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(1)Molecular Diagnosis of Mycobacterium bovis Infection. By Ingrid Kotowski. A Master's Paper submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master of Public Health in the Public Health Leadership Program.. August 2012. _____________________________________ Susan A. Randolph, Advisor. _____________________________________ Julie L. Gauthier, Reader.

(2) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ ABSTRACT. Tuberculosis due to Mycobacterium bovis infection presents a significant public and animal health burden, in particular in economically less developed countries. Distinguishing M. bovis from other mycobacteria, and distinguishing among M. bovis strains, are important for M. bovis control and eradication, and for optimizing treatment plans for patients. Conventional diagnostic methods, such as tuberculin skin tests and microscopic examination of blood smears, are rapid and inexpensive, but generally do not allow differentiation among mycobacteria or mycobacterial strains. A variety of methods have been developed that allow detailed genetic characterization of mycobacteria. These methods are useful for epidemiologic investigation of outbreaks, identifying potential means and patterns of transmission, identifying relationships among infections in various human and animal populations, and investigating the role of wildlife reservoirs in maintenance and spread of infection. These methods can also be used to identify risk factors for transmission, to assess the effectiveness of tuberculosis control and eradication programs, and to assess quality control in laboratory testing. Limitations of these methods include higher costs, and requirements for more specialized equipment, reagents, and expertise than are needed for conventional diagnostic methods. Identifying the most appropriate method or combination of methods to use can be difficult. Factors to consider in selecting a molecular diagnostic approach include the purpose of the investigation, and the structure and molecular characteristics of the M. bovis population under investigation. Use of inappropriate methods can yield misleading results and lead to significant waste of resources. Keywords: Tuberculosis, Mycobacterium bovis, diagnosis. ii.

(3) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ TABLE OF CONTENTS. ABSTRACT ................................................................................................................................... ii TABLE OF CONTENTS ............................................................................................................ iii LIST OF TABLES ....................................................................................................................... vi CHAPTER I .................................................................................................................................. 1 INTRODUCTION......................................................................................................................... 1 Global Impact of Tuberculosis............................................................................................... 1 Impact of Mycobacterium bovis Infection ............................................................................. 2 Importance of Distinguishing M. bovis from Other MTB Complex Members ..................... 3 Importance of M. bovis Strain Typing ................................................................................... 4 CHAPTER II ................................................................................................................................. 7 LITERATURE REVIEW ............................................................................................................ 7 Definition of Tuberculosis ..................................................................................................... 7 Mycobacterium tuberculosis Complex ........................................................................... 7 Mycobacterium bovis ...................................................................................................... 8 Susceptible Species ................................................................................................................ 9 Transmission ........................................................................................................................ 11 Risk Factors for Infection .................................................................................................... 12 Humans ......................................................................................................................... 12 Cattle ............................................................................................................................. 13 Epidemiology ....................................................................................................................... 14 Morbidity ...................................................................................................................... 15 iii.

(4) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ Mortality ....................................................................................................................... 15 Incidence and Prevalence.............................................................................................. 16 Clinical Presentation ............................................................................................................ 18 Treatment ............................................................................................................................. 20 CHAPTER III ............................................................................................................................. 23 PREVENTION AND CONTROL ............................................................................................. 23 Pasteurization ....................................................................................................................... 23 Vaccination .......................................................................................................................... 24 Education ............................................................................................................................. 25 Diagnostic Services .............................................................................................................. 26 Surveillance.......................................................................................................................... 27 Eradication from Livestock.................................................................................................. 30 Eradication of Wildlife Reservoirs ...................................................................................... 31 Legal Requirements ............................................................................................................. 32 CHAPTER IV.............................................................................................................................. 34 DIAGNOSTIC METHODS ....................................................................................................... 34 Clinical ................................................................................................................................. 34 Microbiological .................................................................................................................... 38 Microscopy ................................................................................................................... 38 Culture .......................................................................................................................... 38 Immune Response-Based ..................................................................................................... 40 Tuberculin Skin Test ..................................................................................................... 41 Interferon-Gamma Release Assays ............................................................................... 42 iv.

(5) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ Genetic ................................................................................................................................. 43 IS6110 ........................................................................................................................... 45 Poly(GC)-Rich Sequences (PGRSs) ............................................................................. 47 Direct Repeat (DR) Region........................................................................................... 47 Variable Number Tandem Repeats (VNTRs) ............................................................... 49 Other Methods .............................................................................................................. 50 CHAPTER V ............................................................................................................................... 53 CONCLUSION AND RECOMMENDATIONS ...................................................................... 53 REFERENCES ............................................................................................................................ 57. v.

(6) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ LIST OF TABLES. 4.1. Summary of Select Methods Used to Diagnose M. bovis Infection. ..................................... 35. vi.

(7) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ CHAPTER I INTRODUCTION. Global Impact of Tuberculosis Tuberculosis is one of the most common infectious diseases worldwide, and poses a significant public and animal health burden. Most cases of tuberculosis in humans are caused by infection with Mycobacterium tuberculosis, a member of the Mycobacterium tuberculosis (MTB) complex of mycobacteria (de la Rua-Domenech, 2006; World Health Organization, 2012d). Approximately one-third of humans worldwide are estimated to be infected (Centers for Disease Control and Prevention, 2012e). Most cases of tuberculosis occur in sub-Saharan Africa and Asia (World Health Organization, 2012c). Tuberculosis is preventable, but is one of the leading causes of death worldwide due to infectious disease, second only to human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS) (World Health Organization, 2012d). In 2010, 8.8 million new cases of tuberculosis occurred worldwide (World Health Organization, 2011a). Also in 2010, 1.4 million deaths due to tuberculosis occurred worldwide, equating to an average of more than 3,800 deaths per day (World Health Organization, 2011a). Approximately 95 percent of deaths due to tuberculosis occur in low- or middle-income countries (World Health Organization, 2012d). Tuberculosis is one of the three leading causes of death in women age 1544 years (World Health Organization, 2012a). The number of children who were orphans due to parental tuberculosis deaths was estimated to be almost 10 million in 2009 (World Health Organization, 2011a). Each year, approximately 500,000 children acquire tuberculosis, and 70,000 children die of tuberculosis (World Health Organization, 2012b). In the United States, 1.

(8) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ more than 11,000 cases of tuberculosis were reported in 2010, and more than 500 deaths due to tuberculosis occurred in 2009 (Centers for Disease Control and Prevention, 2011d). Impact of Mycobacterium bovis Infection While the vast majority of tuberculosis cases in humans are caused by infection with M. tuberculosis, a significant proportion of tuberculosis cases in humans, and most cases in cattle, are caused by infection with M. bovis (de la Rua-Domenech, 2006). More than 3 percent of all tuberculosis cases in humans worldwide have been estimated to be due to M. bovis infection, ranging from 1 percent or less in industrialized countries to 10 to 15 percent in economically less developed countries (Ayele, Neill, Zinsstag, Weiss, & Pavlik, 2004; de la Rua-Domenech, 2006). The prevalence of M. bovis infection is expected to increase. In economically well developed countries, increases in prevalence are attributed in part to increases in immigrant populations who can be at higher risk of infection acquired in their countries of origin or through cultural practices that increase their risk, such as consumption of unpasteurized dairy products (Dankner & Davis, 2000; Dankner et al., 1993; de Kantor, LoBue, & Thoen, 2010; LoBue, Betacourt, Peter, & Moser, 2003; Thoen, LoBue, & de Kantor, 2006). In economically less developed countries, increasing prevalence is attributed in part due to increasing intensity of livestock production to meet an increasing demand for food security (Michel, Muller, & van Helden, 2010). The World Health Organization (2011b) has classified tuberculosis due to M. bovis infection as a neglected zoonotic disease, affecting primarily poor rural populations in developing countries who live in close association with livestock. In addition to the public health burden, tuberculosis due to M. bovis infection poses a significant threat to social, economic, and food security, in particular in economically less developed countries. Infection in livestock can threaten livelihoods, income, and social status by 2.

