Relevance of Bovine Tuberculosis Research to the Understanding of Human Disease

Home , Edmond Nocard

Veterinary Immunology and Immunopathology 159 (2014) 113–132

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology

j ournal homepage: www.elsevier.com/locate/vetimm

Relevance of bovine tuberculosis research to the

understanding of human disease: Historical perspectives,

approaches, and immunologic mechanisms

a,∗,1 a b

W. Ray Waters , Mayara F. Maggioli , Jodi L. McGill ,

c a

Konstantin P. Lyashchenko , Mitchell V. Palmer

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 a b s t r a c t

Pioneer studies on infectious disease and immunology by Jenner, Pasteur, Koch, Von

Keywords:

Behring, Nocard, Roux, and Ehrlich forged a path for the dual-purpose with dual ben-

Bovine tuberculosis

eﬁt approach, demonstrating a profound relevance of veterinary studies for biomedical

Central memory T cells

applications. Tuberculosis (TB), primarily due to Mycobacterium tuberculosis in humans and

Multi-functional T cells

␥␦ T cells Mycobacterium bovis in cattle, is an exemplary model for the demonstration of this concept.

IL-17 Early studies with cattle were instrumental in the development of the use of Koch’s tuber-

IP-10 culin as an in vivo measure of cell-mediated immunity for diagnostic purposes. Calmette

M. bovis speciﬁc antibody

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 erad-

ication 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 sever-

ity whereas antibody responses generally correlate with lesion severity. Recently, serologic

tests have emerged for the detection of tuberculous animals, particularly elephants, cap-

tive 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).

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.

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

1. Introduction: historical perspectives to the one use in the control of human TB. These include: (1) stud-

health approach ies demonstrating the safety and efﬁcacy of BCG (including

demonstration that booster doses are generally not beneﬁ-

The dual purpose with dual beneﬁt (One Health) cial), (2) use of tuberculin as an in vivo diagnostic reagent,

approach originated with the onset of research into the and (3) development of interferon (IFN)-␥ release assays

characterization of etiologic agents for infectious diseases, (IGRA) for diagnosis. In 1913 at the Pasteur Institute (Lille,

associated preventive strategies (i.e., vaccines), and the France), Calmette and Guerin vaccinated 9 cows with M.

immune response elicited by the agents. For instance, the bovis (Nocard strain, Edmond Nocard was Guerin’s men-

principle of vaccination was developed through insightful tor) attenuated by serial passage on glycerol soaked potato

observations and experimental studies of Edward Jen- slices in ox bile (i.e., BCG) (Calmette and Guerin, 1911). All

ner and others on the cross protective nature of cowpox 9 animals were protected from challenge with virulent M.

for small pox in humans. Robert Koch and Louis Pasteur, bovis; thereby, demonstrating the potential use of BCG vac-

co-founders of medical microbiology, each worked exten- cination against M. tb infection of humans. In 1921, BCG was

sively on diseases of veterinary and biomedical importance. administered to a newborn child (6 mg orally) and has since

Koch discovered the etiology of anthrax by inoculating been used widely by various administration routes for the

sheep with blood from infected animals while Pasteur control of human TB. Within a few years of the discovery

produced the ﬁrst live attenuated (laboratory produced) of tuberculin by Koch, veterinary investigators in Russia

bacterial vaccine for fowl cholera as well as an effective (Professor Gutman), the UK (John McFadyean), Denmark

anthrax vaccine for cattle. The Spanish physician, Jaime (Bernhard Bang), and the US (Leonard Pearson and Maz’yck

Ferrán, worked on veterinary vaccines prior to developing Ravenel) began using tuberculin as an in vivo diagnostic

the ﬁrst vaccine for immunizing humans against a bacte- reagent for TB in cattle (Marshall, 1932). Clemens von Pir-

rial disease—cholera. With his earnings from the 1st Nobel quet and Charles Mantoux later (circa 1907/1908) adapted

Prize in Medicine and Physiology for the serum theory and improved this technology for use in humans, coinci-

with diptheria and tetanus (von Behring, 1901), Emil Von dently deﬁning the principles of allergy and delayed type

Behring switched the focus of his work almost exclusively hypersensitivity (DTH). During the 1980s, an IGRA was

to the development of a vaccine for tuberculosis (TB) in cat- developed in Australia by Paul Wood, Leigh Corner, James

tle using chemically inactivated M. tb complex strains (e.g., Rothel, Stephen Jones, and others for the diagnosis of TB in

Tuberkulase and Taurovaccine) or human-derived tuber- cattle (Wood et al., 1990); a modiﬁed version of this assay

cle bacilli attenuated by lengthy propagation (designated is now widely used in the diagnosis of both human and

Bovovaccine) (reviewed in Linton, 2005). Concurrent with bovine TB. Most recently, recombinant proteins and pep-

Behring’s studies, McFadyean and colleagues in England tide cocktails of early secretory antigenic target-6 (ESAT-6),

and Pearson and Gilliland in the United States each demon- culture ﬁltrate protein 10 (CFP-10), and various other anti-

strated protective effects of live M. tb vaccination against gens are in use with IGRA’s (reviewed in Vordermeier,

experimental M. bovis infection in cattle (Flexner, 1908). 2010; Vordermeier et al., 2011a,b) and in development

This cross-protective strategy was abandoned, however, for use as skin test antigens in cattle to improve speci-

due to obvious safety concerns and variability in virulence ﬁcity over puriﬁed protein derivative (PPD) preparations

of the human tubercle bacilli in cattle. Use of M. tb for vacci- and as DIVA (differentiate infected from vaccinated ani-

nation of cattle against bovine TB, however, did establish a mals) reagents (Whelan et al., 2010a,b,c). Field studies

precedent followed by Albert Calmette and Camille Guerin with cattle will prove invaluable for the eventual appli-

in their development of an attenuated M. bovis (bacillus of cation of similar DIVA strategies for use in the control

Calmette and Guerin, BCG) for eventual use as a vaccine of human TB. Undoubtedly, advances in basic immunol-

in cattle, humans, and various other species (Fine, 1995; ogy, antigen discovery, comparative mycobacteriology, and

Waters et al., 2012a). vaccine approaches for human TB using animal models and

Research efforts on bovine and human TB have been clinical trials in humans have advanced research efforts

intimately linked beginning as early as the seminal stud- on bovine TB. Together, co-discovery in TB research using

ies on the etiology and pathogenesis of TB performed by both veterinary and biomedical approaches is an exem-

Koch, Jean Antoine Villemin, and others in the mid to late plary model of the one health concept.

1800s (Palmer and Waters, 2011). Initially, Koch postulated

that the tubercle bacilli from cattle and human were iden- 2. Etiology, control measures and experimental

tical, dismissing the studies of Villemin that demonstrated biology approaches

a greater virulence of bovine-origin versus human-origin

isolates in rabbits. Later, studies by Theobald Smith, a 2.1. Etiology and epidemiology

physician scientist working for the Veterinary Division of

the Bureau of Animal Industry, demonstrated deﬁnitive dif- Tuberculosis in animals and humans may result from

ferences between the bovine and human isolates based exposure to bacilli within the M. tb complex (i.e., M. tuber-

upon variable virulence of the two isolates in cattle, rabbits culosis, M. bovis, M. africanum, M. pinnipedii, M. microti, M.

and guinea pigs; as well as morphological differences and caprae, or M. canetti) (Cousins et al., 2003). Mycobacterium

differential growth characteristics of the two organisms on bovis is the species most often isolated from tubercu-

glycerin media. In addition to these contributions, veteri- lous cattle. Signiﬁcant reservoirs exist for bovine TB (i.e.,

nary researchers developed essential tools for the control M. bovis) including white-tailed deer (Odocoileus virgini-

of bovine TB that were concurrently or later adapted for anus, United States), Eurasian Badgers (Meles meles, United

W.R. Waters et al. / Veterinary Immunology and Immunopathology 159 (2014) 113–132 115

Kingdom), brushtail possums (Trichosurus vulpecula, New or residential care facility, etc.). As mentioned previously,

Zealand), wild boar (Sus scrofa, Spain), red deer (Cervus ela- exposure to wildlife reservoirs is a signiﬁcant risk factor

phus, Spain and Canada), African Buffaloes (Syncerus caffer, for transmission of M. bovis to cattle. Primary differences

South Africa) and a few others (Palmer et al., 2012). While between M. bovis and M. tb transmission within their

M. tb may infect a wide variety of animals [e.g., elephants respective primary hosts are: (1) the presence of wildlife

(Elephas maximus), tapir (Tapirus spp.), horses (Equus reservoirs for M. bovis infection of cattle and (2) the

caballus), banded mongooses (Mungos mungo), meerkats increased risk of indirect transmission via environmental

(Suricata suricatta), and numerous other wildlife species, contamination for M. bovis versus M. tb.

particularly in zoological collections], signiﬁcant wildlife Many countries have ofﬁcial bovine TB eradica-

reservoirs for human infection with M. tb are not currently tion/control programs. Prevention and control efforts are

recognized. The comparative virulence of M. bovis versus designed primarily to identify TB affected herds and

M. tb in humans is not completely clear. Prior to wide- remove infected animals or depopulate the entire herd.

scale pasteurization, approximately 20–40% of TB cases in These efforts are generally achieved via routine slaughter

humans resulted from infection with M. bovis, mostly due surveillance to identify TB-affected herds, targeted testing

to ingestion of M. bovis within dairy products (Brockington, schemes to remove TB test positive animals, movement

1937; Francis, 1959; Ravenel, 1933). Transmission of M. restriction of affected herds (with or without movement

bovis between humans, however, is extremely rare (Sunder testing), thorough epidemiologic investigations of reported

et al., 2009). Natural infection of cattle with M. tb has cases to determine risks for other herds (i.e., trace-ins

recently been demonstrated in East Africa (Ameni et al., and trace-outs), and depopulation (i.e., “stamping out”)

