Relevance of bovine tuberculosis research to the understanding of human disease: historical perspectives, approaches, and immunologic mechanisms.

Veterinary Immunology and Immunopathology 159 (2014) 113–132

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Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm

Relevance of bovine tuberculosis research to the understanding of human disease: Historical perspectives, approaches, and immunologic mechanisms W. Ray Waters a,∗,1 , Mayara F. Maggioli a , Jodi L. McGill b , Konstantin P. Lyashchenko c , Mitchell V. Palmer a a b c

Infectious Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Ames, IA, United States Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, Ames, IA, United States Chembio Diagnostic Systems Inc., Medford, NY, United States

a r t i c l e

i n f o

Keywords: Bovine tuberculosis Central memory T cells Multi-functional T cells ␥␦ T cells IL-17 IP-10 M. bovis specific antibody

a b s t r a c t Pioneer studies on infectious disease and immunology by Jenner, Pasteur, Koch, Von Behring, Nocard, Roux, and Ehrlich forged a path for the dual-purpose with dual benefit approach, demonstrating a profound relevance of veterinary studies for biomedical applications. Tuberculosis (TB), primarily due to Mycobacterium tuberculosis in humans and Mycobacterium bovis in cattle, is an exemplary model for the demonstration of this concept. Early studies with cattle were instrumental in the development of the use of Koch’s tuberculin as an in vivo measure of cell-mediated immunity for diagnostic purposes. Calmette and Guerin demonstrated the efficacy of an attenuated M. bovis strain (BCG) in cattle prior to use of this vaccine in humans. The interferon-␥ release assay, now widely used for TB diagnosis in humans, was developed circa 1990 for use in the Australian bovine TB eradication program. More recently, M. bovis infection and vaccine efficacy studies with cattle have demonstrated a correlation of vaccine-elicited T cell central memory (TCM ) responses to vaccine efficacy, correlation of specific antibody to mycobacterial burden and lesion severity, and detection of antigen-specific IL-17 responses to vaccination and infection. Additionally, positive prognostic indicators of bovine TB vaccine efficacy (i.e., responses measured after infection) include: reduced antigen-specific IFN-␥, iNOS, IL-4, and MIP1-␣ responses; reduced antigen-specific expansion of CD4+ T cells; and a diminished activation profile on T cells within antigen stimulated cultures. Delayed type hypersensitivity and IFN-␥ responses correlate with infection but do not necessarily correlate with lesion severity whereas antibody responses generally correlate with lesion severity. Recently, serologic tests have emerged for the detection of tuberculous animals, particularly elephants, captive cervids, and camelids. B cell aggregates are consistently detected within tuberculous lesions of humans, cattle, mice and various other species, suggesting a role for B cells in the immunopathogenesis of TB. Comparative immunology studies including partnerships of researchers with veterinary and medical perspectives will continue to provide mutual benefit to TB research in both man and animals. Published by Elsevier B.V.

∗ Corresponding author at: USDA, ARS, National Animal Disease Center, 1920 Dayton Ave., Ames, IA 50010-0070. Tel.: +1 515 337 7756. E-mail address: [email protected] (W.R. Waters). 1 USDA is an equal opportunity provider and employer. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. http://dx.doi.org/10.1016/j.vetimm.2014.02.009 0165-2427/Published by Elsevier B.V.

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W.R. Waters et al. / Veterinary Immunology and Immunopathology 159 (2014) 113–132

1. Introduction: historical perspectives to the one health approach The dual purpose with dual benefit (One Health) approach originated with the onset of research into the characterization of etiologic agents for infectious diseases, associated preventive strategies (i.e., vaccines), and the immune response elicited by the agents. For instance, the principle of vaccination was developed through insightful observations and experimental studies of Edward Jenner and others on the cross protective nature of cowpox for small pox in humans. Robert Koch and Louis Pasteur, co-founders of medical microbiology, each worked extensively on diseases of veterinary and biomedical importance. Koch discovered the etiology of anthrax by inoculating sheep with blood from infected animals while Pasteur produced the first live attenuated (laboratory produced) bacterial vaccine for fowl cholera as well as an effective anthrax vaccine for cattle. The Spanish physician, Jaime Ferrán, worked on veterinary vaccines prior to developing the first vaccine for immunizing humans against a bacterial disease—cholera. With his earnings from the 1st Nobel Prize in Medicine and Physiology for the serum theory with diptheria and tetanus (von Behring, 1901), Emil Von Behring switched the focus of his work almost exclusively to the development of a vaccine for tuberculosis (TB) in cattle using chemically inactivated M. tb complex strains (e.g., Tuberkulase and Taurovaccine) or human-derived tubercle bacilli attenuated by lengthy propagation (designated Bovovaccine) (reviewed in Linton, 2005). Concurrent with Behring’s studies, McFadyean and colleagues in England and Pearson and Gilliland in the United States each demonstrated protective effects of live M. tb vaccination against experimental M. bovis infection in cattle (Flexner, 1908). This cross-protective strategy was abandoned, however, due to obvious safety concerns and variability in virulence of the human tubercle bacilli in cattle. Use of M. tb for vaccination of cattle against bovine TB, however, did establish a precedent followed by Albert Calmette and Camille Guerin in their development of an attenuated M. bovis (bacillus of Calmette and Guerin, BCG) for eventual use as a vaccine in cattle, humans, and various other species (Fine, 1995; Waters et al., 2012a). Research efforts on bovine and human TB have been intimately linked beginning as early as the seminal studies on the etiology and pathogenesis of TB performed by Koch, Jean Antoine Villemin, and others in the mid to late 1800s (Palmer and Waters, 2011). Initially, Koch postulated that the tubercle bacilli from cattle and human were identical, dismissing the studies of Villemin that demonstrated a greater virulence of bovine-origin versus human-origin isolates in rabbits. Later, studies by Theobald Smith, a physician scientist working for the Veterinary Division of the Bureau of Animal Industry, demonstrated definitive differences between the bovine and human isolates based upon variable virulence of the two isolates in cattle, rabbits and guinea pigs; as well as morphological differences and differential growth characteristics of the two organisms on glycerin media. In addition to these contributions, veterinary researchers developed essential tools for the control of bovine TB that were concurrently or later adapted for

use in the control of human TB. These include: (1) studies demonstrating the safety and efficacy of BCG (including demonstration that booster doses are generally not beneficial), (2) use of tuberculin as an in vivo diagnostic reagent, and (3) development of interferon (IFN)-␥ release assays (IGRA) for diagnosis. In 1913 at the Pasteur Institute (Lille, France), Calmette and Guerin vaccinated 9 cows with M. bovis (Nocard strain, Edmond Nocard was Guerin’s mentor) attenuated by serial passage on glycerol soaked potato slices in ox bile (i.e., BCG) (Calmette and Guerin, 1911). All 9 animals were protected from challenge with virulent M. bovis; thereby, demonstrating the potential use of BCG vaccination against M. tb infection of humans. In 1921, BCG was administered to a newborn child (6 mg orally) and has since been used widely by various administration routes for the control of human TB. Within a few years of the discovery of tuberculin by Koch, veterinary investigators in Russia (Professor Gutman), the UK (John McFadyean), Denmark (Bernhard Bang), and the US (Leonard Pearson and Maz’yck Ravenel) began using tuberculin as an in vivo diagnostic reagent for TB in cattle (Marshall, 1932). Clemens von Pirquet and Charles Mantoux later (circa 1907/1908) adapted and improved this technology for use in humans, coincidently defining the principles of allergy and delayed type hypersensitivity (DTH). During the 1980s, an IGRA was developed in Australia by Paul Wood, Leigh Corner, James Rothel, Stephen Jones, and others for the diagnosis of TB in cattle (Wood et al., 1990); a modified version of this assay is now widely used in the diagnosis of both human and bovine TB. Most recently, recombinant proteins and peptide cocktails of early secretory antigenic target-6 (ESAT-6), culture filtrate protein 10 (CFP-10), and various other antigens are in use with IGRA’s (reviewed in Vordermeier, 2010; Vordermeier et al., 2011a,b) and in development for use as skin test antigens in cattle to improve specificity over purified protein derivative (PPD) preparations and as DIVA (differentiate infected from vaccinated animals) reagents (Whelan et al., 2010a,b,c). Field studies with cattle will prove invaluable for the eventual application of similar DIVA strategies for use in the control of human TB. Undoubtedly, advances in basic immunology, antigen discovery, comparative mycobacteriology, and vaccine approaches for human TB using animal models and clinical trials in humans have advanced research efforts on bovine TB. Together, co-discovery in TB research using both veterinary and biomedical approaches is an exemplary model of the one health concept. 2. Etiology, control measures and experimental biology approaches 2.1. Etiology and epidemiology Tuberculosis in animals and humans may result from exposure to bacilli within the M. tb complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedii, M. microti, M. caprae, or M. canetti) (Cousins et al., 2003). Mycobacterium bovis is the species most often isolated from tuberculous cattle. Significant reservoirs exist for bovine TB (i.e., M. bovis) including white-tailed deer (Odocoileus virginianus, United States), Eurasian Badgers (Meles meles, United


