Mycobacterium bovis Infection of Cattle and White-Tailed Deer: Translational Research of Relevance to Human Tuberculosis.

ILAR Journal, 2015, Vol. 56, No. 1, 26–43 doi: 10.1093/ilar/ilv001 Article

Mycobacterium bovis Infection of Cattle and White-Tailed

W. Ray Waters and Mitchell V. Palmer Dr. W. Ray Waters, DVM, PhD, is a veterinary medical officer in the TB Research Project in the Infectious Bacterial Diseases of Livestock Research Unit at the National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture (USDA), Ames, Iowa, and a collaborator/assistant professor of veterinary microbiology and preventive medicine at Iowa State University, Ames, Iowa. Dr. Mitchell V. Palmer, DVM, PhD, is a veterinary medical officer in the TB Research Project in the Infectious Bacterial Diseases of Livestock Research Unit at the National Animal Disease Center, Agricultural Research Service, USDA, Ames, Iowa, and a collaborator/ assistant professor of veterinary pathology at Iowa State University, Ames, Iowa. Address correspondence and reprint requests to Dr. Ray Waters, National Animal Disease Center, 1920 Dayton Avenue, Ames, IA 50010 or email: ray.waters@ ars.usda.gov.

Abstract Tuberculosis (TB) is a premier example of a disease complex with pathogens primarily affecting humans (i.e., Mycobacterium tuberculosis) or livestock and wildlife (i.e., Mycobacterium bovis) and with a long history of inclusive collaborations between physicians and veterinarians. Advances in the study of bovine TB have been applied to human TB, and vice versa. For instance, landmark discoveries on the use of Koch’s tuberculin and interferon-γ release assays for diagnostic purposes, as well as Calmette and Guérin’s attenuated M. bovis strain as a vaccine, were first evaluated in cattle for control of bovine TB prior to widescale use in humans. Likewise, recent discoveries on the role of effector/memory T cell subsets and polyfunctional T cells in the immune response to human TB, particularly as related to vaccine efficacy, have paved the way for similar studies in cattle. Over the past 15 years, substantial funding for development of human TB vaccines has led to the emergence of multiple promising candidates now in human clinical trials. Several of these vaccines are being tested for immunogenicity and efficacy in cattle. Also, the development of population-based vaccination strategies for control of M. bovis infection in wildlife reservoirs will undoubtedly have an impact on our understanding of herd immunity with relevance to the control of both bovine and human TB in regions of the world with high prevalence of TB. Thus, the one-health approach to research on TB is mutually beneficial for our understanding and control of TB in humans, livestock, and wildlife. Key words: cattle; deer; immunology; mycobacteria; one-health; pathogenesis; tuberculosis; vaccines

Introduction Bovine tuberculosis (TB) results primarily from infection with Mycobacterium bovis, which is a member of the Mycobacterium tuberculosis (tb) complex consisting of M. tb, M. africanum, M. bovis, M. caprae, M. pinnipedii, M. canetii, M. microti, M. mungi,

M. orygis, M. suricattae, and the dassie bacillus (Tortoli 2014; van Ingen et al. 2012). The most recent common ancestor of the M. tb complex likely evolved from a Mycobacterium kansasii-like opportunistic pathogen through horizontal gene transfer that resulted in transformation of the mycobacterium to a

Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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Deer: Translational Research of Relevance to Human Tuberculosis

ILAR Journal, 2015, Vol. 56, No. 1

Wildlife Reservoirs of M. bovis Infection Tuberculosis due to M. bovis has been reported in a number of wild ruminant species worldwide, including the following: • elk (Cervus elaphus nelsoni) in the United States (O’Brien et al. 2006) • red deer (Cervus elaphus) in New Zealand, Spain, England, France, and Austria (Beatson 1985; Delahay et al. 2007; Hellstrom 1979; Schoepf et al. 2012; Zanella et al. 2008b) • sika deer (Cervus nippon) in Ireland and New Zealand (Coleman and Cooke 2001; Dodd 1984) • white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus) in the United States (Rhyan et al. 1995; Schmitt et al. 1997) • fallow deer (Dama dama) in England and Spain (Aranaz et al. 2004; Delahay et al. 2007) • axis deer (Axis axis) in the United States (Sawa et al. 1974) • roe deer (Capreolus capreolus) in England, Spain, Italy, Switzerland, and France (Balseiro et al. 2009; Bischofgberger and Nabholz 1964; Delahay et al. 2007; Zanella et al. 2008a) • muntjac deer (Muntiacus reevesi) in England (Delahay et al. 2007) • reindeer (Rangifer tarandus) in the former Soviet Union (Syroechkovskii 1995) • moose (Alces alces) in Canada (Wobeser 2009) • African buffalo (Syncerus caffer), greater kudu (Tragelaphus strepsiceros), eland (Taurotragus oryx), impala (Aepyceros melampus), bushbuck, nyala, waterbuck, and wildebeest in South Africa (Bengis et al. 1996; Hlokwe et al. 2014; Michel et al. 2006) • lechwe antelope (Kobus lechwe) in Zambia (Munyeme et al. 2008) • feral water buffalo (Bubalis bubalis), wildebeest, and topi (Damaliscus kiorrigum) in Tanzania (Cleaveland et al. 2005) • bison (Bison bison) and wood bison (Bison bison athabascae) in the United States and Canada, respectively (Kaneene et al. 2010). Although susceptible to infection with M. bovis, most of these species are incapable of sufficient intraspecies transmission to maintain infection within their respective populations. However, in an important few, adequate intraspecies transmission occurs to maintain infection within the wildlife population and even allow interspecies transmission to other susceptible hosts. These species are known as reservoir hosts, and in some countries eradication of TB from domestic cattle has been impeded by the presence of M. bovis in wildlife reservoirs (ruminant and nonruminant) with persistent wildlife-to-cattle transmission. As the prevalence of TB in cattle decreases, the relative importance of M. bovis-infected wildlife increases, and disease-control measures are required for both livestock and wildlife. The most recognized wildlife reservoir hosts include the Eurasian badger (Meles meles) in the United Kingdom and Republic of Ireland, brushtail possum (Trichosurus vulpeculus) in New Zealand, wild boar (Sus scrofa) in Spain, and white-tailed deer in the United States (CS Gortazar and DJ O’Brien, personal communication, 2014). In all cases, the presence of wildlife reservoirs of M. bovis obstructs the success of long-standing and costly TB eradication efforts in cattle. In its simplest form, a single wildlife reservoir serves as the source of infection for cattle. This is illustrated by the example of white-tailed deer in Michigan, USA. However, in some countries, multiple species are epidemiologically linked and may include multiple reservoir hosts and multiple routes of transmission (Caley and Hone 2005). Moreover, each species


