Logo of cdiJournal's HomeManuscript SubmissionAims and ScopeAuthor GuidelinesEditorial BoardHome
Clin Dev Immunol. 2011; 2011: 768542.
Published online Dec 6, 2010. doi:  10.1155/2011/768542
PMCID: PMC3004413

Tuberculosis Immunity: Opportunities from Studies with Cattle

Abstract

Mycobacterium tuberculosis and M. bovis share >99% genetic identity and induce similar host responses and disease profiles upon infection. There is a rich history of codiscovery in the development of control measures applicable to both human and bovine tuberculosis (TB) including skin-testing procedures, M. bovis BCG vaccination, and interferon-γ release assays. The calf TB infection model offers several opportunities to further our understanding of TB immunopathogenesis. Recent observations include correlation of central memory immune responses with TB vaccine efficacy, association of SIRPα+ cells in ESAT-6:CFP10-elicited multinucleate giant cell formation, early γδ T cell responses to TB, antimycobacterial activity of memory CD4+ T cells via granulysin production, association of specific antibody with antigen burden, and suppression of innate immune gene expression in infected animals. Partnerships teaming researchers with veterinary and medical perspectives will continue to provide mutual benefit to TB research in man and animals.

1. Introduction: History of Codiscovery

Three essential tools developed in cattle and used for the control of human tuberculosis (TB) include (1) vaccination with an attenuated Mycobacterium bovis Bacillus Calmette Guerin (BCG, [1]), (2) use of tuberculin as an in vivo diagnostic reagent, and (3) antigen-induced interferon- (IFN-) γ as an in vitro biomarker of TB infection. In 1913 at the Pasteur Institute (Lille, France), Albert Calmette and Camille Guerin vaccinated 9 cows with M. bovis (Nocard strain) attenuated by serial passage on glycerol-soaked potato slices in ox bile (i.e., BCG) [1]. All 9 animals were protected from challenge with virulent M. bovis, thereby, demonstrating the potential use of BCG vaccination against M. tuberculosis infection of humans. In 1921, BCG was administered to a newborn child (6 mg orally) and has since been used widely for the control of human TB. Within a few years of the discovery of tuberculin by Robert Koch, veterinary investigators in Russia (Professor Gutman), the UK (John McFadyean), Denmark (Bernhard Bang), and the US (Leonard Pearson and Maz'yck Ravenel) were administering tuberculin to cattle as an in vivo diagnostic reagent (infection indicated by a rise in temperature within 24 hours) [2]. Clemens von Pirquet and Charles Mantoux later (circa 1907/1908) adapted and improved (e.g., subcutaneous to intradermal) this technology for application in the diagnosis of TB in humans, coincidently defining the principles of allergy and delayed type hypersensitivity. During the 1980s, an in vitro IFN-γ release assay was developed for the diagnosis of TB in cattle [3]; a modified version of this assay is now widely used in the diagnosis of both human and bovine TB. Together, these findings demonstrate the mutual benefit for cooperative veterinary and medical research. As stated by Emil von Behring in his Nobel Prize acceptance speech [4], I need hardly add that the fight against cattle tuberculosis only marks a stage on the road which leads finally to the effective protection of human beings against the disease.” The current review highlights recent observations on immunity to bovine TB of relevance for understanding the disease, both in cattle and humans.

2. The Neonatal Calf as a Model for the Study of TB

2.1. Mycobacterium bovis

Mycobacterium bovis, a member of the M. tuberculosis complex, has a wide host range as compared to other species in this disease complex, is infectious to humans, and is the species most often isolated from tuberculous cattle. Prior to implementation of widescale pasteurization, it is estimated that 20–40% of TB cases in humans resulted from infection with M. bovis [57]. An explanation, not apparent at the time, suggests a difference in the capacity of M. tuberculosis and M. bovis to infect and cause disease in cattle. Genome comparisons show that M. bovis and M. tuberculosis evolved into two clades from a common prototypic ancestor some 40,000 years ago: clades defined by presence or absence of M. tuberculosis deletion 1 (TbD1) [8]. The data suggest that both clades arose in humans, with the TbD1 clade 1 coevolving mainly in humans and the TbD1+ clade 2 coevolving in humans, ruminants, and other species. The difference in host range shows that evolution of M. bovis and M. tuberculosis has included development of a difference in virulence and the capacity to cause disease in different species. This difference may prove useful in comparative studies designed to elucidate the mechanisms of immunopathogenesis and development of vaccines. Approximately 90% of humans exposed to M. tuberculosis develop an immune response that controls but does not eliminate the pathogen. Immune control of this persistent (latent) stage of infection may persist for a lifetime or become dysregulated, allowing for disease progression. It is not clear whether a comparable proportion of humans infected with M. bovis develop an immune response that controls infection. Recent direct comparison of M. bovis and M. tuberculosis infection in cattle has demonstrated that M. tuberculosis is less virulent for cattle; however, the M. tuberculosis strain used for these studies was a laboratory-adapted strain (H37 Rv) [9]. However, experimental transmission studies (conducted in the late 1800s by Theobald Smith (physician scientist) and veterinarians Austin Peters and Langdon Frothingham using calves experimentally inoculated with sputum from humans with tuberculosis), demonstrated that human bacilli possess a low virulence for cattle [10]. Other studies clearly demonstrated that nonlaboratory-adapted strains of M. tuberculosis were less virulent in cattle than those of M. bovis (reviewed by Whelan et al., 2010 [9]). Analysis of the difference in the immune response to the two pathogens may provide insight into the mechanisms used by both bacteria to circumvent protective immunity [11].

2.2. Aerosol Infection Model

Aerosol inoculation of M. bovis to calves results in a respiratory tract infection (i.e., lungs and lung-associated lymph nodes), severity is dose-dependent, and the disease closely mimics natural infection of cattle [12]. As related to human disease, studies with neonatal calves are particularly relevant as this is the primary target population for human vaccination and exposure to TB often occurs at a very young age. For calf vaccine studies, parameters to demonstrate efficacy include quantitative and qualitative mycobacterial culture, gross and microscopic disease scoring, radiographic morphometry of lung lesions, and disease-associated immune parameters. Opportunities afforded by use of the calf model include (1) large numbers of affordable age-, gender-, and breed-matched animals available throughout the year (nonhuman primates (NHPs) are seasonal breeders and are costly), (2) cattle are out-bred species, thus, experimental variance is similar to what is expected for NHP and humans, (3) size allows dose titration studies and full immunologic assessment via frequent collection of large volumes of blood which facilitates studies on immune response kinetics, (4) the nutritional status (e.g., vitamin deficiencies and protein malnutrition) can be manipulated to achieve similar levels of deficiency as may occur in target human populations in the developing world, (5) serves as an additional safety screen for evaluation of vaccines, adjuvants, or other administered biologics, and (6) feasibility of duration of immunity studies.

2.3. Parallel Testing of Vaccine Candidates in the Primate and Calf Model-mc26030

A double deletion mutant of M. tuberculosis H37 Rv (i.e., ΔRD1-the primary attenuating mutation of BCG and ΔpanCD-deletion of pantothenate synthesis genes) was evaluated in parallel with BCG (Copenhagen strain) in both the calf model and adolescent Cynomologus monkeys (Macaca fascicularis). The M. tuberculosis ΔRD1 X panCD mutant (mc26030) undergoes limited replication in mice and is safer than BCG in immunocompromised mice (i.e., SCID, IFN-γ receptor-deficient and CD4-deficient mice) [13]. Immunization with this mutant strain of H37 Rv induces prolonged protective immune responses that promote survival of both wild type and CD4-deficient mice against an aerosol challenge with virulent M. tuberculosis. Antibody depletion and adoptive transfer studies revealed that protection in CD4-deficient mice was mediated in the absence of CD4+, CD8+, γδ+, and NK1.1+ T cells, thus, indicating a surprising capacity for protection to be elicited by CD4CD8 TCR−αβ+ cells [14]. For second tier testing, mc26030 was evaluated for efficacy in the newborn calf model and adolescent Cynomologus monkeys. In both calves and monkeys, the vaccine was ineffective [15, 16]; thus, in this instance, responses in mice were not predictive of efficacy in models using natural hosts of infection. For cattle, attenuated M. tuberculosis mutants may be less immunogenic as compared to those produced on a virulent M. bovis or BCG strain; thus, cattle may not be as useful as other models (e.g., monkeys) for the study of vaccine efficacy using M. tuberculosis mutants. Further studies are required to directly compare immunogenicity and virulence of M. tuberculosis versus M. bovis background mutants in cattle.

