Bovine Tuberculosis

W. Ray Waters

■ Definition, Etiology, and Epidemiology Tuberculosis (TB) in animals and humans may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M.

In most countries with active control programs, affected cattle herds contain few infected animals suggestive of low rates of cattle-to-cattle transmission and a slowly progressive course of disease.⁸ Transmission is generally by direct contact with TB-infected animals, as the organism may occur in exhaled droplets, saliva, feces, milk, urine, vaginal discharges, semen, or exudate from tuberculous lesions (e.g., lymph nodes with draining tracts that communicate to the exterior). Infection of the upper respiratory tract, pharyngeal area, and GI tract may also result from ingestion of infected feeds.⁹ Housing (e.g., milking parlors, cattle sheds) and crowding increase the contact of naive animals with infected animals and enhance the spread of this disease. M. bovis is also transmitted by indirect contact through contaminated feed and water, feeding and watering equipment, cleaning equipment, or movements of personnel (i.e., anything that mechanically transfers the organ­ism between locations). Movement of infected animals resulting from transfer of ownership, sharing of breeding animals, and fence-line contact with other herds is a common means of transferring the disease between herds and regions.

Primary control strategies for bovine TB include diagnosis via tuberculin skin test and/or interferon-gamma release assays, isolation of affected herds, subisolation of infected animals within affected herds (i.e., Bang method), slaughter of infected animals, slaughter surveillance, and movement or border testing policies.¹⁰ In addition to these traditional methods, antibody­based tests have recently emerged to detect M. tuberculosis- infected elephants,^11,12 M. bovis-infected captive cervids,¹³ and on occasion M. bovis-infected cattle.¹⁴ Also, molecular-based tools such as mycobacterial interspersed repetitive-unit-variable- number tandem-repeat (MIRU-VNTR), spoligotyping, and restriction fragment length polymorphism (RFLP) analysis of mycobacterial DNA are used to characterize mycobacterial isolates for epidemiologic investigations to determine potential links between new cases and prior outbreaks as well as the likely origin of infection. Most recently, high-throughput whole genome sequencing and rapid sequence analysis tools have been developed for a more thorough comparison of related­ness between M. tuberculosis complex isolates obtained from livestock, wildlife, humans, and zoo species; this technology is rapidly replacing MIRU-VNTR, spoligotyping, and RFLP for determining relatedness of M. tuberculosis complex strains from livestock and wildlife.¹⁵

■ Clinical Signs and Antemortem Diagnosis In most animals the disease is slowly progressive; however, bovine TB occasionally follows a more rapid, fulminating course with early dissemination. Most animals do not show clinical signs of M. bovis infection until late in the course of the disease. Clinical signs, when present, are often nonspecific (e.g., weight loss, inappetence, low-grade fluctuating fever) and vary accord­ing to the organs involved.

The tuberculin skin test remains the primary tool for the diagnosis of bovine TB. Specific applications of the tuberculin skin test, however, vary according to regional and country directives.¹⁰ Primary screening tests may use a single injection of M. bovis purified protein derivative (PPD) applied either to the skin fold at the base of the tail (e.g., caudal fold test used in New Zealand, the United States, Canada, and Mexico) or to the skin of the midcervical region (e.g., in many European Union member states). Alternatively, a dual injection of M. avium and M. bovis PPD may be applied to separate midcervical sites as the primary screening test (in Great Britain, Ireland, and Portugal) or as a confirmatory test (in the United States and Canada). An interferon-gamma release assay (e.g., Bovigam™ [Prionics, Schlieren, Switzerland]; ID-Vet, Montpel­lier, France) is also used as a confirmatory test in many countries.^10,16 Bovine TB test specificity differs regionally, most likely due to variations in application techniques (including differences in PPD usage), environmental factors, and co­infection (e.g., parasite burden¹⁷).