Kingdom), brushtail possums (Trichosurus vulpecula, New Zealand), wild boar (Sus scrofa, Spain), red deer (Cervus elaphus, Spain and Canada), African Buffaloes (Syncerus caffer, South Africa) and a few others (Palmer et al., 2012). While M. tb may infect a wide variety of animals [e.g., elephants (Elephas maximus), tapir (Tapirus spp.), horses (Equus caballus), banded mongooses (Mungos mungo), meerkats (Suricata suricatta), and numerous other wildlife species, particularly in zoological collections], significant wildlife reservoirs for human infection with M. tb are not currently recognized. The comparative virulence of M. bovis versus M. tb in humans is not completely clear. Prior to widescale pasteurization, approximately 20–40% of TB cases in humans resulted from infection with M. bovis, mostly due to ingestion of M. bovis within dairy products (Brockington, 1937; Francis, 1959; Ravenel, 1933). Transmission of M. bovis between humans, however, is extremely rare (Sunder et al., 2009). Natural infection of cattle with M. tb has recently been demonstrated in East Africa (Ameni et al., 2011; Berg et al., 2009; Kankya et al., 2011) and China (Chen et al., 2009; Du et al., 2011), likely due to human-to-cattle transmission. Especially in developing countries, diagnostic laboratories rarely discriminate mycobacterial isolates obtained from tuberculous cattle and humans beyond the M. tb complex designation, thereby, hindering our knowledge of the relative prevalence of M. bovis in humans and M. tb in cattle. Direct comparisons of M. bovis and M. tb infection in cattle indicate that M. tb is less virulent for cattle (Ernst, 1895; Whelan et al., 2010a,b,c). Thus, host adaptation due to transmission capacity likely led to the divergence and speciation of M. bovis and M. tb from a common ancestor (Smith et al., 2009). 2.2. Transmission and control measures In most countries with active control programs, TBaffected cattle herds generally contain few infected animals suggestive of low transmission rates and a slowly progressive course of disease (Palmer and Waters, 2006). Transmission of bovine TB is primarily by direct contact yet indirect transmission via exposure to contaminated feed, water, or premises also occurs. Animals with advanced lesions may be particularly infectious (Khatri et al., 2012). Housing, especially in confined spaces such as milking parlors and cattle sheds, and crowding enhances the spread of disease. Movement of infected animals and fence-line contact with other herds is a common means of transferring the disease between herds and regions. Additionally, global trade agreements are increasingly being implemented to promote international trade of livestock; thereby, significantly increasing risks for inter-regional spread and distribution of bovine TB over great distances. Transmission of TB in humans is primarily by aerosol, with less evidence for indirect transmission. As with bovine TB, crowding and movement of infected individuals are population risk factors for transmission. Significant individual risk factors for human TB include: HIV infection, diabetes, kidney disease, alcohol or drug abuse, immune suppression (e.g., certain treatments for autoimmune disease), and exposure to infected persons (e.g., travel to endemic countries, health care workers, living in a shelter

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or residential care facility, etc.). As mentioned previously, exposure to wildlife reservoirs is a significant risk factor for transmission of M. bovis to cattle. Primary differences between M. bovis and M. tb transmission within their respective primary hosts are: (1) the presence of wildlife reservoirs for M. bovis infection of cattle and (2) the increased risk of indirect transmission via environmental contamination for M. bovis versus M. tb. Many countries have official bovine TB eradication/control programs. Prevention and control efforts are designed primarily to identify TB affected herds and remove infected animals or depopulate the entire herd. These efforts are generally achieved via routine slaughter surveillance to identify TB-affected herds, targeted testing schemes to remove TB test positive animals, movement restriction of affected herds (with or without movement testing), thorough epidemiologic investigations of reported cases to determine risks for other herds (i.e., trace-ins and trace-outs), and depopulation (i.e., “stamping out”) of affected herds if feasible (Schiller et al., 2010). The tuberculin skin test, currently applied either as a comparative test using both M. avium and M. bovis PPD’s injected into separate sites or as a single test with injection of M. bovis PPD only, remains after >100 years as the primary ante-mortem test for the detection of tuberculous cattle in many countries. Interferon-␥ release assays are also used in many countries (e.g., USA, UK, Ireland, New Zealand, and others) primarily as confirmatory or complementary tests in conjunction with skin test(s); although, certain countries are considering use of IGRA’s as a primary test on a limited basis. In addition to these traditional methods, antibody-based tests have recently emerged for use in the detection of tuberculous elephants (Connell and Vanderklok, 2010; Greenwald et al., 2009; primarily M. tb infection in elephants), captive cervids (Buddle et al., 2010; Waters et al., 2011a), camelids (Lyashchenko et al., 2011; Rhodes et al., 2012), and cattle (Green et al., 2009; Waters et al., 2006a, 2011b; Whelan et al., 2008a,b, 2010a,b,c). Also, molecular-based tools [e.g., mycobacterial interspersed repetitive-unit-variable number tandem repeat (MIRU-VNTR), spoligotyping, restriction fragment length polymorphism (RFLP), and high throughput whole genome sequencing of mycobacterial DNA] are used to characterize mycobacterial isolates for epidemiologic investigations to determine potential links between new cases and prior outbreaks as well as the likely origin of infection. Postmortem diagnosis is based upon gross and microscopic evaluation for tuberculous lesions, detection of the organism by mycobacterial culture of tissues with follow-up confirmation/characterization of isolates using molecular techniques, and detection of M. tb complex specific DNA by PCR on histologic sections. Surveillance and control of M. bovis infection in wildlife reservoirs, as well as mitigation efforts to limit spread from wildlife to livestock, is also critical for bovine TB control. As with cattle, skin test (not as a comparative test, M. tb PPD only) and IGRA’s are both routinely used as primary screening test(s) for TB infection/exposure in humans. The control of human TB in non-endemic regions is primarily via preventative public health measures such as surveillance and reporting of disease, diagnostic testing

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(primarily using skin test or IGRA’s with chest X-ray on positive individuals) and treatment of affected individuals, epidemiologic investigations to determine other at-risk individuals, isolation of infectious individuals, directly observed treatment strategies when warranted, treatment of confounding illness, and appropriate public health policies to prevent further transmission. In M. tb endemic regions, a wider range of control measures are implemented such as: BCG vaccination, preventive therapy (treatment of latent infection), treatment of multidrugresistant TB (MDR-TB) using both first- and second-line drugs, and use of a wider range of diagnostic tests (e.g., sputum smear microscopy and culture, molecular diagnostics, antibody-based tests, and emerging technologies), and efforts to minimize socio-economic and environmental risk factors (e.g., tobacco smoke, air pollution, malnutrition, overcrowding, and poor access to health services) (reviewed in Dye and Floyd, 2006). Exposure to environmental non-tuberculous Mycobacteria sp. (NTM) is widely recognized as a confounding factor for TB diagnosis in cattle (Barry et al., 2011); however, this concept is less widely recognized for TB diagnosis in humans as evidenced by the continued reliance on tuberculin skin test for TB screening in humans that does not differentiate M. tb infection from NTM sensitization/infection. Current testing strategies for cattle utilize differential diagnostic procedures (i.e., comparisons between CMI responses to M. avium and M. bovis PPD’s), typically not used with humans. Intensive research efforts have concentrated on the development of specific antigens for the differential diagnosis of TB infection in cattle from NTM sensitization/infection, BCG vaccination, and/or Johne’s disease vaccination (reviewed in Vordermeier et al., 2011a,b). As with human TB, recombinant proteins or synthetic peptides of ESAT-6 and CFP-10, whose genes are deleted in all BCG strains, have been most widely utilized as antigens to differentiate M. bovis-infected from NTM-exposed animals and as DIVA reagents (Buddle et al., 1999; Pollock and Andersen, 1997; van Pinxteren et al., 2000; Vordermeier et al., 2001; Waters et al., 2004). More recently, elucidation of the genomes of multiple strains of M. tb and M. bovis (both BCG and virulent strains) and other mycobacterial species has allowed a rational genome-wide approach to antigen mining. Based on these comparative genomic/transcriptomic studies, additional genes that are not transcribed or proteins that are not secreted by BCG, such as Rv3615c, have been shown to complement ESAT-6 and CFP-10 by increasing specificity without compromising sensitivity in CMI-based assays (Sidders et al., 2008; Millington et al., 2011). Interestingly, the antigen repertoire recognized by M. bovis-infected cattle and M. tb-infected humans overlap considerably (Mustafa et al., 2006; Sidders et al., 2008). The bovine model can therefore be used for antigen discovery with results applicable to the development of reagents and diagnostic tools for the control of human TB, as well as for other livestock and wildlife species. These antigens may be applied to blood based CMI assays (such as IGRA’s, reviewed in Vordermeier et al., 2011a), antibody-based tests, and skin test protocols (Pollock et al., 2003; Whelan et al., 2010a,b,c).