3. Bovine DCs and Macrophages

The role of signal regulatory protein alpha-expressing (SIRPα+) cells in the adaptive response to tuberculous mycobacteria via interactions with ESAT-6/CFP-10. Multiple functions are proposed for the RD1 proteins ESAT-6 and CFP-10 [17, 18]. For instance, ESAT-6 interacts with biomembranes after dissociation from its putative CFP-10 chaperone within the acidic phagolysosome [19], thereby affording a “phagolysosome escape” mechanism for the pathogen. ESAT-6 deletion mutants of M. tuberculosis have reduced tissue invasiveness, likely due to loss of cytolytic activity [20]. In addition, use of the M. marinum/zebrafish granuloma model demonstrates that RD1 components are required for efficient recruitment of macrophages to granulomas “creating new bacterial growth niches” [21, 22]. RD-1 proteins, including ESAT-6/CFP-10, likely elicit more rapid granuloma formation offering a distinct growth advantage for the pathogen [22]. In addition to enhancing recruitment of cells susceptible to infection, the stable ESAT-6/CFP-10 complex binds to host cells [23]; thereby, modulating the host response favourably for the pathogen via down-regulation of host cell killing mechanisms and immune cell activation [24].

A specific receptor (TLR2) for ESAT-6 has been identified using mouse monocyte/macrophage cell lines [25]. Studies with leukocytes obtained from cattle have also demonstrated a specific interaction of the ESAT-6/CFP-10 complex with CD172a (SIRPα)-expressing cells [26]. Stimulation of peripheral blood mononuclear cell cultures from M. bovis-infected calves with ESAT-6/CFP-10 results in the specific expansion of SIRPα+ cells, with binding of the fusion protein bound to the surface of SIRPα+ cells [26]. SIRPα-CD47 interactions are essential for efficient migration of DCs to skin [27] and secondary lymphoid organs [28]. Thus, ESAT-6/CFP-10-induced expansion of SIRPα-expressing cells may favour migration of DC's/macrophages to infection sites, thereby, promoting efficient granuloma formation and early dissemination of M. tuberculosis complex mycobacteria [22]. With the bovine TB model, intradermal injection of rESAT-6:CFP-10 elicits granuloma formation with infiltration of numerous T cells, SIRPα+ cells, and CD14+ cells in M. bovis-infected calves, further supporting a role for ESAT-6/CFP-10 in the recruitment of naïve cells to sites of granuloma formation [26]. A unique aspect of the cellular infiltrates at rESAT-6:CFP-10 injection sites in cattle is the presence of numerous multinucleated giant cells. Multinucleated giant cell formation is mediated, in part, by SIRPα (also termed macrophage fusion receptor). Cell surface expression of SIRPα is strongly and transiently induced upon giant cell formation. As opposed to phagocytosis, SIRPα-CD47 interactions provide “self recognition” signals that prevent killing of internalized (i.e., fused) cells. Thus, cattle may serve as a useful model for the molecular characterization of multinucleate giant cell formation within tuberculous granulomas. Additionally, comparative studies examining ESAT-6/CFP-10 interactions with SIRPα and TLR2 in mouse, human, and bovine tissues are needed to determine if host factors affect ESAT-6/CFP-10 specificity.

4. T Cell Subsets and Effector Mechanisms

4.1. Cell-Mediated Immunity (CMI)

Ongoing studies have shown the adaptive immune system of cattle is quite similar to the human system [29]. Importantly, comparisons at the genomic level indicate that genes encoding cytokines known to play a role in regulating immune responses in humans are present in cattle, including cytokines not found in mice (e.g., IL-26). Cell-mediated immunity is essential for protection against bovine and human TB and there appears to be significant similarity in the primary mechanisms of antimycobacterial CMI between humans (as reviewed in [30]) and cattle (as reviewed in [31]). Similarities include roles of T cell subsets (e.g., CD4+, CD8+, and γδ TCR+), protective function and cellular sources of IFNγ and cytotoxic granule proteins; the reduction of mycobacterial numbers within macrophages by cytotoxic T cells and NK cells, the relative levels of antigen specific Th1 and Th2 cytokines, and expression of memory markers by antigen specific T cells [3242]. Widely utilized for TB diagnosis, antigen-specific release of IFN-γ is clearly an important function of the CMI response to TB in cattle and humans [43, 44]. In contrast to rodents, human and bovine immunity to TB appears to be less reliant on antigen-specific IFN-γ activation of macrophages [45, 46] and may employ cytotoxic immune cells in a more active role.

Protective immunity to TB in cattle, as in human and other animal models of TB, correlates to the induction of Type 1 T cell cytokines following antigen specific stimulation [45, 47]. The balance of cytokines (IFN-γ, IL-4) elicited by mycobacterial antigens in bovine T cells, however, is more similar to cytokine profiles observed following immunization of humans than of mice. Relative levels of IFN-γ and IL-4 expressed by lymph node T cells correlate to tissue pathology and bacterial load in vaccination/challenge studies of bovine TB [15, 48]. In BCG-vaccinated neonatal calves, antigen-specific expression of IL-2 and IFN-γ by peripheral blood leukocytes correlated with clinical protection following challenge with virulent M. bovis [48]. A potential role for the Th2/Th17 cytokine IL-21 in protection against mycobacterial disease was recently identified for the first time in a calf model of TB [35]. IL-21 is a member of the common gamma chain family of cytokines (IL-2, IL-7, IL-15) and is secreted primarily by CD4+ T cells [49]. Following vaccination with BCG, CD4+ T cells from immunized cattle expressed IL-21 upon in vitro stimulation with PPD [35]. Expression of IL-21 in these studies correlated with cytotoxic activity and effector molecule expression by antigen-stimulated CD4+ T cells and occurred late in the antigen-specific response, similar to perforin. The role of IL-21 and other key regulatory factors for maintenance and induction of IFN-γ (IL-12, IL-18, IL-23, and IL-27) and NK cell function in protective immunity to TB is an important avenue of investigation in the efforts to develop a vaccine for humans and cattle.

4.2. γδ T Cells in Bovine Tuberculosis

γδ T cells may form a key component linking innate and adaptive responses to M. bovis infection in cattle given their active release of IFN-γ and relatively high prevalence in the blood of young calves as compared to adults [50]. There are two phenotypically distinct subsets of γδ T cells in cattle, workshop cluster 1 (WC1)+ CD2 and WC1 CD2+γδ T cells. Unique to Artiodactyla, WC1 is a member of the scavenger cysteine rich gene family that includes CD5 and CD6 [51]. Differences in abundance, tissue distribution, patterns of circulation, and TCR gene usage suggest that the two major γδ T cell subsets (WC1+ and WC1) play different roles in host defense [5255]. Orthologues of WC1 have only been identified in pigs and camelids [51, 5659] but there is no known orthologue of WC1 in primates. Isoforms of WC1 are encoded by a cluster of thirteen genes distributed between two loci in cattle [51, 5658] and studies have demonstrated that multiple gene products from the two loci are coexpressed forming two essentially mutually exclusive populations with an apparent dichotomy in function [60, 61]. These two subsets—referred to as WC1.1+ and WC1.2+—are defined by differential staining with specific monoclonal antibodies. Functional studies have demonstrated that the subset expressing WC1.1 is a major source of IFN-γ following antigenic stimulation [55] and is likely an early source of perforin and granulysin. The relative proportions of WC1.1+and WC1.2+cells change with age, with a predominance of WC1.1+γδ T cells in young cattle [62]. In addition, we showed that WC1+γδ T cells from young cattle had a greater capacity for IFNγ secretion, compared to WC1+γδ T cells from adult cattle, and that this was due to a higher number of WC1.1+ cells in the young calves [63].