(9) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ negatively impacting livestock productivity and trade in animals and animal products (Michel et al., 2010; World Health Organization, 2011b). Infection in wildlife can negatively impact ecosystems, and complicates control and eradication programs (Michel et al., 2006; Michel et al., 2010; Nishi, Shury, & Elkin, 2006; Nugent, 2011; O'Brien, Schmitt, Fitzgerald, Berry, & Hickling, 2006). Indirectly, the morbidity, mortality, and decreased livestock productivity associated with M. bovis infection in livestock can threaten the health and economic resilience of low-income individuals who rely on the sale of livestock to raise funds for emergency health care (World Health Organization, 2011b). With fewer or no healthy livestock animals to sell, individuals are unable to pay for essential care or can become trapped in debt. Importance of Distinguishing M. bovis from Other MTB Complex Members Understanding the epidemiology and the public health, social, and economic burdens of M. bovis infection is essential to developing effective control and eradication programs, assessing the effectiveness of programs already in place, and building social, political, and financial support for program success. In addition, distinguishing M. bovis from other MTB complex members can be important in designing effective treatment (González-Duarte, Ponce de León, & Osornio, 2011; LoBue & Moser, 2005; Murray & Jacobs, 2004). However, the full extent of the M. bovis infection burden is unknown, and epidemiologic data on M. bovis infection are sparse (Grange, 2001; Michel et al., 2010; Wedlock, Skinner, de Lisle, & Buddle, 2002). Barriers to gaining a broader understanding of M. bovis infection are numerous. Routine screening and diagnostic tests for tuberculosis generally do not distinguish among members of the MTB complex (Thoen, LoBue, et al., 2006). Definitive identification of M. bovis requires its isolation and molecular characterization (de Kantor et al., 2010). In many cases, distinguishing among members of the MTB complex is not a public health priority (de 3.

(10) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ Kantor et al., 2010). Definitive tests can be laborious, time consuming, and expensive, and can require sophisticated laboratory equipment and reagents. The necessary expertise or other resources for M. bovis identification are not always available (de Kantor et al., 2010). Many laboratories, particularly in economically less developed countries, routinely use culture media that do not adequately support the growth of M. bovis (Thoen, LoBue, et al., 2006; Wedlock et al., 2002). Even genetic tests that are widely used do not necessarily distinguish among MTB complex members (Thoen, LoBue, et al., 2006). Importance of M. bovis Strain Typing Beyond simply distinguishing M. bovis from other MTB complex members, distinguishing among M. bovis strains is essential for understanding the epidemiology of M. bovis infection, developing effective control and eradication programs, and monitoring their progress (Michel et al., 2010). However, very little is known about the global distribution patterns of M. bovis genotypes, and in many cases, even country- or region-level epidemiologic data are not available (Ayele et al., 2004; de Kantor et al., 2010; Smith, Gordon, de la RuaDomenech, Clifton-Hadley, & Hewinson, 2006; Thoen, LoBue, et al., 2006). Strain differentiation cannot be used to definitively determine the source and direction of infection, but is a useful tool in epidemiologic investigations, complementing other epidemiologic information gathered (Thoen, Steele, & Gilsdorf, 2006). It can be used to generate testable hypotheses in investigations, and insights into the population genetics and evolution of M. bovis (Smith, Gordon, et al., 2006). This information in turn is useful in investigating the maintenance and spread of M. bovis infection, identifying risk factors for infection, and developing better vaccines and diagnostic tests (Smith, Gordon, et al., 2006).. 4.

(11) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ Strain differentiation is useful for investigating the origins of outbreaks, identifying potential sources of infection, and means and patterns of transmission, and investigating the relationships among infection in various human, domesticated animal, and wildlife populations (Skuce & Neill, 2001). Mycobacterium bovis typing was used to identify the Australian brushtail possum (Trichosurus vulpecula) as a major wildlife reservoir of M. bovis (Collins, 2011). This analysis also revealed geographic localization of strain types, which was useful in investigating the source of M. bovis infection in livestock and humans. In New Zealand, M. bovis strain typing is an integral part of control programs for M. bovis infection, and is used to inform decisions on levels of herd testing and wildlife control, and to define the extent of spread in wildlife (Skuce & Neill, 2001). In the United Kingdom, M. bovis typing revealed strong geographic localization of M. bovis strains (Smith, Gordon, et al., 2006). This information has been used to identify potential sources of infection, such as movement of cattle from one geographic region to another, and indicated the need for pre-movement testing to prevent movement of infected animals. Typing of M. bovis also provided evidence of M. bovis transmission between cattle and badgers (Smith, Gordon, et al., 2006). In laboratory applications, strain typing is useful in monitoring quality control and in investigating potential cross-contamination (Skuce & Neill, 2001). Strain typing data can also be used to improve M. bovis diagnostics, by allowing better matching of the antigens used in testing to the strains that are most common in the population being tested (Smith, Gordon, et al., 2006). Similarly, typing data can be used to increase the effectiveness of vaccines. Evidence has been reported that M. bovis strains differ in their ability to overcome vaccine-induced immunity; typing data would enable testing of the efficacy of candidate vaccines on the M. bovis strains that are prevalent in the target population (Smith, Gordon, et al., 2006). 5.

(12) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ Finally, strain-level typing can be used to more effectively combat the high prevalence of multidrug resistant and extensively drug resistant strains of mycobacteria (World Health Organization, 2011c). The estimated prevalence of multidrug resistant tuberculosis worldwide in 2010 was 650,000 (World Health Organization, 2012a). Approximately 25,000 new cases of extensively drug resistant cases of tuberculosis occur each year (World Health Organization, 2011c). The emergence of drug resistant tuberculosis is attributed primarily to mismanagement of tuberculosis treatment (World Health Organization, 2011c). Strain data can be used to improve the design of treatment regimens, and to better match infected individuals with the most effective treatment regimen for them (World Health Organization, 2011c).. 6.

(13) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ CHAPTER II LITERATURE REVIEW. Definition of Tuberculosis Tuberculosis is an acute or chronic disease caused by infection with any of several bacteria of the genus Mycobacterium (de la Rua-Domenech, 2006; Yancey, 2008). Mycobacteria are acid-fast, acidophilic, aerobic, non-motile, non-sporulating, rod-shaped bacteria approximately 2 to 4 micrometers long (Rastogi, Legrand, & Sola, 2001; Soler, Brieva, & Ribón, 2009; Yancey, 2008). They are members of the taxonomic family Mycobacteriaceae (Rastogi et al., 2001). More than 50 types of mycobacteria have been identified (Yancey, 2008). Mycobacteria that cause tuberculosis in mammals are referred to collectively as members of the MTB complex (de la Rua-Domenech, 2006). Several characteristics of members of the MTB complex and differences between M. bovis and other members of the MTB complex are discussed. Mycobacterium tuberculosis Complex Eight members of the MTB complex have been described: M. tuberculosis, M. canettii, M. africanum, M. pinnipedii, M. microti, M. caprae, M. bovis, and M. mungi (Alexander et al., 2010; Smith, Gordon, et al., 2006). Recently, Mycobacterium orygis was proposed as a ninth member of the MTB complex (van Ingen et al., 2012). The nomenclature and taxonomic relationships among MTB complex members have been a source of confusion and dispute (de la Rua-Domenech, 2006). For example, the members of the MTB complex have been proposed to all be members of the same species, and been distinguished variously as distinct subspecies, strains, or ecotypes (Aranaz et al., 1999; Grange, 2001; Smith, Gordon, et al., 2006; Smith, 7.