professional intracellular pathogen (Behr 2013). The various species within the complex then evolved from this common ancestor primarily through deletion of unnecessary DNA (Behr 2013). Comparative analyses of M. tb and M. bovis genomes indicate that M. bovis evolved from an ancestral M. tb strain, as the M. bovis genome is smaller than that of M. tb (Smith et al. 2006). Host adaptation and differential transmissibility potential are distinguishing traits for the M. tb complex species. For instance, human-to-human transmission of M. bovis is rare (Evans et al. 2007), whereas M. tb readily transmits between humans. And host preference, including diversity of susceptible hosts and the primary host(s) for each species, varies between the tubercle bacilli. Mycobacterium bovis has a wide host range, is infectious to humans, and causes significant economic hardship for livestock farmers, with estimates of more than 50 million cattle infected worldwide, costing $3 billion annually. Zoonotic transmission of M. bovis occurs primarily via ingestion of unpasteurized dairy products or close contact with infected cattle (Cosivi et al. 1998). It is estimated that, prior to wide-scale pasteurization, approximately 20–40% of TB cases in humans resulted from infection with M. bovis (Francis 1959; Ravenel 1933). The current proportion of human TB cases due to M. bovis is low (global median proportion ≤1.4%), yet higher rates are reported for continental Africa (∼2.8%), Mexico (∼7.6%), Turkey (∼5.3%), and the West Bank of Palestine (∼6.5%) (Müller et al. 2013). From 2001 to 2005, there were 19 deaths due to M. bovis infection in San Diego County, California; and from 1994 to 2005, M. bovis accounted for 45% (62/138) of all culture-positive TB cases in children and 6% (203/3,153) in adults in this population (Rodwell et al. 2008). Of the human TB cases caused by M. bovis in San Diego County, ∼96% of the patients were of Hispanic ethnicity and 60% of Mexican origin. A major risk factor for this population was consumption of unpasteurized dairy products, primarily cheese, produced from milk obtained from TB-endemic regions of Mexico. In countries with a low prevalence of bovine TB, affected cattle herds generally contain few infected animals, suggestive of a slowly progressive course of disease with low rates of cattle-tocattle transmission (Palmer and Waters 2006). Transmission is often by direct contact with TB-infected animals, especially those with advanced lesions (Khatri et al. 2012). Ingestion of infected feeds may also lead to infection originating in the upper respiratory tract, pharyngeal area, or gastrointestinal tract (Palmer et al. 2004). Housing (e.g., milking parlors and sheds) and crowding (e.g., particularly gathering of animals from different sources such as heifer-rearing facilities and ports of entry) increase the contact of naïve animals with infected animals and enhance the spread of this disease. Veterinarians in the 1890s incorporated “open-air” policies for dairies (Palmer and Waters 2011), reminiscent of TB sanatoria for humans, potentially to decrease the level of M. bovis exposure. M. bovis is also transmitted by indirect contact through contaminated feed, water, equipment, personnel, or anything that mechanically transfers the organism between locations. Movement of infected animals and fence-line contact with other herds are common means of transferring the disease between regions and herds. Increasingly, infection of cattle also results from direct or indirect exposure to M. bovis-infected wildlife (Palmer 2013). There is an increasing risk of inter-regional spread of TB due to economic globalization. Transmission of M. tb in humans is generally by aerosol exposure, and there are no known animal reservoirs of M. tb infection. There appears to be a lower risk of indirect transmission with M. tb infection of humans as compared with M. bovis infection of livestock and wildlife.

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may differ in terms of disease prevalence, pathology, ecology, and behavior (Power and Mitchell 2004). These relationships create complex patterns of intra- and interspecies transmission (Nugent 2011). This is illustrated in Spain, where wild boar, red deer, cattle, and domestic pigs create a multihost system of persistent M. bovis infection (CS Gortazar, personal communication, 2014, and DJ O’Brien, personal communication, 2014).

Experimental Biology Approaches—Cattle and WhiteTailed deer

Pathology Bovine TB manifests as a chronic, caseonecrotic, granulomatous, inflammatory response primarily affecting lungs and lymph nodes. In cattle, the lesion characteristics are well known from natural and experimental infection studies. Generally, with a few exceptions, these characteristics can be applied to M. bovis infection in other ruminants such as sheep, goats, and deer. In deer, however, tuberculous lesions have commonly been described as liquefied or abscess-like in contrast to the caseous (i. e., cheesy) nature of lesions in cattle (Beatson 1985; Delahay et al. 2007; Fitzgerald and Kaneene 2013; Friend et al. 1963). So common is this abscess-like appearance that some authors suggest that all abscess-like lesions in deer should be regarded as suspicious and processed by bacteriological culture for mycobacteria (Beatson 1985). In addition to a liquefactive appearance, lesions in deer may also be caseous, with or without dystrophic mineralization. The likely route of transmission can be inferred from the distribution of lesions, particularly when only a single lesion is present (Biet et al. 2005). Hence, it is presumed that animals with lesions restricted to the respiratory tract have been infected by inhalation of aerosolized M. bovis, and that lesions restricted to the alimentary tract arise from infection through ingestion. However, these assumptions may not be entirely reliable. For example, in a study of experimental oral infection of cattle (i.e., infection through consumption of M. bovis containing corn), the preponderance of lesions were in the lungs and pulmonary lymph nodes (Palmer et al. 2004). Retropharyngeal lymph nodes, a common site for lesion development in cattle, were not affected. In another study, it was shown that of 36 bovine calves fed raw milk from M. bovis-infected cows, all developed lesions of the lungs and associated lymph nodes with no evidence of infection of mesenteric lymph nodes or intestine (Edwards 1937).

Natural Infection In naturally infected cattle, as in humans, tuberculous lesions are most commonly noted in the lungs and pulmonary lymph nodes. This combination of lesions at the initial site of infection (lungs) and corresponding draining lymph nodes is known as a primary complex of lesions. Within the lung, lesion distribution is not uniform. In humans, there is a predilection for granuloma development in the apical lung lobes (Samuelson and von Litchenberg 1994). In cattle, 90% of pulmonary lesions are found in the diaphragmatic lobes, 50% of which are located in the distal one third (McKay 1959; McIlroy et al. 1986; Stamp 1948). In cattle, a higher ventilation/perfusion ratio exists in cranioventral regions of the lung than in caudodorsal regions, creating a relative hypoxia in the caudodorsal lung regions (Robinson 1982; Ruiz et al. 1974). Such hypoxia may result in suboptimal macrophage function. Lesions in ruminants have been described as caseous and, to a lesser degree, liquefactive. This conversion of a solid or semisolid caseous lesion to one of a more liquid nature is similar to the liquefaction of pulmonary lesions described in humans and experimentally infected rabbits and due to the action of various


Aerosol and intratracheal inoculation are the most common routes currently used to infect cattle with virulent M. bovis. Both routes result in disease and lesion distribution similar to that seen in natural infection, with lesions most commonly found in the lungs and pulmonary lymph nodes (tracheobronchial and mediastinal); moreover, lesion severity is dose and time dependent (Buddle et al. 1994, 2005; Palmer et al. 2002b). Experimental approaches permit disease confirmation with laboratory analysis by histopathology and bacterial culture, defining the relationship between dose and route of infection, time, immune response, and pathogenesis. The neonatal calf model has proved useful in evaluation of candidate TB vaccines (Endsley et al. 2009) and development or evaluation of diagnostic assays. The bovine experimental infection model also provides opportunities to investigate the basis of genetic susceptibility and impacts of nutrition, parasite burden (Flynn et al. 2009), and viral infections (Kao et al. 2007) on pathogenesis and diagnostic techniques. It is generally thought that the majority of humans infected with M. tb have clinically latent infection, meaning they are infected and skin test positive, but do not exhibit clinical signs and are not contagious to others. In approximately 5–10% of latently infected humans the infection will reactivate, resulting in active TB. In contrast, latency and reactivation are not considered common characteristics of M. bovis infection of cattle. M. bovis infection of cattle usually results in a slowly progressive disease. Occasionally, M. bovis is detected by culture of tissues from animals with no visible lesions; however, it is uncertain if animals with no visible lesions would eventually develop progressive disease. Experimentally, M. tb H37Rv or M. bovis AN5 or Ravenel (all three are laboratory-adapted strains) infection of cattle results in colonization with minimal to no lesions 4 to 5 months after challenge, yet with robust cell-mediated immune (CMI) responses (Whelan et al. 2010). It is uncertain, however, if this experimental system could be manipulated to reproduce latency or reactivation. In humans and most species of animals, M. bovis infection is acquired through exposure to infectious aerosols. However, lesion distribution in naturally infected white-tailed deer suggests that oral exposure through ingestion is an important route of exposure (Palmer et al. 2000). In naturally infected deer, lesions are most commonly seen in the retropharyngeal lymph nodes, followed by the lungs and associated lymph nodes. In species where aerosol exposure dominates, lesions are focused on the lungs and associated lymph nodes with cranial lymph nodes less commonly involved (Palmer et al. 2002b). It is interesting to note that M. bovis infection in humans often results in cervical lymphadenitis (scrofula). More common in M. bovis-infected humans, these extrapulmonary lesions are rarely seen in M. tbinfected humans. One accepted reason for this difference is that a major route of M. bovis exposure in humans is through the consumption of milk from M. bovis-infected cows. Inoculation of deer through instillation of inoculum in the palatine tonsillar crypt results in a pattern of disease similar to

that seen in nature, one that is focused on the retropharyngeal lymph nodes with common but lesser involvement of the lungs and associated lymph nodes. This experimental model of infection has been used to evaluate pathogenesis, interspecies and intraspecies transmission, immune responses, and vaccine efficacy (Griffin and Buchan 1994).