2011; Berg et al., 2009; Kankya et al., 2011) and China (Chen of affected herds if feasible (Schiller et al., 2010). The

et al., 2009; Du et al., 2011), likely due to human-to-cattle tuberculin skin test, currently applied either as a compara-

transmission. Especially in developing countries, diagnos- tive test using both M. avium and M. bovis PPD’s injected

tic laboratories rarely discriminate mycobacterial isolates into separate sites or as a single test with injection of

obtained from tuberculous cattle and humans beyond the M. bovis PPD only, remains after >100 years as the pri-

M. tb complex designation, thereby, hindering our knowl- mary ante-mortem test for the detection of tuberculous

edge of the relative prevalence of M. bovis in humans and cattle in many countries. Interferon-␥ release assays are

M. tb in cattle. Direct comparisons of M. bovis and M. tb also used in many countries (e.g., USA, UK, Ireland, New

infection in cattle indicate that M. tb is less virulent for Zealand, and others) primarily as conﬁrmatory or comple-

cattle (Ernst, 1895; Whelan et al., 2010a,b,c). Thus, host mentary tests in conjunction with skin test(s); although,

adaptation due to transmission capacity likely led to the certain countries are considering use of IGRA’s as a pri-

divergence and speciation of M. bovis and M. tb from a mary test on a limited basis. In addition to these traditional

common ancestor (Smith et al., 2009). methods, antibody-based tests have recently emerged for

use in the detection of tuberculous elephants (Connell and

2.2. Transmission and control measures Vanderklok, 2010; Greenwald et al., 2009; primarily M. tb

infection in elephants), captive cervids (Buddle et al., 2010;

In most countries with active control programs, TB- Waters et al., 2011a), camelids (Lyashchenko et al., 2011;

affected cattle herds generally contain few infected animals Rhodes et al., 2012), and cattle (Green et al., 2009; Waters

suggestive of low transmission rates and a slowly pro- et al., 2006a, 2011b; Whelan et al., 2008a,b, 2010a,b,c).

gressive course of disease (Palmer and Waters, 2006). Also, molecular-based tools [e.g., mycobacterial inter-

Transmission of bovine TB is primarily by direct contact yet spersed repetitive-unit-variable number tandem repeat

indirect transmission via exposure to contaminated feed, (MIRU-VNTR), spoligotyping, restriction fragment length

water, or premises also occurs. Animals with advanced polymorphism (RFLP), and high throughput whole genome

lesions may be particularly infectious (Khatri et al., 2012). sequencing of mycobacterial DNA] are used to character-

Housing, especially in conﬁned spaces such as milking par- ize mycobacterial isolates for epidemiologic investigations

lors and cattle sheds, and crowding enhances the spread of to determine potential links between new cases and prior

disease. Movement of infected animals and fence-line con- outbreaks as well as the likely origin of infection. Post-

tact with other herds is a common means of transferring mortem diagnosis is based upon gross and microscopic

the disease between herds and regions. Additionally, global evaluation for tuberculous lesions, detection of the organ-

trade agreements are increasingly being implemented to ism by mycobacterial culture of tissues with follow-up

promote international trade of livestock; thereby, sig- conﬁrmation/characterization of isolates using molecular

niﬁcantly increasing risks for inter-regional spread and techniques, and detection of M. tb complex speciﬁc DNA

distribution of bovine TB over great distances. Trans- by PCR on histologic sections. Surveillance and control of

mission of TB in humans is primarily by aerosol, with M. bovis infection in wildlife reservoirs, as well as mitiga-

less evidence for indirect transmission. As with bovine tion efforts to limit spread from wildlife to livestock, is also

TB, crowding and movement of infected individuals are critical for bovine TB control.

population risk factors for transmission. Signiﬁcant indi- As with cattle, skin test (not as a comparative test, M. tb

vidual risk factors for human TB include: HIV infection, PPD only) and IGRA’s are both routinely used as primary

diabetes, kidney disease, alcohol or drug abuse, immune screening test(s) for TB infection/exposure in humans.

suppression (e.g., certain treatments for autoimmune dis- The control of human TB in non-endemic regions is pri-

ease), and exposure to infected persons (e.g., travel to marily via preventative public health measures such as

endemic countries, health care workers, living in a shelter surveillance and reporting of disease, diagnostic testing

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

(primarily using skin test or IGRA’s with chest X-ray on Similarities in bovine and human TB control programs

positive individuals) and treatment of affected individuals, include: a primary focus on diagnosis of infection using

epidemiologic investigations to determine other at-risk similarly applied diagnostic tools (e.g., skin test and IGRA’s),

individuals, isolation of infectious individuals, directly intensive development of vaccines and associated DIVA

observed treatment strategies when warranted, treatment strategies, and an emphasis on epidemiologic investi-

of confounding illness, and appropriate public health poli- gations to determine sources of infection and exposed

cies to prevent further transmission. In M. tb endemic contacts. Differences in human versus bovine TB control

regions, a wider range of control measures are imple- programs include: complications in control strategies for

mented such as: BCG vaccination, preventive therapy bovine TB resulting from the presence of wildlife reser-

(treatment of latent infection), treatment of multidrug- voirs, a necessity for the differentiation of latent versus

resistant TB (MDR-TB) using both ﬁrst- and second-line active disease in humans, the option for anti-mycobacterial

drugs, and use of a wider range of diagnostic tests (e.g., treatment in TB-infected humans, the option for a host

sputum smear microscopy and culture, molecular diag- removal strategy with bovine TB (i.e., slaughter of infected

nostics, antibody-based tests, and emerging technologies), animals and, in certain instances, depopulation of affected

and efforts to minimize socio-economic and environmental herds), and a greater emphasis on differentiation of M.

risk factors (e.g., tobacco smoke, air pollution, malnutri- bovis-infected versus NTM-exposed animals with veteri-

tion, overcrowding, and poor access to health services) nary approaches.

(reviewed in Dye and Floyd, 2006).

Exposure to environmental non-tuberculous Mycobac- 2.3. Pathogenesis

teria sp. (NTM) is widely recognized as a confounding factor

for TB diagnosis in cattle (Barry et al., 2011); however, M. tb complex bacilli infect cells primarily of mono-

this concept is less widely recognized for TB diagno- cyte/macrophage lineage and may also be associated with

sis in humans as evidenced by the continued reliance type II alveolar epithelial cells, dendritic cells, neutrophils,

on tuberculin skin test for TB screening in humans that and a few other cell types. The classic lesion of the M. tb

does not differentiate M. tb infection from NTM sen- complex, the granuloma, forms in response to virulence

sitization/infection. Current testing strategies for cattle factors and chronic antigen stimulation due to persistent

utilize differential diagnostic procedures (i.e., comparisons infection. Granulomas in cattle are characterized by a cen-

between CMI responses to M. avium and M. bovis PPD’s), tral core of caseous, often mineralized material, surrounded

typically not used with humans. Intensive research efforts by inﬁltrates of epithelioid macrophages, Langhan’s type

have concentrated on the development of speciﬁc anti- multinucleated giant cells and lymphocytes; all of which

gens for the differential diagnosis of TB infection in cattle are often surrounded by a discreet, thickened ﬁbrous cap-

from NTM sensitization/infection, BCG vaccination, and/or sule, The level of ﬁbrous encapsulation varies depending

Johne’s disease vaccination (reviewed in Vordermeier et al., on the rate of development and chronicity of infection (i.e.

2011a,b). As with human TB, recombinant proteins or syn- more chronic lesions generally contain a greater degree of

thetic peptides of ESAT-6 and CFP-10, whose genes are ﬁbrosis). Morphologically, tuberculous granulomas in cat-

deleted in all BCG strains, have been most widely uti- tle resemble those seen in humans. With both bovine and

lized as antigens to differentiate M. bovis-infected from human TB, acid-fast bacilli are generally present in low

NTM-exposed animals and as DIVA reagents (Buddle et al., numbers (i.e., paucibacillary). In humans, caseonecrotic

1999; Pollock and Andersen, 1997; van Pinxteren et al., lesions may become liquefactive and form cavities within

2000; Vordermeier et al., 2001; Waters et al., 2004). More affected lung. Cavitation is uncommon in cattle. In contrast

recently, elucidation of the genomes of multiple strains to caseous lesions, liquefactive lesions can contain large

of M. tb and M. bovis (both BCG and virulent strains) numbers of acid-fast bacilli (multibacillary).

and other mycobacterial species has allowed a rational From the initial site of entry (often lungs, especially

genome-wide approach to antigen mining. Based on these with human TB due to M. tb), the organism invades local

comparative genomic/transcriptomic studies, additional lymph nodes often causing granulomas. The granuloma is

genes that are not transcribed or proteins that are not thought to limit the spread of the tubercle bacilli within the

secreted by BCG, such as Rv3615c, have been shown to host by walling-off the infection. The combination of the

complement ESAT-6 and CFP-10 by increasing speciﬁcity initial site of infection and the regional lymph node con-

without compromising sensitivity in CMI-based assays stitute the primary complex commonly referred to as the

(Sidders et al., 2008; Millington et al., 2011). Interestingly, Ghon’s complex with pulmonary M. tb infection of humans.

the antigen repertoire recognized by M. bovis-infected Dissemination from the primary complex occurs via both

cattle and M. tb-infected humans overlap considerably lymphatic and hematogenous spread. Determination of

(Mustafa et al., 2006; Sidders et al., 2008). The bovine the primary complex (indicative of the route of infection)

model can therefore be used for antigen discovery with may be impossible, especially in cases of prolonged or

results applicable to the development of reagents and diag- severe disseminated disease. Post-primary dissemination

nostic tools for the control of human TB, as well as for may occur leading to spread of the organism to other lymph

other livestock and wildlife species. These antigens may be nodes and organs. Diffuse miliary TB may occur in severe

applied to blood based CMI assays (such as IGRA’s, reviewed cases. Latency (i.e. infection and limited primary lesion for-

in Vordermeier et al., 2011a), antibody-based tests, and mation without disease progression) is a common sequela

skin test protocols (Pollock et al., 2003; Whelan et al., with M. tb infection of humans; however, latency is typ-

2010a,b,c). ically not considered a common stage of disease with M.

