Jody Willis, DVM; Julie L. Webb, DVM; Bruce E. LeRoy, DVM, PhD, Dipl. ACVP; Frederick D. Quinn, PhD

Class of 2007 (Willis), Department of Pathology (Webb, LeRoy), and Department of Infectious Diseases (Quinn), College of Veterinary Medicine, University of Georgia, Athens, GA, 30602-7388

Introduction

Tuberculosis (TB) is a common and potentially deadly disease caused by infection with bacteria of the genus Mycobacterium (most commonly M. tuberculosis). A variety of species can be infected with M. tuberculosis bacilli including cattle, elephants, psittacines, non-human primates (NHPs) and humans.¹ TB is a highly infectious disease that can be spread between species (humans to animals and vice-versa). Approximately 1/3 of the world's human population, mostly in developing countries, is infected with M. tuberculosis bacilli giving the disease extreme economic and public health significance. The majority of human cases are subclinical, latent infections but 1 in 10 infections become active and, if left untreated, has a mortality rate of 50%.² The AIDS epidemic and its associated immunosuppression has led to a resurgence of active TB. Tuberculosis is one of the oldest known diseases: fossil records have found evidence of TB in bison and humans as far back as 4000 BC.

Tuberculosis in NHPs is not a common disease in the wild but it poses an insidious threat to Old World monkey research colonies.¹ Old World monkeys are genera that originated in Africa and Asia as compared to New World monkeys that originated in South and Central America. Asian Old World monkeys (macaques) are highly susceptible to infection with M. tuberculosis bacilli while New World monkeys (tamarins, capuchins, marmosets) appear to be fairly resistant.¹ African Old World monkeys (baboons and great apes) have intermediate susceptibility. Monkeys in research colonies have much closer and prolonged contact with each other than those in the wild. This level of contact allows infection to spread easily from one animal to another. Also, monkeys in colonies come into close, frequent contact with humans, another source of infection.

Of the Old World monkeys, Macaca mulatta (rhesus monkey) and Macaca fascicularis (cynomolgus monkey) represent the two most common NHP species used in biomedical research.¹ Research uses for macaques include the study of mechanisms of immunodeficiency disease, HIV/AIDS vaccine development, reproductive biology, aging, atherosclerosis, alcoholism and diabetes. In addition, these two species are used as models for human TB since their clinical signs, gross pathology and outcomes of infection closely parallels that of humans.⁴ These two NHP species will be the focus of this paper.

Etiology

Mycobacterium spp. are aerobic, facultative, intracellular bacilli. Two species of Mycobacterium cause TB in macaques: M. tuberculosis and M. bovis. Clinical signs, gross pathology and outcomes are identical with both organisms.¹ Other species (M. avium, M. arctoides, M. scrofulaceum and M. kansasii) cause different, non-TB, disease entities in macaques.

Pathogenesis

After inhalation, the organism settles in the lung and induces a marked, but somewhat delayed, inflammatory reaction. The most common manifestations of disease in macaques are similar to humans and include pulmonary granulomas with calcification, caseation, cavitation, and infection of draining hilar lymph nodes. Pulmonary lesions first develop 4 weeks after infection. Hilar lymphadenomegaly is first observed 3 weeks after infection but microscopic lesions are not observed in the nodes until 5 weeks after infection.⁵

Rhesus monkeys develop extensive pulmonary disease and subsequent hematogenous spread to the liver, spleen and kidneys following aerosol infection.^6,7 One study showed that rhesus monkey infected experimentally with 500 colony forming units resulted in 100% mortality in 3-8 months.⁶ In natural infections, 100% mortality was observed within 12 months. Cynomologus monkeys can develop pulmonary disease following intratracheal infection, but can also experience a latent infection.^4,8 Experimentally, 40% of cynomologus monkeys develop active infection and 60% develop latent infections. Extrapulmonary spread is less common in cynomologus monkeys.⁴

The clinical signs of M. tuberculosis bacterial infection in macaques range from nonspecific signs such as weight loss, lethargy, and unthriftiness, to respiratory signs such as persistent cough, and exertional dyspnea. Other signs include diarrhea, peripheral lymph node enlargement (with or without draining tracts), and enlarged liver or spleen on palpation.

These signs may be insidious, with only slight behavioral changes being noticed, followed by anorexia and lethargy, or animals may die suddenly while appearing to be in good condition.⁹

The most common hematologic abnormalities associated with TB are a normocytic, normochromic, nonregenerative anemia and a leukocytosis. The leukocytosis is inflammatory in nature and is characterized by a neutrophilia, monocytosis and lymphopenia. Elevated serum globulins and an increased erythrocyte sedimentation rate are other clinicopathologic abnormalities often observed. These changes are all indicators of a chronic inflammatory process and are not specific for TB.³

The gross lesions associated with tuberculosis in macaques include caseous nodules found in the hilar lymph nodes and lung, and large cavitary lesions within the lung, which can coalesce and extend into the pulmonary pleura. If the disease is allowed to progress, there may be secondary spread to the liver, spleen, lymph nodes and kidney as either multifocal, miliary disease or larger foci of caseation (Figure 1). In addition, tuberculous nodules may also be found in the central nervous system, mesentery, uterus and skin, although these manifestations are less common.³

Figure 1: Multiple yellow caseous Mycobacteria nodules in a liver. Image courtesy of Noah's Arkive.

