Continuity in ovine Johne's disease vaccination practices despite a decline in clinical disease.

The Gudair® vaccine has been commercially available in Australia for almost two decades for the control of ovine Johne's disease, but concerns have been raised about potential discontinuation of vaccination by producers after a decline in the incidence of clinical disease. An online questionnaire was distributed to Australian sheep producers to identify the proportion of respondents discontinuing the Gudair vaccine and reasons for discontinuation. Results revealed that 88% of sheep producers surveyed have continued to vaccinate their sheep with Gudair, with continuation greater for predominantly Merino sheep flocks. Reasons for discontinuing vaccination stemmed from management, economic or health concerns. These results suggest that Gudair is still widely used by Australian sheep producers and concerns about large-scale discontinuation are unfounded. These findings have implications for ovine Johne's disease control programs in Australia.

The immunogenicity and tissue reactivity of Mycobacterium avium subsp paratuberculosis inactivated whole cell vaccine is dependent on the adjuvant used.

Johne's disease (JD) is a chronic enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). Current commercial vaccines are effective in reducing the occurrence of clinical disease although vaccinated animals can still become infected and transmit MAP. Many vaccinated sheep develop severe injection site lesions. In this study a range of adjuvants (Montanide TM ISA 50V, ISA 50V2, ISA 61VG, ISA 70 M VG, ISA 71 VG, ISA 201 VG and Gel 01 PR) formulated with heat-killed MAP were tested to determine the incidence of injection site lesions and the types of immune profiles generated in sheep. All the novel formulations produced fewer injection site lesions than a commercial vaccine (Gudair®). The immune profiles of the sheep differed between treatment groups, with the strength of the antibody and cell mediated immune responses being dependant on the adjuvant used. One of the novel vaccines resulted in a reduced IFN-γ immune response when a second "booster" dose was administered. These findings have significance for JD vaccine development because it may be possible to uncouple protective immunity from excessive tissue reactivity, and apparently poorly immunogenic antigens may be re-examined to determine if an appropriate immune profile can be established using different adjuvants. It may also be possible to formulate vaccines that produce targeted immunological profiles suited to protection against other pathogens, i.e. those for which a bias towards cellular or humoral immunity would be advantageous based on understanding of pathogenesis.

Sheep and cattle exposed to Mycobacterium avium subspecies paratuberculosis exhibit altered total serum cholesterol profiles during the early stages of infection.

Pathogenic mycobacteria such as Mycobacterium tuberculosis are capable of utilising cholesterol as a primary carbon-based energy source in vitro but there has been little research examining the significance of cholesterol in vivo. Johne's disease is a chronic enteric disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). This study sought to evaluate the levels of total serum cholesterol in the host following exposure to MAP. Blood samples were collected from both sheep and cattle prior to experimental challenge with MAP and at monthly intervals post-challenge. Total serum cholesterol levels in sheep challenged with MAP were significantly elevated at 9 weeks post-inoculation (wpi) in comparison to controls. When stratified based on disease outcome, there was no significant difference in serum cholesterol at the timepoints examined between MAP exposed sheep that were susceptible and those that were resistant to Johne's disease. There was a similar elevation in serum cholesterol at 9 wpi in cattle with histopathological gut lesions associated with disease or those with an early high IFN-γ response. Total serum cholesterol in exposed cattle was significantly lower when compared to controls at 13 wpi. Taken together, these results demonstrate changes in serum cholesterol following MAP exposure and disease progression which could reflect novel aspects of the pathogenesis and immune response associated with MAP infection in both sheep and cattle.

Case definition terminology for paratuberculosis (Johne's disease).

Paratuberculosis (Johne's disease) is an economically significant condition caused by Mycobacterium avium subsp. paratuberculosis. However, difficulties in diagnosis and classification of individual animals with the condition have hampered research and impeded efforts to halt its progressive spread in the global livestock industry. Descriptive terms applied to individual animals and herds such as exposed, infected, diseased, clinical, sub-clinical, infectious and resistant need to be defined so that they can be incorporated consistently into well-understood and reproducible case definitions. These allow for consistent classification of individuals in a population for the purposes of analysis based on accurate counts. The outputs might include the incidence of cases, frequency distributions of the number of cases by age class or more sophisticated analyses involving statistical comparisons of immune responses in vaccine development studies, or gene frequencies or expression data from cases and controls in genomic investigations. It is necessary to have agreed definitions in order to be able to make valid comparisons and meta-analyses of experiments conducted over time by a given researcher, in different laboratories, by different researchers, and in different countries. In this paper, terms are applied systematically in an hierarchical flow chart to enable classification of individual animals. We propose descriptive terms for different stages in the pathogenesis of paratuberculosis to enable their use in different types of studies and to enable an independent assessment of the extent to which accepted definitions for stages of disease have been applied consistently in any given study. This will assist in the general interpretation of data between studies, and will facilitate future meta-analyses.