γδ T cells respond to mycobacterial antigens including crude and defined antigens, heat shock proteins, and other nonproteinaceous components which may be expressed relatively early in infection with M. bovis or other mycobacterial species [6466]. The effector contribution of the WC1+γδ T cells to M. bovis immunity in cattle is not fully elucidated but early roles postinfection and postvaccination are indicated from a number of studies in cattle and in mice. Vaccination of calves with BCG induced an early increase in circulating WC1+γδ T cells which was associated with an increase in the secretion of antigen-specific IFN-γ [67]. We also showed that intranasal administration of BCG induced significant increases in WC1.1+ T cells in the lungs of immunised calves and that these cells clustered with DC in tissues [50]. In M. bovis-infected cattle, WC1+γδ T cells were detected within lesions early in the course of infection [68, 69]. Concurrently, numbers of circulating WC1+γδ T cells decrease shortly after infection, followed by a rapid increase [66]. In vivo depletion of WC1+γδ T cells from cattle prior to infection did not alter the course of disease [70] but, rather, significantly influenced the immune bias of the antigen-specific response. These data suggest that WC1+ cells may be involved in directing immune bias through IFN-γ secretion, as the WC1+γδ T cell-depleted cattle had increased IL-4 expression and altered immunoglobulin isotype profile as compared to nondepleted, M. bovis-infected controls [70]. Studies in mice have also revealed roles for γδ T cells in immune responses to Mycobacteria spp.. The WC1-bearing γδ T -cell population was shown to have an essential role in regulating inflammation in both the liver and the spleen of M. bovis-infected SCID-bovine heterochimeric (mouse-bovine) chimeras [37]. A similar regulatory effect of γδ T lymphocytes in inflammatory responses induced by M. tuberculosis was also reported to influence bacterial survival within tissues [71]. The early presence of γδ T cells in tuberculous lesions likely promotes containment of M. bovis via cytokine (e.g., IL-12 and IFN-γ), granulysin, and chemokine release stimulating macrophage activation and T cell recruitment [70, 72, 73]. However, recent studies in the SCID-bovine heterochimeric mouse model have shown that γδ T cells are not a primary source of chemokines in response to various agonists but may influence other cells types (including macrophages) in the production of chemokines and granuloma formation [74]. Treatment of mice with an anti-WC1 monoclonal antibody resulted in an apparent loss of control of the inflammatory response confirming important roles for WC1+γδ T cells in vivo in regulation of the immune response. Similar findings were obtained with mycobacterial infection of γδ TCR knockout mice [75, 76]. Interestingly other studies have also shown that it is the bovine WC1.1+ and WC1.2+γδ T cells which act as T regulatory cells, not CD4+CD25+FoxP3+ cells as has been observed in other species [77]. The available data suggest that bovine WC1+γδ T cells have multiple roles and can be both regulatory and stimulatory through the expression of cytokines. Their exact roles in M. bovis immunity remain to be fully revealed.

4.3. Cytotoxic Lymphocytes (CTLs) and Biomarkers for Effector Mechanisms in Bovine TB

Human and murine CD4+, CD8+, and γδ T cells exhibit CTL activity against mycobacterial-infected targets, indicating a role in immunity to TB [7881]. Production of granulysin by CTL reduces intracellular mycobacteria and appears to require perforin to gain access to the interior of the infected cell. Bovine T cells express a homologue of human granulysin, a potent antimicrobial protein stored with perforin in cytotoxic granules [34]. Memory CD4+ T cells (CD45R0+) from BCG-vaccinated animals are efficient at reducing colony forming units of BCG in infected macrophages following antigen specific stimulation [35]. This antimycobacterial activity correlates with expression of perforin, granulysin, and IFN-γ by the same memory subset. Expression of the bovine granulysin gene can be induced in CD4+, CD8+, and γδ T cells resulting in antimycobacterial activity similar to human granulysin [34, 35]. Using laser capture microdissection, granulysin mRNA was detected in the lymphocytic cuff of a forming granuloma, simultaneous with M. bovis DNA, in an experimentally challenged calf [34]. Granulysin and perforin gene expression are upregulated in peripheral blood CD4+ and CD8+ T cells in both BCG- and M. bovis ΔRD1-vaccinated calves (protected) as compared with nonvaccinated (not protected) calves [82], demonstrating the potential of these biomarkers as correlates of protection for prioritizing vaccine candidates. To date, a murine and guinea pig orthologue of granulysin has not been identified, precluding studies of the full repertoire of lytic granule proteins in rodent models of TB. Further studies of protective biomarkers in peripheral blood and granulomas of vaccinated and experimentally infected cattle have significant potential for testing strategies for augmenting immunity by vaccination.

Bovine NK cells have been identified with a mAb specific for CD335 [83, 84] and numbers of NK cells in peripheral blood were found to be highest in young calves [83, 85]. The population is comprised of CD2+ and CD2 subsets expressing combinations of killer immunoglobulin-like receptors (KIRs), leukocyte-receptor complex (LRC) CD94/NKG2C (inhibitory) and CD94/NKG2A (activating), NKG2D, and lectin-like receptors Ly49, CD69, NKP-R1, and KLRJ (reviewed in [86]). Initial studies suggest that NK cells play a significant role in the innate response to mycobacterial pathogens [33, 8789]. They are an initial source of IFN-γ, IL-17, and IL-22 that play a role in the inflammatory response to intracellular pathogens, including M. tuberculosis, and a source of perforin and granulysin. Bovine neonatal NK cells were shown to be a major source of IFNγ through interactions with BCG-infected DCs and this may be a pivotal early influence in vivo [88].

NKT cells have not been identified in cattle. Analysis of the CD1 family of proteins involved in antigen presentation to NK and NKT cells suggests NKT cells may not be present in cattle or that receptor usage differs markedly from that in humans and mice. The CD1 family in cattle is comprised of genes encoding CD1a, multiple CD1b molecules, and CD1e. An orthologue of the human CD1c gene is not present in the bovine genome. Both identified CD1d genes are pseudogenes supporting the possibility that NKT cells may be absent [90].

5. Immunological Parameters as Correlates of Protection versus Indicators of Disease

5.1. T Cell Central Memory (TcM) Immune Responses

Costly and protracted efficacy studies using various and often multiple animal models are currently used to evaluate human TB vaccine candidates [91]. Vaccine-elicited immune parameters (i.e., correlates of protection) are very much needed to prioritize the multitude of candidates. Several vaccine studies with cattle have demonstrated that TcM responses [92] negatively correlate with mycobacterial burden [93] and TB-associated pathology [94]; thus, TcM responses are positive correlates of protection. With the TcM assay, short-term T cell lines are generated via stimulation of peripheral blood mononuclear cells with specific antigens including Ag85A/TB10.4 and M. bovis PPD. Early effector T cell responses wane over time and memory cells are maintained via addition of IL-2 and fresh medium. On the final day of culture (~13d), cells are moved to plates containing autologous antigen presenting cells and antigen for elicitation of TcM responses as measured by IFN-γ ELISPOT. With two independent vaccine efficacy trials [93, 94], protected calves had greater TcM responses as compared to nonprotected calves. As with TcM responses, IL-17 responses (as measured by real time RT-PCR) also correlated with protection [94]. Recent findings indicate that IL-21 and IL-22 may also be good candidates for further evaluation (Davis and Waters, unpublished observations). These data demonstrate the potential for defining a protective signature elicited by vaccination to prioritize candidates for efficacy testing within calves.

5.2. Immune Activation as a Positive Indicator of Disease and Negative Indicator of Vaccine Efficacy

Positive prognostic indicators (i.e., as measured after challenge) for vaccine efficacy include reduced antigen-specific IFN-γ, iNOS, IL-4, and MIP1-α (CCL3) responses, reduced expansion of CD4+ cells in culture, and a diminished activation profile (i.e, ↓ expression of CD25 and CD44 and ↑ expression of CD62L) on T cells with antigen-stimulated cultures [15]. In particular, reduced responses to ESAT-6/CFP-10 upon experimental challenge are positive prognostic indicators for vaccine efficacy [44, 93, 95] whereas robust or increasing cellular immune responses (with the exception of IL-17 [94]) to ESAT-6/CFP-10 are generally a negative prognostic indicator of vaccine efficacy. These findings are consistent with the diagnostic capacity for ESAT-6/CFP-10 as antigens in cellular immune assays. Prognostic indicators offer ante-mortem monitoring techniques for vaccine efficacy studies.