Similarities in bovine and human TB control programs include: a primary focus on diagnosis of infection using similarly applied diagnostic tools (e.g., skin test and IGRA’s), intensive development of vaccines and associated DIVA strategies, and an emphasis on epidemiologic investigations to determine sources of infection and exposed contacts. Differences in human versus bovine TB control programs include: complications in control strategies for bovine TB resulting from the presence of wildlife reservoirs, a necessity for the differentiation of latent versus active disease in humans, the option for anti-mycobacterial treatment in TB-infected humans, the option for a host removal strategy with bovine TB (i.e., slaughter of infected animals and, in certain instances, depopulation of affected herds), and a greater emphasis on differentiation of M. bovis-infected versus NTM-exposed animals with veterinary approaches. 2.3. Pathogenesis M. tb complex bacilli infect cells primarily of monocyte/macrophage lineage and may also be associated with type II alveolar epithelial cells, dendritic cells, neutrophils, and a few other cell types. The classic lesion of the M. tb complex, the granuloma, forms in response to virulence factors and chronic antigen stimulation due to persistent infection. Granulomas in cattle are characterized by a central core of caseous, often mineralized material, surrounded by infiltrates of epithelioid macrophages, Langhan’s type multinucleated giant cells and lymphocytes; all of which are often surrounded by a discreet, thickened fibrous capsule, The level of fibrous encapsulation varies depending on the rate of development and chronicity of infection (i.e. more chronic lesions generally contain a greater degree of fibrosis). Morphologically, tuberculous granulomas in cattle resemble those seen in humans. With both bovine and human TB, acid-fast bacilli are generally present in low numbers (i.e., paucibacillary). In humans, caseonecrotic lesions may become liquefactive and form cavities within affected lung. Cavitation is uncommon in cattle. In contrast to caseous lesions, liquefactive lesions can contain large numbers of acid-fast bacilli (multibacillary). From the initial site of entry (often lungs, especially with human TB due to M. tb), the organism invades local lymph nodes often causing granulomas. The granuloma is thought to limit the spread of the tubercle bacilli within the host by walling-off the infection. The combination of the initial site of infection and the regional lymph node constitute the primary complex commonly referred to as the Ghon’s complex with pulmonary M. tb infection of humans. Dissemination from the primary complex occurs via both lymphatic and hematogenous spread. Determination of the primary complex (indicative of the route of infection) may be impossible, especially in cases of prolonged or severe disseminated disease. Post-primary dissemination may occur leading to spread of the organism to other lymph nodes and organs. Diffuse miliary TB may occur in severe cases. Latency (i.e. infection and limited primary lesion formation without disease progression) is a common sequela with M. tb infection of humans; however, latency is typically not considered a common stage of disease with M.


bovis infection of cattle. With that said, M. bovis may be isolated from tissues without visible gross lesions, indicating a potential for ‘latent’ infection. However, in most cases, it is not certain if these infections represent early lesions without visible gross lesions or persistent infections without disease progression. 2.4. Experimental biology approaches Aerosol and intratracheal inoculation are the most common routes currently used to experimentally infect cattle with virulent M. bovis (reviewed in Hewinson et al., 2003; Pollock et al., 2006; Waters et al., 2012a). These routes result primarily in a respiratory tract infection (i.e., lungs and lung-associated lymph nodes), severity is dose and time dependent, and the disease closely mimics natural infection of cattle (Buddle et al., 1994; Palmer et al., 2002). Experimental infection of cattle with virulent strains of M. bovis elicits robust cellular immune responses [e.g., IFN-␥, TNF␣, IP-10, and DTH responses (Pollock et al., 2001; Waters et al., 2003a, 2012b)] beginning as early as 2–3 wks after challenge and humoral immune responses (both IgM and IgG) typically 2–4 weeks later (Waters et al., 2006a). Experimental approaches permit disease confirmation through postmortem examination and the capacity to quantitatively measure the severity of gross and microscopic lesions (Wangoo et al., 2005). Additionally, tissues are collected for assessment of mycobacterial colonization (qualitative and quantitative measures). Thus, experimental variables such as prior vaccination, immune response, and dose/strain may be correlated with lesion severity and mycobacterial colonization. In contrast to virulent field strains of M. bovis, inoculation of cattle with M. bovis AN5 or M. bovis Ravenel (both are laboratoryadapted strains) results in robust cell-mediated immune (CMI) responses and colonization, yet only minimal to no tuberculous lesions 4–5 months after challenge (Martin Vordermeier, AHVLA, personal communication; Waters et al., unpublished observations), similar to infection of cattle with M. tb H37Rv (Whelan et al., 2010a,b,c). Thus, as with experimental biology approaches using M. tb in various animal models, strain selection and laboratory maintenance of isolates are critical variables for consideration when using M. bovis. Experimental protocols are also developed for infection/sensitization of cattle with NTM for comparative immunology, pathogenesis and diagnostic studies (Waters et al., 2004, 2006b). Interactions of NTM exposure on TB vaccine efficacy may also be evaluated. In a study in which cattle were naturally exposed to environmental mycobacteria prior to vaccination, BCG vaccination did not induce protection against TB, while vaccination with two other attenuated strains of M. bovis induced protection (Buddle et al., 2002). Particularly in bovine TB endemic regions, large-scale field trials are possible with cattle for vaccine efficacy and diagnostic test evaluation. Field trials provide a unique opportunity for evaluation of vaccines and diagnostic tests with the intended target population; however, variables such as NTM exposure, co-infection, stress, and limitations on sampling (particularly with necropsy limitations and timing of blood sampling) may confound interpretation of results. With humans, experimental

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biology approaches are generally limited to in vitro analysis and opportunistic studies with samples obtained from naturally infected individuals. Field studies in humans are particularly useful, however, for evaluation of emerging diagnostic approaches (Walzl et al., 2011). In regions with official bovine TB control programs, vaccines are rarely used for the control of bovine TB, primarily due to the potential for interference with traditional ante-mortem testing strategies using PPD as antigen (i.e., tuberculin skin test and IGRA’s) (Moodie, 1977). With that said, multiple countries are seriously considering vaccine approaches to control bovine TB in cattle, particularly in regions with wildlife reservoirs (e.g., England, Wales) or lack of an effective surveillance program (e.g., Latin America). Vaccine efficacy studies in cattle provide a unique opportunity to evaluate safety, immunogenicity, and efficacy of emerging TB vaccines, with relevance for efforts to develop TB vaccines intended for humans. Additionally, efficacy studies are easily performed with neonatal calves, a target population for both cattle and human TB vaccines (Endsley et al., 2009). Numerous experimental and field vaccine efficacy studies have demonstrated that BCG is effective in cattle, although the level of protection varies dramatically across studies – as occurs in humans (reviewed in Waters et al., 2012a). Multiple vaccine platforms have been tested with cattle such as attenuated mutants, subunit, DNA, heterologous prime-boost strategies, and several adjuvant preparations (reviewed in Vordermeier, 2010). Cattle have been the first natural host for TB where prime-boost or combinations of BCG and DNA, protein or virus-vectored vaccines have been shown to induce better protection against TB than does BCG alone (Wedlock et al., 2003; Skinner et al., 2003a, 2005; Vordermeier et al., 2009). Recent experimental trials with cattle have demonstrated that adenoviral-vectored Ag85A may boost immunity elicited by BCG in cattle, BCG is particularly protective when administered to neonates, and DIVA approaches are feasible in cattle using both in vitro or in vivo methods (reviewed in Vordermeier et al., 2011b and Waters et al., 2012a). BCG vaccination of calves within 12 h of birth induces a high level of protection against subsequent M. bovis challenge, whereas BCG vaccination at birth and booster vaccination at 6 weeks of age induces significantly less protection compared to a single vaccination (Buddle et al., 2003). Thus, cattle offer a unique model to study not only the safety and efficacy of candidate TB vaccines in a natural host pathogen system but also to evaluate potential interactions such as exposure to NTM’s, age (i.e., neonatal immunology), dose, co-infection (e.g., parasites), nutritional status, and combinations of the various approaches. 3. Cell-mediated immunity 3.1. IFN- and delayed type hypersensitivity (DTH) responses An essential component of the immune response to TB (bovine and human) is the production of IFN-␥ by T helper 1 (Th1) CD4 T cells (Cooper and Torrado, 2012; Torrado and Cooper, 2011). Immune deficiencies affecting CD4 T cells,

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as with AIDS patients, and IL-12/IFN-␥/STAT1 signaling pathways results in more severe disease in TB-affected individuals (Diedrich and Flynn, 2011; Cooper et al., 2007). Given the importance of Th1 cells in the response to TB, it is not surprising that IFN-␥ (i.e., IGRA’s) and DTH (i.e., skin test) responses are useful correlates to infection (reviewed by Schiller et al., 2010 for cattle and Walzl et al., 2011 for humans). Most effective TB vaccines also elicit specific IFN-␥ and DTH responses (Black et al., 2002), but not all vaccines that induce IFN-␥ and DTH responses are protective against TB. Additionally, the amount of IFN-␥ produced in response to vaccination does not necessarily correlate with the level of protection afforded by the vaccine (Abebe, 2012; Mittrücker et al., 2007; Waters et al., 2012a). For instance, cattle vaccinated with BCG-Danish have similar levels of protection as cattle vaccinated with BCG-Pasteur, despite having significantly lower vaccine-elicited IFN␥ production to M. bovis PPD (Wedlock et al., 2007). In addition to post vaccination (pre-challenge) parameters, immune responses elicited after infection may be compared with efficacy indicators to determine post challenge correlates of protection. After M. bovis challenge, responses with diagnostic potential, such as IFN-␥ responses, are generally inversely correlated with responses that predict vaccine efficacy. For instance, a robust ESAT-6/CFP-10specific IFN-␥ response after challenge is a negative prognostic indicator of vaccine efficacy as these responses have been shown to positively correlate with TB-associated pathology (Dietrich et al., 2005; Vordermeier et al., 2002; Waters et al., 2007, 2009). These findings are not surprising as it would be expected that failed vaccine approaches would result in increasing antigen burden and associated pathological changes; thus, evoking immune responses indicative of infection. With that said, there is no indication that the level of DTH responses detectable after M. bovis challenge correlates with the level of pathological changes in cattle or vaccine-elicited protection; however, analysis of DTH responses is often confounded by responses evoked by attenuated live vaccines (Waters et al., 2009). Examples of positive prognostic indicators of vaccine efficacy as measured after challenge include: reduced antigen-specific IFN-␥, iNOS, IL-4, and MIP1-␣ (CCL3) responses; reduced antigen-specific 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 within antigen stimulated cultures from protected versus non-protected cattle (Waters et al., 2003b,c, 2007). Recent RNA-seq and microarray studies with samples from M. bovis-infected cattle have identified additional candidates (IL-22, LT-␣, granzyme A and B, CXCL-9, and CXCL-10) for further evaluation as biomarkers of infection and vaccine efficacy in cattle (Aranday-Cortes et al., 2012a,b; Bhuju et al., 2012; Thacker et al., 2011). 3.2. Effector and T cell central memory (Tcm) immune responses Memory T cell responses are generally considered essential for vaccine-elicited protection against intracellular infectious agents; however, the relationship between effector T cell responses and long lasting T cell memory