Progression of Disease upon Experimental Infection— White-Tailed Deer Experimental administration of M. bovis by aerosol, intranasal, intratracheal, subcutaneous, and intravenous routes results in disease in red deer and white-tailed deer, but the resulting lesion distribution pattern is unlike that seen in naturally infected animals (Mackintosh et al. 1993; Palmer et al. 2003). However, instillation of M. bovis into the palatine tonsilar crypts results in disease and lesion distribution similar to that seen in natural infection in both red deer and white-tailed deer (Griffin et al. 2006; Mackintosh et al. 1993, 1995; Palmer et al. 1999, 2002a). Of particular note, the generally considered optimal challenge dose for vaccine efficacy studies using intratonsilar inoculation in both red deer and white-tailed deer is 100–500 colony-forming units (CFU); whereas, a tenfold higher dose (1000–5000 CFU) is used for intratracheal/bronchial and aerosol inoculation in cattle (Vordermeier et al. 2009; Waters et al. 2007; Wedlock et al. 2008). In experimental infections, tuberculous granulomas develop from a collection of epithelioid macrophages, multinucleated giant cells and few neutrophils and progress to a lesion characterized by macrophages, multinucleated giant cells, infiltrates of neutrophils and central necrosis. Multiple granulomas may coalesce to form a large granuloma with multiple centers of necrosis (Palmer et al. 2002a). The necrotic caseum may liquefy, a change not often seen in cattle, and the reason that many lesions in deer resemble abscesses. After instillation of M. bovis in the tonsilar crypts of whitetailed deer, microscopic lesions can be seen as early as 28 days in the medial retropharyngeal lymph nodes and 42 days in the lungs (Palmer et al. 2002a). Granulomas begin as collections of macrophages and multinucleated giant cells at 28 days, but by 42 days there is a focal area of necrosis surrounded by infiltrates of macrophages, multinucleated giant cells, and lymphocytes. Granulomas increase in size primarily due to an expanding

zone of central necrosis with a smaller contribution in size due to increased cellular infiltrates. Progressing lesions eventually coalesce, partially mineralize and become surrounded by a thin capsule of fibrous connective tissue. The character of the necrotic material is initially caseous, but with time may liquefy, resulting in an abscess-like gross appearance. In experimentally infected deer, liquefaction was first noted 328 days after inoculation (Palmer et al. 2002a). Using a similar intratonsilar inoculation approach, microscopic lesions could be seen in the retropharyngeal lymph nodes of cattle as early as 15 days after inoculation, with gross lesions evident by 28 days in not only the retropharyngeal lymph nodes but also the pulmonary lymph nodes (tracheobronchial and mediastinal). Gross lesions in the lungs were seen by 42 days.

Vaccine Research from Biocontainment to the Field Historical Perspectives: Tuberculosis Vaccines for Cattle There is a rich history of codiscovery with TB vaccines designed for cattle, wildlife and humans (reviewed in Waters et al. 2012a). The first effective vaccine used in humans, M. bovis BCG (bacillus of Calmette and Guérin), was tested in cattle at the Pasteur Institute in Lille, France, circa 1911–1914, prior to its first use in a human infant in 1921. After winning the first Nobel Prize in Medicine and Physiology in 1901, Emil Von Behring set out on a series of studies to develop a vaccine to prevent TB in cattle. Vaccines developed and tested by Behring at the turn of the 20th century included: • bovovaccine: a live vaccine consisting of human-derived tubercle bacilli attenuated by lengthy propogation; • taurin: an attenuated strain of bovine origin tubercle bacilli; • tuberkulase: tubercle bacilli inactivated with chloral hydrate; and • taurovaccine: chemically attenuated strains of tubercle bacilli (Behring 1901; Francis 1947). While Behring’s efforts were not productive, they were a testimony to his faith and leadership in the philosophy and study of specific etiological prophylaxis followed later by Calmette and Guérin in their development of BCG. At this time, McFadyean and colleagues in England, as well as Pearson and Gilliland in the United States, each demonstrated that vaccination with live human-derived TB bacilli provided protection against bovine TB (Flexner 1908). This approach, however, was abandoned due to the variability in virulence of human tubercle bacilli in cattle, occasionally resulting in dissemination and shedding of the live M. tb vaccine. Likewise, other attempts to immunize cattle against bovine TB were carried out in the early 20th century in Great Britain, France, the Netherlands, Italy, Argentina, the United States, and Japan by renowned researchers such as Koch, Roux, and Shiga— as well as Behring’s colleagues Paul Romer and Carl Siebert (Linton 2005). Strategies included injection of inactivated mycobacterial extracts, including various tuberculins, as well as mycobacterial isolates of fowl, bovine, equine, and human origin. Other than BCG, none of these efforts proved effective and practical. As early as 1920, field efficacy trials demonstrated that BCGvaccinated cattle had reduced numbers of lung lesions as compared to nonvaccinates. Haring and colleagues (1930) demonstrated that live BCG is required for protection. In 1939, Buxton and Glover reported that low-dose (5 mg) oral BCG fails to protect, whereas higher doses (50 mg) provide moderate protection


proteases and nucleases (Converse et al. 1996). Liquefactive necrosis of large sections of lung transform into cavities as portions of the necrotic mass become fissured and fragmented and are coughed out (Hunter 2011). Within the necrotic mass are large numbers of tubercle bacilli, making humans with cavitary tuberculous lesions highly contagious. Although liquefaction of lesions occurs in cattle and other domestic and wild ruminants, cavity formation as described in humans has not been observed. Formerly, TB of the tonsil and cervical lymph nodes in humans was recognized as a primary complex of lesions from which M. bovis was often isolated (Cowan and Jones 1972). In the late 18th and early 19th centuries, this primary complex of lesions, manifested as cervical lymphadenitis, was often seen in children consuming milk from M. bovis-infected cows. This distribution of lesions substantiated the importance of the oral route of exposure. In contrast to cattle, the most common site for tuberculous lesions in deer are the retropharyngeal lymph nodes. As efferent lymphatics from the tonsil drain to the retropharyngeal lymph nodes (Saar and Getty 1975), the tonsils and retropharyngeal lymph nodes may also be viewed as a primary complex of lesions in deer. Accordingly, in white-tailed deer, 76% of deer with lesions in the retropharyngeal lymph nodes also had lesions compatible with TB in the palatine tonsils. Likewise, in deer with palatine tonsilar lesions, 90% had lesions in the retropharyngeal lymph nodes (Palmer et al. 2002a). In naturally infected red deer, M. bovis was isolated from 61% of palatine tonsil samples (Lugton et al. 1998).

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(Buxton and Glover 1939). Buddle and colleagues (2011) recently confirmed these findings in their efforts to develop a nonsensitizing vaccine (i.e., a vaccine that does not elicit a skin test response) for cattle. Schellner and Gaggermeier (1955) demonstrated that BCG vaccination, combined with segregation of vaccinates, can be used to clear infection in TB-affected herds. By 1972, it was determined that host genetic factors affect vaccine efficacy, as BCG provides greater protection in purebred Zebu cattle than in crossbred Zebu cattle (Ellwood and Waddington 1972). During the mid to late 1900s, numerous experimental and field trials were performed to examine the efficacy of BCG in cattle. Primary findings included (1) a single dose of BCG provides nonsterile immunity to subsequent experimental challenge with virulent M. bovis; (2) field vaccination results in variable efficacy (0–80%); (3) live bacilli are required for protection; and (4) revaccination does not improve efficacy (reviewed in Francis 1958).