Variation in susceptibility of different breeds of sheep to Mycobacterium avium subspecies paratuberculosis following experimental inoculation.

Exposure to Mycobacterium avium subspecies paratuberculosis (MAP) does not always lead to Johne's disease. Understanding differences in disease susceptibility of individual animals is a key aspect to controlling mycobacterial diseases. This study was designed to examine the susceptibility or resistance of various breeds of sheep to MAP infection. Merino, Suffolk first cross Merino, Border Leicester, and Poll Dorset sheep were orally inoculated with MAP and monitored for 14 months. Clinical disease occurred more frequently in the Merino (42%) and Suffolk first cross Merino (36%) compared to the Border Leicester (12%) and Poll Dorset (11%) breeds. Infection risk, as determined by culture of gut and associated lymphoid tissues, ranged from 75% for the Suffolk first cross Merino to 47% for the Poll Dorset sheep. Significant differences were identified in the site in the intestines of the most severe histopathological lesions and the immune responses to infection between the breeds. However, there was no difference in faecal MAP shedding by clinical cases between breeds. All breeds tested were susceptible to MAP infection, as determined by infection and clinical disease development, although there were differences in the proportions of diseased animals between the breeds. Poll Dorset and Border Leicester sheep were more resilient to MAP infection but there was evidence that more animals could have developed disease if given more time. These findings provide evidence of potential differential disease susceptibility between breeds, further our understanding of disease pathogenesis and risks of disease spread, and may have an influence on control programs for paratuberculosis.

Applying the One Health Concept to Mycobacterial Research - Overcoming Parochialism.

Mycobacterial infections remain a public health problem. Historically important, globally ubiquitous and with a wide host range, we are still struggling to control mycobacterial infections in humans and animals. While previous reviews have focused on individual mycobacterial infections in either humans or animals, a comprehensive review of the zoonotic aspect of mycobacteria in the context of the One Health initiative is lacking. With the purpose of providing a concise and comprehensive resource, we have collated literature to address the zoonotic potential of different mycobacterial species and elaborate on the necessity for an inter-sectorial approach to attain a new vision to combat mycobacterial infections.

Specific faecal antibody responses in sheep infected with Mycobacterium avium subspecies paratuberculosis.

Many studies have examined the serum antibody response to Mycobacterium avium subspecies paratuberculosis (MAP) infection in cases of Johne's disease (JD), but there are no reports on the mucosal antibody response. Faecal immunoglobulin (Ig) G and IgA ELISA responses were examined from sheep experimentally inoculated with MAP for up to 23 months post inoculation (PI). Corresponding serum IgG responses and the presence of viable MAP shed in faeces were also examined. The sheep were divided into three groups: (i) "un-inoculated controls" (n=10), (ii) "clinical cases" (n=8) which were inoculated animals that developed clinical disease and had moderate to high levels of MAP shedding and (iii) "survivors" (n=11) which were inoculated animals from which MAP could not be cultured from tissues at the conclusion of the trial. Serum IgG responses gradually increased in all inoculated animals, peaking at 12-16 months PI. A significant increase in the levels of MAP-specific faecal IgG and IgA was measured in the survivors at 16 and 17 months PI, while levels in the un-inoculated controls and clinical cases remained at baseline levels. The detection of faecal Ig in the survivors coincided with the removal of sheep that developed clinical disease. The data suggest that some sheep produced MAP-specific IgG and IgA in the intestinal mucosa, which was released into their faeces. We hypothesise that the survivors produced faecal Ig as a direct response to ingestion of MAP associated with environmental contamination from clinical cases. Thus MAP specific mucosal antibodies may play a previously unreported role as part of a protective response triggered by environmental exposure.

Histopathological Characterization of Cutaneous Delayed-type Hypersensitivity and Correlations with Intestinal Pathology and Systemic Immune Responses in Sheep with Paratuberculosis.

Cell-mediated immunity has been exploited historically in the diagnosis of mycobacterial diseases through elicitation of a delayed-type hypersensitivity (DTH) reaction following intradermal injection of an antigen. Here we describe the histopathological features of the cutaneous DTH reaction and its association with intestinal pathology and systemic immune responses in sheep with Mycobacterium avium subspecies paratuberculosis (MAP) infection. A mixed mononuclear cellular infiltrate dominated the DTH reaction and was present in perivascular and periadnexal patterns. Multiple multinucleate giant cells were present in the cellular infiltrate in one sheep while plasma cells were an obvious feature in six others. Sheep with paucibacillary intestinal lesions had the greatest degrees of cutaneous induration, more severe cellular infiltration in DTH lesions and high systemic interferon (IFN)-γ production. In contrast, sheep with multibacillary intestinal lesions, and particularly those with dissemination of MAP to extra-intestinal tissues, had minimal cutaneous induration, nil to mild cellular infiltration in DTH lesions and high serum anti-MAP antibody levels. Systemic IFN-γ production generally was augmented following skin sensitization. In general, the gross and histopathological features of the cutaneous DTH response matched the stage of paratuberculosis reflected by intestinal pathology and systemic measures of humoral and cellular immunity.