■ Pathophysiology, Necropsy Findings, and Dif­ferential Diagnosis While often viewed as a pulmonary disease in humans, TB is primarily a disease of the lymphatic system. With ruminants, tuberculous lesions are most commonly detected within pulmonary and cranial lymph nodes as well as lungs. In naturally infected cattle, 67% of tuberculous lesions are detected in lungs and pulmonary lymph nodes, 39% in cranial lymph nodes, and 8% in mesenteric lymph nodes.²⁰ Lesions are most commonly detected in the medial retropha­ryngeal lymph nodes of tuberculous white-tailed deer²¹; lungs, pulmonary lymph nodes, and pleura are also common sites of lesions in deer. In pigs (i.e., Eurasian wild boar and free-ranging domestic black pigs), tuberculous lesions are most commonly detected in the cranial lymph nodes, and dissemination is common.^22,23 With experimental infection, infectious dose affects disease severity and progression; animals exposed to high doses develop an earlier onset of lesions with greater severity than animals exposed to lower doses. The infectious dose of M. bovis for ruminants is quite low. For instance, intratracheal administration of one colony-forming unit (~6 to 10 bacilli) may cause disease.²⁴

M.

Postmortem diagnosis is based on (1) gross and microscopic evaluation for tuberculous lesions, (2) detection of the organism by mycobacterial culture within tissues, with follow-up con­firmation or characterization using molecular techniques, (3) direct detection of M. tuberculosis complex DNA within tuberculous lesions, and (4) detection of M. tuberculosis complex-specific DNA by PCR on histologic sections. Often multiple lesions are detectable on examination, although infected cattle may display no grossly visible lesions or only a single visible lesion. Macroscopic lesions appear as firm, encapsulated nodules and vary in size from barely visible to greater than 100 mm in diameter. Lesions may contain thick, white to yellow-orange, creamy to caseous purulent material. Lesions may be calcified. Lymph nodes, organs, and pleural or peritoneal serosa may be riddled with small miliary tubercles, and in the lungs these lesions may coalesce into a suppurative broncho­pneumonia. Granulomas along the pleural serosa (i.e., pearl disease) often appear glossy. Chronic lesions are characterized by a discrete, thickened fibrous to calcified capsule, often containing thick caseous material. Lesion cavitation due to M. bovis infection in cattle is rare. Microscopically, lesions in lymph nodes or organs are granulomatous, generally consisting of caseous necrosis surrounded by infiltrates of epithelioid macrophages, Langhans-type multinucleated giant cells, and lymphocytes. Lesions with necrotic cores may contain dystrophic calcification. Detection of acid-fast bacilli within granulomatous lesions in tissue sections provides a preliminary diagnosis of mycobacterial infection that must be confirmed by mycobacterial culture and PCR. Acid-fast bacilli are often few in number and most commonly detected within the caseum of the necrotic core. Resolving lesions are thought to be rare. Lesion develop­ment can be divided into four different stages primarily based on the level of necrosis, fibrous encapsulation, and mineraliza­tion: stage 1 (no necrosis), stage 2 (minimal central necrosis, thin connective tissue capsule), stage 3 (central caseous necrosis with complete fibrous encapsulation), and stage 4 (extensive multicentric caseous necrosis with mineralization). Differentia­tion between the thickness of the fibrous capsule and the histopathologic stage of development may assist in determining the chronicity of the infection.

Mycobacterial culture is the gold standard for TB diagnosis. M. bovis grows slowly, and cultures are generally incubated for 8 weeks; growth usually becomes visible within 3 to 6 weeks. The identity of the organism in cultures may be determined with biochemical tests, colony morphology, and M. tuberculosis complex-specific PCR. Potential pathogens for consideration in differential diagnosis include nontuberculous Mycobacteria spp., Nocardia spp., Arcanobacterium pyogenes, Corynebacterium pseudotuberculosis (caseous lymphadenitis in goats and sheep), and mycotic infections. In particular, nontuberculous myco­bacteria (e.g., M. avium subsp. paratuberculosis, M. avium subsp. avium, M. kansasii, and many others) are common isolates within tissues obtained from domestic livestock and wildlife. Johne's disease, caused by M. avium subsp. paratuberculosis, is common in domestic livestock, causing a mild to severe granulomatous enteritis. Other nontuberculous mycobacteria occasionally cause clinical disease resulting in granulomatous lesions in lymph nodes, lungs, and visceral organs.