(14) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ Kremer, et al., 2006). Alternatively, they are referred to as distinct species (Centers for Disease Control and Prevention, 2011d; Grange, 2001; Hlavsa et al., 2008; LoBue, Enarson, & Thoen, 2010a). Adding to the confusion are differences in classification criteria, ranging from differences in drug sensitivity or host tropism, to genetic characteristics (Grange, 2001). Members of the MTB complex differ widely in host preference, phenotype, and pathogenicity (Brosch et al., 2002; Mathema, Kurepina, Bifani, & Kreiswirth, 2006). For example, M. tuberculosis, M. canettii, and M. africanum are found most frequently in humans, whereas M. microti is generally found in voles and shrews, M. pinnipedii in marine mammals, M. caprae in goats, and M. bovis in cattle (Brosch et al., 2002; Smith, Kremer, et al., 2006). Examples of phenotypic differences between M. tuberculosis and M. bovis are differences in colony morphology, carbon source requirements, growth inhibition by glycerol, and drug resistance (Kaneene, Miller, de Kantor, & Thoen, 2010; Rowe & Donaghy, 2008). In contrast, genetically, members of the MTB complex are extremely similar, exhibiting 99.9% similarity at the nucleotide level and near identity in their 16S rRNA sequences (Brosch et al., 2002; Mathema et al., 2006; Smith, Gordon, et al., 2006). Mycobacterium bovis Phylogenetic evidence suggests that members of the MTB complex evolved from a common progenitor, with M. bovis placed evolutionarily more distant than M. tuberculosis from this progenitor (Brosch et al., 2002; Garnier et al., 2003). Results of genetic deletion analysis suggest that M. bovis evolved as a distinct clone with distinct host preference, contradicting earlier hypotheses that M. tuberculosis represents a human-adapted descendant of M. bovis (Brosch et al., 2002; Garnier et al., 2003; Smith, Gordon, et al., 2006). Consistent with the high degree of sequence conservation among members of the MTB complex, the average nucleotide 8.

(15) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ sequence divergence between M. bovis and M. tuberculosis is less than 0.05 percent (Smith, Gordon, et al., 2006). In comparison, two strains of Escherichia coli were found in one study to have an average sequence divergence of 1.6 percent (Smith, Gordon, et al., 2006). Phenotypically, M. bovis differs from other members of the MTB complex in that its growth requires pyruvate and is inhibited by glycerol (de Kantor et al., 2010; Hewinson, Vordermeier, Smith, & Gordon, 2006; Kaneene et al., 2010). This is attributed to a defect in glycerol metabolism, caused by an inactivating mutation in pyruvate kinase, an enzyme essential for glycolysis (Hewinson et al., 2006; Keating et al., 2005). Susceptible Species The host spectrum of M. bovis is broad. Most mammals are thought to be susceptible to M. bovis infection (Center for Food Security and Public Health, 2009). Pigeons and crows have been infected experimentally (Center for Food Security and Public Health, 2009). Among domesticated animals, the primary hosts of M. bovis are cattle (Center for Food Security and Public Health, 2009). Mycobacterium bovis was first isolated from domesticated animals, and cattle remain the epidemiologically most important livestock reservoir host (Thoen, Steele, et al., 2006). Tuberculosis caused by M. bovis infection is often referred to as bovine tuberculosis, regardless of host species. Susceptible species can be generally categorized as (1) maintenance or reservoir hosts, that is, species in which M. bovis infection is present and maintained in the absence of transmission from other species (Naranjo, Gortazar, Vicente, & de la Fuente, 2008); or (2) incidental or spillover hosts, that is, species from which M. bovis has been isolated but which are not generally recognized as reservoir hosts (Kaneene et al., 2010). Several wildlife species, 9.

(16) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ primarily ungulates, have been identified as M. bovis reservoirs: African buffalo (Syncerus caffer), wood bison (Bison bison athabascae), North American bison (Bison bison), white-tailed deer (Odocoileus virginianus), mule deer (Odocoileus hemonius), lechwe (Kobus lechwe), red deer (Cervus elaphus), and wapiti (Cervus canadensiselaphus) (Kaneene et al., 2010). In Ireland and the United Kingdom, European badgers (Meles meles) are reservoirs (Corner, O'Meara, Costello, Lesellier, & Gormley, 2012). In New Zealand, the brushtail possum (Trichosurus vulpecula) is a reservoir (Nugent, 2011). Feral swine and wild boar (Sus scrofa) have been identified as M. bovis reservoirs in Europe (Naranjo et al., 2008). Numerous other wildlife species have been identified as potential reservoirs of M. bovis. These include bush pigs (Potamochoerus porcus), warthogs (Phacochoerus africanus), various types of deer (Axis axis, Dama dama, Capreolus capreolus, Cervus nippon), European wild goats (Capra aegagrus), greater and lesser kudu (Tragelaphus strepsiceros and Tragelaphus imberbis), llamas (Lama glama), muntjacs (Muntiacus spp.), giraffes (Giraffa camelopardalis), water buffalo (Bubalis bubalis), tapirs (Tapirus terrestris), impalas (Aepyceros melampus), Bactrian camels (Camelus ferus), wildebeest (Connochaetes spp.), topi (Damalisus korrigum), yaks (Bos grunniens), and eland (Taurotragus spp.) (Kaneene et al., 2010). Mycobacterium bovis has been isolated from a wide range of carnivores and scavengers, including large cats (Panthera leo, Panthera tigris, Panthera pardus, Acinonyx jubatus, Uncia uncial, Lynx pardinus, and Felix rufus), foxes (Vulpes vulpes), coyotes (Canis latrans), wolves (Canis lupus), hyenas (Crocuta spp.), black bear (Ursus americanus), raccoons (Procyon lotor), opossums (Didelphis virginiana), mustelids (Mustela spp.), and meerkats (Suricata suricatta). Other small mammals from which M. bovis has been isolated include moles (Scalopus aquaticus), voles (Clethrionomys spp. and Microtus spp.), rats (Rattus spp.), squirrels (Sciurus 10.