W.R. Waters et al. / Veterinary Immunology and Immunopathology 159 (2014) 113–132 117

bovis infection of cattle. With that said, M. bovis may be iso- biology approaches are generally limited to in vitro anal-

lated from tissues without visible gross lesions, indicating a ysis and opportunistic studies with samples obtained from

potential for ‘latent’ infection. However, in most cases, it is naturally infected individuals. Field studies in humans are

not certain if these infections represent early lesions with- particularly useful, however, for evaluation of emerging

out visible gross lesions or persistent infections without diagnostic approaches (Walzl et al., 2011).

disease progression. In regions with ofﬁcial bovine TB control programs,

vaccines are rarely used for the control of bovine TB, pri-

2.4. Experimental biology approaches marily due to the potential for interference with traditional

ante-mortem testing strategies using PPD as antigen (i.e.,

Aerosol and intratracheal inoculation are the most com- tuberculin skin test and IGRA’s) (Moodie, 1977). With that

mon routes currently used to experimentally infect cattle said, multiple countries are seriously considering vaccine

with virulent M. bovis (reviewed in Hewinson et al., 2003; approaches to control bovine TB in cattle, particularly in

Pollock et al., 2006; Waters et al., 2012a). These routes regions with wildlife reservoirs (e.g., England, Wales) or

result primarily in a respiratory tract infection (i.e., lungs lack of an effective surveillance program (e.g., Latin Amer-

and lung-associated lymph nodes), severity is dose and ica). Vaccine efﬁcacy studies in cattle provide a unique

time dependent, and the disease closely mimics natural opportunity to evaluate safety, immunogenicity, and efﬁ-

infection of cattle (Buddle et al., 1994; Palmer et al., 2002). cacy of emerging TB vaccines, with relevance for efforts

Experimental infection of cattle with virulent strains of to develop TB vaccines intended for humans. Addition-

M. bovis elicits robust cellular immune responses [e.g., ally, efﬁcacy studies are easily performed with neonatal

␥

IFN- , TNF␣, IP-10, and DTH responses (Pollock et al., calves, a target population for both cattle and human TB

2001; Waters et al., 2003a, 2012b)] beginning as early as vaccines (Endsley et al., 2009). Numerous experimental

2–3 wks after challenge and humoral immune responses and ﬁeld vaccine efﬁcacy studies have demonstrated that

(both IgM and IgG) typically 2–4 weeks later (Waters BCG is effective in cattle, although the level of protection

et al., 2006a). Experimental approaches permit disease varies dramatically across studies – as occurs in humans

conﬁrmation through postmortem examination and the (reviewed in Waters et al., 2012a). Multiple vaccine

capacity to quantitatively measure the severity of gross platforms have been tested with cattle such as atten-

and microscopic lesions (Wangoo et al., 2005). Addition- uated mutants, subunit, DNA, heterologous prime-boost

ally, tissues are collected for assessment of mycobacterial strategies, and several adjuvant preparations (reviewed

colonization (qualitative and quantitative measures). Thus, in Vordermeier, 2010). Cattle have been the ﬁrst natural

experimental variables such as prior vaccination, immune host for TB where prime-boost or combinations of BCG

response, and dose/strain may be correlated with lesion and DNA, protein or virus-vectored vaccines have been

severity and mycobacterial colonization. In contrast to vir- shown to induce better protection against TB than does

ulent ﬁeld strains of M. bovis, inoculation of cattle with BCG alone (Wedlock et al., 2003; Skinner et al., 2003a, 2005;

M. bovis AN5 or M. bovis Ravenel (both are laboratory- Vordermeier et al., 2009). Recent experimental trials with

adapted strains) results in robust cell-mediated immune cattle have demonstrated that adenoviral-vectored Ag85A

(CMI) responses and colonization, yet only minimal to may boost immunity elicited by BCG in cattle, BCG is par-

no tuberculous lesions 4–5 months after challenge (Mar- ticularly protective when administered to neonates, and

tin Vordermeier, AHVLA, personal communication; Waters DIVA approaches are feasible in cattle using both in vitro

et al., unpublished observations), similar to infection of cat- or in vivo methods (reviewed in Vordermeier et al., 2011b

tle with M. tb H37Rv (Whelan et al., 2010a,b,c). Thus, as with and Waters et al., 2012a). BCG vaccination of calves within

experimental biology approaches using M. tb in various ani- 12 h of birth induces a high level of protection against sub-

mal models, strain selection and laboratory maintenance sequent M. bovis challenge, whereas BCG vaccination at

of isolates are critical variables for consideration when birth and booster vaccination at 6 weeks of age induces

using M. bovis. Experimental protocols are also developed signiﬁcantly less protection compared to a single vaccina-

for infection/sensitization of cattle with NTM for compar- tion (Buddle et al., 2003). Thus, cattle offer a unique model

ative immunology, pathogenesis and diagnostic studies to study not only the safety and efﬁcacy of candidate TB

(Waters et al., 2004, 2006b). Interactions of NTM exposure vaccines in a natural host pathogen system but also to eval-

on TB vaccine efﬁcacy may also be evaluated. In a study uate potential interactions such as exposure to NTM’s, age

in which cattle were naturally exposed to environmen- (i.e., neonatal immunology), dose, co-infection (e.g., para-

tal mycobacteria prior to vaccination, BCG vaccination did sites), nutritional status, and combinations of the various

not induce protection against TB, while vaccination with approaches.

two other attenuated strains of M. bovis induced protec-

tion (Buddle et al., 2002). Particularly in bovine TB endemic 3. Cell-mediated immunity

regions, large-scale ﬁeld trials are possible with cattle for

vaccine efﬁcacy and diagnostic test evaluation. Field trials 3.1. IFN- and delayed type hypersensitivity (DTH)

provide a unique opportunity for evaluation of vaccines and responses

diagnostic tests with the intended target population; how-

ever, variables such as NTM exposure, co-infection, stress, An essential component of the immune response to TB

and limitations on sampling (particularly with necropsy (bovine and human) is the production of IFN-␥ by T helper

limitations and timing of blood sampling) may confound 1 (Th1) CD4 T cells (Cooper and Torrado, 2012; Torrado and

interpretation of results. With humans, experimental Cooper, 2011). Immune deﬁciencies affecting CD4 T cells,

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

␥

as with AIDS patients, and IL-12/IFN- /STAT1 signaling is not completely understood in either humans (Todryk

pathways results in more severe disease in TB-affected et al., 2009) or cattle (Waters et al., 2009). Immunolog-

individuals (Diedrich and Flynn, 2011; Cooper et al., 2007). ical memory develops naturally as a result of infection.

Given the importance of Th1 cells in the response to TB, it Vaccinations usually aim at mimicking an infection to gen-

is not surprising that IFN-␥ (i.e., IGRA’s) and DTH (i.e., skin erate memory cells capable of responding more rapidly and

test) responses are useful correlates to infection (reviewed efﬁciently upon subsequent infection. Pathogens and their

by Schiller et al., 2010 for cattle and Walzl et al., 2011 derivatives are generally transported to lymphoid organs

for humans). Most effective TB vaccines also elicit speciﬁc by APCs for initiation of T cell responses. Initiation of this

IFN-␥ and DTH responses (Black et al., 2002), but not all vac- primary response may take days to weeks to develop rely-

cines that induce IFN-␥ and DTH responses are protective ing on exposure of naive T cells to antigens in secondary

against TB. Additionally, the amount of IFN-␥ produced in lymphoid organs, expansion of antigen-speciﬁc cells, and

response to vaccination does not necessarily correlate with homing of effector cells to the site of infection (Mackay

the level of protection afforded by the vaccine (Abebe, et al., 1990). With TB, this 2–3 week delay in the response

2012; Mittrücker et al., 2007; Waters et al., 2012a). For at the primary site of infection seems to be advantageous

instance, cattle vaccinated with BCG-Danish have similar for the agent, and is a feature of the infection in cattle,

levels of protection as cattle vaccinated with BCG-Pasteur, humans, and mice (Ernst, 2012). The recirculation feature

despite having signiﬁcantly lower vaccine-elicited IFN- of naive T cells is mediated, in part, by expression of CD62L

␥

production to M. bovis PPD (Wedlock et al., 2007). In and CCR7, required for migration from blood into lymph

addition to post vaccination (pre-challenge) parameters, nodes across the high endothelial venules, and the lack of

immune responses elicited after infection may be com- the homing/chemokine receptor combinations required for

pared with efﬁcacy indicators to determine post challenge entry into lymphoid sites (Butcher and Picker, 1996; von

correlates of protection. After M. bovis challenge, responses Andrian and Mackay, 2000; Bromley et al., 2008).

with diagnostic potential, such as IFN-␥ responses, are Experiments with mice have demonstrated that the

generally inversely correlated with responses that predict numbers of Ag-speciﬁc T cells increase up to ∼10,000-

vaccine efﬁcacy. For instance, a robust ESAT-6/CFP-10- fold after initial encounter with cognate antigen and

speciﬁc IFN-␥ response after challenge is a negative subsequent proliferation/activation (Hou et al., 1994;

prognostic indicator of vaccine efﬁcacy as these responses Murali-Krishna et al., 1998; Whitmire et al., 1998). Besides

have been shown to positively correlate with TB-associated the clonal expansion, activated T cells differentiate and

pathology (Dietrich et al., 2005; Vordermeier et al., 2002; exhibit effector functions, characterized by expression of

Waters et al., 2007, 2009). These ﬁndings are not surpris- important mediators of pathogen control (e.g. IFN-␥, TNF-