Prior to 1994, there had been isolated case reports of TB in whitetailed deer or mule deer (Odocoileus hemionus) in the United States (Belli 1962; Friend et al. 1963; Levine 1934; Rhyan et al. 1995). All reports involved only one to three deer and were seen in hunter-killed deer or cases of accidental death. At the time, it was postulated that M. bovis had spilled over from tuberculous cattle in the region; however, no surveys were conducted and no strain comparisons were made to confirm such a hypothesis. In 1975, a wild white-tailed deer in northern Michigan was diagnosed with TB due to M. bovis (Schmitt et al. 1997). This was thought to be an anomaly, and no surveys of other wild deer in the area were conducted. Approximately 20 years later (i.e., 1994), a hunter-killed wild white-tailed deer was identified with TB due to M. bovis. This deer was found only 13 km from the site where the tuberculous deer had been identified in 1975 (Schmitt et al. 1997). Subsequent surveys identified a focus of M. bovis infection in wild white-tailed deer in northeastern Michigan. This represented the first known reservoir of M. bovis in wildlife in the United

Vaccine Efficacy Studies in Cattle Overthe Past 15 years A number of TB vaccines have been tested in cattle for efficacy against experimental infection with virulent M. bovis over the past 10 years (Table 1). Currently, no subunit TB vaccines have provided protection exceeding that of BCG, but prime–boost

Table 1 Recent vaccines evaluated for protection against tuberculosis in cattle Type of vaccine

Description/Rationale

Reference

Attenuated live M. bovis

M. bovis Δmce2, parent strain is a virulent strain (NCTC 10772) with targeted deletion of two genes that encode proteins mediating cell entry/ attenuation without loss of other key immune stimulatory antigens lacking in BCG BCG overexpressing Ag85B; provides enhanced immunogenicity to M. bovis cell wall synthesis proteins BCG + modified vaccinia Ankara or adenovirus-vectored vaccines expressing Ag85A booster vaccination; provides boosted immunogenicity to M. bovis cell wall synthesis proteins M. bovis ΔRD1, parent strain is an attenuated strain (Ravenel); targeted deletion of RD1, which is the primary attenuating defect of BCG M. bovis culture filtrate protein with adjuvant and BCG; provides additional immunogenicity to culture filtrate proteins M. tb ΔRD1 x ΔpanCD double deletion mutant of strain H37Rv; targeted deletions of primary attenuating defect of BCG (RD1) and pantothenate synthesis ( panCD) M. bovis ΔleuD of M. bovis (Ravenel strain); targeted deletion of leucine synthesis provides attenuation Mycobacterial DNA with or without BCG/DNA vaccines offer high stability, cost effectiveness, and simple purification process

Blanco et al. (2013)

Enhanced BCG BCG + viral-vectored subunit booster Attenuated live M. bovis Adjuvanted protein vaccines + BCG Attenuated live M. tb

Attenuated live M. bovis DNA ± BCG

Abbreviation: BCG, Bacille Calmette Guerin.

Rizzi et al. (2012) Vordermeier et al. (2009)

Waters et al. (2009) Wedlock et al. (2008) Waters et al. (2007)

Khare et al. (2007) Cai et al. (2005); Maue et al. (2004, 2007); Skinner et al. (2003, 2005)


Historical Perspectives: The Need for a Tuberculosis Vaccine for Use in Wild White-Tailed Deer

States and the first known epizootic of TB in white-tailed deer in the world. It also represented a serious threat to domestic livestock and the bovine TB eradication effort in the United States. Michigan officials have continued surveillance efforts via two principal sources: harvest by licensed hunters and disease control permits issued to livestock producers in the affected area. Hunter-harvested deer, which account for 91% of all deer tested since 1994, are voluntarily submitted for testing during hunting seasons (O’Brien et al. 2006). Most commonly, heads are submitted, although other tissues and entire carcasses are also accepted. In addition to surveillance, two principal management strategies were implemented beginning in 1996. The first was to decrease deer population densities via hunting to biological carrying capacity. More liberal issuance of hunting permits and additional hunting seasons resulted in a 50% decrease in the deer population in the affected area by 2009 (O’Brien et al. 2009). The second strategy was to implement restrictions on supplemental feeding of deer. This approach was based on research demonstrating the ability of tuberculous deer to indirectly transmit M. bovis to other deer or cattle through sharing of feed (Palmer et al. 2004). Feeding restrictions in Michigan have ranged from voluntary to mandatory according to region and are inconsistently enforced; however, large-scale feeding of deer in this region has been dramatically reduced despite these shortcomings. Although progress has been made in decreasing the prevalence of TB among deer in northeastern Michigan, it is believed that current management strategies alone are unlikely to result in disease eradication. Vaccination of wild deer has been proposed as an additional management strategy in Michigan, and considerable research efforts are underway to develop methods to vaccinate wild deer in this region.


combinations of BCG with DNA, protein, or viral-vectored vaccines may elicit better protection than does BCG alone (reviewed in Vordermeier et al. 2014). In addition, recent studies have more clearly defined the parameters that may affect the efficacy of BCG in cattle (reviewed by Buddle et al. 2011, 2013). Most notably:

Recently, Parlane and colleagues (2014) demonstrated that revaccination of cattle two years after the first BCG vaccination (i.e., when immunity has waned) significantly boosts the level of protection afforded by a single dose of BCG. Additionally, immunogenicity trials have identified several vaccines as priority candidates for vaccination/M. bovis challenge studies (i.e., BCG Danish Δzmp1 [Khatri et al. 2014]); Ad5-TBF that expresses Ag85A, Rv0287, Rv0288, and Rv0251c (Dean et al. 2014); and ID83, which is a fusion of Rv1813, Rv3620, and Rv2608 proteins along with toll-like receptor agonists (Jones et al. 2013)]. While BCG is the only licensed TB vaccine for humans, numerous new vaccines are in Phase I–IIb human clinical trials (reviewed in Buddle et al. 2013). The most promising of these vaccine targets will likely end up in the pipeline, if not already, for efficacy testing in cattle.

Development of BCG for Use in White-Tailed Deer Vaccines for wildlife have been used, or considered for use, in diseases that affect public health; impact livestock health/commerce; or negatively impact iconic, protected, or endangered species (Cross et al. 2007). These include rabies in skunks, raccoons, foxes, and other mammals; Lyme disease in mice; plague in black-tailed prairie dogs; brucellosis in bison and elk; classical swine fever in European wild boar; anthrax in cheetah and black rhinoceros; and TB in African buffalo, deer, badgers, brushtail possums, and wild boar. Generally, oral vaccines, in the form of baits, are considered the most feasible means of vaccinating wildlife. However, under certain circumstances, hand-injected and pneumatic dart-administered vaccines have also been used successfully (Olsen and Johnson 2012). In the case of TB, the human vaccine, M. bovis BCG, has been the most widely investigated and tested in various species, including deer. In all species tested, BCG reduced disease severity and in some trials also protected against infection (Griffin et al. 1999, 2006; Palmer et al. 2007, 2009). Early efficacy studies demonstrated that a single dose of BCG given to 3-month-old red deer reduced disease severity but was not protective against infection (Griffin and Mackintosh 2000). By contrast, prime–boost with low (104 CFU) to moderate (106 CFU) doses of BCG protected against both disease and infection. Subsequent studies (Griffin et al. 2006) showed that the interval between prime and boost was important. Animals boosted at 8- or 16-week intervals generated protective immunity, while animals boosted at a 43-week