■ Treatment, Prevention, and Control Bovine TB is rarely, if ever, treated in domestic livestock. Particularly in developed countries, affected animals are slaughtered to limit potential transmission to other animals and to limit the duration and severity of regulatory restrictions. Antimycobacterial therapy may be indicated for rare or endangered animals or for those considered valuable as zoologic exhibits.¹¹ Antituberculous first- line chemotherapies include isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin. These agents have the greatest activity with the least undesirable side effects. Second-line agents include capreomycin, ethionamide, cycloserine, and thiacetazone. The fluoroquinolones (moxifloxacin, ciprofloxa­cin, levofloxacin, and enrofloxacin), although not considered first-line agents, have significant bactericidal activity against M. tuberculosis complex species. Outside of these rare cases, treatment for TB in animals is generally not considered practical on a cost basis.

Many countries have official bovine TB eradication or control programs, including uniform methods and rules for application of the programs. Prevention and control efforts are designed to identify affected herds and remove infected animals or depopulate the entire herd (stamping out). These efforts are generally achieved via routine slaughter surveillance to identify affected herds, targeted testing schemes to remove TB test-positive animals (reactors), movement restriction of affected herds, thorough epidemiologic investigations of reported cases to determine risks for other affected herds, and depopulation of affected herds. In addition, movement testing with certification of a negative TB tests may be required prior to entry of animals into certain countries or regions. In certain instances, entry of animals from known TB-affected regions may not be allowed. Specific regulations and rules concerning bovine TB control are provided within specific country and regional directives.^1,26,2' Due to limitations in the sensitivity of antemortem diagnostic tests for tuberculosis, depopulation of infected herds is thought to be the most effective means to ensure eradication of the disease from the target population. Surveillance and control of M. bovis infec­tion in wildlife reservoirs, as well as mitigation efforts to limit spread from wildlife to livestock, are also critical for bovine TB control.

In instances where herds are depopulated and the owner intends to repopulate in the future, it is important to remove or thoroughly clean and disinfect facilities and equipment potentially exposed to the organism. The ability of M. bovis to survive for an extended period in the environment, and the ineffectiveness of disinfection of organic materials, necessitates that pastures and fields to be used in repopulation schemes be left vacant for a period prior to use. Instances of infection that are thought to originate from wildlife sources may precipitate changes in facilities, management and feeding practices, or wildlife control to prevent reinfection. Current experience with endemic M. bovis infections in wildlife in numerous countries has demonstrated that eradication of TB in free-ranging wildlife is difficult and a long-term proposition. Vaccines are rarely used for the control of bovine TB in regions with official control programs, primarily due to the potential for interference with traditional antemortem testing using PPD as antigen (i.e., tuberculin skin test and interferon-gamma release assays).²⁸ Bacillus Calmette-Guerin (BCG) is an attenuated M. bovis developed by Albert Calmette and Camille Guerin circa 1911 at the Pasteur Institute in Lille, France. Field efficacy trials

performed in the early twentieth century demonstrated the partial effectiveness of BCG for the control of bovine TB.²⁸ Recent experimental trials with cattle have demonstrated that (1) subunit vaccines may boost immunity elicited by BCG in cattle, (2) BCG is particularly protective when administered to neonates, and (3) differentiation of infected and vaccinated animals is feasible in cattle using in vitro or in vivo methods.^18,28 In regard to wildlife reservoirs, the efficacy of BCG delivered orally has been demonstrated for brushtail possums (in field trials) as well as for Eurasian badgers, wild boar, and white-tailed deer (each in experimental challenge studies). The most practical route of vaccine delivery to wildlife reservoirs of M. bovis is via oral-delivered baits, although a parenteral route is being used for badgers in England. Additional research is needed in the areas of diagnostic testing, environmental sampling, vaccine development, and management practices that may decrease the risk of disease spillover from wildlife reservoirs to cattle.