␣

ing as it would be expected that failed vaccine approaches , IL-17, and cytotoxic granules). These cells down regulate

would result in increasing antigen burden and associated the expression of CD62L and CCR7, while up regulating

pathological changes; thus, evoking immune responses non-lymphoid homing receptors, such as CXCR3 and CCR5

indicative of infection. With that said, there is no indication (Reinhardt et al., 2001). If the immune system successfully

that the level of DTH responses detectable after M. bovis controls the infection, 90–95% of the Ag-speciﬁc T cell pop-

challenge correlates with the level of pathological changes ulation undergoes apoptosis and only a few memory cells

in cattle or vaccine-elicited protection; however, analysis remain (Totté et al., 2010; Wilkinson et al., 2009). Sallusto

of DTH responses is often confounded by responses evoked et al. (1999) identiﬁed two functionally distinct subsets

− +

by attenuated live vaccines (Waters et al., 2009). Exam- of memory CD4 T cells in humans (CD45RA /CD45RO )

ples of positive prognostic indicators of vaccine efﬁcacy as based on expression of the lymphoid homing receptors

measured after challenge include: reduced antigen-speciﬁc CD62L and CCR7: (1) central memory T (Tcm) cells which

␥ + +

IFN- , iNOS, IL-4, and MIP1-␣ (CCL3) responses; reduced are CD62L CCR7 and preferentially localize to lymphoid

antigen-speciﬁc expansion of CD4 cells in culture; and a tissues and (2) effector memory T (Tem) cells which are

− −

diminished activation proﬁle (i.e, ↓ expression of CD25 and CD62L CCR7 and preferentially localize to peripheral

CD44 and ↑expression of CD62L) on T cells within antigen tissues. Tcm cells show great proliferation and IL-2 pro-

stimulated cultures from protected versus non-protected duction capabilities (Champagne et al., 2001; Sallusto et al.,

cattle (Waters et al., 2003b,c, 2007). Recent RNA-seq and 2010). Tem cells may remain blood associated, either cir-

microarray studies with samples from M. bovis-infected culating or contained within splenic red pulp or hepatic

cattle have identiﬁed additional candidates (IL-22, LT-␣, sinusoids. Tem cells have immediate effector functions and

granzyme A and B, CXCL-9, and CXCL-10) for further eval- may maintain preformed cytotoxic granules for rapid cytol-

uation as biomarkers of infection and vaccine efﬁcacy in ysis of infected host cells (Woodland and Kohlmeier, 2009).

cattle (Aranday-Cortes et al., 2012a,b; Bhuju et al., 2012; However, upon restimulation, Tem cells undergo relatively

Thacker et al., 2011). little proliferation and secrete minimal IL-2 (Sallusto et al.,

2004, 2010; Champagne et al., 2001).

3.2. Effector and T cell central memory (Tcm) immune The effector response to mycobacterial antigens is often

responses accessed on diagnostic tests (e.g., IFN-␥ production by

blood leukocytes via Bovigam®, Quantiferon®, or ELISPOT

Memory T cell responses are generally considered assays) demonstrating that this response is a good correlate

essential for vaccine-elicited protection against intracellu- to infection. Effector IFN-␥ responses to ESAT-6 and CFP-10

lar infectious agents; however, the relationship between during the early stages of experimental M. tb infection posi-

effector T cell responses and long lasting T cell memory tively correlate to disease severity in non-human primates

W.R. Waters et al. / Veterinary Immunology and Immunopathology 159 (2014) 113–132 119

␥

(Lin et al., 2009). Monitoring effector IFN- responses to the migration pattern of ␥␦ T cells, Vrieling et al. (2012)

ESAT-6 and CFP-10 is also a good measure of TB exposure recently demonstrated that an anti-human CCR7 antibody

in humans (Doherty et al., 2002). HIV infection is a pri- cross-reacts with bovine CCR7 molecules. Utilizing this

mary risk factor for developing active TB. HIV-1 infected antibody, our group has identiﬁed the primary contribu-

individuals show not only lower levels of CD4 cells but tion of Tcm cells, with a minor contribution by Tem cells,

also impairment of T cell function. Disease progression in the long-term cultured IFN-␥ ELISPOT response to M.

and increase in HIV-1 load correlate with a loss of IL- bovis-infection of cattle (Maggioli et al., 2012). Data on

2 secretory function by CD4 T cells (Day et al., 2008). the response to vaccination and subsequent challenge that

Combined antiretroviral therapy restores immunity and make it possible to access the correlation between Tcm and

reduces the risk of TB in HIV-infected individuals, primarily Tem responses to protection/pathology are still lacking.

via enhancement of Tcm responses (Wilkinson et al., 2009). However, these data demonstrate the potential for deﬁning

␥

Additionally, assessment of long-term IFN- production a protective signature elicited by vaccination to prioritize

(i.e., as a surrogate to Tcm responses) may be used to detect candidates for efﬁcacy testing within calves.

past M. tb infection in patients with negative short-term

culture assays (i.e., standard IGRA’s measuring effector and 3.3. IL-17 responses

Tem responses) (Butera et al., 2009). Thus, while measure

of effector responses is as a correlate of TB infection, mea- Recently, there has been considerable interest in the

surement of Tcm responses may also provide diagnostic role of IL-17 producing cells in the immune response to

beneﬁt. TB (Cooper, 2010). M. tb infection of mice and humans

Upon stimulation by mycobacterial antigens (e.g., (Jurado et al., 2012; Khader and Cooper, 2008) and M.

rESAT-6:CFP-10 or M. bovis PPD), bovine peripheral blood bovis infection of cattle (Vordermeier et al., 2009) results

␥␦

CD4, CD8, and T cells from M. bovis-infected cat- in a signiﬁcant IL-17 response. Early expression of IL-17

tle proliferate and display an activated phenotype (i.e., is required for rapid accumulation of protective memory

↑

↑ ↑

CD25, CD26, CD44) after 3-6 days in culture (Waters cells in TB infection of mice (Khader et al., 2007); how-

et al., 2003b; Maue et al., 2005). Mycobacterial antigen- ever, the absence of IL-17 during infection only modestly

activated CD4 cells also decrease expression of CD62L and alters the inﬂammatory response (Khader et al., 2005;

increase the expression of CD45RO (associated with mem- Umemura et al., 2007). With aerosol BCG infection of mice,

+ +

ory T cells) while decreasing the expression of CD45R IL-17A produced by V␥4 and V␥6 ␥␦ T cells are neces-

(associated with naive T cells phenotype) (Maue et al., sary for appropriate maturation of granulomas (Okamoto

2005). The variant splices A, B and C of CD45 receptor are Yoshida et al., 2010). IL-17 is produced primarily by ␥␦ and

not described for cattle (Bembridge et al., 1995). These other non-CD4 T cells during M. tb infection in mice; and,

ﬁndings demonstrate that cattle exhibit an expected T early IL-17 produced by ␥␦ T cells occurs prior to ␣␤ T

cell effector phenotype upon antigen activation within cell priming, thus, biasing the ensuing adaptive response

short-term cultures. Less, however, is known about the (Lockhart et al., 2006). However, excessive IL-17 responses

phenotype of Tem and Tcm populations in cattle. Vac- may be detrimental. Cruz et al. (2010) demonstrated that

cine efﬁcacy studies with cattle have demonstrated that repeated BCG vaccination of M. tb-infected mice exac-

long-term T cell responses (Whelan et al., 2008a,b) nega- erbates inﬂammation due to infection. This exaggerated

tively correlate with mycobacterial burden (Waters et al., response, however, is not detected in mice genetically

2009) and TB-associated pathology (Vordermeier et al., deﬁcient in IL-23p19 or in mice treated with anti-IL-17

2009). Additionally, BCG vaccination of neonatal calves blocking antibody, demonstrating the dependence of IL-17

induces signiﬁcant protection against M. bovis challenge on the damaging response (Cruz et al., 2010). The excessive

at 12 months but not at 24 months after vaccination; inﬂammation in these mice is associated with increased

and, loss of efficacy correlates with a significant reduc- numbers of IL-17 producing T cells and a substantial influx

␥

tion in the numbers of antigen-speciﬁc IFN- -secreting of granulocytes (Gr1+ cells) as well as increased amounts

cells within long-term PBMC cultures (Thom et al., 2012). of MIP2, TNF-␣, and IL-6 within affected lungs. Together,

Studies with samples from humans have demonstrated these ﬁndings suggest that the timing and amount of IL-

that the responding cells within these long-term cultures 17 produced in response to TB infection is critical for the

(up to 14 days) are mainly Tcm’s and that this response, balance between responses that support control of the

in contrast to effector responses, correlates with better bacilli versus detrimental inﬂammatory responses. Indeed,

infection outcomes (Todryk et al., 2009). With the long- retinoic acid receptor-related orphan receptor ␥ (ROR␥)

term culture assay for cattle, T cell lines are generated inhibitors are in consideration for inclusion in treatment

via stimulation of PBMC with speciﬁc antigens including regimens for M. tb infection of humans (Baures, 2012). A

Ag85A, TB10.4 and M. bovis PPD. Effector T cell responses presumed mechanism of action of ROR␥-inhibitors is to

wane over time and memory cells are sustained via addi- promote a more favorable IL-17/IFN-␥ balance via inhibi-

tion of IL-2 and fresh medium. After 13 days of culture, tion of IL-17 production.

cells are washed, transferred to plates containing autolo- With both M. bovis infection and BCG plus viral-

gous APCs, cultured overnight, and the ensuing response vectored Ag85A vaccination of cattle, stimulation of PBMC

␥

measured by IFN- ELISPOT. Tcm’s likely contribute to the cultures with either M. bovis PPD or Ag85A elicits IL-17

long-term cultured ELISPOT response to BCG vaccination mRNA expression (Vordermeier et al., 2009). Vaccine-

by cattle, although a lack of an anti-bovine CCR7 antibody elicited IL-17 responses to Ag85A stimulation 10 wks after

has hindered this characterization. In the assessment of vaccination and prior to challenge negatively correlate

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

with TB-associated pathology (Vordermeier et al., 2009). cysteine rich (SCRC) superfamily, which includes CD163,

Likewise, Rizzi et al. (2012) recently demonstrated that CD5, CD6 and DMBT1 (Sarrias et al., 2004). The majority of

IL-17 mRNA expression is increased in response to M. bovis peripheral blood bovine ␥␦ T cells express WC1, and it is

PPD stimulation in cattle vaccinated with a BCG strain the WC1 subpopulation that has been shown to respond

over-expressing Ag85B; and, this post-vaccination/pre- to M. bovis infection; although, recent studies indicate that

−

challenge IL-17 response negatively correlates with lesion WC1 ␥␦ T cells are also responsive (McGill et al., 2012).