(17) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ carolinensis), hedgehogs (Erinaceous europaeus), rabbits (Orytolagus cuniculus cuniculus), and hares (Lepus europaeus). Elephants (Elephas maximus and Loxodonta africana), otters (Lutra lutra), and marine mammals are also susceptible to M. bovis infection, as are a variety of primate species, including humans (Center for Food Security and Public Health, 2009; Kaneene et al., 2010; Mikota & Maslow, 2011). Spillover hosts among domesticated livestock include sheep, goats, horses, and pigs (Center for Food Security and Public Health, 2009). Transmission Mycobacterium bovis can be transmitted by inhalation or ingestion, or through breaks in the skin (Center for Food Security and Public Health, 2009; Grange, 2001). The epidemiologic importance of these routes varies by species, and the role of each host species in transmission varies with transmission route, host abundance, and host interactions (Nugent, 2011). Humans are most commonly infected through inhalation of contaminated aerosols and consumption of contaminated milk or dairy products (Grange, 2001). Cattle are infected primarily through inhalation of contaminated aerosols (de la Rua-Domenech, 2006). Carnivores can be infected through ingestion of contaminated carcasses (Kaneene et al., 2010). Humans and animals can also be infected through contact with contaminated fomites, such as thermometers, masks, cages, and food or feed containers (Kaneene et al., 2010). An epidemiologically important means of interspecies transmission is ingestion of food, feed, or water that is contaminated with M. boviscontaining excretions or secretions from infected animals, or through ingestion of infected carcasses (Kaneene et al., 2010). Transmission occurs most frequently between animals of the same or different species, and from animals to humans (de la Rua-Domenech, 2006). Reported sources of animal-to-human transmission include cattle, elk, seals, and rhinoceros (Grange, 2001). Cases of human-to-animal 11.

(18) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ transmission are less common; most or all cases reported to date have involved transmission or suspected transmission from humans to cattle (Fritsche, Engel, Buhl, & Zellweger, 2004; Grange, 2001). Human-to-human transmission is rare, but does occur (de Kantor et al., 2010; Etchechoury et al., 2010; Evans et al., 2007; LoBue et al., 2010a; Sunder et al., 2009). In addition, several cases of M. bovis infection in humans have been attributed to injection of contaminated chemotherapeutics (Vos et al., 2003; Waecker, Stefanova, Cave, Davis, & Dankner, 2000). The minimum infectious dose of M. bovis is unknown. For humans, it has been estimated to be tens of hundreds of cells for respiratory transmission and millions of cells for transmission through ingestion (de la Rua-Domenech, 2006). Risk Factors for Infection Humans One of the primary risk factors for M. bovis infection in humans is consumption of unpasteurized dairy products, including both soft and hard cheeses (de la Rua-Domenech, 2006). Mycobacterium bovis can survive for up to two weeks in cream cheese and up to 100 days in butter (de la Rua-Domenech, 2006). Other risk factors include employment in the livestock industry, residence in rural areas, and residence in countries in which dairy pasteurization is not widely practiced (Cordova et al., 2012; de la Rua-Domenech, 2006). An occupational risk factor for M. bovis infection in humans is working with M. bovis-infected animals; individuals at high risk are cattle and deer farmers, stockyard workers, veterinarians, meat inspectors, slaughter plant workers, butchers, hunters, zookeepers, game wardens, and laboratory personnel (de la Rua-Domenech, 2006; Ingram, Bremner, Inglis, Murray, & Cousins, 2010). In one study, birth outside the United States, Hispanic origin, age less than 15 years, and human immunodeficiency. 12.

(19) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ virus (HIV) infection were each associated with increased risk of infection with M. bovis compared with M. tuberculosis (Hlavsa et al., 2008). Immunosuppression is associated with a greater likelihood of developing active tuberculosis after M. bovis infection (Dankner et al., 1993; Grange, 2001; LoBue et al., 2010a). High rates of co-infection with M. bovis and HIV have been reported (Barnes et al., 1999; Cicero, Olivera, Hernández-Solis, Ramírez-Casanova, & Escobar-Gutiérrez, 2009; Dankner et al., 1993; Esteban, Robles, Soledad Jiménez, & Fernández Guerrero, 2005; Grange, 2001). Also, HIV infection increases the risk of developing active M. bovis infection among infants and children who undergo Bacillus Calmette-Guérin (BCG) vaccination for tuberculosis (Nuttall & Eley, 2011). An additional risk factor for iatrogenic infection is injection with chemotherapeutics (Vos et al., 2003; Waecker et al., 2000). Individuals in economically less developed countries tend to be at higher risk of M. bovis infection than individuals in other countries (Michel et al., 2010). In many cases, individuals in economically less developed countries are exposed simultaneously to multiple risk factors, such as living in poverty in close proximity to livestock, having a family member with a history of diagnosed tuberculosis, living in a rural area, lack of widespread pasteurization of dairy products, and living in a country in which the prevalence of human immunodeficiency virus infection is high (Michel et al., 2010). Cattle Risk factors for tuberculosis due to M. bovis infection in cattle can be categorized as animal-level, herd-level, and geographic region-level risk factors (Humblet, Boschiroli, & Saegerman, 2009). Animal-level risk factors include age, gender, breed, body condition, and M. bovis infection status of the dam (Humblet et al., 2009). Older age can be associated with higher 13.

(20) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ risk due to increasing duration of exposure, or potential for exposure, over time. Gender-related risk can be influenced by age and management practices; male cattle have been reported to be at higher risk where they were maintained longer than female cattle for field work, whereas female cattle have been reported to be at higher risk than males in dairies, where they are maintained longer than males due to their use for calving and milking. In Africa, imported breeds of cattle have been reported to be at higher risk than indigenous breeds; differences in risk have been attributed to differences in management practices or tuberculosis test sensitivity among breeds. Poor body condition has been linked to increased likelihood of M. bovis infection, as has being born to an infected dam. Herd-level risk factors include a history of M. bovis infection in the herd, and herd maintenance in close proximity to M. bovis-infected humans. Larger herd size and management practices such as herd maintenance outdoors, at high density, or in proximity to infected wildlife have also been linked to a higher risk of M. bovis infection (Cleaveland et al., 2007; Humblet et al., 2009). Other herd-level risk factors that have been reported are lack of adequate testing for M. bovis infection, introduction of new cattle, and low cull rates (Humblet et al., 2009). Geographic region-level risk factors include a higher prevalence of M. bovis infection, geographic contiguity with an infected herd, and introduction of cattle from regions that are not officially designated as free of M. bovis infection. Epidemiology Reliable epidemiologic data regarding M. bovis infection are scarce. Reported numbers are likely to be underestimates (de la Rua-Domenech, 2006; Rowe & Donaghy, 2008). Underdiagnosis of M. bovis infection is common (Drobniewski, Strutt, Smith, Magee, & Flanagan, 2003). Reasons for underdiagnosis include lack of resources for laboratory 14.

(21) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ diagnostics, especially in economically less developed countries, the difficulty of culturing M. bovis, or use of diagnostic methods that cannot detect M. bovis infection or have insufficient discriminatory power to distinguish M. bovis from other members of the MTB complex (Centers for Disease Control and Prevention, 2011d; Drobniewski et al., 2003; Rowe & Donaghy, 2008). Often, epidemiologic data are reported for tuberculosis in general, with no subcategorization by etiologic agent to a level more specific than Mycobacterium. Morbidity Most humans with mycobacterial infection do not develop active disease. Only 5 to 20 percent of humans who test positive for exposure by skin test are estimated to develop active tuberculosis in their lifetime (Raviglione, 2010). Most cases of active tuberculosis in humans occur within the first two years of infection. Infected cattle can remain asymptomatic, or develop clinical signs only in old age or under physical stress, or rapidly develop debilitating and eventually fatal disease (Center for Food Security and Public Health, 2009). Among cattle, up to 40 percent of susceptible contacts of an infected animal have been estimated to become infected, with up to 10 percent developing gross lesions (Center for Food Security and Public Health, 2009). In other maintenance hosts, the severity of clinical signs varies by host species (Center for Food Security and Public Health, 2009). Most infected badgers are asymptomatic, whereas infected brushtail possums usually develop progressive, fatal disease (Center for Food Security and Public Health, 2009; Ryan et al., 2006). Mortality Reliable data regarding mortality due specifically to M. bovis infection are scarce. Tuberculosis in general is one of the top ten causes of death in humans worldwide (Raviglione, 2010). In economically well developed countries with well developed tuberculosis control 15.