is not completely understood in either humans (Todryk et al., 2009) or cattle (Waters et al., 2009). Immunological memory develops naturally as a result of infection. Vaccinations usually aim at mimicking an infection to generate memory cells capable of responding more rapidly and efficiently upon subsequent infection. Pathogens and their derivatives are generally transported to lymphoid organs by APCs for initiation of T cell responses. Initiation of this primary response may take days to weeks to develop relying on exposure of naive T cells to antigens in secondary lymphoid organs, expansion of antigen-specific cells, and homing of effector cells to the site of infection (Mackay et al., 1990). With TB, this 2–3 week delay in the response at the primary site of infection seems to be advantageous for the agent, and is a feature of the infection in cattle, humans, and mice (Ernst, 2012). The recirculation feature of naive T cells is mediated, in part, by expression of CD62L and CCR7, required for migration from blood into lymph nodes across the high endothelial venules, and the lack of the homing/chemokine receptor combinations required for entry into lymphoid sites (Butcher and Picker, 1996; von Andrian and Mackay, 2000; Bromley et al., 2008). Experiments with mice have demonstrated that the numbers of Ag-specific T cells increase up to ∼10,000fold after initial encounter with cognate antigen and subsequent proliferation/activation (Hou et al., 1994; Murali-Krishna et al., 1998; Whitmire et al., 1998). Besides the clonal expansion, activated T cells differentiate and exhibit effector functions, characterized by expression of important mediators of pathogen control (e.g. IFN-␥, TNF␣, IL-17, and cytotoxic granules). These cells down regulate the expression of CD62L and CCR7, while up regulating non-lymphoid homing receptors, such as CXCR3 and CCR5 (Reinhardt et al., 2001). If the immune system successfully controls the infection, 90–95% of the Ag-specific T cell population undergoes apoptosis and only a few memory cells remain (Totté et al., 2010; Wilkinson et al., 2009). Sallusto et al. (1999) identified two functionally distinct subsets of memory CD4 T cells in humans (CD45RA− /CD45RO+ ) based on expression of the lymphoid homing receptors CD62L and CCR7: (1) central memory T (Tcm) cells which are CD62L+ CCR7+ and preferentially localize to lymphoid tissues and (2) effector memory T (Tem) cells which are CD62L− CCR7− and preferentially localize to peripheral tissues. Tcm cells show great proliferation and IL-2 production capabilities (Champagne et al., 2001; Sallusto et al., 2010). Tem cells may remain blood associated, either circulating or contained within splenic red pulp or hepatic sinusoids. Tem cells have immediate effector functions and may maintain preformed cytotoxic granules for rapid cytolysis of infected host cells (Woodland and Kohlmeier, 2009). However, upon restimulation, Tem cells undergo relatively little proliferation and secrete minimal IL-2 (Sallusto et al., 2004, 2010; Champagne et al., 2001). The effector response to mycobacterial antigens is often accessed on diagnostic tests (e.g., IFN-␥ production by blood leukocytes via Bovigam®, Quantiferon®, or ELISPOT assays) demonstrating that this response is a good correlate to infection. Effector IFN-␥ responses to ESAT-6 and CFP-10 during the early stages of experimental M. tb infection positively correlate to disease severity in non-human primates


(Lin et al., 2009). Monitoring effector IFN-␥ responses to ESAT-6 and CFP-10 is also a good measure of TB exposure in humans (Doherty et al., 2002). HIV infection is a primary risk factor for developing active TB. HIV-1 infected individuals show not only lower levels of CD4 cells but also impairment of T cell function. Disease progression and increase in HIV-1 load correlate with a loss of IL2 secretory function by CD4+ T cells (Day et al., 2008). Combined antiretroviral therapy restores immunity and reduces the risk of TB in HIV-infected individuals, primarily via enhancement of Tcm responses (Wilkinson et al., 2009). Additionally, assessment of long-term IFN-␥ production (i.e., as a surrogate to Tcm responses) may be used to detect past M. tb infection in patients with negative short-term culture assays (i.e., standard IGRA’s measuring effector and Tem responses) (Butera et al., 2009). Thus, while measure of effector responses is as a correlate of TB infection, measurement of Tcm responses may also provide diagnostic benefit. Upon stimulation by mycobacterial antigens (e.g., rESAT-6:CFP-10 or M. bovis PPD), bovine peripheral blood CD4, CD8, and ␥␦ T cells from M. bovis-infected cattle proliferate and display an activated phenotype (i.e., ↑CD25, ↑CD26, ↑CD44) after 3-6 days in culture (Waters et al., 2003b; Maue et al., 2005). Mycobacterial antigenactivated CD4 cells also decrease expression of CD62L and increase the expression of CD45RO (associated with memory T cells) while decreasing the expression of CD45R (associated with naive T cells phenotype) (Maue et al., 2005). The variant splices A, B and C of CD45 receptor are not described for cattle (Bembridge et al., 1995). These findings demonstrate that cattle exhibit an expected T cell effector phenotype upon antigen activation within short-term cultures. Less, however, is known about the phenotype of Tem and Tcm populations in cattle. Vaccine efficacy studies with cattle have demonstrated that long-term T cell responses (Whelan et al., 2008a,b) negatively correlate with mycobacterial burden (Waters et al., 2009) and TB-associated pathology (Vordermeier et al., 2009). Additionally, BCG vaccination of neonatal calves induces significant protection against M. bovis challenge at 12 months but not at 24 months after vaccination; and, loss of efficacy correlates with a significant reduction in the numbers of antigen-specific IFN-␥-secreting cells within long-term PBMC cultures (Thom et al., 2012). Studies with samples from humans have demonstrated that the responding cells within these long-term cultures (up to 14 days) are mainly Tcm’s and that this response, in contrast to effector responses, correlates with better infection outcomes (Todryk et al., 2009). With the longterm culture assay for cattle, T cell lines are generated via stimulation of PBMC with specific antigens including Ag85A, TB10.4 and M. bovis PPD. Effector T cell responses wane over time and memory cells are sustained via addition of IL-2 and fresh medium. After 13 days of culture, cells are washed, transferred to plates containing autologous APCs, cultured overnight, and the ensuing response measured by IFN-␥ ELISPOT. Tcm’s likely contribute to the long-term cultured ELISPOT response to BCG vaccination by cattle, although a lack of an anti-bovine CCR7 antibody has hindered this characterization. In the assessment of

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the migration pattern of ␥␦ T cells, Vrieling et al. (2012) recently demonstrated that an anti-human CCR7 antibody cross-reacts with bovine CCR7 molecules. Utilizing this antibody, our group has identified the primary contribution of Tcm cells, with a minor contribution by Tem cells, in the long-term cultured IFN-␥ ELISPOT response to M. bovis-infection of cattle (Maggioli et al., 2012). Data on the response to vaccination and subsequent challenge that make it possible to access the correlation between Tcm and Tem responses to protection/pathology are still lacking. However, these data demonstrate the potential for defining a protective signature elicited by vaccination to prioritize candidates for efficacy testing within calves. 3.3. IL-17 responses Recently, there has been considerable interest in the role of IL-17 producing cells in the immune response to TB (Cooper, 2010). M. tb infection of mice and humans (Jurado et al., 2012; Khader and Cooper, 2008) and M. bovis infection of cattle (Vordermeier et al., 2009) results in a significant IL-17 response. Early expression of IL-17 is required for rapid accumulation of protective memory cells in TB infection of mice (Khader et al., 2007); however, the absence of IL-17 during infection only modestly alters the inflammatory response (Khader et al., 2005; Umemura et al., 2007). With aerosol BCG infection of mice, IL-17A produced by V␥4+ and V␥6+ ␥␦ T cells are necessary for appropriate maturation of granulomas (Okamoto Yoshida et al., 2010). IL-17 is produced primarily by ␥␦ and other non-CD4+ T cells during M. tb infection in mice; and, early IL-17 produced by ␥␦ T cells occurs prior to ␣␤ T cell priming, thus, biasing the ensuing adaptive response (Lockhart et al., 2006). However, excessive IL-17 responses may be detrimental. Cruz et al. (2010) demonstrated that repeated BCG vaccination of M. tb-infected mice exacerbates inflammation due to infection. This exaggerated response, however, is not detected in mice genetically deficient in IL-23p19 or in mice treated with anti-IL-17 blocking antibody, demonstrating the dependence of IL-17 on the damaging response (Cruz et al., 2010). The excessive inflammation in these mice is associated with increased numbers of IL-17 producing T cells and a substantial influx of granulocytes (Gr1 + cells) as well as increased amounts of MIP2, TNF-␣, and IL-6 within affected lungs. Together, these findings suggest that the timing and amount of IL17 produced in response to TB infection is critical for the balance between responses that support control of the bacilli versus detrimental inflammatory responses. Indeed, retinoic acid receptor-related orphan receptor ␥ (ROR␥) inhibitors are in consideration for inclusion in treatment regimens for M. tb infection of humans (Baures, 2012). A presumed mechanism of action of ROR␥-inhibitors is to promote a more favorable IL-17/IFN-␥ balance via inhibition of IL-17 production. With both M. bovis infection and BCG plus viralvectored Ag85A vaccination of cattle, stimulation of PBMC cultures with either M. bovis PPD or Ag85A elicits IL-17 mRNA expression (Vordermeier et al., 2009). Vaccineelicited IL-17 responses to Ag85A stimulation 10 wks after vaccination and prior to challenge negatively correlate