␥␦

severity after experimental infection. IL-17 responses to M. T cells expand signiﬁcantly in the blood of mice infected

bovis PPD detected after challenge, however, do not corre- with BCG (Janis et al., 1989) and humans with active tuber-

late with protection as there is a ∼20 fold increase in IL-17 culosis (Meraviglia et al., 2011). In the bovine, ␥␦ T cells

gene expression detected in samples from non-vaccinated, also undergo dynamic changes, with a marked decrease in

vaccinated/protected, and vaccinated/non-protected the circulating population shortly after M. bovis infection

groups (Vordermeier et al., 2009). Similar IL-17 responses (Cassidy et al., 1998; Pollock et al., 1996). The initial drop in

are detected post-challenge, regardless of vaccine- the frequency of peripheral ␥␦ T cells has been attributed to

evoked protective immunity. Thus, vaccine-elicited IL-17 movement out of circulation to the site of infection. Indeed,

responses are either boosted post-challenge or persist, WC1 ␥␦ T cells are amongst the ﬁrst cells to accumulate

despite protection. IL-17 protein is also detectable within at the site of DTH responses following PPD injection of M.

M. bovis PPD or rESAT-6:CFP-10-stimulated whole blood bovis infected cattle (Doherty et al., 1996), and at the site of

or PBMC cultures from M. bovis-infected cattle (McGill early lesion development in the lungs and lymph nodes of

et al., 2012). Additionally, IL-17 expression (mRNA, 60 virulent M. bovis infected cattle (Cassidy et al., 1998; Palmer

and 90 days after experimental infection) in response to et al., 2007). Following intranasal BCG vaccination, bovine

M. bovis PPD correlates with the presence of gross tuber- ␥␦ T cells also rapidly accumulate, inﬁltrating all lobes of

culous lesions, suggesting that IL-17 may prove useful the lungs, the pharyngeal tonsils and the lymph nodes of

as a biomarker of infection (Blanco et al., 2011). Using the head (Price et al., 2010). In mice, BCG inoculation also

laser capture microdissection followed by qPCR, Aranday- results in a rapid recruitment of ␥␦ T cells to the lungs

Cortes et al. demonstrated increased IL-17A and IL-22 and pulmonary lymph nodes (Dieli et al., 2003). Following

expression within tuberculous granulomas as compared to the initial decrease in circulation, WC1 cells from M. bovis

non-affected tissues from experimentally infected cattle infected cattle undergo signiﬁcant expansion in the blood,

(Aranday-Cortes et al., 2012a,b). Expression of IL-17A with a concomitant increase in activation as marked by

and IL-22 was greatest in early lesions with decreasing up-regulated CD25 expression, indicating that the cells are

expression in more advanced lesions (as deﬁned by undergoing an active response to infection (Pollock et al.,

increasing ﬁbrosis/necrosis upon microscopic evaluation). 1996).

A reason for this differential expression in early versus Early studies by Smyth et al. and Rhodes et al. reported

advanced lesions is unclear; however, it may indicate a that ␥␦ T cells from M. bovis infected cattle responded

temporal connection for these cytokines in the response in vitro to complex antigens of M. bovis including PPD, M.

to TB. Further studies with bovine TB should prove useful bovis sonic extract and M. bovis culture ﬁltrate proteins

for delineating potential roles for IL-17A and other Th17 with proliferation (Smyth et al., 2001; Rhodes et al., 2001)

cytokines (e.g., IL-22, Bhuju et al., 2012) in protective and and robust IFN-␥ production (Rhodes et al., 2001). It has

detrimental responses to the tubercle bacillus in cattle. subsequently been shown that bovine ␥␦ T cells recog-

nize protein antigens of M. bovis, with robust responses

3.4. ı T cells to Ag85 and ESAT-6 (Rhodes et al., 2001; Waters et al.,

2006a,b), both known to elicit robust CD4 and CD8 T cell

Accumulating evidence suggests that ␥␦ T cells play a responses, and minor responses to the protein antigens

critical role in the early response to M.tb and M. bovis, and MPB83, MPB70 and hsp16.1 (Rhodes et al., 2001). Human

may be key in bridging innate and adaptive immunity fol- and murine ␥␦ T cells recognize both protein and non-

lowing infection. The frequency of ␥␦ T cells circulating in protein phosphoantigens of M.tb (Born et al., 1990; Fournie

humans and mice is rare, representing 5–10% of the circu- and Bonneville, 1996; Haregewoin et al., 1989; Morita et al.,

lating peripheral lymphocyte population (Kabelitz, 2011). 1995) and the same also appears true for bovine ␥␦ T cells.

In contrast, ␥␦ T cells are signiﬁcantly more abundant in Welsh et al. (2002) demonstrated that while proteinase-K

ruminant species, where they constitute up to 70% of the treatment of M. bovis sonic extracts ablates the ability of

circulating peripheral blood lymphocytes in very young CD4 T cells to respond, ␥␦ T cells retain their ability to pro-

animals (Hein and Mackay, 1991; Jutila et al., 2008). The liferate and produce IFN-␥. A subsequent study by Vesosky

increased frequency of ␥␦ T cells in cattle indicate a crit- et al. (2004) identiﬁed mycolylarabinogalactan peptido-

ical role for this population in the immune system of the glycan, a component of the mycobacterial cell wall, as a

ruminant and, compared to species with rare circulating primary non-protein antigen for bovine ␥␦ T cells.

frequencies (e.g., mice and humans), make it an excellent Through in vitro and in vivo reports, it is evident

model for dissecting the role of ␥␦ T cells in the response that bovine ␥␦ T cells are responding to M. bovis infec-

to infections such as Mycobacterium spp. Populations of tion; however, the physiologic role of this response in

bovine ␥␦ T cells are commonly divided based upon their immune protection is still unclear. ␥␦ T-cell deﬁcient mice

expression of Workshop Cluster 1 (WC1) (Clevers et al., are able to control BCG (Ladel et al., 1995) and low-

1990; Machugh et al., 1997; Mackay et al., 1989; Morrison dose M. tuberculosis infection (D’Souza et al., 1997), but

and Davis, 1991), a member of the scavenger receptor exhibit signiﬁcantly larger and less-organized granulomas

W.R. Waters et al. / Veterinary Immunology and Immunopathology 159 (2014) 113–132 121

in both cases, suggesting a role for ␥␦ T cells in granu- following M. tb infection in mice recruiting neutrophils, NK

loma formation. In agreement, depletion of WC1 ␥␦ T cells, monocytes, and lymphocytes via speciﬁc receptors

cells from SCID-bo mice prior to M. bovis infection signiﬁ- such as CCR2/5 and CXCR1/CXCR2. In response to inﬂam-

cantly alters the architecture of the developing granuloma mation, T and B cells up-regulate CXCR3, CCR5, and CCR6

(Smith et al., 1999). However, depletion of WC1 ␥␦ T and accumulate in TB-affected lung tissues. Lung dendritic

cells from M. bovis-infected cattle has no effect on disease cells carrying M. tb up-regulate CCR7 and migrate along

severity or granuloma formation (Kennedy et al., 2002). chemokine gradients to lymphoid organs where they inter-

Instead, these animals exhibit a signiﬁcant reduction in act with T cells, promoting cytokine production.

␥

early IFN- production and an increased skewing towards T cells, monocytes, macrophages, basophils and imma-

Th2 type immunity, suggesting a role for ␥␦ T cell-derived ture dendritic cells each express CCR2. Following aerosol

cytokines in establishing Th1 immunity. Along those lines, M. tb infection, immune cell migration into lungs and

it has been described that reciprocal interactions between pulmonary lymph nodes is delayed in mice genetically deﬁ-

murine and human ␥␦ T cells and M. tb-infected DC can cient in CCR2 (Peters et al., 2001; Scott and Flynn, 2002).

lead to enhanced IFN-␥ and IL-12p70 production, respec- Mycobacterial colonization, however, is similar between

−/−

tively (Dieli et al., 2004; Martino et al., 2007; Meraviglia CCR2 and wild type mice after low dose M. tb infec-

et al., 2010). This DC-␥␦ T cell cross-talk promotes the tion whereas challenge with moderate to high doses results

−/−

development of robust Th1 responses. Work by Price and in more severe disease in CCR2 versus wild type mice

Hope (2009) recently demonstrated that the same recip- (Peters et al., 2001). These ﬁndings suggest that a sufﬁ-

rocal interactions occur with bovine DC and ␥␦ T cells. cient number of macrophages are able to migrate to the

+ −/−

Incubation of WC1 ␥␦ T cells with M. bovis-infected DC lung to control low dose M. tb infection in CCR2 mice

induced signiﬁcantly enhanced expression of MHC II and whereas macrophage numbers are not sufﬁcient to control

CD25, as well as increased secretion of IFN-␥ by the ␥␦ T the disease with higher doses of M. tb. Thus, a “normal”

cells (Price and Hope, 2009). In turn, DC produced enhanced macrophage response may actually be exaggerated with

levels of biologically active IL-12 when incubated with ␥␦ low dose M. tb infection of mice. CCR5 is a chemokine

