental infection models for Johne's disease in farm and laboratory animals was conducted. A total of 73 references were admitted. They were published between 1938 and 2006 and covered species as diverse as cattle, sheep, goats, deer, mice, pigs and others. The factors that appeared to influence the outcome of experimental infections with Mycobacterium avium subsp. paratuberculosis (Mptb) were the species, breed and age of subject used for the infection, the route of infection, and the strain, dose and number of doses of Mptb used to inoculate the subjects. Natural paratuberculosis infection passes through stages, generally over a period measured in years. However, the endpoints chosen by researchers using experimental infections have been determined by the need for immunological, microbiological, pathological or clinical outcomes, and these were the likely factors determining the duration of the trials. Studies have been lacking in the use of a defined type strain of Mptb in pure culture prepared from an archived seed stock of Mptb that can be used at the same passage level in a later trial. Replication of experimental groups has been very uncommon, temporal replication equally rare, as have sufficiently long time scales so as to be able to observe a full range of immunological and pathological changes at different stages of the disease process. While it may be difficult to develop a satisfactory experimental infection model, there is room for improvement in the way experiments have been designed and carried out to date. Choice of animal species/breed of host and strain of Mptb used in an experimental model should be based on the purpose of the study (for example, vaccine efficacy trial, diagnostic test evaluation, pathogenesis study) and local needs. The strain of Mptb used should be typed using IS900 RFLP analysis, IS1311 sequence analysis and other genotypic methods, and preferably be from an archived low passage pure culture with viable bacteria enumerated using a sensitive method rather than from an uncharacterised and unrepeatable tissue homogenate. It is generally agreed that the faecal-oral route is the most important natural route of exposure and the oral route is therefore the preferred route of experimental inoculation to achieve Johne's disease that closely resembles natural infection.

A national serosurvey to determine the prevalence of paratuberculosis in cattle in Bhutan following detection of clinical cases.

Johne's disease is an economically important ruminant disease predominantly affecting cattle, sheep and goats. The economic losses are due to early culling, reduced growth rate, progressive weight loss and reduced production. It is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Johne's disease was reported in cattle in Bhutan, based on clinical signs and histopathology; in the late 1990s samples from one mithun that was suspected to have died due to this disease was confirmed by molecular testing at the Faculty of Veterinary Science, University of Sydney, Australia. However, no detailed study on prevalence of JD has been attempted in Bhutan. Objective of this study was to conduct serosurveillance to determine the national prevalence of Johne's disease in cattle for the period 2013-2014 to provide the basis for planning a future control strategy. A national serosurvey was conducted wherein a two-stage sampling procedure was used with 95% confidence and an error level of ±0.05. The sample size required for the survey was calculated using the software-Survey Toolbox for Livestock Diseases, available as Epitools at http://www.ausvet.com.au. A total of 1123 serum samples were collected from an administrative structure of 52 villages, 40 sub-districts and 15 districts. Serum samples were tested using commercially available antibody enzyme linked immunosorbent assay. Statistical analysis was performed using GraphPad Prism 5.0. Illustration such as maps was produced using QGIS version 2.18 'Las Palmas. The mean national apparent prevalence of Johne's disease was found to be 2.31 (26/1123) (95% CI: 0.80-4.50) with an estimated true prevalence was found to be 8.00 (95% CI: 2.00-17.00). Trongsa district had the highest prevalence (12.96) followed by Zhemgang (4.34), Lhuntse (4.25), Sarpang (3.89), Bumthang (3.60), Trashigang (2.67) and Haa (2.63). Prevalence for all other districts was 2.00 or below. Seropositive samples were reported from all over the country with varying levels of sero-positivity. In the recent past many more cattle were imported from India to boost dairy production. Nevertheless, the wide distribution of seroreactive JD cattle all over the country is a concern for future control. Therefore, in future, a detailed study on the impact of cattle import with regard to disease incursion such as Johne's disease and other diseases should be undertaken.

Defining resilience to mycobacterial disease: Characteristics of survivors of ovine paratuberculosis.

Paratuberculosis is an insidious, chronic disease of ruminants that has significant animal welfare implications and reduces on-farm profitability globally. Not all animals exposed to the causative pathogen, Mycobacterium avium subspecies paratuberculosis (MAP), succumb to disease and this unique, long-term trial was designed to track animals that were resilient. The advantages of understanding immune protection include the management option to retain resilient individuals in a herd/flock and the potential for deliberate manipulation of the host immune response using novel vaccines. Twenty sheep experimentally exposed to MAP and 10 controls were monitored for 2.5 years during which the condition progressed, resembling natural disease development. Cellular and humoral immune parameters and faecal MAP shedding were examined regularly and disease outcomes were classified at necropsy, based on the presence of viable MAP and histopathological lesions in intestinal tissues, either at the termination of the trial or when animals were culled due to weight loss. There were distinct characteristics, such as an early strong IFNγ response, that differentiated resilient sheep from susceptible individuals prior to the onset of clinical disease. Faecal MAP shedding and serum antibody level, commonly used to diagnose disease, were more ambiguous. The former was transient in the majority of resilient animals and therefore should not be used for diagnosis of MAP infection in younger animals. Remarkably, the serum antibody level in some resilient animals was higher than the usual positive-negative cut-off for disease diagnosis at multiple samplings throughout the trial. Consequently the antibody response in resistance to paratuberculosis requires further investigation.