(22) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ programs, mortality due to tuberculosis is rare. In the United States, the tuberculosis mortality rate per 100,000 persons decreased from more than 70 in 1930 to 0.2 in 2010 (Centers for Disease Control and Prevention, 2011d; Schlossberg, 2006). The decrease is attributed primarily to the introduction of widespread pasteurization and effective chemotherapy (Schlossberg, 2006). Worldwide, the mortality rate per 100,000 persons was estimated to be 29 in 2002, with a range from 6.2 in the Americas to 83 in Africa (Schlossberg, 2006). Natural mortality due to tuberculosis or M. bovis infection in animals is difficult to investigate. Farmed animals in which infection is detected are generally culled in an effort to control spread of the disease. Estimates of mortality rates in wild animals are complicated by lack of sufficient knowledge of the size of the infected population or of the definitive causes of death, or both. Incidence and Prevalence Reliable data regarding the incidence and prevalence of M. bovis infection are scarce (Wedlock et al., 2002). The incidence rate of tuberculosis of any cause per 100,000 persons in 2002 was estimated to be 141 worldwide, with a range from 43 in the Americas to 350 in Africa (Schlossberg, 2006). In 2010, the estimated global incidence rate was 128 cases per 100,000 population (World Health Organization, 2012a). The estimated prevalence rate of tuberculosis per 100,000 persons in 2002 showed a similar geographic distribution pattern, with a worldwide prevalence rate of 112 and a range from 25 in the Americas to 224 in Africa (Schlossberg, 2006). In most industrialized countries, approximately 1 percent or less of tuberculosis cases in humans are estimated to be due to M. bovis infection (de la Rua-Domenech, 2006; Hlavsa et al., 2008; Ingram et al., 2010; Thoen & LoBue, 2007). In 2010, slightly more than 11,000 cases of tuberculosis occurred in the United States as reported to the Centers for Disease Control and 16.

(23) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ Prevention (2011d). The incidence rate of tuberculosis in humans has been estimated at 4.3 per100,000 in the United States and Canada, 126 per 100,000 in Peru, 155 per 100,000 in Bolivia, and 306 per 100,000 in Haiti (de Kantor et al., 2010). The incidence of M. bovis infection in humans depends in part on the prevalence of M. bovis infection in cattle, the effectiveness of control programs for M. bovis infection in cattle, and the extent of consumption of unpasteurized dairy products (de la Rua-Domenech, 2006). From 1993 to 2003, the number of culture-positive cases of M. bovis infection in humans in England, Wales, and Northern Ireland ranged from 12 to 41 per year (Jalava et al., 2007). In 2010, 133 cases of M. bovis infection were reported in the 27 European Union member states (European Food Safety Authority and European Center for Disease Prevention and Control, 2012). In Latin America, an estimated 7000 new cases of M. bovis infection occur each year (Michel et al., 2010). The prevalence of M. bovis infection can be higher in some geographic regions or populations. In one study, approximately 7 percent of culture-positive cases of tuberculosis in patients in San Diego, California in 1994 through 2000 were found to be due to M. bovis infection (de Kantor et al., 2010). In one region of Mexico, almost 14 percent of tuberculosis cases were attributed to M. bovis infection (de Kantor et al., 2010). In a separate study in Baja California, 11 percent of tuberculosis cases, and 34 percent of culture-positive tuberculosis cases, were attributed to M. bovis infection (Dankner & Davis, 2000). In one study in China, less than 0.5 percent of tuberculosis cases in humans were found to be due to M. bovis infection (Chen et al., 2009). The proportion of tuberculosis cases due to M. bovis infection in Tanzania and Uganda has been estimated at 18 to 30 percent (Michel et al., 2010).. 17.

(24) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ Generally, cattle are diagnosed as tuberculosis positive by methods that do not include identification of the etiologic agent to a level more specific than Mycobacterium. In countries with effective control programs for mycobacterial infections in cattle, the incidence and prevalence of infection in cattle are generally low. The United States Department of Agriculture recognizes all or part of 49 States plus Puerto Rico and the United States Virgin Islands as free of bovine tuberculosis (United States Department of Agriculture, 2012a). In Canada, only one cattle herd was found infected with M. bovis in 2011 (Canadian Food Inspection Agency, 2012). In contrast, in 2005 the prevalence of M. bovis infection in cattle in Tanzania was found to be 2.5 percent (Durnez et al., 2009). In pastoral cattle herds in Ethiopia in 2008, the prevalence was estimated to be approximately five percent at the individual animal level, and 42 percent at the herd level (Gumi et al., 2011). In maintenance hosts other than cattle, the prevalence of M. bovis infection varies by host species (Center for Food Security and Public Health, 2009). For example, in culled badgers in Ireland, the prevalence of M. bovis infection has been estimated at up to 50 percent (Murphy et al., 2011). In brushtail possums in New Zealand, the prevalence estimates of M. bovis infection generally range from 1 to 10 percent, but are more than 50 percent for some areas (Center for Food Security and Public Health, 2009). In white-tailed deer in Michigan, the prevalence generally ranges from 2 to 4 percent. In wild elk in some regions of Canada, the prevalence ranges from 1 percent overall to almost 5 percent in mature males (Center for Food Security and Public Health, 2009). Clinical Presentation Most M. bovis infections remain latent, or clinically inapparent, for years (Rowe & Donaghy, 2008). In 5 to 10 percent of infected humans, clinical signs begin to appear only 18.

(25) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ months or years after infection, with the disease manifesting as general wasting and decline in health. Symptoms include unexplained weight loss, loss of appetite, night sweats, fever, and fatigue (Centers for Disease Control and Prevention, 2011g). Symptoms of pulmonary tuberculosis include coughing for at least 3 weeks, coughing up blood, and chest pain (Centers for Disease Control and Prevention, 2011g). In a minority of cases, clinical signs resolve to localized and self-limiting pathology. In some individuals, in particular immunocompromised individuals, the clinical course progresses rapidly to death. In cases of tuberculosis with central nervous system involvement, M. bovis infection is associated with a worse prognosis than is M. tuberculosis infection (González-Duarte et al., 2011). The clinical presentation of M. bovis infection is generally indistinguishable from that of M. tuberculosis infection, and varies by means of transmission (de la Rua-Domenech, 2006). Tissue lesions that develop are avascular, nodular granulomas known as tubercles (World Organization for Animal Health, 2011). Cutaneous or mucosal transmission can result in development of localized lesions in the skin, tendons, and lymph nodes; otitis; and conjunctivitis (de la Rua-Domenech, 2006). Aerosol transmission is associated with the development of gross lesions primarily in the lungs and thoracic lymph nodes, whereas transmission by ingestion is associated with the development of lesions in the lymph tissue associated with the gastrointestinal tract (Thoen, Steele, et al., 2006). Mycobacterium bovis infection is more likely to cause non-pulmonary disease than is M. tuberculosis infection, due to differences in primary route of infection (Grange, 2001). In cattle, M. bovis infection manifests primarily as a respiratory disease, with development of granulomas mainly in the lymph nodes of the lung (de la Rua-Domenech, 2006; Wedlock et al., 2002). As in humans, infections in cattle can become dormant and reactivate with 19.