T cells. receptor on T cells, macrophages, and dendritic cells. Unex-

In addition to the studies above, ␥␦ T cells have also been pectedly, mice genetically deﬁcient in CCR5 are able to

described to fulﬁll other functions in the immune response control M. tb infection, producing organized granulomas

to mycobacteria, including IL-17 production (Lockhart with increased numbers of T cell lung inﬁltrates as com-

et al., 2006; Umemura et al., 2007) and direct cytoxicity pared to wild type mice without a corresponding increase

(Skinner et al., 2003b; Stenger et al., 1998); however, the in mycobacterial numbers (Algood et al., 2004). With

role of these functions in cattle during M. bovis infection are non-human primates, chemokine expression patterns are

not well deﬁned. Together, it is obvious that ␥␦ T cells play similar to those of mice in response to M. tb infection, yet

a critical role in bridging innate and adaptive immunity less fully characterized (Mehra et al., 2010). With sam-

and promoting the development of a robust, and appropri- ples from humans, numerous studies have demonstrated

ate, adaptive immune response to M. bovis. Future studies various patterns of chemokine and chemokine receptor

should be aimed at more clearly deﬁning the role of ␥␦ expression by various cell types (e.g., macrophages, alve-

T cells in vivo during M. bovis infection and importantly, oloar and bronchial epithelial cells, neutrophils) and from

future attempts at vaccine development should make note cells within bronchial alveolar lavage ﬂuid following in vitro

of the contributions of ␥␦ T cells during mycobacterial stimulation with mycobacterial antigens (reviewed by

infections and strive to engage this population in protective Algood et al., 2003). Additionally, polymorphisms in sev-

immune responses. eral chemokines (e.g., CXCL10, RANTES, IL-8, MCP-1) are

variably associated with TB susceptibility in humans (Azad

3.5. Chemokines et al., 2012).

␥

IFN- -induced Protein 10 (IP-10, also termed CXCL10)

Chemotactic cytokines (chemokines) are essential for is detectable within tuberculous granulomas and in DTH

granuloma formation with mycobacterial infections (Slight reactions in response to mycobacterial antigens of humans

and Khader, 2012). As early as 12–21 days after low (Ferrero et al., 2003; Kaplan et al., 1987). IP-10 attracts acti-

dose aerosol infection with M. tb, mRNA for numerous vated T cells and monocytes to inﬂammatory foci (Farber,

chemokines and their receptors are up-regulated within 1997), has antimicrobial functions (Cole et al., 2001), and

lungs of infected mice, correlating with recruitment of promotes Th1 responses (Sauty et al., 1999). IP-10 levels

immune cells into the lung (Kang et al., 2011). The early are elevated within tissues and serum of leprosy patients

accumulation of NK cells, ␥␦ T cells, and macrophages (Scollard et al., 2011; Stefani et al., 2009) and IP-10 is

as well as slightly later CD4 T cells, CD8 T cells, den- detectable in pleural effusions and plasma of TB-infected

dritic cells, and B cells coincide with the induction of humans (Azzurri et al., 2005; Juffermans et al., 1999). In a

chemokine expression within infected lung tissues, with multicenter evaluation, a Luminex-based IP-10 assay per-

speciﬁc chemokines being associated with speciﬁc cell formed similarly to commercial IGRA’s (Ruhwald et al.,

types. Chemokines such as CXCL13, CCL21, and CCL19 2011) for the diagnosis of TB in humans. Conclusions from

appear to at least partially mediate the spatial local- this study were that antigen-speciﬁc IP-10 is produced in

ization of immune cells within the granuloma, thereby, response to TB infection, the IP-10-based TB test has accu-

participating in mycobacterial control (Khader et al., racy comparable to commercial IGRA’s, IP-10 appears to

␥

2009). Inﬂammatory chemokines are induced very early be less affected by non-TB infections than IFN- , and IP-10

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

is detected in more patients with relevant TB risk factors 2005). Similarly, during TB treatment of HIV-1 co-infected

than IFN-␥ (indicative of either a higher sensitivity or lower individuals, chemokine plasma levels and CCR5/CXCR4

speciﬁcity of the IP-10 assay). IP-10 is also a candidate expression on CD4 levels is increased (Wolday et al.,

biomarker for detection of TB exposure in children; how- 2005). Thus, with humans and cattle, non-speciﬁc eleva-

ever, as with IGRA’s, IP-10-based assays cannot be used to tions (i.e., without recall stimulation in vitro) in peripheral

discriminate between active and latent TB within this pop- chemokines and chemokine receptors are detected, con-

ulation (Whittaker et al., 2008). And, HIV infection does not founding interpretation for speciﬁc diagnostic applications.

appear to impair TB-speciﬁc IP-10 responses as severely as Further studies are necessary to determine the poten-

that observed with IFN-␥-based tests (Aabye et al., 2010; tial for use of chemokines, such as IP-10, as biomarkers

Kabeer et al., 2010). Also, given its acute-phase reactant for use in bovine TB diagnostic tests. Additionally, cattle

properties, IP-10 may be a useful candidate for monitoring may serve as a useful model to better deﬁne sequential

treatment responses, as well as for predicting mortality and expression of chemokines and their respective recep-

morbidity of TB patients (Ruhwald et al., 2012). tors within tissues after experimental infection with M.

Aranday-Cortes et al. demonstrated that CXCL9 and IP- bovis as granuloma morphology and mycobacterial col-

10 are highly expressed (mRNA) within granulomas of onization (i.e., paucibacillary) more closely mimic the

tuberculous cattle (Aranday-Cortes et al., 2012a,b). Simi- disease of humans as compared to mouse models, particu-

larly, CXCL9 and IP-10 are expressed at very high levels in larly with use of laser capture microdissection to deﬁne

cells surrounding granulomas in M. tb-infected macaques local variations in the response (Aranday-Cortes et al.,

(Fuller et al., 2003). With cattle, M. bovis PPD stimulation 2012a,b).

elicits IP-10 mRNA expression by PBMC as early as 28 days

after aerosol M. bovis infection and these responses corre-

late (r = 0.87) to IFN-␥ mRNA expression within the same 3.6. Polyfunctional T cells

samples (Waters et al., 2012b). A whole blood quantitative

rtPCR approach has been described for the detection of TB Polyfunctional antigen-speciﬁc T cells simultaneously

and other infectious diseases of humans (Kasprowicz et al., produce two or more cytokines and higher frequencies of

2011). For the TB assay, MIG (monokine-induced by IFN-␥) these cells are correlated with control of chronic infec-

and IP-10 mRNA responses to ESAT-6 and CFP-10 peptide tions such as HIV, hepatitis C, leishmaniasis, malaria, and

pools correlate with IFN-␥ (protein) responses as detected TB (reviewed in Caccamo and Dieli, 2012; Wilkinson and

by an ELISPOT assay (Kasprowicz et al., 2011). Also, con- Wilkinson, 2010). After infection or vaccination, the ensu-

current analysis of CXCL8 (IL-8), IL-12B, and FoxP3 mRNA ing immune response may consist of a balance of single

expression in ESAT-6-stimulated PBMC cultures has been cytokine and polyfunctional T cell responses, based upon

proposed as a method to distinguish active from latent antigen load and chronicity of infection. For instance, with

TB in humans (Wu et al., 2007). Studies with IP-10 and chronic viral infections, polyfunctional CD4 and CD8 T cells

␥

IFN- mRNA responses in cattle (Waters et al., 2012b) sug- are more likely to be correlated with protection (when anti-

gest that a similar approach using chemokine and cytokine gen load is low) than single-cytokine producing T cells,

mRNA proﬁles may be useful for the detection of tubercu- while single IFN-␥-secreting T cells are characteristic of

lous cattle. Experimental infection trials with cattle may acute infection (when antigen load is high) (Harari et al.,

also be useful to determine the kinetics of the response 2006). If chronic infection persists after failure of immune

as well as interactions of prior vaccination (e.g., BCG or control, the balance tends to shift from polyfunctional T

novel attenuated mutants of M. bovis) and potential inter- cells back to single IFN-␥-secreting cells (Wilkinson and

ference of NTM’s on the response. Such studies would Wilkinson, 2010). With both primary and chronic phases

likely provide information valuable for the application of HIV-1 infection, single IFN-␥-secreting effector cells

of mRNA-based techniques for use with the diagnosis of predominate the response; however, an increased poly-

human TB, particularly with the development of ﬁeld ready functional T cell response occurs after a positive response to

tests. antiretroviral therapy, similar to that observed in individ-

After aerosol M. bovis challenge of cattle, IP-10 (protein) uals who spontaneously control HIV-1 replication (Harari

responses to mycobacterial antigens (e.g., rESAT-6:CFP-10, et al., 2004; Day et al., 2008). M. tb-speciﬁc polyfunc-

M. bovis PPD) exceed pre-infection responses begin- tional T cells secreting IFN-␥/TNF-␣ or IFN-␥/IL-2 are also

ning as early as 7 days (Waters et al., 2012b). Despite detected in M. tb/HIV co-infected individuals (Wilkinson

signiﬁcant responses to mycobacterial antigens upon infec- and Wilkinson, 2010). With these individuals, the fre-

tion, IP-10 concentrations in non-stimulated whole blood quencies of M. tb-speciﬁc IL-2-secreting (IFN-␥/IL-2 and

cultures are similar to those in antigen-stimulated cul- IL-2 only) CD4 T cells negatively correlate with HIV viral

tures. With the same sample set, IFN-␥ responses to load; whilst, there is a positive correlation between the

mycobacterial antigens exceeded corresponding responses frequencies of IFN-␥ single positive CD4 T cells and HIV-

in non-stimulated cultures; thus, IP-10 protein appears 1 viral load (Day et al., 2008). Thus, M. tb-speciﬁc T

to be increased within the plasma of TB-infected cattle cells secreting IL-2, with or without IFN-␥, may be pro-

non-speciﬁcally. High levels of IP-10 are also detected portionally diminished when HIV-1 viral load is high,

in non-stimulated whole blood samples from children corresponding with greater susceptibility for development

with TB (latently infected > active TB) (Whittaker et al., of active TB. Therefore, CD4 T cells secreting IL-2 alone

2008) and in the plasma of adults with active TB, possi- or with other cytokines may correlate with protection to

bly due to chronic TB-induced inﬂammation (Azzurri et al., TB.