5.3. Association of Antibody Responses to Antigen Burden

Antibody responses generally correlate to mycobacterial-elicited pathology [96] in accordance with the belief that Mycobacteria spp. induce antibody primarily late in the course of infection. To further evaluate the correlation of antibody responses to disease expression, calves were inoculated with M. bovis, M. kansasii, or M. tuberculosis and immune responses evaluated [9, 11]. Disease expression ranged from mycobacterial colonization with associated pathology (M. bovis), colonization without pathology (M. tuberculosis), to no colonization or pathology (M. kansasii). Antibody responses were associated with antigen burden; cellular responses (i.e., to PPD) correlated with infection but not necessarily with pathology or bacterial burden; exposure to mycobacterial antigens (in this case, injection of PPD for skin test) boosted antibody responses in presensitized animals. Thus, evaluation of antibody response to mycobacterial infections may be useful for correlation to antigen burden and prior mycobacterial sensitization. Further studies are warranted.

6. Global Strategies for Discovery: Gene Expression Profiling

One argument for use of rodent models in human tuberculosis research was the widespread availability of reagents to detect cytokines, chemokines, and for differentiation of immune cells. The advent of functional genomics, proteomics, and completion of the bovine genome sequence have dramatically improved our ability to study immunopathogenesis of TB in cattle. In addition, there has been a steady increase in numbers of antibody reagents either prepared directly against bovine proteins or validated for cross-reactivity against bovine orthologues. To date, functional genomics studies of M. bovis infection in cattle have been concentrated in two main areas: (1) in vitro studies aimed at defining changes in macrophage gene expression profiles following infection with various M. bovis isolates and (2) ex-vivo studies to define a “gene expression signature” that could be used to detect M. bovis-infected cattle. Many of these studies have relied upon early renditions of cDNA microarrays focused on bovine genes encoding proteins known to be important in immunity [97100].

Wedlock et al. compared gene expression patterns in primary bovine alveolar macrophages infected with a virulent M. bovis strain and its attenuated isogenic counterpart [101]. This study employed a cDNA microarray containing over 20,000 bovine-expressed sequence tags (ESTs) as well as amplicons representing various bovine and cervine cytokines [102]. While virulent and attenuated M. bovis isolates grew at comparable rates in the alveolar macrophages, initial analyses suggested that as many as 45% of the ESTs were differentially expressed between the two infection groups [101]. Of the 20 most differentially expressed genes, IL-8, monocyte chemotactant protein (MCP)-1 (CCL2), epithelial cell inflammatory protein-1, Groα (CXCL1), CDC-like kinase 3, and fibrinogen-like protein-2 were prominent. Wedlock et al. concluded that alveolar macrophages infected with virulent M. bovis adopted a much more proinflammatory gene expression profile than similar cells infected with the attenuated isogenic strain [101]. Another study using microarray technology found that lower levels of chemokines were expressed by M. bovis-infected alveolar macrophages than M. tuberculosis-infected cells, highlighted by the authors as a potential mechanism by which M. bovis can circumvent activation of the host chemotactic response and evade killing [103]. Of note, there appears to be species differences in the response of macrophages to M. bovis as human monocytes and monocyte-derived macrophages (MDM cells) infected with M. bovis do not produce significant IL-8 [104]. In addition, human cells infected with virulent M. tuberculosis produce large amounts of IL-10 [104], which was not observed in M. bovis-infected alveolar macrophages. However, as acknowledged by the authors, it remains a possibility that these discrepancies are due to differences between MDM cells and primary alveolar macrophages rather than actual species differences. Experiments to refine these observations have not been reported to date.

Meade et al. conducted a series of studies aimed at defining the gene expression profiles of bovine peripheral blood mononuclear cells (PBMCs) from M. bovis-infected cattle following stimulation with antigens of M. bovis (PPD-B) in vitro [99]. Samples were collected throughout a 24 hour time course of stimulation. Comparisons were made primarily between PPD-B-stimulated and -unstimulated PBMC mRNA profiles. This study utilized a bovine-specific cDNA microarray (BOTL-4) designed primarily for immune studies in cattle and containing over 1300 ESTs and amplicons spotted in triplicate [105, 106]. Statistical analysis revealed that of the >1300 genes present on the BOTL-4 microarray, 224 (~17%) were differentially expressed in PPD-B-stimulated PBMCs when compared to unstimulated PBMCs from the same chronically infected animals [99]. Major ontological classes of genes that showed significant differential expression included those encoding proteins involved in metabolism, cell communication, response to biotic stimulus, death, and development. Molecular functions most affected by stimulation were catalytic activity, protein binding, and nucleic acid binding. During the 24-hour stimulation time course, the authors observed a cyclical gene expression pattern with larger numbers of transcripts differentially expressed at 3-hour and 12-hour post stimulation relative to 6-hour and 24-hour time points. Although this could be related to cyclical receptor signalling, the fact that very few transcripts were commonly affected throughout the time course suggests that it is due to activation of early response transcripts followed by a lag (possibly due to transcription/translation of early response transcripts) followed by a secondary wave of gene expression. Alternatively, this pattern could be due to immediate response to mycobacterial antigens through, for example, Toll-like receptor signalling followed by a secondary stimulation after uptake, processing, and antigen presentation. The authors suggested that this early response pattern might represent a signature of M. bovis infection [99].

In a series of subsequent studies, Meade et al. pursued the idea that a unique and rapid gene expression pattern in PBMCs could be used to reliably detect M. bovis-infected cattle [107, 108]. Biomarker discovery using genomics and proteomics has been applied to infections with other pathogens in several host species, including humans and cattle [109112]. Meade et al. demonstrated that total leukocytes from late-stage M. bovis-infected cattle contained more lymphocytes (72%) than similar samples from control healthy cattle (43%) and that healthy controls contained more neutrophils (40%) than cells from late-stage M. bovis-infected cattle (14%) [113]. Given such differences, one would expect that total leukocyte gene expression profiles from late-stage M. bovis-infected cattle would be different than those from healthy controls. Indeed, of the >1,300 genes represented on the BOTL-5 microarray, 378 (27%) showed significant differential expression (P <  .05) between total leukocytes from healthy and infected cattle. Importantly, the suppression of innate immune gene expression detected in chronically infected animals [113] could be one mechanism by which M. bovis infection persists [31], and current diagnostics fail to identify all infected animals. Of importance, application of a hierarchical clustering algorithm identified a subset of 15 genes whose combined expression pattern appeared to be indicative of infection status [99]. It may also be the case that the early and transient profile of differential gene expression detected in these studies, could shed light on the mechanisms and kinetics of the shift in the immune system toward a nonprotective, antibody-mediated response associated with progression to chronic disease [31, 47]. It is also possible that expression patterns of this gene subset could be used to diagnose M. bovis infection in cattle. Unfortunately, this was not rigorously tested using a blinded set of samples in the present study. However a recent review from this group suggests that work in this area is on-going [114]. It waits to be seen if changes in expression of such a gene subset are specific to M. bovis infection or are a common response to bacterial invasion.

In order to address the issue of an M. bovis specific gene expression pattern, Meade et al. performed another time course study using M. bovis antigen stimulation to elicit changes in gene expression that would not necessarily be common with any other bacterial infection [107, 108]. This study revealed a subset of 18 genes that showed opposite expression changes (up- or downregulated) in cells from healthy control cattle and M. bovis-infected cattle. In keeping with previous results and relevant literature, the nature of the differentially expressed genes suggested a significant role for Toll-like receptor signalling in response to M. bovis antigens [107, 108, 113, 114]. This is postulated to have significant consequences for the development of disease in humans [115], and the same may hold true in cattle.