(26) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ increasing time or during periods of physiological stress (Center for Food Security and Public Health, 2009). The clinical severity of M. bovis infection in cattle ranges from acute and rapidly progressive in some cases to more frequently chronic and debilitating disease (Center for Food Security and Public Health, 2009). Mycobacterium bovis infection in wild animals is generally clinically inapparent (Kaneene et al., 2010). Cytopathologically, tuberculosis is associated with the development of granulomatous lesions in lymph nodes associated with the lungs, and in other organs (Wedlock et al., 2002). Lesions occur more frequently in lymph nodes than in any other tissue, due to the function of lymph nodes in the host’s cellular immune response system (Thoen, Steele, et al., 2006). Lesions can be firm and localized, encapsulated with fibrous connective tissue, or include cell death and destruction leading to caseous necrosis and liquefaction (Thoen, Steele, et al., 2006). Lesion distribution can vary by host species. For example, in farmed and wild deer, lesions associated with M. bovis infection are located primarily in the lymph nodes of the head, whereas in badgers, lesions are frequently found associated with bite wounds (Wedlock et al., 2002). In cattle, possums and badgers, lesions occur primarily in the lungs and thoracic lymph nodes (Wedlock et al., 2002). Treatment Prompt, effective treatment of tuberculosis can decrease the likelihood of progression to active disease, and can decrease the likelihood of transmission (Centers for Disease Control and Prevention, 2011k; Thoen, Steele, et al., 2006). Design of effective treatment protocols is complicated by resistance of some mycobacterial strains to one or more antimicrobial drugs, by interstrain variability in antimicrobial drug susceptibility, by the variability in clinical expression of mycobacterial infection, and by the long duration of treatment necessary for maximal 20.

(27) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ effectiveness (Centers for Disease Control and Prevention, 2011k; Raviglione, 2010). Other considerations important in selecting an appropriate treatment regimen include patient age, comorbidities, immune status, and pregnancy status. In the United States, ten drugs are currently approved by the United States Food and Drug Administration for treatment of tuberculosis (Centers for Disease Control and Prevention, 2011k). Four standard treatment regimens are recommended as first-line options for treatment of latent infection (Centers for Disease Control and Prevention, 2011h). These regimens vary in terms of the number and type of drugs, duration (three to nine months), dose, and target population. The drugs recommended in these regimens are isoniazid, alone or in combination with rifapentine; and rifampin. Recommended treatment regimens for individuals with active tuberculosis are more complex (Centers for Disease Control and Prevention, 2011i). The basic regimen recommended as a first-line option consists of an initial 8-week phase of isoniazid, rifampin, pyrazinamide, and ethambutol administration, followed by an 18-week continuation phase of isoniazid and rifampin administration. Other treatment regimens are recommended for treatment of patients with drugresistant or multidrug-resistant tuberculosis, or for HIV-infected patients. Ensuring treatment adherence among patients can be a challenge, especially in cases of latent infection, in which the patient is clinically asymptomatic (Centers for Disease Control and Prevention, 2011e). Barriers to adherence include inconvenient appointment hours, misinformation about tuberculosis or treatment, health beliefs and practices, low level of financial resources, co-existing medical conditions, medication side effects, language barriers, and real or perceived stigma (Centers for Disease Control and Prevention, 2011e). One strategy used to increase adherence is directly observed therapy, in which ingestion of the therapeutic 21.

(28) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ agent is directly observed by a designated individual, such as a public health worker, medical professional, or family member (Centers for Law and the Public's Health, 2009). Treatment of M. bovis infection is complicated in many cases by lack of knowledge of whether the patient is infected with M. bovis, and by resistance of most M. bovis strains to pyrazinamide, one of the drugs of first choice in tuberculosis treatment (Centers for Disease Control and Prevention, 2011b). Pyrazinamide resistance can prevent the use of a short-course treatment regimen, which can impact patient compliance and treatment completion (LoBue & Moser, 2005). The effectiveness of treatment of latent tuberculosis, measured in terms of decreased incidence of tuberculosis complications, has been estimated to range from 50 percent to more than 90 percent, depending in part on level of patient compliance and history of chemotherapeutic treatment (Schlossberg, 2006). In one study of treatment outcomes in San Diego in 1994 to 2003, the death rate among M. bovis patients (15 percent) was more than twice that of M. tuberculosis patients (7 percent) (LoBue & Moser, 2005). Considerations in selecting appropriate treatment options for animals include differences in responsiveness to various antibiotics among host species, and ease or practicality of treatment administration. Treatment has been implemented for rare or high-value animals, but is not routinely used at the herd level or in wildlife (Michel et al., 2010; Mikota & Maslow, 2011).. 22.

(29) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ CHAPTER III PREVENTION AND CONTROL. Efforts to prevent or control M. bovis infection have historically been implemented to limit or avoid the potentially severe animal and public health as well as economic consequences of infection. In the early 20th century, tuberculosis was one of the leading causes of death in the United States, accounting for an estimated 1 in 9 deaths in 1900 (Olmstead & Rhode, 2004). The prevalence of infection in cattle in 1917 was estimated at approximately 5 percent. In 1917, United States Secretary of Agriculture D.F. Houston called tuberculosis “the greatest problem confronting the live-stock industry of the country” (Olmstead & Rhode, 2004, p. 28). Approximately 10 percent of tuberculosis cases in humans in the early 20th century are estimated to have been due to M. bovis infection. Economic losses related to tuberculosis in 1906 are estimated to have exceeded $1.1 billion (Olmstead & Rhode, 2004). Control of M. bovis infection is difficult, due to the very broad host range of the pathogen, the variety of means by which it can be transmitted, the moderate effectiveness of vaccination, and challenges to accurate diagnosis (Thoen, Steele, et al., 2006). The perception in the early 20th century that eradication was necessary and feasible grew with the awareness of tuberculosis as a zoonosis, and with the development of methods to test for infection (Olmstead & Rhode, 2004). Prevention and control efforts have evolved over time as knowledge of the etiology and epidemiology of tuberculosis and M. bovis infection have increased. Pasteurization Pasteurization has been one of the most effective means of preventing and controlling M. bovis infection in humans (de la Rua-Domenech, 2006). Mycobacterium bovis is inactivated by 23.

(30) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ heating at 63.5°C for 30 minutes, or 72°C for 15 seconds (Rowe & Donaghy, 2008). Introduction of widespread pasteurization has resulted in steep declines in the incidence of M. bovis infection in many countries (Olmstead & Rhode, 2004; Rowe & Donaghy, 2008). In these countries, new cases of active tuberculosis related to dairy product consumption often occur in very old individuals and represent reactivation of infection that remained latent for decades since before pasteurization was widely practiced, or such cases occur primarily in individuals who have personal or cultural preferences for raw dairy products (Rowe & Donaghy, 2008). Vaccination The only vaccine widely used against tuberculosis is M. bovis bacillus Calmette–Guérin (BCG) (McShane, 2011). This is a live attenuated strain of M. bovis that was first produced in the early 1900s by Calmette and Guérin. The BCG vaccine decreases the risk of tuberculosisassociated meningitis and has an estimated effectiveness of 80 percent in preventing disseminated tuberculosis in children (Checkley & McShane, 2011; McShane, 2011). However, its effectiveness against pulmonary tuberculosis is highly variable, and BCG vaccination can complicate interpretation of tuberculin skin test results (Centers for Disease Control and Prevention, 2011f). Occasionally, BCG vaccination can cause localized or disseminated tuberculosis, in particular in immunocompromised individuals (Grange, 2001). BCG vaccination of neonates in high-risk populations is carried out as part of mass immunization programs sponsored by the World Health Organization in countries in which tuberculosis is prevalent (McShane, 2011). However, BCG vaccine is not widely used in the United States and other countries with comparatively low risks of infection. In the United States, BCG vaccination is recommended only for high-risk individuals, such as health care workers who have a high risk of exposure to drug-resistant strains of Mycobacteria, or for children who 24.