W.R. Waters et al. / Veterinary Immunology and Immunopathology 159 (2014) 113–132 123

Conversely, the majority of studies with human cell responses to disease severity and protective responses

TB indicate that polyfunctional T cell responses are in TB vaccine efﬁcacy trials.

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 lev- 3.7. Cytotoxic T lymphocytes (CTL’s)

els in latently infected individuals. The anti-mycobacterial

response by latently infected patients was mainly due Cytolysis of infected cells can result in direct killing

to IFN-␥ single and IFN-␥/IL-2 dual secreting CD4 T of Mycobacterium spp. or release of bacilli for eventual

cells. Additionally, the frequency of polyfunctional T cells killing via other mechanisms (reviewed by Flynn and Chan,

in TB patients decreased to undetectable levels after 6 2001). CD8 T cells are the primary cell type perform-

months of curative TB treatment, suggesting a correlation ing CTL functions; however, other lymphocyte populations

␥␦

of bacterial load to functional patterns of the CD4 T cell such NK cells, CD4 T cells, and T cells also have

response. Sutherland et al. (2009) also found higher fre- cytolytic capacity (Stenger et al., 1998; Canaday et al.,

quencies of CD4 T cells simultaneously secreting IFN-␥, IL-2 2001). As related to TB infection, primary effector func-

␥

and TNF-␣ in TB patients (sputum smear positive) com- tions of CD8 T cells are production of IFN- necessary for

pared to household contacts. In regard to the protective macrophage activation and lysis of infected macrophages

role that polyfunctional T cells may have on human TB, (Einarsdottir et al., 2009). Perforin and granulysin are

Streitz et al. (2011) found TNF-␣ production in response essential for CTL function via pore formation and antimi-

to mycobacterial antigens as the strongest predictor of crobial functions, respectively. Recent studies indicate

␥

active TB, while CD4 T cells secreting IFN-␥ and IL-2 pre- that IFN- from CD4 T cells is required for effective

dominated in successfully treated, latently infected, and CD8 T cell responses (Green et al., 2013). Thus, CTL

BCG-vaccinated individuals. Using a whole-blood ﬂow- functions are essential for the control of mycobacterial

cytometric assay, Sester et al. (2011) demonstrated that infections and CD4 T cell responses are supportive of this

patients with active TB had lower percentages of PPD- response.

reactive dual cytokine-secreting cells as compared to that With M. bovis infection of cattle, activated CD8 T

of patients with non-active disease—suggestive of a pro- cells are detectable within the lymphocytic outer core of

tective role for polyfunctional T cells. Additionally, several early stage (i.e., stage I and II) tuberculous granulomas,

TB vaccines (including live attenuated, subunit, plas- indicating a potential role for these cells in the initial

mid DNA, recombinant proteins, peptides, viral vectored, containment of the bacilli (Liebana et al., 2007). Antigen-

protein-in-adjuvant vaccines, and primeboost immuniza- speciﬁc CD8 T cells from cattle are capable of causing

tions platforms) elicit polyfunctional T cell responses release of viable M. bovis from infected macrophages,

considered protective (Thakur et al., 2012). Nevertheless, presumably indicating CTL activity (Liebana et al., 2000).

given the diversity of disease states in infants, adolescents, Bovine T cells express a homologue of human gran-

and HIV-infected adults; the various vaccine candidates ulysin, a potent antimicrobial protein stored in association

being developed; various parameters being analyzed; and with perforin in cytotoxic granules (Endsley et al., 2004).

the differences in techniques—it is unlikely that a sin- Antigenic stimulation of peripheral CD4 T cells from BCG-

gle, simple immune correlate relative to single-cytokine vaccinated cattle results in enhanced anti-mycobacterial

or polyfunctional response exists across all these different activity against BCG-infected macrophages linked with

variables (Caccamo and Dieli, 2012). Hopefully, additional increased perforin and granulysin transcription (Endsley

studies may at least provide some clarity for this complex et al., 2007). Expression of the bovine granulysin gene

+ +

response. can be induced in CD4 , CD8 , and ␥␦ T cells resulting in

In cattle, the contribution of polyfunctional T cells anti-mycobacterial activity similar to human granulysin

was until recently missing due to the lack of monoclonal (Endsley et al., 2004, 2007). Using laser capture microdis-

antibodies that recognize biologically active bovine IL-2. section, granulysin and granzyme A mRNA are detectable

Whelan et al. (2011), using a recombinant human anti- within granulomas of M. bovis-infected cattle (Endsley

body fragment that detects the expression of bovine IL-2, et al., 2004; Aranday-Cortes et al., 2012b). Granzymes

recently described the development of an assay to detect are a group of serine proteases released by CD8 T cells

polyfunctional CD4 T cells in cattle using cells from cattle and NK cells in cytoplasmic granules along with per-

naturally infected with M. bovis. Bovine polyfunctional CD4 forin. Granulysin and perforin gene expression are also

hi lo + + +

T cells exhibited a characteristic CD44 CD62L CD45RO T up-regulated in peripheral blood CD4 and CD8 T cells

cell effector memory (TEM) phenotype. Additionally, Kaveh in both BCG- and M. bovis RD1-vaccinated calves (pro-

et al. (2012) demonstrated that the duration of the intracel- tected) as compared with non-vaccinated (not protected)

lular staining culture, pre-stimulation period, and cytokine calves (Capinos Scherer et al., 2009) demonstrating the

accumulation period substantially inﬂuence the cytokine potential of these biomarkers as correlates of protection

repertoire and frequency of antigen-speciﬁc polyfunctional for prioritizing vaccine candidates. Clearly, CTL’s are an

CD4 T cell subsets in cattle. Thus, the tools are now available important aspect of the bovine immune response to M.

to evaluate polyfunctional T cell responses in cattle and fur- bovis infection and vaccine responses; however, further

ther investigations are warranted to determine the kinetics studies are required to identify their exact roles, both

of the polyfunctional T cell response to M. bovis infection in protective immunity and response to persistent infec-

in cattle as well as a comparison of bovine polyfunctional T tion.

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

4. B cells studies demonstrate the potential for antibody-mediated

immunity to modulate the course of infection. Supportive

4.1. Role of B cells: therapeutic sera, supportive of this notion, is the consistent observation of B cell

functions, and ectopic B cell aggregates aggregates associated with tuberculous lesions in mice

(Gonzalez-Juarrero et al., 2001), humans (Ulrichs et al.,

B cell responses are essential for protection against 2004), cattle (Aranday-Cortes et al., 2012a,b; Johnson

a wide variety of microbial pathogens; however, et al., 2006), and other host species (García-Jiménez et al.,

speciﬁc roles for B cells in the immune response to 2012; Phuah et al., 2012) infected with M. tb complex

TB have remained elusive and controversial—generally organisms. These tertiary lymphoid structures contain

being considered supportive rather essential in nature aggregates of B cells (naïve, memory, and plasma cells)

+ +

(reviewed by Glatman-Freedman and Casadevall, 1998; as well as intermixed CD4 and CD8 T cells, follicular

Maglione and Chan, 2009). Since the late 19th century, dendritic cells, and mycobacteria-laden APC’s (Ulrichs

numerous studies have examined the possibility for use et al., 2004). Intradermal injection of rESAT-6:CFP-10

of ‘therapeutic sera’ (i.e., immune sera given to inactivate fusion protein into calves experimentally infected with M.

or clear the bacillus from infected individuals) in the bovis elicits a DTH response involving B cells, in addition to

+ +

treatment of TB—with mixed and unconvincing results predominating CD4 and CD8 cells found in the skin inﬁl-

(Glatman-Freedman and Casadevall, 1998). The use of trates (Waters et al., 2009). Using immunohistochemistry,

therapeutic sera, and associated efﬁcacy studies, fell out Aranday-Cortes et al. (2012a,b) demonstrated the presence

of favor with the advent of successful pharmacologic of CD79a-stained cells, a B cell marker, within granulomas

anti-mycobacterial compounds (e.g., streptomycin) in of tuberculous cattle. In that study, early granulomas

the mid 20th century. Recent studies with monoclonal (stages I and II) displayed scattered B cells, whereas more

antibodies speciﬁc for mycobacterial arabinomannan, advanced granulomas (stages III and IV) showed satellite

16 kDa ␣-crystallin protein, MPB83 protein or 28 kDa nests of CD79a cells located peripherally and outside of

heparin-binding hemagglutinin have shown protective the ﬁbrous capsule. Non-human primate models of M.

efﬁcacy in mouse models of TB caused by M. tb or M. tb infection demonstrated signiﬁcant numbers of B cells

bovis (Glatman-Freedman and Casadevall, 1998; Glatman- (CD20 ) within lung granulomas, which were organized

Freedman, 2006; reviewed by Abebe and Bjune, 2009). into discrete clusters with characteristics of germinal

With that said, antibodies elicited by infection with M. centers including numerous activated B cells (Phuah et al.,

tb complex bacilli are typically not considered protective 2012). In mice, formation of B cell follicles within infected

for the control of TB (reviewed in Glatman-Freedman and lung tissues is dependent upon IL-23 and CXCL13; and,