Does a Th1 over Th2 dominancy really exist in the early stages of Mycobacterium avium subspecies paratuberculosis infections?

The immune response of ruminants to Johne's disease has been long associated with a cell mediated immune (CMI) response in the early stages of infection with a switch to an antibody response later as the disease manifests. This study examines the immune response in sheep to Mycobacterium avium subspecies paratuberculosis (Map) infections, specifically the antigen-specific interferon gamma (IFN-γ) and antibody responses as surrogates of T helper-1 (Th1) and Th2 immunity. The difference in IFN-γ production between paucibacillary and multibacillary diseased animals was also examined. The results show that sheep are more likely to have a combined antibody and IFN-γ response (seen in 50% of the animals) rather than a switch from an IFN-γ to antibody response (39%). Multibacillary diseased animals were found to have a decrease in functional ability to produce IFN-γ from cells stimulated with MAP-specific antigens and non-specific mitogens. This indicates that the immune responses to Map infections are more complex than thought, where both antibody and cellular immunity may play key roles in the early stages of disease manifestation or resistance. The loss of the cellular response in multibacillary animals may be an indication that the entire immune response is dysfunctional, with the cell mediated responses becoming affected first.

Reversal of experimental allergic encephalomyelitis with non-mitogenic, non-depleting anti-CD3 mAb therapy with a preferential effect on T(h)1 cells that is augmented by IL-4.

This study examined whether therapy with a non-mitogenic, non-activating anti-CD3 mAb (G4.18) alone, or in combination with the T(h)2 cytokines, could inhibit induction or facilitate recovery from experimental allergic encephalomyelitis (EAE) in Lewis rats. G4.18, but not rIL-4, rIL-5 or anti-IL-4 mAb, reduced the severity and accelerated recovery from active EAE. A combination of rIL-4 with G4.18 was more effective than G4.18 alone. The infiltrate of CD4(+) and CD8(+) T cells, B cells, dendritic cells, and macrophages in the brain stem was less with combined G4.18 and IL-4 than G4.18 therapy or no treatment. Residual cells had preferential sparing of T(r)1 cytokines IL-5 and transforming growth factor-beta with loss of T(h)1 markers IL-2, IFN-gamma and IL-12Rbeta2, and the T(h)2 cytokine IL-4 as well as macrophage cytokines IL-10 and tumor necrosis factor-alpha. Lymph nodes draining the site of immunization had less mRNA for T(h)1 cytokines, but T(h)2 and T(r)1 cytokine expression was spared. Treatment with G4.18, rIL-4 or rIL-5 from the time of immunization had no effect on the course of active EAE. MRC OX-81, a mAb that blocks IL-4, delayed onset by 2 days, but had no effect on severity of active EAE. G4.18 also inhibited the ability of activated T cells from rats with active EAE to transfer passive EAE. This study demonstrated that T cell-mediated inflammation was rapidly reversed by a non-activating anti-CD3 mAb that blocked effector T(h)1 cells, and spared cells expressing T(h)2 and T(r)1 cytokines.

Induction of specific tolerance to allografts in rats by therapy with non-mitogenic, non-depleting anti-CD3 monoclonal antibody: association with TH2 cytokines not anergy.

BACKGROUND: Anti-CD3 monoclonal antibodies (mAb) are potent immunosuppressives in transplantation but most do not induce tolerance. They induce anergy in Th1 cells but, if they bind to Fc receptors on antigen presenting cells, they activate T cells to release cytokines. METHODS: This study examined the mechanisms of transplant tolerance induction to PVG fully allogeneic grafts in dark agouti rats by G4.18, a mouse immunoglobulinG3 anti-rat CD3 mAb that does not bind rat Fc receptors. Evidence of T cell activation was assayed by flow cytometry, reverse transcription (RT)-polymerase chain reaction (PCR) for cytokine mRNA, and responsiveness in mixed lymphocyte culture. RESULTS: G4.18 treatment modulated T cell receptor/CD3 and CD2 and depleted T cells by <20% but did not induce activation surface markers. mRNA for interleukin (IL)-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and IL-4 in the lymph node, spleen, and thymus was not increased, and IFN-gamma mRNA was reduced. G4.18-treated and naive rat cells had similar proliferation and expression of IL-2, IFN-gamma, and IL-4 in vitro. G4.18-treated allograft recipients had no induction of mRNA for IL-2, IFN-gamma, TNF-alpha, TNF-beta, IL-4, IL-5, IL-10, perforin, and granzyme A & B in the spleen or grafts, with levels similar to those in isografts. The IL-4 and IL-5 mRNA levels in the spleen but not the graft of G4.18-treated recipients were higher than in rejecting and naive animals. Cells from G4.18-treated graft recipients proliferated more rapidly to the donor than to the third party and had increased IL-4 expression. CONCLUSIONS: G4.18 induced transplant tolerance by a combination of modulation and blocking of the TCR/CD3, associated with increased Th2 cytokines, without depletion, induction of anergy, or nonspecific activation of T cells.