While there may be some species differences between the response of humans and cattle to mycobacterial infections, as discussed elsewhere in this article, there are fewer significant differences between cattle and humans than between mice and humans. This can have significant advantages for translational understanding of the mechanisms of pathogenesis [116]. Detailed pan-genomic transcriptomic analyses will also aid the understanding and potential therapeutic targeting of M. bovis infections in other natural infection models, including wildlife species that pose threats as reservoirs of infection [117119]. Significant progress has been made in defining a gene expression signature that may be diagnostic for M. bovis infection in cattle. In many cases, eradication of M. bovis is difficult because currently used diagnostic tests do not detect all infected cattle. Development of an improved test, based on genomic or proteomic biomarkers, would be a great improvement. Particular focus also needs to be paid to the development of early stage indicators of infection in order to minimize the losses and risks associated with advanced or chronic infection. Evidence for differential cytokine gene expression, found to be associated with pathology in M. bovis infected cattle [120], and a pre-existing gene expression profile in infected animals holds promise for such a resolution [108, 113]. In fact a recent study has made considerable progress in this area using comparative proteomic analysis to identify biomarkers of subclinical (latent) M. bovis infection [121]. Serum levels from experimentally infected animals showed marked increases of alpha-1-microglobulin/bikunin precursor (AMBP) protein, alpha-1-acid glycoprotein, alpha-1-microglobulin and fetulin proteins in M. bovis infected animals, which were absent from animals challenged with other closely related mycobacteria. As yet, genomic and proteomic tests have not been subjected to rigorous field testing, presumably because funding sources for a large blinded study have not been available. In addition, transfer of genomics information from M. bovis infected cattle to M. tuberculosis (and M. bovis) infections in humans has not been forthcoming. However, functional genomics has added new dimensions to our understanding of the bovine immune response to M. bovis infection, and the advent of new technologies including next-generation sequencing holds significant potential for the development of novel intervention strategies against this recalcitrant disease.

7. Summary

The advent of genomic resources for cattle has dramatically improved our ability to use this species in studies of immunity to a variety of diseases, including zoonotic infections such as M. bovis. Although the mouse model has proven useful in comparative studies of tuberculosis, recent advances in the characterization of the immune system of cattle now afford an opportunity to gain further insight into the mechanisms of immunopathogenesis utilizing a natural host/pathogen relationship. It is clear from recent investigations that the Th1/Th2 paradigm must be expanded to include the newly identified CD4 and CD8 T cells subsets that comprise the effector and regulatory subsets responding to mycobacterial antigens during different stages of infection. Further studies are needed to determine the relative contribution of Treg and Th17 subsets in the response of cattle to M. bovis infection/vaccination. Efforts to elucidate differences between the immune response profile of individuals with latent infection and patients with progressive disease suggest that latency is associated with maintenance of subsets of CD4 T cells [117]. The calf TB model affords a unique opportunity for evaluation of neonatal immune responses to vaccination/infection. Promising TB vaccines may also be evaluated in the calf (safety and efficacy) prior to testing in costly NHPs (thereby prioritizing candidates), and as presented in this review, important mechanisms of immunity may be uncovered by the use of the calf for the study of TB.