(31) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ are continually exposed to adults who have untreated tuberculosis or are infected with drugresistant strains of Mycobacteria (Centers for Disease Control and Prevention, 2011f). BCG vaccination is not recommended for individuals who are, or are expected to become, immunocompromised, such as HIV infected individuals or organ transplant candidates (Centers for Disease Control and Prevention, 2011f). Efforts to develop more effective tuberculosis vaccines have focused on development of BCG replacements, or BCG-based booster vaccines (McShane, 2011). Replacement candidates in development include recombinant BCG strains designed to elicit a stronger immune response, attenuated M. tuberculosis strains, and inactivated M. vaccae strains. Booster vaccine development is focused on selection of appropriate antigenic subunits, and optimizing delivery systems. BCG vaccine is of variable effectiveness in livestock and wildlife (Buddle, Wedlock, Denis, Vordermeier, & Hewinson, 2011; Kaneene et al., 2010). Vaccination in these populations can hinder surveillance and control efforts, by complicating the interpretation of diagnostic test results and by the shedding of the BCG strain by some animals after vaccination (Kaneene et al., 2010; Palmer, Thacker, & Waters, 2009). In addition, efficient delivery of vaccine to livestock and wildlife remains a challenge. Oral administration of BCG vaccine has been shown to decrease the severity of disease in various animal species experimentally infected with M. bovis, and to prevent M. bovis infection of wild possums (Buddle et al., 2011). Education Appropriately designed educational components are key to effective tuberculosis prevention and control programs (Advisory Council for the Elimination of Tuberculosis, 1995). Educational efforts can be broad or narrowly focused, and can be designed to address a variety of 25.

(32) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ target audiences and issues. Goals of efforts aimed at the general public can include increasing awareness of tuberculosis symptoms, transmission risks, and prevention strategies (Centers for Disease Control and Prevention, 2012d). Educational efforts aimed at private practitioners and hospital staff, who are often the first contacts of symptomatic individuals seeking treatment, can similarly be aimed at increasing knowledge of tuberculosis symptoms and treatment options, as well as treatment guidelines and reporting requirements. Examples of more narrowly focused educational efforts include campaigns aimed at infected individuals or their health care providers to increase patient adherence to treatment regimens, or to increase knowledge of issues surrounding coinfection with HIV. Other examples are programs aimed at addressing tuberculosis risk in homeless individuals, or in specific cultural or ethnic groups (Kirtland, Lopez-De Fede, & Harris, 2006; Rayner, 2000; Working Group on Tuberculosis Among Foreign-Born Persons, 1998). Challenges include gaining enough accurate information about target groups to adequately inform the design of an effective educational campaign, tailoring messages to specific cultural, ethnic, or regional needs, and ensuring that the appropriate messages reach and are understood and applied by the target populations. Educational methods can be as varied as the messages. They can range from informational leaflets or radio and television public service announcements, to face-to-face counseling, formal continuing education programs for health care providers, and outreach by regulatory agencies to specific groups such as livestock producers and wildlife managers. Diagnostic Services A robust, reliable system of diagnostic services is essential for an effective tuberculosis prevention and control program (Advisory Council for the Elimination of Tuberculosis, 1995). Accurate diagnosis is a prerequisite for assessment of the scope and epidemiology of infection, 26.

(33) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ which in turn is necessary for effective program planning and implementation, and evaluation of program effectiveness. Important diagnostic services for humans include chest radiography and interpretation, for initial screening for active pulmonary tuberculosis (Schlossberg, 2006). Mycobacteriology laboratory services are essential for diagnostic testing, identification of the etiologic agent, and evaluating patient response to treatment, for both humans and animals. Laboratory services can range from acid-fast bacillus smear examination to culture, isolation, and characterization of the pathogen, including drug-susceptibility testing. Advanced laboratory services are essential for detection of M. bovis infection. Other important diagnostic services include determining whether patients who are receiving tuberculosis treatment are showing signs of drug toxicity. Information gained can be used in decision making related to patient care and treatment regimens. Finally, given the strong association between tuberculosis and HIV infection, and the importance of considering HIV infection status in treatment decisions, HIV testing and counseling services are often available within the scope of tuberculosis diagnostic services. Surveillance The Centers for Disease Control and Prevention has defined public health surveillance as follows: The ongoing, systematic collection, analysis, and interpretation of health data is essential to the planning, implementation, and evaluation of public health practice, closely integrated with the timely dissemination of these data to those who need to know. The final link in the surveillance chain is the application of these data to prevention and. 27.

(34) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ control. A surveillance system includes a functional capacity for data collection, analysis, and dissemination linked to public health programs. (Lee & Thacker, 2011, p. 19) Surveillance in domesticated animals and wildlife can be defined similarly, with a shift in focus from disease in humans to disease in animals. Both public and animal health surveillance can serve as a mechanism for early warning of disease outbreaks, to inform disease control program planning and monitoring, and support compliance with international disease reporting guidelines. Animal health surveillance information additionally is used to manage risks associated with domestic and international trade in animals and animal products, and, in the case of livestock health surveillance, is used in assessing animal production efficiency (Australian Biosecurity Cooperative Research Center for Emerging Infectious Disease, 2009). In most countries, including the United States, no comprehensive national system is in place for surveillance for M. bovis infection in humans. Surveillance specific for M. bovis requires sufficient capacity and resources for advanced laboratory testing, because tuberculosis caused by M. bovis is indistinguishable clinically, radiographically, and pathologically from tuberculosis caused by other members of the MTB complex, and because of the high level of genetic similarity among members of the MTB complex (Hlavsa et al., 2008). In the United States, the national surveillance system for tuberculosis in humans is based on laboratory diagnostic criteria for detection of MTB complex, with acknowledgment that most routine laboratory tests do not distinguish among members of the MTB complex, and most cases of tuberculosis in humans in the United States are expected to be due to M. tuberculosis infection (Centers for Disease Control and Prevention, 2011d).. 28.

(35) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ The Centers for Disease Control and Prevention (2004) introduced the National TB Genotyping Service, a laboratory service aimed at genotyping MTB complex isolates from patients in the United States with culture-confirmed tuberculosis. Only tuberculosis programs that have been approved to participate are eligible to submit samples, and sample submission is voluntary. The primary goal of this service was to allow more detailed epidemiologic investigation of M. tuberculosis infection, by linking surveillance to genotype data (Hlavsa et al., 2008). However, the service has also allowed, for the first time, national-level epidemiologic study of M. bovis infection. Efforts at surveillance for M. bovis infection in animals face similar obstacles in that definitive diagnosis requires advanced laboratory testing. However, because of the role of cattle and other livestock as maintenance hosts for M. bovis and as a primary source of human infection, surveillance programs targeted specifically at M. bovis infection in livestock are generally more well developed than are surveillance programs for M. bovis infection in humans. The primary goal of M. bovis surveillance in livestock in the United States is to support eradication (United States Department of Agriculture, 2001). Specific objectives are to measure progress of the eradication program, demonstrate freedom from disease or low risk to trading partners, and rapidly detect infection in the event of introduction in geographic regions that were previously free of infection (United States Department of Agriculture, 2001). The surveillance system consists of both slaughter surveillance and live-animal testing (Kaneene, Miller, & Meyer, 2006; United States Department of Agriculture, 2011). Tissue samples from carcasses of animals in which lesions are detected at slaughter, or from animals that test positive by tuberculin skin test, are submitted to the National Veterinary Services Laboratories for pathogen isolation and characterization. Federal surveillance efforts are supplemented by state 29.