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with TB-associated pathology (Vordermeier et al., 2009). Likewise, Rizzi et al. (2012) recently demonstrated that IL-17 mRNA expression is increased in response to M. bovis PPD stimulation in cattle vaccinated with a BCG strain over-expressing Ag85B; and, this post-vaccination/prechallenge IL-17 response negatively correlates with lesion severity after experimental infection. IL-17 responses to M. bovis PPD detected after challenge, however, do not correlate with protection as there is a ∼20 fold increase in IL-17 gene expression detected in samples from non-vaccinated, vaccinated/protected, and vaccinated/non-protected groups (Vordermeier et al., 2009). Similar IL-17 responses are detected post-challenge, regardless of vaccineevoked protective immunity. Thus, vaccine-elicited IL-17 responses are either boosted post-challenge or persist, despite protection. IL-17 protein is also detectable within M. bovis PPD or rESAT-6:CFP-10-stimulated whole blood or PBMC cultures from M. bovis-infected cattle (McGill et al., 2012). Additionally, IL-17 expression (mRNA, 60 and 90 days after experimental infection) in response to M. bovis PPD correlates with the presence of gross tuberculous lesions, suggesting that IL-17 may prove useful as a biomarker of infection (Blanco et al., 2011). Using laser capture microdissection followed by qPCR, ArandayCortes et al. demonstrated increased IL-17A and IL-22 expression within tuberculous granulomas as compared to non-affected tissues from experimentally infected cattle (Aranday-Cortes et al., 2012a,b). Expression of IL-17A and IL-22 was greatest in early lesions with decreasing expression in more advanced lesions (as defined by increasing fibrosis/necrosis upon microscopic evaluation). A reason for this differential expression in early versus advanced lesions is unclear; however, it may indicate a temporal connection for these cytokines in the response to TB. Further studies with bovine TB should prove useful for delineating potential roles for IL-17A and other Th17 cytokines (e.g., IL-22, Bhuju et al., 2012) in protective and detrimental responses to the tubercle bacillus in cattle. 3.4. ı T cells Accumulating evidence suggests that ␥␦ T cells play a critical role in the early response to M.tb and M. bovis, and may be key in bridging innate and adaptive immunity following infection. The frequency of ␥␦ T cells circulating in humans and mice is rare, representing 5–10% of the circulating peripheral lymphocyte population (Kabelitz, 2011). In contrast, ␥␦ T cells are significantly more abundant in ruminant species, where they constitute up to 70% of the circulating peripheral blood lymphocytes in very young animals (Hein and Mackay, 1991; Jutila et al., 2008). The increased frequency of ␥␦ T cells in cattle indicate a critical role for this population in the immune system of the ruminant and, compared to species with rare circulating frequencies (e.g., mice and humans), make it an excellent model for dissecting the role of ␥␦ T cells in the response to infections such as Mycobacterium spp. Populations of bovine ␥␦ T cells are commonly divided based upon their expression of Workshop Cluster 1 (WC1) (Clevers et al., 1990; Machugh et al., 1997; Mackay et al., 1989; Morrison and Davis, 1991), a member of the scavenger receptor

cysteine rich (SCRC) superfamily, which includes CD163, CD5, CD6 and DMBT1 (Sarrias et al., 2004). The majority of peripheral blood bovine ␥␦ T cells express WC1, and it is the WC1+ subpopulation that has been shown to respond to M. bovis infection; although, recent studies indicate that WC1− ␥␦ T cells are also responsive (McGill et al., 2012). ␥␦ T cells expand significantly in the blood of mice infected with BCG (Janis et al., 1989) and humans with active tuberculosis (Meraviglia et al., 2011). In the bovine, ␥␦ T cells also undergo dynamic changes, with a marked decrease in the circulating population shortly after M. bovis infection (Cassidy et al., 1998; Pollock et al., 1996). The initial drop in the frequency of peripheral ␥␦ T cells has been attributed to movement out of circulation to the site of infection. Indeed, WC1+ ␥␦ T cells are amongst the first cells to accumulate at the site of DTH responses following PPD injection of M. bovis infected cattle (Doherty et al., 1996), and at the site of early lesion development in the lungs and lymph nodes of virulent M. bovis infected cattle (Cassidy et al., 1998; Palmer et al., 2007). Following intranasal BCG vaccination, bovine ␥␦ T cells also rapidly accumulate, infiltrating all lobes of the lungs, the pharyngeal tonsils and the lymph nodes of the head (Price et al., 2010). In mice, BCG inoculation also results in a rapid recruitment of ␥␦ T cells to the lungs and pulmonary lymph nodes (Dieli et al., 2003). Following the initial decrease in circulation, WC1+ cells from M. bovis infected cattle undergo significant expansion in the blood, with a concomitant increase in activation as marked by up-regulated CD25 expression, indicating that the cells are undergoing an active response to infection (Pollock et al., 1996). Early studies by Smyth et al. and Rhodes et al. reported that ␥␦ T cells from M. bovis infected cattle responded in vitro to complex antigens of M. bovis including PPD, M. bovis sonic extract and M. bovis culture filtrate proteins with proliferation (Smyth et al., 2001; Rhodes et al., 2001) and robust IFN-␥ production (Rhodes et al., 2001). It has subsequently been shown that bovine ␥␦ T cells recognize protein antigens of M. bovis, with robust responses to Ag85 and ESAT-6 (Rhodes et al., 2001; Waters et al., 2006a,b), both known to elicit robust CD4 and CD8 T cell responses, and minor responses to the protein antigens MPB83, MPB70 and hsp16.1 (Rhodes et al., 2001). Human and murine ␥␦ T cells recognize both protein and nonprotein phosphoantigens of M.tb (Born et al., 1990; Fournie and Bonneville, 1996; Haregewoin et al., 1989; Morita et al., 1995) and the same also appears true for bovine ␥␦ T cells. Welsh et al. (2002) demonstrated that while proteinase-K treatment of M. bovis sonic extracts ablates the ability of CD4 T cells to respond, ␥␦ T cells retain their ability to proliferate and produce IFN-␥. A subsequent study by Vesosky et al. (2004) identified mycolylarabinogalactan peptidoglycan, a component of the mycobacterial cell wall, as a primary non-protein antigen for bovine ␥␦ T cells. Through in vitro and in vivo reports, it is evident that bovine ␥␦ T cells are responding to M. bovis infection; however, the physiologic role of this response in immune protection is still unclear. ␥␦ T-cell deficient mice are able to control BCG (Ladel et al., 1995) and lowdose M. tuberculosis infection (D’Souza et al., 1997), but exhibit significantly larger and less-organized granulomas