References

1. Calmette A, Guerin C. Recherches expérimentales sur la defense de l’organisme contre l’infection tuberculose. Annals Institut Pasteuer. 1911;25:625–641.
2. Marshall CJ. Progress in controlling bovine tuberculosis. Journal of the American Veterinary Association. 1932;80:625–633.
3. Wood PR, Corner LA, Plackett P. Development of a simple, rapid in vitro cellular assay for bovine tuberculosis based on the production of gamma interferon. Research in veterinary science. 1990;49(1):46–49. [PubMed]
4. von Behring E. Nobel Lectures Physiology or Medicine 1901–1921. River Edge, NJ, USA: World Scientific; 1901. Serum therapy in therapeutics and medical science.
5. Brockington CF. The evidence for compulsory pasteurization of milk. British Medical Journal. 1937;1:667–668. [PMC free article] [PubMed]
6. Francis J. The work of the British Royal Commission on tuberculosis, 1901–1911. Tubercle. 1959;40(2):124–132. [PubMed]
7. Ravenel MP. Tuberculosis of bovine origin. American Journal of Public Health and the Nation's Health. 1933;23:316–318. [PMC free article] [PubMed]
8. Wirth T, Hildebrand F, Allix-Béguec C, et al. Origin, spread and demography of the Mycobacterium tuberculosis complex. PLoS Pathogens. 2008;4(9) Article ID e1000160. [PMC free article] [PubMed]
9. Whelan AO, Coad M, Cockle PJ, Hewinson G, Vordermeier M, Gordon SV. Revisiting host preference in the Mycobacterium tuberculosis complex: experimental infection shows M. tuberculosis H37Rv to be avirulent in cattle. PLoS One. 2010;5(1, article e8527) [PMC free article] [PubMed]
10. Ernst HC. Infectiousness of Milk: Result of Investigations Made for the Trustees of the Massachusetts Society for Promoting Agriculture. Cambridge, UK: The Riverside Press; 1895.
11. Waters WR, Whelan AO, Lyashchenko KP, et al. Immune responses in cattle inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii. Clinical and Vaccine Immunology. 2010;17(2):247–252. [PMC free article] [PubMed]
12. Palmer MV, Waters WR, Whipple DL. Aerosol delivery of virulent Mycobacterium bovis to cattle. Tuberculosis. 2002;82(6):275–282. [PubMed]
13. Sambandamurthy VK, Wang X, Chen B, et al. A pantothenate auxotroph of Mycobacterium tuberculosis is highly attenuated and protects mice against tuberculosis. Nature Medicine. 2002;8(10):1171–1174. [PubMed]
14. Derrick SC, Evering TH, Sambandamurthy VK, et al. Characterization of the protective T-cell response generated in CD4-deficient mice by a live attenuated Mycobacterium tuberculosis vaccine. Immunology. 2007;120(2):192–206. [PMC free article] [PubMed]
15. Waters WR, Palmer MV, Nonnecke BJ, et al. Failure of a Mycobacterium tuberculosis ΔRD1 ΔpanCD double deletion mutant in a neonatal calf aerosol M. bovis challenge model: comparisons to responses elicited by M. bovis bacille Calmette Guerin. Vaccine. 2007;25(45):7832–7840. [PubMed]
16. Larsen MH, Biermann K, Chen B, et al. Efficacy and safety of live attenuated persistent and rapidly cleared Mycobacterium tuberculosis vaccine candidates in non-human primates. Vaccine. 2009;27(34):4709–4717. [PMC free article] [PubMed]
17. Swaim LE, Connolly LE, Volkman HE, Humbert O, Born DE, Ramakrishnan L. Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity. Infection and Immunity. 2006;74(11):6108–6117. [PMC free article] [PubMed]
18. Swaim LE, Connolly LE, Volkman HE, Humbert O, Born DE, Ramakrishnan L. Erratum: mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity (Infection and Immunity (2006) 74, 11 (6108–6117)) Infection and Immunity. 2007;75(3):p. 1540. [PMC free article] [PubMed]
19. de Jonge MI, Pehau-Arnaudet G, Fretz MM, et al. ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity. Journal of Bacteriology. 2007;189(16):6028–6034. [PMC free article] [PubMed]
20. Hsu T, Hingley-Wilson SM, Chen B, et al. The primary mechanism of attenuation of bacillus Calmette-Guérin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proceedings of the National Academy of Sciences of the United States of America. 2003;100(21):12420–12425. [PMC free article] [PubMed]
21. Volkman HE, Clay H, Beery D, Chang JCW, Sherman DR, Ramakrishnan L. Tuberculous granuloma formation is enhanced by a Mycobacterium virulence determinant. PLoS Biology. 2004;2(11, article e367) [PMC free article] [PubMed]
22. Davis JM, Ramakrishnan L. The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell. 2009;136(1):37–49. [PMC free article] [PubMed]
23. Renshaw PS, Lightbody KL, Veverka V, et al. Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6. EMBO Journal. 2005;24(14):2491–2498. [PMC free article] [PubMed]
24. Ganguly N, Siddiqui I, Sharma P. Role of M. tuberculosis RD-1 region encoded secretory proteins in protective response and virulence. Tuberculosis. 2008;88(6):510–517. [PubMed]
25. Pathak SK, Basu S, Basu KK, et al. Direct extracellular interaction between the early secreted antigen ESAT-6 of Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. Nature Immunology. 2007;8(6):610–618. [PubMed]
26. Waters WR, Palmer MV, Nonnecke BJ, et al. Signal regulatory protein α (SIRPα)+ cells in the adaptive response to ESAT-6/CFP-10 protein of tuberculous mycobacteria. PLoS One. 2009;4(7 article e6414) [PMC free article] [PubMed]
27. Hagnerud S, Manna PP, Cella M, et al. Deficit of CD47 results in a defect of marginal zone dendritic cells, blunted immune response to particulate antigen and impairment of skin dendritic cell migration. Journal of Immunology. 2006;176(10):5772–5778. [PubMed]
28. Van VQ, Lesage S, Bouguermouh S, et al. Expression of the self-marker CD47 on dendritic cells governs their trafficking to secondary lymphoid organs. EMBO Journal. 2006;25(23):5560–5568. [PMC free article] [PubMed]
29. Goddeeris BM. Immunology of cattle. In: Pastoret PP, Griebel P, Bazin H, Govaerts A, editors. Handbook of Vertebrate Immunology. San Diego, Calif, USA: Academic Press; 1998. pp. 439–484.
30. Ottenhoff THM, Verreck FAW, Hoeve MA, Van De Vosse E. Control of human host immunity to mycobacteria. Tuberculosis. 2005;85(1-2):53–64. [PubMed]
31. Pollock JM, Neill SD. Mycobacterium bovis infection and tuberculosis in cattle. Veterinary Journal. 2002;163(2):115–127. [PubMed]
32. Pollock JM, McNair J, Welsh MD, et al. Immune responses in bovine tuberculosis. Tuberculosis. 2001;81(1-2):103–107. [PubMed]
33. Endsley JJ, Endsley MA, Estes DM. Bovine natural killer cells acquire cytotoxic/effector activity following activation with IL-12/15 and reduce Mycobacterium bovis BCG in infected macrophages. Journal of Leukocyte Biology. 2006;79(1):71–79. [PubMed]
34. Endsley JJ, Furrer JL, Endsley MA, et al. Characterization of bovine homologues of granulysin and NK-lysin. Journal of Immunology. 2004;173(4):2607–2614. [PubMed]
35. Endsley JJ, Hogg A, Shell LJ, et al. Mycobacterium bovis BCG vaccination induces memory CD4+ T cells characterized by effector biomarker expression and anti-mycobacterial activity. Vaccine. 2007;25(50):8384–8394. [PubMed]
36. Cassidy JP, Bryson DG, Gutiérrez Cancela MM, Forster F, Pollock JM, Neill SD. Lymphocyte subtypes in experimentally induced early-stage bovine tuberculous lesions. Journal of Comparative Pathology. 2001;124(1):46–51. [PubMed]
37. Smith RA, Kreeger JM, Alvarez AJ, et al. Role of CD8+ and WC-1+ γ/δ T cells in resistance to Mycobacterium bovis infection in the SCID-bo mouse. Journal of Leukocyte Biology. 1999;65(1):28–34. [PubMed]
38. Villarreal-Ramos B, McAulay M, Chance V, Martin M, Morgan J, Howard CJ. Investigation of the role of CD8+ T cells in bovine tuberculosis in vivo. Infection and Immunity. 2003;71(8):4297–4303. [PMC free article] [PubMed]
39. Waters WR, Nonnecke BJ, Foote MR, et al. Mycobacterium bovis bacille Calmette-Guerin vaccination of cattle: activation of bovine CD4+ and γδ TCR+ cells and modulation by 1,25-dihydroxyvitamin D3. Tuberculosis. 2003;83(5):287–297. [PubMed]
40. Waters WR, Palmer MV, Whipple DL, Carlson MP, Nonnecke BJ. Diagnostic implications of antigen-induced gamma interferon, nitric oxide, and tumor necrosis factor alpha production by peripheral blood mononuclear cells from Mycobacterium bovis-infected cattle. Clinical and Diagnostic Laboratory Immunology. 2003;10(5):960–966. [PMC free article] [PubMed]
41. Liébana E, Aranaz A, Welsh M, Neill SD, Pollock JM. In vitro T-cell activation of monocyte-derived macrophages by soluble messengers or cell-to-cell contact in bovine tuberculosis. Immunology. 2000;100(2):194–202. [PMC free article] [PubMed]
42. Skinner MA, Parlane N, Mccarthy A, Buddle BM. Cytotoxic T-cell responses to Mycobacterium bovis during experimental infection of cattle with bovine tuberculosis. Immunology. 2003;110(2):234–241. [PMC free article] [PubMed]