interval showed no protection. The age of animals at primary vaccination may also be important, as the prime–boost effect seen in 3-month-old deer was not evident in cattle, where a single vaccination at 1 day of age provided optimal protection (Buddle et al. 2003). Studies in white-tailed deer demonstrated that both oral and parenterally administered BCG provided the same degree of protection (Nol et al. 2008). Simulation modeling has examined the potential role that vaccination might play both in the eradication of bovine TB from free-ranging deer in Michigan and in control programs aimed at minimizing cattle herd breakdowns. Vaccination frequency, the proportion of susceptible deer vaccinated, and whether or not baiting was occurring during the hunting season all affected the probability of bovine TB eradication (Ramsey et al. 2014). Using a vaccine with 90% efficacy, annual vaccination of 90% of susceptible deer was necessary to achieve a 95% probability of eradication within 30 years. That same level of exposure was predicted to bring about a 95% probability of having no infected cattle herds in the bovine TB core area within 14 years. Exposing 50% (rather than 90%) of susceptible deer reached the same threshold in about 20 years. Vaccinating 50–90% of the susceptible deer within a 5-km radius of cattle farms achieved a 95% probability of having zero cattle herd breakdowns in 15 to 18 years (Ramsey et al. 2014). Recent studies provide evidence of vaccine shedding with transmission of BCG from vaccinated deer to in-contact unvaccinated deer (Nol et al. 2013; Palmer et al. 2009, 2010). It is as yet unclear whether such secondary transmissions confer protection from challenge with virulent M. bovis. If they do, modeled projections of time to bovine TB eradication are likely to be overstated, as vaccine transmission would increase vaccine coverage (i.e., percentage of deer vaccinated), thereby reducing the estimated time to eradication. In addition to being efficacious, a vaccine must also be safe. This is of special relevance with live vaccines such as BCG. Safety is of concern in terms of the animal being vaccinated, other animals (including humans) that may consume a vaccinated animal, and animals not intended for vaccination but nevertheless having contact with vaccine bait (i.e., nontarget species). M. bovis BCG was first used as a human vaccine in 1921. Since that time, billions of doses have been administered, many of which were given to newborn infants. With such a long history, there is a great deal of information concerning the safety of BCG in humans. It is considered to have an excellent safety record and to be nonvirulent in immunocompetent individuals. Additionally, BCG has been used in many small animal models of human TB; therefore, much is known concerning safety in mice, rats, guinea pigs, and rabbits. Another potential concern is the shedding of BCG from vaccinated deer to cattle resulting in responsiveness to purified protein derivatives (PPDs) used in interferon-γ release assays and skin tests; however, initial studies indicate that this scenario is unlikely (Nol et al. 2013; Palmer et al. 2010). Regardless, if field studies are implemented, the risk of BCG shedding from vaccinated deer to cattle will need to be evaluated given the significant consequences to producers and regulatory officials. People often consume hunter-killed deer, so there is potential for humans to contact BCG through consumption of venison. Although disease is not likely to result from human consumption of BCG, false positive results on tuberculin skin tests could interfere with public health monitoring for human TB. BCG has been shown to persist for 3 to 12 months in lymphoid tissues of deer vaccinated parenterally or orally (Palmer et al. 2014, 2010). Importantly, in studies of vaccinated deer, BCG has never been isolated


1. 104–106 CFU BCG induces similar levels of protection, and this dose range appears optimal for parenteral immunization; 2. Pasteur and Danish strains provide similar levels of protection; 3. exposure to M. avium or other nontuberculous Mycobacteria spp. (NTM) affects efficacy both positively and negatively based on multiple variables; 4. oral immunization provides a level of protection similar to that of parenteral routes yet requires a higher dose of BCG; 5. duration of BCG-induced immunity is ≥12 months but ≤24 months; and 6. early (i.e., 6 weeks but not 6 months) revaccination reduces the level of protection over that of a single dose (reviewed in Buddle et al. 2011, 2013; Waters et al. 2012a).

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from those tissues commonly consumed by hunters (i.e., muscle). Moreover, cooking meat products at a minimum temperature of 60°C for at least 10 minutes has been shown to kill >90% of M. bovis that may be present (Merkal and Whipple 1980). In other circumstances, to avert some risk, heat-killed BCG has been used for vaccination of wild boar (Garrido et al. 2011).

Immune Aspects

Correlates of Protection As reviewed by Plotkin (2008), a correlate of protection is defined as “a specific immune response to a vaccine that is closely related to protection against infection, disease, or other defined end point” ( p. 402). Correlates of protection may be absolute (i.e., near complete) or relative (i.e., partial) and may involve multiple synergistic components (i.e., co-correlates). A surrogate of protection is a response that is not in itself protective yet is indicative

Table 2 Relevance of IFN-γ and delayed type hypersensitivity responses to efficacy of bovine tuberculosis vaccines A. Responses to vaccination and prior to challenge Most effective TB vaccines elicit IFN-γ and DTH responses (Black et al. 2002) Not all vaccines inducing IFN-γ and DTH responses are protective (Waters et al. 2007) Protection is not linked with maintenance of these responses (Whelan et al. 2011) The amount of IFN-γ 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) B. Responses after vaccination and after challenge IFN-γ responses are generally inversely correlated with responses that predict vaccine efficacy (Vordermeier et al. 2011) A robust ESAT-6/CFP-10-specific IFN-γ response (ex vivo, effector 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) The level of DTH responses detectable after M. bovis challenge does not correlate with the level of pathological changes in cattle or vaccine-elicited protection (Whelan et al. 2011); however, analysis of DTH responses is often confounded by responses evoked by attenuated live vaccines Abbreviations: BCG, Bacille Calmette Guerin; CFP, culture filtrate protein; DTH, delayed type hypersensitivity; IFN, interferon; Tb, tuberculosis.

Table 3 Candidate surrogates of protection against bovine tuberculosis Surrogate

Supportive finding

References

Central memory T cell responses IL-17A mRNA responses IL-22 mRNA responses

Responses to protective vaccines correlate with reduced mycobacterial burden and associated pathology as well as duration of immunity Responses to protective vaccines correlate with reduced severity of lesions Leading candidate biomarker in RNA-seq analysis of vaccine-elicited responses and level of the response correlates with reduced severity of lesions

Thom et al. (2012); Vordermeier et al. (2009); Waters et al. (2009); Whelan et al. (2008) Rizzi et al. (2012); Vordermeier et al. (2009) Bhuju et al. (2012)


Interferon (IFN)-γ release assays (IGRAs) and delayed-type hypersensitivity (DTH, skin test) responses are widely used for the diagnosis of bovine TB in cattle (reviewed by Schiller et al. 2010). While useful as correlates of infection, the level of these responses does not necessarily correlate with the severity of disease (Blanco et al. 2014; Waters et al. 2012a). With TB vaccine efficacy trials, IGRAs are typically used to monitor responses in cattle (reviewed in Waters et al. 2012a) and humans (Rustomjee et al. 2013; Sutherland et al. 2013). With cattle, DTH assays are generally incorporated into the study design to determine potential interference of vaccines with traditional skin test procedures and as an endpoint measure of infection (Hewinson et al. 2003). The relevance of IFN-γ and DTH responses to efficacy of bovine TB vaccines is summarized in Table 2.

of other true correlates, known or unknown. Correlates of protection are relatively easily defined for vaccines eliciting antibodies that neutralize pathogens/toxins on mucosal surfaces or prior to entry into cells (reviewed by Plotkin 2008; Thakur et al. 2012). The identification of distinct immune correlates, however, is difficult with diseases such as TB due to the complexity of the host/pathogen interaction (Marinova et al. 2013; Rustomjee et al. 2014; Thakur et al. 2012; Wallis et al. 2009). With bovine TB, specific immune responses have been identified that appear to be associated with protection (Table 3); however, it is not clear whether these responses are merely substitutes for other as yet undefined protective responses (i.e., surrogates) or if they are actually responsible for protection either alone (i.e., correlates) or as a part of a multifaceted response (i.e., co-correlates). With human TB, the identification of immune correlates of protection has become a priority; and extensive planning and resources have been allocated for this effort in ongoing and future phase II/III vaccine trials (Rustomjee et al. 2013). Identification of immune correlates/ surrogates of protection would greatly benefit the field as the numerous vaccine candidates proven effective in rodent models could be prioritized for costly efficacy trials in cattle and humans. Upon M. bovis challenge, increasing antigen burden and associated pathological changes in nonprotected cattle (i.e., nonvaccinated and animals receiving ineffectual vaccines) elicit adaptive immune responses detectable within short term in vitro assays, such as increased antigen-specific: (1) IFN-γ (and various other cytokine and chemokine) production, (2) expansion of lymphocytes ( particularly CD4+ and γδ TCR+ cells, McGill et al. 2014), and (3) expression of activation markers on T cells (Waters et al. 2007). In particular, an increase in ESAT-6 specific IFN-γ responses after challenge is generally an indicator of vaccine failure, as these responses have been shown to positively correlate with TB-associated pathology (Vordermeier et al. 2002). A possi-