in both cases, suggesting a role for ␥␦ T cells in granuloma formation. In agreement, depletion of WC1+ ␥␦ T cells from SCID-bo mice prior to M. bovis infection significantly alters the architecture of the developing granuloma (Smith et al., 1999). However, depletion of WC1+ ␥␦ T cells from M. bovis-infected cattle has no effect on disease severity or granuloma formation (Kennedy et al., 2002). Instead, these animals exhibit a significant reduction in early IFN-␥ production and an increased skewing towards Th2 type immunity, suggesting a role for ␥␦ T cell-derived cytokines in establishing Th1 immunity. Along those lines, it has been described that reciprocal interactions between murine and human ␥␦ T cells and M. tb-infected DC can lead to enhanced IFN-␥ and IL-12p70 production, respectively (Dieli et al., 2004; Martino et al., 2007; Meraviglia et al., 2010). This DC-␥␦ T cell cross-talk promotes the development of robust Th1 responses. Work by Price and Hope (2009) recently demonstrated that the same reciprocal interactions occur with bovine DC and ␥␦ T cells. Incubation of WC1+ ␥␦ T cells with M. bovis-infected DC induced significantly enhanced expression of MHC II and CD25, as well as increased secretion of IFN-␥ by the ␥␦ T cells (Price and Hope, 2009). In turn, DC produced enhanced levels of biologically active IL-12 when incubated with ␥␦ T cells. In addition to the studies above, ␥␦ T cells have also been described to fulfill other functions in the immune response to mycobacteria, including IL-17 production (Lockhart et al., 2006; Umemura et al., 2007) and direct cytoxicity (Skinner et al., 2003b; Stenger et al., 1998); however, the role of these functions in cattle during M. bovis infection are not well defined. Together, it is obvious that ␥␦ T cells play a critical role in bridging innate and adaptive immunity and promoting the development of a robust, and appropriate, adaptive immune response to M. bovis. Future studies should be aimed at more clearly defining the role of ␥␦ T cells in vivo during M. bovis infection and importantly, future attempts at vaccine development should make note of the contributions of ␥␦ T cells during mycobacterial infections and strive to engage this population in protective immune responses. 3.5. Chemokines Chemotactic cytokines (chemokines) are essential for granuloma formation with mycobacterial infections (Slight and Khader, 2012). As early as 12–21 days after low dose aerosol infection with M. tb, mRNA for numerous chemokines and their receptors are up-regulated within lungs of infected mice, correlating with recruitment of immune cells into the lung (Kang et al., 2011). The early accumulation of NK cells, ␥␦ T cells, and macrophages as well as slightly later CD4 T cells, CD8 T cells, dendritic cells, and B cells coincide with the induction of chemokine expression within infected lung tissues, with specific chemokines being associated with specific cell types. Chemokines such as CXCL13, CCL21, and CCL19 appear to at least partially mediate the spatial localization of immune cells within the granuloma, thereby, participating in mycobacterial control (Khader et al., 2009). Inflammatory chemokines are induced very early

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following M. tb infection in mice recruiting neutrophils, NK cells, monocytes, and lymphocytes via specific receptors such as CCR2/5 and CXCR1/CXCR2. In response to inflammation, T and B cells up-regulate CXCR3, CCR5, and CCR6 and accumulate in TB-affected lung tissues. Lung dendritic cells carrying M. tb up-regulate CCR7 and migrate along chemokine gradients to lymphoid organs where they interact with T cells, promoting cytokine production. T cells, monocytes, macrophages, basophils and immature dendritic cells each express CCR2. Following aerosol M. tb infection, immune cell migration into lungs and pulmonary lymph nodes is delayed in mice genetically deficient in CCR2 (Peters et al., 2001; Scott and Flynn, 2002). Mycobacterial colonization, however, is similar between CCR2−/− and wild type mice after low dose M. tb infection whereas challenge with moderate to high doses results in more severe disease in CCR2−/− versus wild type mice (Peters et al., 2001). These findings suggest that a sufficient number of macrophages are able to migrate to the lung to control low dose M. tb infection in CCR2−/− mice whereas macrophage numbers are not sufficient to control the disease with higher doses of M. tb. Thus, a “normal” macrophage response may actually be exaggerated with low dose M. tb infection of mice. CCR5 is a chemokine receptor on T cells, macrophages, and dendritic cells. Unexpectedly, mice genetically deficient in CCR5 are able to control M. tb infection, producing organized granulomas with increased numbers of T cell lung infiltrates as compared to wild type mice without a corresponding increase in mycobacterial numbers (Algood et al., 2004). With non-human primates, chemokine expression patterns are similar to those of mice in response to M. tb infection, yet less fully characterized (Mehra et al., 2010). With samples from humans, numerous studies have demonstrated various patterns of chemokine and chemokine receptor expression by various cell types (e.g., macrophages, alveoloar and bronchial epithelial cells, neutrophils) and from cells within bronchial alveolar lavage fluid following in vitro stimulation with mycobacterial antigens (reviewed by Algood et al., 2003). Additionally, polymorphisms in several chemokines (e.g., CXCL10, RANTES, IL-8, MCP-1) are variably associated with TB susceptibility in humans (Azad et al., 2012). IFN-␥-induced Protein 10 (IP-10, also termed CXCL10) is detectable within tuberculous granulomas and in DTH reactions in response to mycobacterial antigens of humans (Ferrero et al., 2003; Kaplan et al., 1987). IP-10 attracts activated T cells and monocytes to inflammatory foci (Farber, 1997), has antimicrobial functions (Cole et al., 2001), and promotes Th1 responses (Sauty et al., 1999). IP-10 levels are elevated within tissues and serum of leprosy patients (Scollard et al., 2011; Stefani et al., 2009) and IP-10 is detectable in pleural effusions and plasma of TB-infected humans (Azzurri et al., 2005; Juffermans et al., 1999). In a multicenter evaluation, a Luminex-based IP-10 assay performed similarly to commercial IGRA’s (Ruhwald et al., 2011) for the diagnosis of TB in humans. Conclusions from this study were that antigen-specific IP-10 is produced in response to TB infection, the IP-10-based TB test has accuracy comparable to commercial IGRA’s, IP-10 appears to be less affected by non-TB infections than IFN-␥, and IP-10

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is detected in more patients with relevant TB risk factors than IFN-␥ (indicative of either a higher sensitivity or lower specificity of the IP-10 assay). IP-10 is also a candidate biomarker for detection of TB exposure in children; however, as with IGRA’s, IP-10-based assays cannot be used to discriminate between active and latent TB within this population (Whittaker et al., 2008). And, HIV infection does not appear to impair TB-specific IP-10 responses as severely as that observed with IFN-␥-based tests (Aabye et al., 2010; Kabeer et al., 2010). Also, given its acute-phase reactant properties, IP-10 may be a useful candidate for monitoring treatment responses, as well as for predicting mortality and morbidity of TB patients (Ruhwald et al., 2012). Aranday-Cortes et al. demonstrated that CXCL9 and IP10 are highly expressed (mRNA) within granulomas of tuberculous cattle (Aranday-Cortes et al., 2012a,b). Similarly, CXCL9 and IP-10 are expressed at very high levels in cells surrounding granulomas in M. tb-infected macaques (Fuller et al., 2003). With cattle, M. bovis PPD stimulation elicits IP-10 mRNA expression by PBMC as early as 28 days after aerosol M. bovis infection and these responses correlate (r = 0.87) to IFN-␥ mRNA expression within the same samples (Waters et al., 2012b). A whole blood quantitative rtPCR approach has been described for the detection of TB and other infectious diseases of humans (Kasprowicz et al., 2011). For the TB assay, MIG (monokine-induced by IFN-␥) and IP-10 mRNA responses to ESAT-6 and CFP-10 peptide pools correlate with IFN-␥ (protein) responses as detected by an ELISPOT assay (Kasprowicz et al., 2011). Also, concurrent analysis of CXCL8 (IL-8), IL-12B, and FoxP3 mRNA expression in ESAT-6-stimulated PBMC cultures has been proposed as a method to distinguish active from latent TB in humans (Wu et al., 2007). Studies with IP-10 and IFN-␥ mRNA responses in cattle (Waters et al., 2012b) suggest that a similar approach using chemokine and cytokine mRNA profiles may be useful for the detection of tuberculous cattle. Experimental infection trials with cattle may also be useful to determine the kinetics of the response as well as interactions of prior vaccination (e.g., BCG or novel attenuated mutants of M. bovis) and potential interference of NTM’s on the response. Such studies would likely provide information valuable for the application of mRNA-based techniques for use with the diagnosis of human TB, particularly with the development of field ready tests. After aerosol M. bovis challenge of cattle, IP-10 (protein) responses to mycobacterial antigens (e.g., rESAT-6:CFP-10, M. bovis PPD) exceed pre-infection responses beginning as early as 7 days (Waters et al., 2012b). Despite significant responses to mycobacterial antigens upon infection, IP-10 concentrations in non-stimulated whole blood cultures are similar to those in antigen-stimulated cultures. With the same sample set, IFN-␥ responses to mycobacterial antigens exceeded corresponding responses in non-stimulated cultures; thus, IP-10 protein appears to be increased within the plasma of TB-infected cattle non-specifically. High levels of IP-10 are also detected in non-stimulated whole blood samples from children with TB (latently infected > active TB) (Whittaker et al., 2008) and in the plasma of adults with active TB, possibly due to chronic TB-induced inflammation (Azzurri et al.,

2005). Similarly, during TB treatment of HIV-1 co-infected individuals, chemokine plasma levels and CCR5/CXCR4 expression on CD4 levels is increased (Wolday et al., 2005). Thus, with humans and cattle, non-specific elevations (i.e., without recall stimulation in vitro) in peripheral chemokines and chemokine receptors are detected, confounding interpretation for specific diagnostic applications. Further studies are necessary to determine the potential for use of chemokines, such as IP-10, as biomarkers for use in bovine TB diagnostic tests. Additionally, cattle may serve as a useful model to better define sequential expression of chemokines and their respective receptors within tissues after experimental infection with M. bovis as granuloma morphology and mycobacterial colonization (i.e., paucibacillary) more closely mimic the disease of humans as compared to mouse models, particularly with use of laser capture microdissection to define local variations in the response (Aranday-Cortes et al., 2012a,b).