43. Buddle BM, Ryan TJ, Pollock JM, Andersen P, De Lisle GW. Use of ESAT-6 in the interferon-γ test for diagnosis of bovine tuberculosis following skin testing. Veterinary Microbiology. 2001;80(1):37–46. [PubMed]
44. Vordermeier HM, Chambers MA, Cockle PJ, Whelan AO, Simmons J, Hewinson RG. Correlation of ESAT-6-specific gamma interferon production with pathology in cattle following Mycobacterium bovis BCG vaccination against experimental bovine tuberculosis. Infection and Immunity. 2002;70(6):3026–3032. [PMC free article] [PubMed]
45. Buddle BM, Skinner MA, Wedlock DN, De Lisle GW, Vordermeier HM, Hewinson RG. Cattle as a model for development of vaccines against human tuberculosis. Tuberculosis. 2005;85(1-2):19–24. [PubMed]
46. Flynn JL. Immunology of tuberculosis and implications in vaccine development. Tuberculosis. 2004;84(1-2):93–101. [PubMed]
47. Pollock JM, Welsh MD, McNair J. Immune responses in bovine tuberculosis: towards new strategies for the diagnosis and control of disease. Veterinary Immunology and Immunopathology. 2005;108(1-2):37–43. [PubMed]
48. Buddle BM, Pollock JM, Skinner MA, Wedlock DN. Development of vaccines to control bovine tuberculosis in cattle and relationship to vaccine development for other intracellular pathogens. International Journal for Parasitology. 2003;33(5-6):555–566. [PubMed]
49. Leonard WJ, Spolski R. Interleukin-21: a modulator of lymphoid proliferation, apoptosis and differentiation. Nature Reviews Immunology. 2005;5(9):688–698. [PubMed]
50. Price S, Davies M, Villarreal-Ramos B, Hope J. Differential distribution of WC1+γδ TCR+ T lymphocyte subsets within lymphoid tissues of the head and respiratory tract and effects of intranasal M. bovis BCG vaccination. Veterinary Immunology and Immunopathology. 2010;136(1-2):133–137. [PubMed]
51. Ahn JS, Konno A, Gebe JA, et al. Scavenger receptor cysteine-rich domains 9 and 11 of WC1 are receptors for the WC1 counter receptor. Journal of Leukocyte Biology. 2002;72(2):382–390. [PubMed]
52. Jutila MA, Holderness J, Graff JC, Hedges JF. Antigen-independent priming: a transitional response of bovine gammadelta T-cells to infection. Animal Health Research Reviews. 2008;9(1):47–57. [PubMed]
53. Wilson E, Aydintug MK, Jutila MA. A circulating bovine γδ T cell subset, which is found in large numbers in the spleen, accumulates inefficiently in an artificial site of inflammation: correlation with lack of expression of E-selectin ligands and L-selectin. Journal of Immunology. 1999;162(8):4914–4919. [PubMed]
54. Wilson E, Walcheck B, Davis WC, Jutila MA. Preferential tissue localization of bovine γδ T cell subsets defined by anti-T cell receptor for antigen antibodies. Immunology Letters. 1998;64(1):39–44. [PubMed]
55. Blumerman SL, Herzig CTA, Rogers AN, Telfer JC, Baldwin CL. Differential TCR gene usage between WC1 and WC1+ ruminant γδ T cell subpopulations including those responding to bacterial antigen. Immunogenetics. 2006;58(8):680–692. [PubMed]
56. Carr MM, Howard CJ, Sopp P, Manser JM, Parsons KR. Expression on porcine γδ lymphocytes of a phylogenetically conserved surface antigen previously restricted in expression to ruminant γδ T lymphocytes. Immunology. 1994;81(1):36–40. [PMC free article] [PubMed]
57. Davis WC, Heirman LR, Hamilton MJ, et al. Flow cytometric analysis of an immunodeficiency disorder affecting juvenile llamas. Veterinary Immunology and Immunopathology. 2000;74(1-2):103–120. [PubMed]
58. Morrison WI, Davis WC. Differentiation antigens expressed predominantly on CD4- CD8- T lymphocytes (WC1, WC2) Veterinary Immunology and Immunopathology. 1991;27(1–3):71–76. [PubMed]
59. Davis WC, Hamilton MJ. Use of flow cytometry to characterize immunodeficiency syndromes in camelids. Small Ruminant Research. 2006;61(2-3):187–193.
60. Davis WC, Brown WC, Hamilton MJ, et al. Analysis of monoclonal antibodies specific for the γδ TcR. Veterinary Immunology and Immunopathology. 1996;52(4):275–283. [PubMed]
61. MacHugh ND, Wijngaard PLJ, Clevers HC, Davis WC. Clustering of monoclonal antibodies recognizing different members of the WC1 gene family. Veterinary Immunology and Immunopathology. 1993;39(1–3):155–160. [PubMed]
62. Rogers AN, Vanburen DG, Hedblom E, Tilahun ME, Telfer JC, Baldwin CL. Function of ruminant γδ T cells is defined by WC1.1 or WC1.2 isoform expression. Veterinary Immunology and Immunopathology. 2005;108(1-2):211–217. [PubMed]
63. Price SJ, Sopp P, Howard CJ, Hope JC. Workshop cluster 1+ γδ T-cell receptor+ T cells from calves express high levels of interferon-γ in response to stimulation with interleukin-12 and -18. Immunology. 2006;120(1):57–65. [PMC free article] [PubMed]
64. Waters WR, Palmer MV, Pesch BA, Olsen SC, Wannemuehler MJ, Whipple DL. Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative. Veterinary Immunology and Immunopathology. 2000;77(3-4):257–273. [PubMed]
65. Welsh MD, Kennedy HE, Smyth AJ, Girvin RM, Andersen P, Pollock JM. Responses of bovine WC1+γδ T cells to protein and nonprotein antigens of Mycobacterium bovis. Infection and Immunity. 2002;70(11):6114–6120. [PMC free article] [PubMed]
66. Pollock JM, Pollock DA, Campbell DG, et al. Dynamic changes in circulating and antigen-responsive T-cell subpopulations post-Mycobacterium bovis infection in cattle. Immunology. 1996;87(2):236–241. [PMC free article] [PubMed]
67. Buza J, Kiros T, Zerihun A, Abraham I, Ameni G. Vaccination of calves with Mycobacteria bovis Bacilli Calmete Guerin (BCG) induced rapid increase in the proportion of peripheral blood γδ T cells. Veterinary Immunology and Immunopathology. 2009;130(3-4):251–255. [PubMed]
68. McNair J, Welsh MD, Pollock JM. The immunology of bovine tuberculosis and progression toward improved disease control strategies. Vaccine. 2007;25(30):5504–5511. [PubMed]
69. Cassidy JP, Bryson DG, Pollock JM, Evans RT, Forster F, Neill SD. Early lesion formation in cattle experimentally infected with Mycobacterium bovis. Journal of Comparative Pathology. 1998;119(1):27–44. [PubMed]
70. Kennedy HE, Welsh MD, Bryson DG, et al. Modulation of immune responses to Mycobacterium bovis in cattle depleted of WC1+γδ T cells. Infection and Immunity. 2002;70(3):1488–1500. [PMC free article] [PubMed]
71. D’Souza CD, Cooper AM, Frank AA, Mazzaccaro RJ, Bloom BR, Orme IM. An anti-inflammatory role for γδ T lymphocytes in acquired immunity to Mycobacterium tuberculosis. Journal of Immunology. 1997;158(3):1217–1221. [PubMed]
72. Pollock JM, Welsh MD. The WC1+γδ T-cell population in cattle: a possible role in resistance to intracellular infection. Veterinary Immunology and Immunopathology. 2002;89(3-4):105–114. [PubMed]
73. Smyth AJ, Welsh MD, Girvin RM, Pollock JM. In vitro responsiveness of γδ T cells from Mycobacterium bovis-infected cattle to mycobacterial antigens: predominant involvement of WC1+ cells. Infection and Immunity. 2001;69(1):89–96. [PMC free article] [PubMed]
74. Alvarez AJ, Endsley JJ, Werling D, Mark Estes D. WC1+γδ; T cells indirectly regulate chemokine production during mycobacterium bovis Infection in SCID-bo mice. Transboundary and Emerging Diseases. 2009;56(6-7):275–284. [PubMed]
75. Kaufmann SHE, Ladel CH. Role of T cell subsets in immunity against intracellular bacteria: experimental infections of knock-out mice with Listeria monocytogenes and Mycobacterium bovis BCG. Immunobiology. 1994;191(4-5):509–519. [PubMed]
76. Saunders BM, Frank AA, Cooper AM, Orme IM. Role of T cells in immunopathology of pulmonary Mycobacterium avium infection in mice. Infection and Immunity. 1998;66(11):5508–5514. [PMC free article] [PubMed]
77. Hoek A, Rutten VPMG, Kool J, et al. Subpopulations of bovine WC1+γδ T cells rather than CD4+CD25high Foxp3+ T cells act as immune regulatory cells ex vivo. Veterinary Research. 2009;40(1) [PMC free article] [PubMed]
78. Cho S, Mehra V, Thoma-Uszynski S, et al. Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(22):12210–12215. [PMC free article] [PubMed]
79. Dieli F, Troye-Blomberg M, Ivanyi J, et al. Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by V-γ9/Vδ2 T lymphocytes. Journal of Infectious Diseases. 2001;184(8):1082–1085. [PubMed]
80. Stenger S, Hanson DA, Teitelbaum R, et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science. 1998;282(5386):121–125. [PubMed]
81. Stenger S, Mazzaccaro RJ, Uyemura K, et al. Differential effects of cytolytic T cell subsets on intracellular infection. Science. 1997;276(5319):1684–1687. [PubMed]
82. Capinos Scherer CF, Endsley JJ, De Aguiar JB, et al. Evaluation of granulysin and perforin as candidate biomarkers for protection following vaccination with mycobacterium bovis BCG or M. bovisΔRD1. Transboundary and Emerging Diseases. 2009;56(6-7):228–239. [PubMed]
83. Kulberg S, Boysen P, Storset AK. Reference values for relative numbers of natural killer cells in cattle blood. Developmental and Comparative Immunology. 2004;28(9):941–948. [PubMed]