antimicrobial functions, respectively, as well as production of IFN-γ leading to activation and lysis of infected macrophages (Einarsdottir et al. 2009; Flynn 2004). While these functions are described as an element of the response by cattle to bovine TB (Aranday-Cortes et al. 2012; Endsley et al. 2004, 2007; Liebana et al. 2000), definitive studies linking these responses to protection are lacking. With nonhuman primates, CD8 T cells are essential for the control of M. tb and for BCG-elicited immunity (Chen et al. 2009). Protective immunity elicited by pro-apoptotic attenuated strains of M. tb is attributed to enhancement of CD8 T cell responses in mice (Hinchey et al. 2007). Another strategy to evoke CD8 responses is to engineer cytolysins (e.g., listeriolysin) into BCG, enabling phagosomal escape and improved MHC class I presentation of mycobacterial antigens and thereby promoting CD8 responses (Grode et al. 2005). Indeed, a vaccine candidate using this strategy (i.e., VPM1002, rBCGΔUreC::hly containing a urease C [UreC] deletion to improve phagosome acidification and expressing listeriolysin [hly] from Listeria monocytogenes for phagosomal escape) is undergoing Phase IIa clinical trials in humans (Andersen and Kaufmann 2014). Other vaccines, Aeras 401 (a BCG-based strain containing a mutated perfringolysin O and urease deletion) and Aeras 422 (Aeras 401 overexpressing antigen 85A, antigen 85b, and Tb-10.4) were also developed as TB vaccines to elicit potent CD8 responses (Sun et al. 2009). However, Phase I clinical trials with Aeras 422 had to be discontinued due to an unanticipated side effect: reactivation of the varicella-zoster virus (Kupferschmidt 2011). Further studies are warranted to evaluate vaccines, such as pro-apoptotic and phagosomal escape mutants that promote CD8 responses in cattle. Polyfunctional (also called multifunctional) 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 hepatitis C, HIV, leishmaniasis, malaria, and TB (reviewed in Caccamo and Dieli 2012; Prezzemolo et al. 2014; Wilkinson and Wilkinson 2010). The role of polyfunctional T cells in the immune response to human TB is complex, oftentimes leading to disparate results in the various studies (reviewed by Prezzemolo et al. 2014). With that said, multiple human TB vaccines in Phase I or II clinical trials have a demonstrated capacity for eliciting polyfunctional T cells in rodent models (Knudsen et al. 2014) and humans (Grode et al. 2013). Also, several studies indicate a protective role (i.e., cured or latent versus active TB) for a predominance of antigen-specific CD4 T cells secreting IL2 and IFN-γ in human TB (Petruccioli et al. 2013; Streitz et al. 2011). Treatment of TB patients with M. tb detectable within their sputum results in a decline of IFN-γ+IL-2−TNF-α+ CD4 T cells and an increase in IFN-γ+IL-2+TNF-α+ CD4 T cells (Riou et al. 2014), supporting a protective role for IL-2 and the potential for impairment of IL-2 production associated with bacterial load. Likewise, several studies have demonstrated lower frequencies of IL-2+ CD4 T cells (i.e., a higher proportion of mycobacteria-specific IFN-γ+IL-2−TNF-α+ or IFN-γ+IL-2− CD4 T cells) in active TB patients (Millington et al. 2007; Sester et al. 2011; Young et al. 2010), again supporting a protective role for IL-2–secreting cells. With cattle, methods have been developed to evaluate polyfunctional T cell responses to M. bovis infection (Kaveh et al. 2012), and studies are ongoing to evaluate the kinetics of polyfunctional T cells in the response to vaccination and M. bovis infection (Maggioli MF, Palmer MV, Thacker TC, Vordermeier HM, Waters WR, unpublished data). A user-friendly mycobacterial growth inhibition assay was recently adapted as a means of measuring immune responses elicited by vaccination that result in mycobacterial killing or inhibition of growth (i.e., as a correlate of protection) (Burl et al.


ble exception would be use of vaccines designed to elicit ESAT-6specific responses. Vaccine efficacy studies in cattle have demonstrated that long-term cultured IFN-γ ELISPOT (so called, cultured ELISPOT) responses are positive predictors of vaccine efficacy. In other words, cultured ELISPOT responses elicited by protective vaccines correlate with reduced mycobacterial burden and associated pathology (Vordermeier et al. 2009; Waters et al. 2009; Whelan et al. 2008) as well as duration of immunity (Thom et al. 2012) upon subsequent challenge with virulent M. bovis. BCG vaccination of neonatal calves results in significant protection against M. bovis challenge at 12 months but not at 24 months after vaccination. This loss of efficacy correlates with a significant reduction in the numbers of antigen-specific IFN-γ-secreting cells within long-term peripheral blood mononuclear cell (PBMC) cultures (Thom et al. 2012). With humans, the responding cells within these long-term cultures are predominately central memory T cells (Tcm) and this response, in contrast to effector responses, correlates with more effective and durable responses to multiple antigens from various pathogens (Todryk et al. 2009). Recent studies in our laboratory have determined that responding cells within cultured ELISPOT assays from M. bovis-infected cattle are primarily CD4+ Tcms (∼76%), with a lesser contribution by effector memory T cells (Tem, ∼23%) and effector T cells (1%) (Maggioli MF, Palmer MV, Thacker TC, Vordermeier HM, Waters WR, unpublished data). However, the relative contribution of effector/ memory and T cell subpopulations in the response will differ based on antigen selection (e.g., soluble versus particulate versus live), inciting agent (e.g., vaccine versus pathogen), and type of pathogen (e.g., viral versus bacterial versus parasite). As with cattle, there is considerable interest in the role of Tcms as mediators of protection for TB vaccines intended for humans (Vogelzang et al. 2014; reviewed by Andersen and Kaufmann 2014; HenaoTamayo et al. 2014). Several vaccine efficacy studies with cattle have demonstrated that interleukin (IL)-17 responses to vaccination correlate with reduced lesion severity after experimental M. bovis infection (Rizzi et al. 2012; Vordermeier et al. 2009; reviewed by Waters et al. 2014). With cynomolgus macaques, IL-17 mRNA is significantly upregulated in response to PPD 6 weeks after M. tb challenge in monkeys capable of controlling the disease (i.e., BCGvaccinates and a subset of nonvaccinated animals; Wareham et al. 2014). With this study, IL-17 mRNA was also upregulated in BCG-vaccinates 8 weeks following vaccination and prior to challenge, suggesting a role for IL-17 as a potential correlate of protection. In mice, IL-17 is required for protection against hypervirulent Beijing strains of M. tb, but not against low-virulence strains such as H37Rv (Gopal et al. 2014). Using RNA-Seq followed by RT-qPCR analysis, Bhuju and colleagues (2012) demonstrated that IL-22 is strongly upregulated in response to PPD stimulation by BCG vaccinates (i.e., with protected versus nonprotected calves) prior to M. bovis challenge. With humans, antimycobacterial therapy induces an enhanced and sustained antigen-specific IL-22 response (Zhang et al. 2014). Together, these findings support a critical role for IL-17– and IL-22–producing cells in the protective response to TB; however, the timing and nature of the response likely dictates protective versus harmful inflammatory responses associated with progressive disease (Bhuju et al. 2012; Cowan et al. 2012; Dhiman et al. 2009; Gopal and Khader 2013; van Laarhoven et al. 2013; Vordermeier et al. 2009). CD8 T cells are also recognized as a key component of the immune response to bovine and human TB. Primary effector functions of CD8+ T cells are production of perforin and granulysin essential for pathogen killing via pore formation and