3.6. Polyfunctional T cells Polyfunctional antigen-specific T cells simultaneously produce two or more cytokines and higher frequencies of these cells are correlated with control of chronic infections such as HIV, hepatitis C, leishmaniasis, malaria, and TB (reviewed in Caccamo and Dieli, 2012; Wilkinson and Wilkinson, 2010). After infection or vaccination, the ensuing immune response may consist of a balance of single cytokine and polyfunctional T cell responses, based upon antigen load and chronicity of infection. For instance, with chronic viral infections, polyfunctional CD4 and CD8 T cells are more likely to be correlated with protection (when antigen load is low) than single-cytokine producing T cells, while single IFN-␥-secreting T cells are characteristic of acute infection (when antigen load is high) (Harari et al., 2006). If chronic infection persists after failure of immune control, the balance tends to shift from polyfunctional T cells back to single IFN-␥-secreting cells (Wilkinson and Wilkinson, 2010). With both primary and chronic phases of HIV-1 infection, single IFN-␥-secreting effector cells predominate the response; however, an increased polyfunctional T cell response occurs after a positive response to antiretroviral therapy, similar to that observed in individuals who spontaneously control HIV-1 replication (Harari et al., 2004; Day et al., 2008). M. tb-specific polyfunctional T cells secreting IFN-␥/TNF-␣ or IFN-␥/IL-2 are also detected in M. tb/HIV co-infected individuals (Wilkinson and Wilkinson, 2010). With these individuals, the frequencies of M. tb-specific IL-2-secreting (IFN-␥/IL-2 and IL-2 only) CD4 T cells negatively correlate with HIV viral load; whilst, there is a positive correlation between the frequencies of IFN-␥ single positive CD4 T cells and HIV1 viral load (Day et al., 2008). Thus, M. tb-specific T cells secreting IL-2, with or without IFN-␥, may be proportionally diminished when HIV-1 viral load is high, corresponding with greater susceptibility for development of active TB. Therefore, CD4 T cells secreting IL-2 alone or with other cytokines may correlate with protection to TB.


Conversely, the majority of studies with human TB indicate that polyfunctional T cell responses are associated with clinical disease (i.e., as a biomarker of active TB) (Wilkinson and Wilkinson, 2010). Caccamo et al. (2010) found increased frequencies of polyfunctional T cells in active TB patients and almost undetectable levels in latently infected individuals. The anti-mycobacterial response by latently infected patients was mainly due to IFN-␥ single and IFN-␥/IL-2 dual secreting CD4 T cells. Additionally, the frequency of polyfunctional T cells in TB patients decreased to undetectable levels after 6 months of curative TB treatment, suggesting a correlation of bacterial load to functional patterns of the CD4 T cell response. Sutherland et al. (2009) also found higher frequencies of CD4 T cells simultaneously secreting IFN-␥, IL-2 and TNF-␣ in TB patients (sputum smear positive) compared to household contacts. In regard to the protective role that polyfunctional T cells may have on human TB, Streitz et al. (2011) found TNF-␣ production in response to mycobacterial antigens as the strongest predictor of active TB, while CD4 T cells secreting IFN-␥ and IL-2 predominated in successfully treated, latently infected, and BCG-vaccinated individuals. Using a whole-blood flowcytometric assay, Sester et al. (2011) demonstrated that patients with active TB had lower percentages of PPDreactive dual cytokine-secreting cells as compared to that of patients with non-active disease—suggestive of a protective role for polyfunctional T cells. Additionally, several TB vaccines (including live attenuated, subunit, plasmid DNA, recombinant proteins, peptides, viral vectored, protein-in-adjuvant vaccines, and primeboost immunizations platforms) elicit polyfunctional T cell responses considered protective (Thakur et al., 2012). Nevertheless, given the diversity of disease states in infants, adolescents, and HIV-infected adults; the various vaccine candidates being developed; various parameters being analyzed; and the differences in techniques—it is unlikely that a single, simple immune correlate relative to single-cytokine or polyfunctional response exists across all these different variables (Caccamo and Dieli, 2012). Hopefully, additional studies may at least provide some clarity for this complex response. In cattle, the contribution of polyfunctional T cells was until recently missing due to the lack of monoclonal antibodies that recognize biologically active bovine IL-2. Whelan et al. (2011), using a recombinant human antibody fragment that detects the expression of bovine IL-2, recently described the development of an assay to detect polyfunctional CD4 T cells in cattle using cells from cattle naturally infected with M. bovis. Bovine polyfunctional CD4 T cells exhibited a characteristic CD44hi CD62Llo CD45RO+ T cell effector memory (TEM) phenotype. Additionally, Kaveh et al. (2012) demonstrated that the duration of the intracellular staining culture, pre-stimulation period, and cytokine accumulation period substantially influence the cytokine repertoire and frequency of antigen-specific polyfunctional CD4 T cell subsets in cattle. Thus, the tools are now available to evaluate polyfunctional T cell responses in cattle and further investigations are warranted to determine the kinetics of the polyfunctional T cell response to M. bovis infection in cattle as well as a comparison of bovine polyfunctional T

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cell responses to disease severity and protective responses in TB vaccine efficacy trials.

3.7. Cytotoxic T lymphocytes (CTL’s) Cytolysis of infected cells can result in direct killing of Mycobacterium spp. or release of bacilli for eventual killing via other mechanisms (reviewed by Flynn and Chan, 2001). CD8+ T cells are the primary cell type performing CTL functions; however, other lymphocyte populations such NK cells, CD4+ T cells, and ␥␦ T cells also have cytolytic capacity (Stenger et al., 1998; Canaday et al., 2001). As related to TB infection, primary effector functions of CD8+ T cells are production of IFN-␥ necessary for macrophage activation and lysis of infected macrophages (Einarsdottir et al., 2009). Perforin and granulysin are essential for CTL function via pore formation and antimicrobial functions, respectively. Recent studies indicate that IFN-␥ from CD4+ T cells is required for effective CD8+ T cell responses (Green et al., 2013). Thus, CTL functions are essential for the control of mycobacterial infections and CD4+ T cell responses are supportive of this response. With M. bovis infection of cattle, activated CD8+ T cells are detectable within the lymphocytic outer core of early stage (i.e., stage I and II) tuberculous granulomas, indicating a potential role for these cells in the initial containment of the bacilli (Liebana et al., 2007). Antigenspecific CD8 T cells from cattle are capable of causing release of viable M. bovis from infected macrophages, presumably indicating CTL activity (Liebana et al., 2000). Bovine T cells express a homologue of human granulysin, a potent antimicrobial protein stored in association with perforin in cytotoxic granules (Endsley et al., 2004). Antigenic stimulation of peripheral CD4+ T cells from BCGvaccinated cattle results in enhanced anti-mycobacterial activity against BCG-infected macrophages linked with increased perforin and granulysin transcription (Endsley et al., 2007). Expression of the bovine granulysin gene can be induced in CD4+ , CD8+ , and ␥␦ T cells resulting in anti-mycobacterial activity similar to human granulysin (Endsley et al., 2004, 2007). Using laser capture microdissection, granulysin and granzyme A mRNA are detectable within granulomas of M. bovis-infected cattle (Endsley et al., 2004; Aranday-Cortes et al., 2012b). Granzymes are a group of serine proteases released by CD8+ T cells and NK cells in cytoplasmic granules along with perforin. Granulysin and perforin gene expression are also up-regulated in peripheral blood CD4+ and CD8+ T cells in both BCG- and M. bovis RD1-vaccinated calves (protected) as compared with non-vaccinated (not protected) calves (Capinos Scherer et al., 2009) demonstrating the potential of these biomarkers as correlates of protection for prioritizing vaccine candidates. Clearly, CTL’s are an important aspect of the bovine immune response to M. bovis infection and vaccine responses; however, further studies are required to identify their exact roles, both in protective immunity and response to persistent infection.

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4. B cells 4.1. Role of B cells: therapeutic sera, supportive functions, and ectopic B cell aggregates B cell responses are essential for protection against a wide variety of microbial pathogens; however, specific roles for B cells in the immune response to TB have remained elusive and controversial—generally being considered supportive rather essential in nature (reviewed by Glatman-Freedman and Casadevall, 1998; Maglione and Chan, 2009). Since the late 19th century, numerous studies have examined the possibility for use of ‘therapeutic sera’ (i.e., immune sera given to inactivate or clear the bacillus from infected individuals) in the treatment of TB—with mixed and unconvincing results (Glatman-Freedman and Casadevall, 1998). The use of therapeutic sera, and associated efficacy studies, fell out of favor with the advent of successful pharmacologic anti-mycobacterial compounds (e.g., streptomycin) in the mid 20th century. Recent studies with monoclonal antibodies specific for mycobacterial arabinomannan, 16 kDa ␣-crystallin protein, MPB83 protein or 28 kDa heparin-binding hemagglutinin have shown protective efficacy in mouse models of TB caused by M. tb or M. bovis (Glatman-Freedman and Casadevall, 1998; GlatmanFreedman, 2006; reviewed by Abebe and Bjune, 2009). With that said, antibodies elicited by infection with M. tb complex bacilli are typically not considered protective for the control of TB (reviewed in Glatman-Freedman and Casadevall, 1998). While the exact role of B cell responses during mycobacterial diseases remains unclear, generally B cells may influence the ensuing response to infections beyond direct antibody-pathogen interactions. Capturing antigen by specific surface receptors, B cells are efficient APC’s effectively priming memory T cell responses even at low levels of circulating antigen concentration (Lund et al., 2006; Shen et al., 2003). Antibodies, including immune complexes, also regulate APC’s through interactions with Fc␥ receptors. Disruption of stimulatory Fc␥ receptor activity increases susceptibility of mice to M. tb infection whereas genetic deletion of inhibitory Fc␥RIIB improves containment of M. tb, presumably by increasing Th1 polarization (Maglione et al., 2008). Additionally, immune complexes from TB patients down regulate cytokine production by neutrophils while enhancing chemotaxis and phagocytosis (Senbagavalli et al., 2012). Antibody-dependent T cell cytotoxicity is also believed to play a role in protective immunity to mycobacteria (de Vallière et al., 2005). Outcomes of M. tb infection experiments on B cell-deficient mice are variable, ranging from delayed disease progression and diminished immunity to no effects (reviewed in Maglione and Chan, 2009). Genetic ablation of polymeric Ig receptor, however, results in heightened susceptibility of mice to M. tb infection, implying a role for secretory IgA for optimal TB immunity (Tjarnlund et al., 2006). Intranasal inoculation of human monoclonal IgA1 antibody specific to ␣-crystallin in combination with recombinant mouse IFN-␥ inhibits pulmonary M. tb infection in mice transgenic for human CD89 (Balu et al., 2011). While not always definitive, these