84. Storset AK, Kulberg S, Berg I, Boysen P, Hope JC, Dissen E. NKp46 defines a subset of bovine leukocytes with natural killer cell characteristics. European Journal of Immunology. 2004;34(3):669–676. [PubMed]
85. Graham EM, Thom ML, Howard CJ, et al. Natural killer cell number and phenotype in bovine peripheral blood is influenced by age. Veterinary Immunology and Immunopathology. 2009;132(2–4):101–108. [PubMed]
86. Boysen P, Storset AK. Bovine natural killer cells. Veterinary Immunology and Immunopathology. 2009;130(3-4):163–177. [PubMed]
87. Denis M, Keen DL, Parlane NA, Storset AK, Buddle BM. Bovine natural killer cells restrict the replication of Mycobacterium bovis in bovine macrophages and enhance IL-12 release by infected macrophages. Tuberculosis. 2007;87(1):53–62. [PubMed]
88. Hope JC, Sopp P, Howard CJ. NK-like CD8+ cells in immunologically naïve neonatal calves that respond to dendritic cells infected with Mycobacterium bovis BCG. Journal of Leukocyte Biology. 2002;71(2):184–194. [PubMed]
89. Olsen I, Boysen P, Kulberg S, Hope JC, Jungersen G, Storset AK. Bovine NK cells can produce gamma interferon in response to the secreted mycobacterial proteins ESAT-6 and MPP14 but not in response to MPB70. Infection and Immunity. 2005;73(9):5628–5635. [PMC free article] [PubMed]
90. Van Rhijn I, Koets AP, Im JS, et al. The bovine CD1 family contains group 1 CD1 proteins, but no functional CD1d. Journal of Immunology. 2006;176(8):4888–4893. [PubMed]
91. Ly LH, McMurray DN. Tuberculosis: vaccines in the pipeline. Expert Review of Vaccines. 2008;7(5):635–650. [PubMed]
92. Whelan AO, Wright DC, Chambers MA, Singh M, Hewinson RG, Vordermeier HM. Evidence for enhanced central memory priming by live Mycobacterium bovis BCG vaccine in comparison with killed BCG formulations. Vaccine. 2008;26(2):166–173. [PubMed]
93. Waters WR, Palmer MV, Nonnecke BJ, et al. Efficacy and immunogenicity of Mycobacterium bovis ΔRD1 against aerosol M. bovis infection in neonatal calves. Vaccine. 2009;27(8):1201–1209. [PMC free article] [PubMed]
94. Vordermeier HM, Villarreal-Ramos B, Cockle PJ, et al. Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis. Infection and Immunity. 2009;77(8):3364–3373. [PMC free article] [PubMed]
95. Dietrich J, Aagaard C, Leah R, et al. Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy. Journal of Immunology. 2005;174(10):6332–6339. [PubMed]
96. Lyashchenko K, Whelan AO, Greenwald R, et al. Association of tuberculin-boosted antibody responses with pathology and cell-mediated immunity in cattle vaccinated with Mycobacterium bovis BCG and infected with M. bovis. Infection and Immunity. 2004;72(5):2462–2467. [PMC free article] [PubMed]
97. Yao J, et al. Production of high-quality normalized cDNA libraries representing bovine leukocytes and swine muscle. In: Proceedings of the Plant and Animal Genome Conference; 2000; San Diego, Calif, USA.
98. Coussens PM, Colvin CJ, Wiersma K, Abouzied A, Sipkovsky S. Gene expression profiling of peripheral blood mononuclear cells from cattle infected with Mycobacterium paratuberculosis. Infection and Immunity. 2002;70(10):5494–5502. [PMC free article] [PubMed]
99. Meade KG, Gormley E, Park SDE, et al. Gene expression profiling of peripheral blood mononuclear cells (PBMC) from Mycobacterium bovis infected cattle after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD) Veterinary Immunology and Immunopathology. 2006;113(1-2):73–89. [PubMed]
100. Murphy JT, Sommer S, Kabara EA, et al. Gene expression profiling of monocyte-derived macrophages following infection with Mycobacterium avium subspecies avium and Mycobacterium avium subspecies paratuberculosis. Physiological Genomics. 2006;28(1):67–75. [PubMed]
101. Wedlock DN, Kawakami RP, Koach J, Buddle BM, Collins DM. Differences of gene expression in bovine alveolar macrophages infected with virulent and attenuated isogenic strains of Mycobacterium bovis. International Immunopharmacology. 2006;6(6):957–961. [PubMed]
102. Diez-Tascón C, Keane OM, Wilson T, et al. Microarray analysis of selection lines from outbred populations to identify genes involved with nematode parasite resistance in sheep. Physiological Genomics. 2005;21(1):59–69. [PubMed]
103. Widdison S, Watson M, Piercy J, Howard C, Coffey TJ. Granulocyte chemotactic properties of M. tuberculosis versus M. bovis-infected bovine alveolar macrophages. Molecular Immunology. 2008;45(3):740–749. [PubMed]
104. Fietta A, Meloni F, Francioli C, et al. Virulence of Mycobacterium tuberculosis affects interleukin-8, monocyte chemoattractant protein-1 and interleukin-10 production by human mononuclear phagocytes. International Journal of Tissue Reactions. 2001;23(4):113–125. [PubMed]
105. Coussens PM, Jeffers A, Colvin C. Rapid and transient activation of gene expression in peripheral blood mononuclear cells from Johne’s disease positive cows exposed to Mycobacterium paratuberculosis in vitro. Microbial Pathogenesis. 2004;36(2):93–108. [PubMed]
106. Coussens PM, Verman N, Coussens MA, Elftman MD, McNulty AM. Cytokine gene expression in peripheral blood mononuclear cells and tissues of cattle infected with Mycobacterium avium subsp. paratuberculosis: evidence for an inherent proinflammatory gene expression pattern. Infection and Immunity. 2004;72(3):1409–1422. [PMC free article] [PubMed]
107. Meade KG, Gormley E, Doyle MB, et al. Innate gene repression associated with Mycobacterium bovis infection in cattle: toward a gene signature of disease. BMC Genomics. 2007;8, artile 400 [PMC free article] [PubMed]
108. Meade KG, Gormley E, O'Farrelly C, et al. Antigen stimulation of peripheral blood mononuclear cells from Mycobacterium bovis infected cattle yields evidence for a novel gene expression program. BMC Genomics. 2008;9, article 447 [PMC free article] [PubMed]
109. Asselah T, Bièche I, Sabbagh A, et al. Gene expression and hepatitis C virus infection. Gut. 2009;58(6):846–858. [PMC free article] [PubMed]
110. Kagnoff MF, Eckmann L. Analysis of host responses to microbial infection using gene expression profiling. Current Opinion in Microbiology. 2001;4(3):246–250. [PubMed]
111. Manger ID, Relman DA. How the host ’sees’ pathogens: global gene expression responses to infection. Current Opinion in Immunology. 2000;12(2):215–218. [PubMed]
112. Skovgaard K, Grell SN, Heegaard PMH, Jungersen G, Pudrith CB, Coussens PM. Differential expression of genes encoding CD30L and P-selectin in cattle with Johne’s disease: progress toward a diagnostic gene expression signature. Veterinary Immunology and Immunopathology. 2006;112(3-4):210–224. [PubMed]
113. Meade KG, Gormley E, Doyle MB, et al. Innate gene repression associated with Mycobacterium bovis infection in cattle: toward a gene signature of disease. BMC Genomics. 2007;8, article 400 [PMC free article] [PubMed]
114. MacHugh DE, Gormley E, Park SDE, et al. Gene expression profiling of the host response to mycobacterium bovis infection in cattle. Transboundary and Emerging Diseases. 2009;56(6-7):204–214. [PubMed]
115. Doherty TM, Arditi M. TB, or not TB: that is the question—does TLR signaling hold the answer? Journal of Clinical Investigation. 2004;114(12):1699–1703. [PMC free article] [PubMed]
116. Van Rhijn I, Godfroid J, Michel A, Rutten V. Bovine tuberculosis as a model for human tuberculosis: advantages over small animal models. Microbes and Infection. 2008;10(7):711–715. [PubMed]
117. Naranjo V, Gortazar C, Villar M, de la Fuente J. Comparative genomics and proteomics to study tissue-specific response and function in natural Mycobacterium bovis infections. Animal Health Research Reviews. 2007;8(1):81–88. [PubMed]
118. Fernández de Mera IG, Pérez de la Lastra JM, Ayoubi P, et al. Differential expression of inflammatory and immune response genes in mesenteric lymph nodes of Iberian red deer (Cervus elaphus hispanicus) naturally infected with Mycobacterium bovis. Developmental and Comparative Immunology. 2008;32(2):85–91. [PubMed]
119. Gallagher J, Clifton-Hadley RS. Tuberculosis in badgers; a review of the disease and its significance for other animals. Research in Veterinary Science. 2000;69(3):203–217. [PubMed]
120. Thacker TC, Palmer MV, Waters WR. Associations between cytokine gene expression and pathology in Mycobacterium bovis infected cattle. Veterinary Immunology and Immunopathology. 2007;119(3-4):204–213. [PubMed]
121. Seth M, Lamont EA, Janagama HK, et al. Biomarker discovery in subclinical mycobacterial infections of cattle. PLoS One. 2009;4(5) Article ID e5478. [PMC free article] [PubMed]

Articles from Clinical and Developmental Immunology are provided here courtesy of Hindawi Publishing Corporation
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...