(36) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ surveillance, such as Michigan’s program for M. bovis surveillance in white-tailed deer, which are a known reservoir of infection in that state (Kaneene et al., 2006). Eradication from Livestock Control of M. bovis infection in livestock is aimed at decreasing the risk of human infection and minimizing livestock production and trade losses (Wedlock et al., 2002). Most national programs for M. bovis eradication in livestock are based on whole-herd testing, and culling of infected animals (Wedlock et al., 2002). The eradication program in the United States was launched in 1917 and is a cooperative Federal, state, and industry effort (Olmstead & Rhode, 2004). A variety of complementary strategies have formed the basis of the program: targeted testing and slaughter of test-positive animals; payment of indemnity for condemned animals; quarantine of affected herds; and herd and geographic area-based designation of affected status. Eradication programs have been very successful in the United States, Europe, and Australia (Michel et al., 2010; Radunz, 2006; Reviriego Gordejo & Vermeersch, 2006). For example, in the United States, the estimated national prevalence of M. bovis infection in cattle decreased from 5 percent to 0.5 percent within the first 20 years of the program, and every county in the country was designated “free” of bovine tuberculosis (defined as prevalence less than 0.5 percent) by 1941. As noted above, the United States Department of Agriculture (2012a) now recognizes all or part of 49 States plus Puerto Rico and the United States Virgin Islands as free of bovine tuberculosis. Primary benefits of eradication of M. bovis infection in livestock are decreased incidence of human infection and decreased spill-over infections to susceptible wildlife species (Center for Food Security and Public Health, 2009; Kaneene et al., 2010). A primary challenge to eradication of M. bovis infection in livestock is cost. Eradication programs are expensive in terms of loss of livestock and the amount of resources required for 30.

(37) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ program planning, administration, implementation, and evaluation. In general, eradication programs based on a test and slaughter policy are rare in economically less developed countries. In 2001, only 12 countries in Latin America and 7 countries each in Asia and Africa had test and slaughter policies in place (Grange, 2001). Other challenges include the presence of wildlife reservoirs of infection, and public opposition related to animal welfare concerns or failure to recognize the importance of eradication (Moda, 2006). Eradication of Wildlife Reservoirs The presence of wildlife reservoirs of M. bovis infection can complicate or prevent its eradication from livestock (Kaneene et al., 2010; Palmer et al., 2009). In Australia, removal of free-ranging water buffalo and cattle reservoir hosts was a major component of a successful campaign in the late 20th century to eradicate M. bovis infection from farmed livestock (Radunz, 2006). In Ireland, focal culling of badgers is part of the national strategy to control M. bovis infection in cattle; the results of prevalence studies in badgers and cattle indicate that this strategy has been effective (Murphy et al., 2011). In New Zealand, targeted wildlife reservoirs have included brushtail possums, ferrets, deer, and feral swine (Ryan et al., 2006). In several regions of the world, the economic or social value of wildlife reservoirs has complicated their management or elimination. Examples include white-tailed deer in Michigan, wood bison and wapiti in Canada, and several species of wildlife in Kruger National Park in South Africa (Michel et al., 2006; Nishi et al., 2006; O'Brien et al., 2006). An alternative to wildlife culling is vaccination (Ryan et al., 2006). The feasibility and effectiveness of this approach have not yet been demonstrated.. 31.

(38) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ Legal Requirements Legal requirements pertaining to tuberculosis generally are designed to prevent, identify, or manage tuberculosis cases, or safeguard individual rights (Cabrera, Hodge, & Gostin, 2008). Human tuberculosis caused by infection with any member of the MTB complex is reportable in the United States (Centers for Disease Control and Prevention, 2012a). Reporting at the national level is voluntary, and does not routinely include reporting of which member of the MTB complex is the etiologic agent (Centers for Disease Control and Prevention, 2011c). Reporting at the state or tribal level is mandatory in all United States jurisdictions (Centers for Law and the Public's Health, 2009; Hlavsa et al., 2008). Requirements regarding testing and treatment for tuberculosis are enacted at the state and tribal level, and vary by jurisdiction (Centers for Law and the Public's Health, 2009). For example, some jurisdictions require testing as a condition of employment in specified jobs, such as park, playground, or recreational services. In general, individuals cannot be forced to undergo treatment, although courts can require patients who refuse treatment to remain isolated until they no longer pose a threat to public health. Legal requirements for pasteurization of dairy products similarly vary by jurisdiction. In the United States, Federal regulations prohibit shipment across state lines of unpasteurized milk or milk products in final package form for direct human consumption, with few exceptions (United States Department of Health and Human Services, 2012). The sale of unpasteurized milk and milk products within state or tribal jurisdictions is regulated by the respective jurisdiction (Centers for Disease Control and Prevention, 2012b). Approximately half of all States prohibit the sale of unpasteurized milk (Centers for Disease Control and Prevention, 2012b).. 32.

(39) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ Cases of M. bovis infection in bovines, caprines, and ovines are reportable to the United States Department of Agriculture under the Federal National Animal Health Reporting System (United States Department of Agriculture, 2012b). Federal regulations pertaining to control of M. bovis infection in livestock are based on classification of geographic areas (e.g., States or defined parts of States) with respect to herd prevalence of infection (United States Department of Agriculture, 2012a). The regulations restrict interstate movement of animals based on classification status (e.g., modified accredited or accreditation preparatory). In addition, individual jurisdictions impose their own legal requirements, such as regarding testing, reporting, and movement of infected or susceptible animals (Olmstead & Rhode, 2004).. 33.

(40) Molecular Diagnosis of M. bovis Infection. ______________________________________________________________________________ CHAPTER IV DIAGNOSTIC METHODS. Methods of diagnosing tuberculosis can be loosely categorized as clinical, microbiological, immune-response-based, or genetic. This list of categories is not exhaustive, and the methods and categories are not mutually exclusive; often diagnosis is based on a combination of methods, across two or more categories. Definitive identification of M. bovis requires molecular characterization of the disease agent. Selecting an appropriate diagnostic approach requires consideration of several factors, including the health status of the population and the main objective of testing, such as screening or definitive diagnosis (Adams, 2001). The methods described below are summarized in Table 4.1. Clinical Clinical evaluation for suspected active tuberculosis consists primarily of taking a thorough medical history and conducting a physical examination (Centers for Disease Control and Prevention, 2011g). Information considered includes the patient’s history of tuberculosis exposure, infection, and disease; demographic factors; risk factors for exposure; risk factors for progression from latent infection to active disease; and physical health. Chest radiography can be used to detect chest abnormalities such as lesions in the lungs. Clinical findings can be suggestive of tuberculosis, but cannot provide a definitive diagnosis. Latent tuberculosis is generally not associated with any of the clinical findings that are suggestive of active tuberculosis (Centers for Disease Control and Prevention, 2010).. 34.

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