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Determinants of Disease Progression Particularly in resource-poor settings, funds are often not available to compensate producers for removal of all reactor cattle from TB-affected herds; thus, an assay capable of distinguishing animals with advanced disease from animals with less severe disease (Blanco et al. 2014) would be beneficial to prioritize removal of the most infectious animals. Increased IL-17A expression by PBMC in response to M. bovis PPD correlates with the severity of gross tuberculous lesions (Aranday-Cortes et al. 2012; Blanco et al. 2011, 2014). IL-17A mRNA expression is also increased within tuberculous granulomas as compared to nonaffected tissues from experimentally infected cattle (ArandayCortes et al. 2013). With human TB, the level of IL-17 produced by peripheral blood lymphocytes varies between studies, potentially related to confounding variables such as vaccination status, stage of disease, co-infections, and other factors (Bandaru et al. 2014; Jurado et al. 2012; Marín et al. 2012). With bovine TB, IL-22 mRNA is upregulated after natural infection (Aranday-Cortes et al. 2012) and as early as 8 weeks following experimental infection (Bhuju et al. 2012). IL-22 mRNA is also detectable within tuberculous granulomas, with decreasing expression from early- to late-stage granulomas (Aranday-Cortes et al. 2013). Antigen-specific IL-22 responses (i.e., protein) are detectable in high frequencies by PBMC from humans exposed to TB patients, yet lower frequencies are detected in active pulmonary TB patients (Scriba et al. 2008). Also, IL-22 is detected in bronchoalveolar lavage (BAL), pleural, and pericardial fluid of TB patients, possibly mediating pulmonary inflammation (Mathews et al. 2011; Scriba et al. 2008). Increased IL-22–expressing T cells are also detected in M. tb-infected macaques, particularly in BAL fluid and lung tissues (Yao et al. 2010). In contrast, van Laarhoven et al. (2013) demonstrated decreased IL-22 and increased IL-17 production by PBMC in response to whole cell lysates of modern versus ancient Beijing strains, possibly reflecting the increased transmission potential of the modern isolates. Further studies with bovine TB are warranted to delineate roles for IL17 and IL-22 in the response to M. bovis infection/vaccination and their potential use as biomarkers of infection and/or progression of disease.

Polyfunctional CD4 T cells simultaneously producing IL-2, IFN-γ, and TNF-α are associated with active TB in humans (Caccamo et al. 2010; Sester et al. 2011; Sutherland et al. 2009; Young et al. 2010). In contrast, individuals with latent TB have higher frequencies of antigen-specific IFN-γ single and IFN-γ/IL-2 dual secreting CD4 T cells (reviewed by Prezzemolo et al. 2014). The frequency of polyfunctional T cells in TB patients decreases to undetectable levels 6 months after curative TB treatment, suggesting a correlation of bacterial load to functional patterns of the T cell response (Caccamo et al. 2010; Petruccioli et al. 2013). Recent studies indicate that the balance of innate inflammatory responses elicited by M. tb infection is linked with disease resistance or exacerbation. Specifically, IL-1 confers host resistance through induction of eicosanoids that limit excessive type 1 interferon production and fostering bacterial containment whereas reduced IL-1 is linked to eicosanoid imbalance and disease exacerbation (Mayer-Barber et al. 2014). In another study, IL-32 was identified as a potential correlate of protection against active TB in humans (Montoya et al. 2014). IL-32 is a cytokine important for the differentiation of monocytes into dendritic cells with increased ability to cross-present antigen via MHC class I to CD8+ T cells (Schenk et al. 2012) and to promote vitamin D-mediated antimicrobial activity (Montoya et al. 2014). Whereas type 1 interferon is considered a “correlate of risk or pathogenesis” (Wallis et al. 2009), IL-32 may be a correlate of latency (Montoya et al. 2014). Interestingly, repeat skin testing of cattle (i.e., up to four comparative cervical tests) at a 60-day interval results in skin test anergy and an associated decrease in IL-1β production by whole blood leukocytes (Coad et al. 2010). In general, antibodies produced in response to infection with M. tb complex bacilli are not considered protective but are known to modulate the host response (reviewed by Kozakiewicz et al. 2013). As with humans and nonhuman primates, conspicuous aggregates of B cells are detectable within tuberculous lung and lymph node lesions of cattle (Aranday-Cortes et al. 2013; Johnson et al. 2006). In early granulomas (i.e., Stage I and II), few scattered B cells are detectable within the granulomas, whereas B cells form satellite nests located peripherally and outside of the fibrous capsule within Stage III and IV lesions (i.e., more advanced lesions) (Aranday-Cortes et al. 2013). Experimentally, antibody responses correlate with the level of pathology (Waters et al. 2010). Inoculation of cattle with M. tb H37Rv results in colonization with minimal to no tuberculous lesions, whereas inoculation of cattle with virulent M. bovis results in colonization and associated tuberculous lesions (Waters et al. 2010). Mycobacterial-specific antibody is detectable relatively early after M. tb challenge of cattle, yet these responses wane over time. In contrast, antibody responses persist with virulent M. bovis infection, likely due to increasing antigen burden. With naturally infected cattle, diagnostic sensitivity of a commercial antibody-based assay is greater with sera from animals with visible (Waters et al. 2011) and advanced lesions (Perosino et al. 2014) as compared to that from animals without visible lesions. Thus, antibody responses increase in association with the severity of disease.

Antemortem Diagnostic Strategies In 1890, Robert Koch announced that he had discovered a substance, termed paratoloid and now known as tuberculin, which could prevent and cure TB in guinea pigs (Ernst 1891). Soon proven ineffective as a vaccine or therapy, tuberculin later emerged as a diagnostic reagent that revolutionized TB control programs. Early on, it was noted that TB patients injected with tuberculin often developed systemic reactions, including fever. Recognizing


2013; Fletcher et al. 2013). Fletcher and colleagues (2013) demonstrated that PBMC from BCG vaccinates inhibits M. bovis BCG growth and that revaccination does not increase the response. Additionally, the ability of vaccination to limit mycobacterial growth in vivo may be determined in humans by biopsy of intradermal challenge sites. For instance, BCG recovery from biopsy sites is reduced in individuals vaccinated 10 years prior with BCG as compared to nonvaccinated controls (Harris et al. 2014). Other attenuated mycobacterial strains may also be used as the challenge strain to assess the capacity of vaccines to restrict mycobacterial growth in vitro or in vivo. Studies are ongoing to determine the usefulness of these approaches with cattle, including injections into prescapular lymph nodes (VillarrealRamos et al. 2014). In summary, there are several promising candidate correlates of protection for bovine TB; however, additional studies are required to confirm these preliminary findings. A major lesson of the recent failed human TB vaccine efficacy trial in Africa (Tameris et al. 2013) is that, without strong correlates of protection, it will be extremely difficult to develop and evaluate new vaccine candidates for humans (Sakai et al. 2014). This notion is also relevant for the development of bovine TB vaccines because experimental and field efficacy studies are costly and labor intensive.


ensure viability of cells within the blood sample and background responses, respectively. This technology was transferred for application to the detection of human TB (Andersen et al. 2000; reviewed by Pai et al. 2014). There are two commercial IGRAs available: (1) QuantiFERON-TB Gold In-Tube assay (Cellestis/Qiagen, Carnegie, Australia), which uses peptides from ESAT-6, CFP10, and TB7.7 in an in-tube format; and (2) T-SPOT.TB assay (Oxford Immunotec, Abingdon, United Kingdom), which uses ESAT6 and CFP-10 in an ELISPOT format. An ESAT-6/CFP-10-based assay is also commercially available for use in cattle (Bass et al. 2013). Interestingly, the potential use of ESAT-6 as a diagnostic reagent (Pollock and Andersen 1997) and to differentiate infected from vaccinated animals (DIVA) (Buddle et al. 1999) using IGRAs was realized as early as the late 1990s; almost concurrently with the realization of the diagnostic potential of this immunodominant antigen for use in the diagnosis of human TB (Mustafa et al. 1998; Ravn et al. 1999; Ulrichs et al. 1998). Intensive research efforts are ongoing to identify and develop improved antigens for use in bovine TB diagnostic tests (Vordermeier et al. 2011), as well as biomarkers of infection (Aranday-Cortes et al. 2012; Bhuju et al. 2012; Waters et al. 2012b). Examples of biomarkers other than IFN-γ evaluated for use in cattle to diagnose bovine TB include: TNF-α, nitric oxide, IP-10, IL-2, soluble IL-2 receptor, and IL-1 (Cockle et al. 2002; Nualláin et al. 1997; Rhodes et al. 2014; Waters et al. 2003, 2012b). Again, these findings highlight the mutually beneficial aspects of bovine and human TB research, as biomarkers deemed useful for the diagnosis of bovine TB will likely be of relevance for development of similar strategies in humans (Waters et al. 2014).