studies demonstrate the potential for antibody-mediated immunity to modulate the course of infection. Supportive of this notion, is the consistent observation of B cell aggregates associated with tuberculous lesions in mice (Gonzalez-Juarrero et al., 2001), humans (Ulrichs et al., 2004), cattle (Aranday-Cortes et al., 2012a,b; Johnson et al., 2006), and other host species (García-Jiménez et al., 2012; Phuah et al., 2012) infected with M. tb complex organisms. These tertiary lymphoid structures contain aggregates of B cells (naïve, memory, and plasma cells) as well as intermixed CD4+ and CD8+ T cells, follicular dendritic cells, and mycobacteria-laden APC’s (Ulrichs et al., 2004). Intradermal injection of rESAT-6:CFP-10 fusion protein into calves experimentally infected with M. bovis elicits a DTH response involving B cells, in addition to predominating CD4+ and CD8+ cells found in the skin infiltrates (Waters et al., 2009). Using immunohistochemistry, Aranday-Cortes et al. (2012a,b) demonstrated the presence of CD79a-stained cells, a B cell marker, within granulomas of tuberculous cattle. In that study, early granulomas (stages I and II) displayed scattered B cells, whereas more advanced granulomas (stages III and IV) showed satellite nests of CD79a+ cells located peripherally and outside of the fibrous capsule. Non-human primate models of M. tb infection demonstrated significant numbers of B cells (CD20+ ) within lung granulomas, which were organized into discrete clusters with characteristics of germinal centers including numerous activated B cells (Phuah et al., 2012). In mice, formation of B cell follicles within infected lung tissues is dependent upon IL-23 and CXCL13; and, CXCL13 production is dependent upon IL-17A and IL-22 in this response (Khader et al., 2011). The presence of ectopic germinal centers indicates that the M. tb complex – and the ensuing inflammation – induces active B cell clusters that modulate the host response. It is thus believed that these follicles provide at least a partial framework for coordinated immune control of mycobacterial growth in the affected tissues (Ulrichs et al., 2004). 4.2. Emerging serodiagnostic tests for veterinary applications Several antibody-based tests have recently emerged for use in cattle, captive cervids, several wildlife reservoirs of M. bovis, and various zoo species (most notably elephants). Sero-dominant antigens vary by host species, with MPB83 (alone or in combination with MPB70 or 16 kDa ␣-crystallin), ESAT-6, and CFP-10 being commonly recognized targets for many species (Lyashchenko et al., 2006, 2008; Waters et al., 2006a,b, 2011a). Human antibody responses in active TB involve generally variable patterns of serum IgG reactivity, with the 38 kDa protein of M. tb being a major serodiagnostic antigen recognized in sputum smear positive patients (Lyashchenko et al., 1998). Interestingly, this protein elicits poor antibody responses, if any, in most animal hosts including nonhuman primates (Lyashchenko et al., 2007, 2008). In contrast, ESAT-6 and CFP-10 epitopes are predominantly involved in T cell reactivity in both humans and animal species (reviewed in Vordermeier, 2010). The accuracy of antibody-based tests also varies considerably by species (Lyashchenko et al.,


2008), remarkably, approaching 100% sensitivity and >95% specificity for elephants with use of the Elephant TB Stat Pak or Dual Path Platform assays (Chembio Diagnostics Inc, Medford, NY; Greenwald et al., 2009). Antibody responses by elephants with advanced stages of disease are particularly robust, both in the level of the response and diversity of antigen recognition (Greenwald et al., 2009; Lyashchenko et al., 2006, 2012). Antibody responses by elephants typically decline following a favorable outcome with antibiotic therapy or increase shortly before disease recrudescence (Lyashchenko et al., 2008, 2012). These findings demonstrate the potential for real-time serologic monitoring of response to therapy as well as disease staging. For certain cervid and camelid species in which skin test is ineffective for TB diagnosis, serology offers a convenient option for surveillance (Rhodes et al., 2012; Waters et al., 2011a). Thus, lessons learned with serology for various captive-wildlife or livestock species may be applicable for pathogenesis and diagnostic purposes with M. tb infection in humans. 4.3. Booster effect Intradermal tuberculin administration is known to significantly boost antibody responses in tuberculous cattle, cervids, and other hosts, but not in non-infected animals (Lyashchenko et al., 2004, 2007; Harrington et al., 2008; Dean et al., 2009). This phenomenon can be observed from 1-2 weeks to several months after tuberculin injection, depending on the type and dose of PPD, host species, stage of disease, pre-existing antibody levels, antigen reactivity patterns, and immunoassay format used (Chambers et al., 2009; Palmer et al., 2006; our unpublished observations). Although the boosting effect is not well studied, the absence of seroconversion in non-infected animals and the short-lived antibody kinetics with features of an anamnestic response strongly suggest that it is due to memory B cells originally primed by mycobacterial infection that can be quickly activated upon re-stimulation by tuberculin in vivo. For human TB, it remains unknown whether the skin test using much lower doses of PPD can affect the antibody response and hence modify accuracy of serodiagnostic tests. However, based on the animal studies, such possibilities exist and it should be taken into account when new immunodiagnostic assays for human TB are validated in the future. 4.4. Antibody responses: comparisons to CMI as related to pathogenesis Cellular immune responses elicited by mycobacterial infections of cattle generally correlate with infection but not necessarily with the level of pathology (Waters et al., 2010). Inoculation of cattle with M. tb H37 Rv or M. bovis Ravenel results in relatively robust CMI responses and persistent colonization with minimal to no lesions. With M. kansasii inoculation, CMI responses are elicited (although less robust than with M. tb or M. bovis infection) without detection of the organism or associated lesions. As expected, inoculation of cattle with virulent M. bovis (field strains such as 95-1315 or A2122/97) results in robust

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CMI responses, persistent colonization, and associated tuberculous lesions. Thus, regardless of the pathological and mycobacterial burden outcome, CMI responses are elicited. In contrast, antibody responses generally correlate with levels of pathology associated with mycobacterial infections. For instance, mycobacterial-specific antibody is detectable relatively early after M. tb challenge of cattle, yet these responses wane over time, likely coincident with the reduction of M. tb colonization. In contrast, with M. bovis infection that leads to persistent infection and significant pathology, antibody responses persist, likely due to persistently increasing antigen burden. With less virulent mycobacteria (e.g., M. kansasii), antibody responses are elicited and then immediately wane, likely coincident with complete clearance of the pathogen. Regardless of disease expression (e.g., with M. tb, M. bovis, or M. kansasii inoculation), mycobacteria-specific antibody responses may be boosted by re-exposure to mycobacterial antigens (e.g., PPD) or other live mycobacteria. Close association between antibody responses and presence of visible lesions was also reported for non-bovine hosts of M. bovis infection (Lyashchenko et al., 2008; Boadella et al., 2012). In regards to relevance to human TB, M. bovis infection of cattle may be considered as a model of clinical TB, whilst M. tb H37 Rv infection could be similar to latent, subclinical TB and M. kansasii inoculation of cattle a model for TB that has been successfully cleared from its human host. Therefore, these three infection systems could be developed into useful models mimicking different stages of human TB infection.

5. Summary In many aspects, the immune response by cattle to M. bovis is analogous to the response by humans to M. tb. This is not surprising given the similarities of the two pathogens (∼99.95% identity at the nucleotide level) and the extended evolution of the respective host/pathogen relationships. Landmark studies over the past 120 years have demonstrated the usefulness of cattle for the development of tools to control human TB (i.e., BCG, skin test, IGRA’s, and DIVA strategies). More recent studies have demonstrated that immunopathogenesis studies in cattle, including vaccine efficacy trials for correlation to protective responses, are also applicable to our understanding of TB in humans. With cattle, experimental biology approaches (i.e., M. bovis challenge and vaccine efficacy procedures) are refined to determine immune correlates associated with both protective and damaging responses. Tools are available to study in-depth cellular immune responses such as effector/memory populations, T helper subsets, CTL responses, cytokine profiles (including polyfunctional T cells), chemokines, and ␥␦ T cell subpopulations; antibody responses including correlations to protection/pathology; in situ responses using laser capture microscopy and qPCR; and interactions with NTM’s and other co-infections. Finally, findings from experimental biology studies are easily translated into development and validation of new tools for the control of bovine TB, some of which, may then become useful for improved disease management in humans.

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