Anti-CD4 monoclonal antibody-induced tolerance to MHC-incompatible cardiac allografts maintained by CD4+ suppressor T cells that are not dependent upon IL-4.

Anti-CD4 mAb-induced tolerance to transplanted tissues has been proposed as due to down-regulation of Thl cells by preferential induction of Th2 cytokines, especially IL-4. This study examined the role of CD4+ cells and cytokines in tolerance to fully allogeneic PVG strain heterotopic cardiac allografts induced in naive DA rats by treatment with MRC Ox38, a nondepleting anti-CD4 mAb. All grafts survived >100 days but had a minor mononuclear cell infiltrate that increased mRNA for the Thl cytokines IL-2, IFN-gamma, and TNF-beta, but not for Th2 cytokines IL-4 and IL-6 or the cytolytic molecules perforin and granzyme A. These hosts accepted PVG skin grafts but rejected third-party grafts, which were not blocked by anti-IL-4 mAb. Cells from these tolerant hosts proliferated in MLC and produced IL-2, IFN-gamma, and IL-4 at levels equivalent to naive cells. Unfractionated and CD4+ T cells, but not CD8+ T cells, transferred specific tolerance to irradiated heart grafted hosts and inhibited reconstitution of rejection by cotransferred naive cells. This transfer of tolerance was associated with normal induction of IL-2 and delayed induction of IFN-gamma, but not with increased IL-4 or IL-10 mRNA. Transfer of tolerance was also not inhibited by anti-IL-4 mAb. This study demonstrated that tolerance induced by a nondepleting anti-CD4 mAb is maintained by a CD4+ suppressor T cell that is not associated with preferential induction of Th2 cytokines or the need for IL-4; nor is it associated with an inability to induce Th1 cytokines or anergy.

Induction of tolerance with nondepleting anti-CD4 monoclonal antibodies is associated with down-regulation of TH2 cytokines.

BACKGROUND: Induction of tolerance with anti-CD4 has mainly focused on monoclonal antibodies (mAbs) that deplete CD4+ T cells. In this study, the mechanisms by which nondepleting anti-CD4 mAbs induce tolerance in the Dark Agouti to PVG rat heart graft model were examined. METHODS: Five anti-CD4 mAbs were tested. Immunohistology and cytokine mRNA profiles were analyzed within grafts. Effects of combining anti-CD4 therapy with alloantibody (alloAb), interleukin (IL)-4, and anti-IL-4 mAb were also examined. RESULTS: All mAbs tested induced indefinite graft survival (>150 days), with blocking of alloAb production. Exogenous alloAb did not restore rejection. Similar T cell receptor alphabeta+, CD8+, IL-2 receptor+ T cell, macrophage, and natural killer cell infiltration and comparable MHC II and intercellular adhesion molecule-1 levels were seen in rejecting and tolerant grafts. mRNA for IL-2, interferon-gamma, lymphotoxin, tumor necrosis factor-alpha, transforming growth factor-beta, cytolysin, and granzyme-A/B was comparable, although inducible nitric oxide synthase was slightly reduced in tolerant grafts. IL-4 and IL-5 were significantly reduced in tolerant grafts, although IL-6, IL-10, and IL-13 levels were similar; this was consistent with partial T helper (Th)2 response inhibition, which was also manifested by inhibited alloAb. The combination of alloAb, IL-4, or anti-IL-4 mAb with anti-CD4 did not prevent tolerance induction. CONCLUSIONS: This study demonstrated that anti-CD4 mAb therapy did not inhibit activation and infiltration of Th1 and CD8+ effector T cells. Preferential induction of Th2 responses, especially IL-4, was not essential for the induction of tolerance. Our studies also found no evidence to support induction of anergy or transforming growth factor-beta as mechanisms of tolerance induction. These results question whether IL-4 is required for induction of transplantation tolerance.