Conclusions There have been numerous advances in research on bovine TB in cattle and white-tailed deer over the past 15 years. Several new vaccines have been tested including multiple approaches with various attenuated live strains, DNA, subunit, and heterologous prime–boost strategies. Techniques are in place to differentiate infected from vaccinated cattle using immunodominant antigens not included in most of the vaccines tested so far. There have been major advances in assays to determine surrogates of protection and determinants of disease progression. The most promising candidates to date include the so-called cultured ELISPOT as a measure of central memory T cells as well as IL-17 and IL-22 as surrogates of protection. The most promising advances in the development of antemortem diagnostic tests is the increasing acceptance and use of specific antigens for use in both in vitro and in vivo cell-mediated assays (Chen et al. 2014), as well as for emerging serologic tests. Indeed, there is much optimism for the development of a “synthetic PPD” composed of specific antigens such as ESAT-6, CFP10, and Rv3615c to replace the standard PPDs. Development of large-scale production techniques to produce these antigens at a scale and cost comparable to standard tuberculin production has significantly increased the potential for wide-scale use of specific antigens in new bovine TB tests. With wildlife reservoirs of M. bovis infection (such as whitetailed deer), great strides have been made to develop approaches for vaccination of large populations of free-ranging animals with BCG to limit the spread of the disease within the reservoir population and to nearby livestock. These approaches in wildlife will provide useful information on herd immunity, with ramifications on the development of similar approaches for humans and livestock. Finally, intricate and detailed methods have been developed and standardized for the assessment of the immunopathogenesis of M. bovis infection, particularly in cattle but also in


the diagnostic potential, Professor Gutmann of the Veterinary Institute in Dorpat Russia (currently Tartu in Estonia) developed an in vivo technique to diagnose bovine TB by injecting Koch’s tuberculin subcutaneously into cattle and monitoring for a rise in temperature (Marshall 1917). Soon after (1891–1892), Bernhard Bang in Denmark, John McFadyean in England, and Leonard Pearson in the United States followed Gutmann’s approach in their respective countries. Charles Mantoux along with Clemens von Pirquet later (circa 1907/1908) adapted and improved this technology for use in humans using intradermal injection, coincidently defining the principles of allergy and delayed-type hypersensitivity (Menzies 2000). The intradermal test became widely used as the preferred test in cattle about the time of the initiation of bovine TB eradication programs in the United States (1917) and Canada (1923). The codiscovery and co-development of the intradermal skin test for TB diagnosis is a premier example of the one-health approach to infectious disease research. Many countries have official bovine TB eradication/control programs. Prevention and control efforts are designed to identify TB-affected herds for movement restriction to prevent spread of the disease and to remove infected animals or depopulate the entire herd. Based on the prevalence in a particular region, routine slaughter surveillance and/or antemortem testing are used to identify TB-affected herds. Intradermal skin test and interferon-gamma release assays (IGRAs; e.g., Bovigam, Life Technologies) (Schiller et al. 2010) as well as targeted use of serology (i.e., IDEXX M. bovis Ab Test, Portland, Maine; Casal et al. 2014) are approved by the Office International des Epizooties for detection of bovine TB in cattle (Bezos et al. 2014). Surveillance for M. bovis infection in wildlife reservoirs and population control of known wildlife reservoirs are also increasingly utilized as standard features of bovine TB control (Fitzgerald and Kaneene 2013). 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 as the primary antemortem test for the detection of tuberculous cattle in most countries. The comparative test is applied to differentiate cattle infected with M. bovis from those infected with or sensitized to M. avium subsp. Paratuberculosis, or other NTM. Nontuberculous mycobacteria are common in the environment (especially in water), may be fast or slow growing with approximately 150 officially recognized species, are often misdiagnosed, and are occasionally responsible for opportunistic infections in humans and livestock (Biet and Boschiroli 2014; Tortoli 2014). A single tuberculin test is used for humans despite the possibility of false positive reactions resulting from exposure to NTM. In certain populations, NTM may be isolated from a significant proportion of the population. For instance, NTM were isolated from 24.8% of infants enrolled in a TB biomarker study in southern India (Dhanasekaran et al. 2014). Specific antigens —such as ESAT-6, CFP10, and Rv3615c—are increasingly recognized as feasible alternatives to the use of PPD for skin test in cattle (Casal et al. 2012; Chen et al. 2014; Whelan et al. 2010). These immunodominant antigens of M. bovis and M. tb are not present in NTM, with a few notable exceptions (e.g., M. kansasii, M. szulgai, M. marinum; Hur et al. 2014). During the late 1980s, veterinary researchers in Australia developed a whole blood assay based on the detection of IFN-γ secreted by lymphocytes in response to PPD (Wood et al. 1990). The initial and most widely used test relies on differential IFN-γ responses to PPDa and PPDb to distinguish infection/sensitization to NTM (especially, M avium subsp. paratuberculosis) from M. bovis infection (reviewed by Schiller et al. 2010). With this assay, positive (mitogen) and negative (no antigen) controls are included to

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other livestock and wildlife species. This capacity has greatly improved our ability to understand the immune response at the site of infection and to characterize the efficacy of vaccines. Ongoing and future studies with field vaccine efficacy studies in cattle located in TB-endemic regions (e.g., Mexico and Ethiopia; Ameni et al. 2010; Lopez-Valencia et al. 2010) should prove important, not only as a means to evaluate TB vaccines for use in cattle but also for clinical trials in humans.

Acknowledgments 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.

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Human tuberculosis due to Mycobacterium bovis: report of 10 cases.

The role of gamma delta T cells in immunity to Mycobacterium bovis infection in cattle.

Mycobacterium bovis infection in a herd of Japanese Shika deer (Cervus nippon).

Detection of Mycobacterium tuberculosis and Mycobacterium bovis in Sahiwal cattle from an organized farm using ante-mortem techniques.

[Pleural and peritoneal tuberculosis due to Mycobacterium bovis].

Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids.

Virulence of two strains of mycobacterium bovis in cattle following aerosol infection.

Feline tuberculosis caused by Mycobacterium bovis.

Human ULK1 Variation and Susceptibility to Mycobacterium tuberculosis Infection.

Antigen-Specific IP-10 Release Is a Sensitive Biomarker of Mycobacterium bovis Infection in Cattle.

Mycobacterium bovis infection in cats and people.

Assessment of BCG and inactivated Mycobacterium bovis vaccines in an experimental tuberculosis infection model in sheep.

Mycobacterium bovis infection in cats and people.

Molecular identification of Mycobacterium bovis from cattle and human host in Mali: expanded genetic diversity.

Mycobacterium bovis in Burkina Faso: epidemiologic and genetic links between human and cattle isolates.

Evaluation of Immunogenicity and Protective Efficacy Elicited by Mycobacterium bovis BCG Overexpressing Ag85A Protein against Mycobacterium tuberculosis Aerosol Infection.

Occurrence and Distribution of bovine tuberculosis (Mycobacterium bovis) in Slaughtered cattle in the abattoirs of Bauchi State, Nigeria.

A bloody evidence: Is Mycobacterium bovis bacteraemia frequent in cattle?!

A mathematical model for Mycobacterium bovis excretion from tuberculous cattle.

Extent of Mycobacterium bovis infection in dairy cattle herds subject to partial culling as determined by an interferon-gamma assay.

Pulsed field gel electrophoresis of representatives of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains.

Intranasal administration of Mycobacterium bovis BCG induces superior protection against aerosol infection with Mycobacterium tuberculosis in mice.

Nitrate reductase activity of Mycobacterium tuberculosis and Mycobacterium bovis in the presence of electron donors.

In vitro anti-mycobacterial activity of selected medicinal plants against Mycobacterium tuberculosis and Mycobacterium bovis strains.