Casadevall, 1998). While the exact role of B cell responses CXCL13 production is dependent upon IL-17A and IL-22 in

during mycobacterial diseases remains unclear, generally this response (Khader et al., 2011). The presence of ectopic

B cells may inﬂuence the ensuing response to infections germinal centers indicates that the M. tb complex – and

beyond direct antibody-pathogen interactions. Capturing the ensuing inﬂammation – induces active B cell clusters

antigen by speciﬁc surface receptors, B cells are efﬁcient that modulate the host response. It is thus believed that

APC’s effectively priming memory T cell responses even these follicles provide at least a partial framework for

at low levels of circulating antigen concentration (Lund coordinated immune control of mycobacterial growth in

et al., 2006; Shen et al., 2003). Antibodies, including the affected tissues (Ulrichs et al., 2004).

immune complexes, also regulate APC’s through interac-

tions with Fc␥ receptors. Disruption of stimulatory Fc␥ 4.2. Emerging serodiagnostic tests for veterinary

receptor activity increases susceptibility of mice to M. tb applications

infection whereas genetic deletion of inhibitory Fc␥RIIB

improves containment of M. tb, presumably by increasing Several antibody-based tests have recently emerged for

Th1 polarization (Maglione et al., 2008). Additionally, use in cattle, captive cervids, several wildlife reservoirs

immune complexes from TB patients down regulate of M. bovis, and various zoo species (most notably ele-

cytokine production by neutrophils while enhancing phants). Sero-dominant antigens vary by host species, with

chemotaxis and phagocytosis (Senbagavalli et al., 2012). MPB83 (alone or in combination with MPB70 or 16 kDa

␣

Antibody-dependent T cell cytotoxicity is also believed to -crystallin), ESAT-6, and CFP-10 being commonly recog-

play a role in protective immunity to mycobacteria (de nized targets for many species (Lyashchenko et al., 2006,

Vallière et al., 2005). Outcomes of M. tb infection experi- 2008; Waters et al., 2006a,b, 2011a). Human antibody

ments on B cell-deﬁcient mice are variable, ranging from responses in active TB involve generally variable patterns

delayed disease progression and diminished immunity of serum IgG reactivity, with the 38 kDa protein of M. tb

to no effects (reviewed in Maglione and Chan, 2009). being a major serodiagnostic antigen recognized in spu-

Genetic ablation of polymeric Ig receptor, however, results tum smear positive patients (Lyashchenko et al., 1998).

in heightened susceptibility of mice to M. tb infection, Interestingly, this protein elicits poor antibody responses,

implying a role for secretory IgA for optimal TB immunity if any, in most animal hosts including nonhuman primates

(Tjarnlund et al., 2006). Intranasal inoculation of human (Lyashchenko et al., 2007, 2008). In contrast, ESAT-6 and

monoclonal IgA1 antibody speciﬁc to ␣-crystallin in CFP-10 epitopes are predominantly involved in T cell reac-

combination with recombinant mouse IFN-␥ inhibits tivity in both humans and animal species (reviewed in

pulmonary M. tb infection in mice transgenic for human Vordermeier, 2010). The accuracy of antibody-based tests

CD89 (Balu et al., 2011). While not always deﬁnitive, these also varies considerably by species (Lyashchenko et al.,

W.R. Waters et al. / Veterinary Immunology and Immunopathology 159 (2014) 113–132 125

2008), remarkably, approaching 100% sensitivity and >95% CMI responses, persistent colonization, and associated

speciﬁcity for elephants with use of the Elephant TB Stat tuberculous lesions. Thus, regardless of the pathological

Pak or Dual Path Platform assays (Chembio Diagnostics Inc, and mycobacterial burden outcome, CMI responses are

Medford, NY; Greenwald et al., 2009). Antibody responses elicited. In contrast, antibody responses generally corre-

by elephants with advanced stages of disease are par- late with levels of pathology associated with mycobacterial

ticularly robust, both in the level of the response and infections. For instance, mycobacterial-speciﬁc antibody is

diversity of antigen recognition (Greenwald et al., 2009; detectable relatively early after M. tb challenge of cattle,

Lyashchenko et al., 2006, 2012). Antibody responses by yet these responses wane over time, likely coincident with

elephants typically decline following a favorable outcome the reduction of M. tb colonization. In contrast, with M.

with antibiotic therapy or increase shortly before dis- bovis infection that leads to persistent infection and sig-

ease recrudescence (Lyashchenko et al., 2008, 2012). These niﬁcant pathology, antibody responses persist, likely due

ﬁndings demonstrate the potential for real-time serologic to persistently increasing antigen burden. With less viru-

monitoring of response to therapy as well as disease stag- lent mycobacteria (e.g., M. kansasii), antibody responses are

ing. For certain cervid and camelid species in which skin elicited and then immediately wane, likely coincident with

test is ineffective for TB diagnosis, serology offers a conve- complete clearance of the pathogen. Regardless of disease

nient option for surveillance (Rhodes et al., 2012; Waters expression (e.g., with M. tb, M. bovis, or M. kansasii inocu-

et al., 2011a). Thus, lessons learned with serology for vari- lation), mycobacteria-speciﬁc antibody responses may be

ous captive-wildlife or livestock species may be applicable boosted by re-exposure to mycobacterial antigens (e.g.,

for pathogenesis and diagnostic purposes with M. tb infec- PPD) or other live mycobacteria. Close association between

tion in humans. antibody responses and presence of visible lesions was

also reported for non-bovine hosts of M. bovis infection

4.3. Booster effect (Lyashchenko et al., 2008; Boadella et al., 2012). In regards

to relevance to human TB, M. bovis infection of cattle may

Intradermal tuberculin administration is known to sig- be considered as a model of clinical TB, whilst M. tb H37Rv

niﬁcantly boost antibody responses in tuberculous cattle, infection could be similar to latent, subclinical TB and M.

cervids, and other hosts, but not in non-infected animals kansasii inoculation of cattle a model for TB that has been

(Lyashchenko et al., 2004, 2007; Harrington et al., 2008; successfully cleared from its human host. Therefore, these

Dean et al., 2009). This phenomenon can be observed from three infection systems could be developed into useful

1-2 weeks to several months after tuberculin injection, models mimicking different stages of human TB infection.

depending on the type and dose of PPD, host species, stage

of disease, pre-existing antibody levels, antigen reactiv-

ity patterns, and immunoassay format used (Chambers 5. Summary

et al., 2009; Palmer et al., 2006; our unpublished observa-

tions). Although the boosting effect is not well studied, the In many aspects, the immune response by cattle to M.

absence of seroconversion in non-infected animals and the bovis is analogous to the response by humans to M. tb.

short-lived antibody kinetics with features of an anamnes- This is not surprising given the similarities of the two

∼

tic response strongly suggest that it is due to memory pathogens ( 99.95% identity at the nucleotide level) and

B cells originally primed by mycobacterial infection that the extended evolution of the respective host/pathogen

can be quickly activated upon re-stimulation by tuberculin relationships. Landmark studies over the past 120 years

in vivo. For human TB, it remains unknown whether the have demonstrated the usefulness of cattle for the devel-

skin test using much lower doses of PPD can affect the opment of tools to control human TB (i.e., BCG, skin test,

antibody response and hence modify accuracy of serodi- IGRA’s, and DIVA strategies). More recent studies have

agnostic tests. However, based on the animal studies, such demonstrated that immunopathogenesis studies in cattle,

possibilities exist and it should be taken into account when including vaccine efﬁcacy trials for correlation to protective

new immunodiagnostic assays for human TB are validated responses, are also applicable to our understanding of TB

in the future. in humans. With cattle, experimental biology approaches

(i.e., M. bovis challenge and vaccine efﬁcacy procedures)

4.4. Antibody responses: comparisons to CMI as related are reﬁned to determine immune correlates associated

to pathogenesis with both protective and damaging responses. Tools are

available to study in-depth cellular immune responses

Cellular immune responses elicited by mycobacterial such as effector/memory populations, T helper subsets, CTL

infections of cattle generally correlate with infection but responses, cytokine proﬁles (including polyfunctional T

␥␦

not necessarily with the level of pathology (Waters et al., cells), chemokines, and T cell subpopulations; antibody

2010). Inoculation of cattle with M. tb H37Rv or M. bovis responses including correlations to protection/pathology;

Ravenel results in relatively robust CMI responses and per- in situ responses using laser capture microscopy and

sistent colonization with minimal to no lesions. With M. qPCR; and interactions with NTM’s and other co-infections.

kansasii inoculation, CMI responses are elicited (although Finally, ﬁndings from experimental biology studies are

less robust than with M. tb or M. bovis infection) with- easily translated into development and validation of new

out detection of the organism or associated lesions. As tools for the control of bovine TB, some of which, may

expected, inoculation of cattle with virulent M. bovis (ﬁeld then become useful for improved disease management in

strains such as 95-1315 or A2122/97) results in robust humans.

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

Conﬂict of interest vaccinated cattle revealed a dominant role of IL-22 for protection

against bovine tuberculosis. PLoS Pathogens 8 (12), e1003077.

Black, G.F., Weir, R.E., Floyd, S., Bliss, L., Warndorff, D.K., Crampin, A.C.,

The authors of this manuscript do not have any com-

Ngwira, B., Sichali, L., Nazareth, B., Blackwell, J.M., Branson, K., Chag-

mercial or other associations that might pose a conﬂict of uluka, S.D., Donovan, L., Jarman, E., King, E., Fine, P.E., Dockrell,

H.M., 2002. BCG-induced increase in interferon-gamma response to

interest for this manuscript.

mycobacterial antigens and efﬁcacy of BCG vaccination in Malawi

and the UK: two randomised controlled studies. Lancet 359 (9315),

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Research funds were provided by: USDA, ARS (CRIS

associated with pathology in a bovine model of tuberculosis. Tuber-

#3625-32000-104) and Project #2011-67015-30736 under culosis (Edinb) 91 (1), 57–63.

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