Diagnosis

Tuberculin Skin Test (TST)

The cornerstone of the antemortem diagnosis of tuberculosis in nonhuman primates used in biomedical research is the intradermal tuberculin test. This is a routine test performed in quarantine and preventive medicine protocols. The tuberculin test is done in the skin of the eyelids of anesthetized animals, whereby a dilution of mammalian tuberculin is injected intradermally. A reaction is considered positive when there is swelling, edema, erythema and ptosis. The test is based on the principle of Type IV (delayed-type) hypersensitivity reactions: animals that have been previously exposed to (ie., infected with) with M. tuberculosis bacilli will react strongly to the injected tuberculin. Animals that have not been previously exposed will not react to the injection. TSTs become positive approximately 5 weeks after an animal is infected.⁵ Unfortunately, skin tests are subject to a fair amount of subjective assessment as the reaction is graded on a visual scale. In addition, the test is prone to false positive and false negative reactions. False positive reactions may occur when a monkey has been exposed to environmental, nonpathogenic Mycobacterium spp.¹⁰ As a species, orangutans appear to be extremely sensitive to the TST and have a high rate of false positives.¹ False negatives can occur late in the disease process, after treatment with isoniazid and in immunosuppressed animals.³

Serologic Tests

In response to the difficulties associated with the TST, serologic tests have been developed with the hopes of providing a more objective and reliable method of diagnosis. These tests involve stimulating whole blood or isolated lymphocytes with tuberculin and measuring the amount of interferon gamma (IFNγ) produced. The theory behind the tests is similar to that of the TST: blood or lymphocytes from animals previously exposed to M. tuberculosis bacilli will react strongly to the tuberculin and produce larger amounts of IFNγ compared to animals that have not been previously exposed. Reliability of the serologic tests appears to be equal to, if not better than, the TST but they are also subject to occasional false positive and false negative results.¹⁰

Diagnostic Imaging

Radiographs are often used as a non-invasive way to confirm the presence of tuberculous nodules in the lungs. However, radiographs do not distinguish TB from other cavitary-causing diseases such as cryptococcosis or nocardiosis.³

Mycobacteria do not stain with traditional Romanowsky cytologic stains. Organisms may be visualized as rod-shaped negative images within macrophages or free against a stained background (Figure 2). Organisms will stain red with acid-fast staining (Figure 3). After colonizing the respiratory tract via inhalation or digestive tract via ingestion, mycobacteria can be detected with acid fast staining in bronchial lavage samples, gastric fluid, feces and blood.⁸

Figure 2: Numerous Mycobacteria sp. visualized as negative rod-shaped images with a Romanowsky stain (arrows). Organisms are both free in the background and within a macrophage. 100X objective.	Figure 3: Mycobacteria organisms stain red with an acid-fast stain (arrow). Image courtesy of Dr. Frederick Quinn, University of Georgia.

Histopathology

The microscopic lesions associated with M. tuberculosis and M. bovis bacilli are unencapsulated granulomas of varying size. There is a necrotic core, which is surrounded by a layer of epithelioid macrophages, neutrophils and multinucleated giant cells. Occasionally, early in the disease process, the necrotic core is not present and the entire granuloma is composed of inflammatory cells (Figures 4 and 5).⁵ Calcification of the granuloma is rare. Using acid-fast stain, the bacteria may be seen both within and outside of cells.³

Figure 4: A cellular tuberculosis granuloma. An inner core of epithelioid macrophages (larger light pink cells in the center) is surrounded by an outer ring of lymphocytes and neutrophils (smaller dark purple cells). H&E, 20X objective.	Figure 5: A mixture of epithelioid macrophages (large arrows) and neutrophils (small arrows) in a caseous tuberculosis lesion. H&E, 50X objective.

Culture

Culture of bronchial and gastric lavage samples is a screening test occasionally performed in research colonies along with the TST. Positive results are highly specific for TB but a negative culture does not rule out infection. As a general rule, animals with active infection are far more likely to have positive culture results than animals with subclinical infection.⁵

Treatment/Control

The presence of tuberculosis in a colony of macaques usually requires that the infected animals be euthanized. Therefore, prevention of tuberculosis in biomedical research is the focus rather than treatment. Control measures include periodic intradermal tuberculin testing along with thoracic radiography.¹¹ Animals that have positive intradermal tuberculin reactions or present with suspicious radiographic lung lesions will undergo further diagnostic tests as discussed above.

1. Isaza R. Tuberculosis in all taxa. In: Fowler ME, Miller RE, eds. Zoo and Wild Animal Medicine. 5 th ed. St. Louis, MO: Elsevier; 2003:689-696.

2. World Health Organization Tuberculosis Site. http://www.who.int/tb/en/

3. Bernacky BJ, Gibson SV, Keeling ME, et al. Non-human primates. In: Laboratory Animal Medicine. 2 nd ed. Boston, MA: Academic Press; 2002:738-742.

4. Capuano SV, Croix DA, Pawar S, et al. Experimental mycobacterium tuberculosis infection of cynomolgus macaques closely resembles the various manifestations of human m. tuberculosis infection. Infect Immun. 2003;71(10):5831-5844.

5. Lin PL, Pawar S, Myers A, et al. Early events in mycobacterium tuberculosis infection in cynomolgus macaques. Infect Immun. 2006;74(7):3790-3803.

6. Good RC. Biology of the mycobacterioses. Simian tuberculosis: Immunologic aspects. Ann. N.Y. Acad. Sci. 1968;154(1):200-213.

7. Barclay WR, Anacker RL, Brehmer W, et al. Aerosol-induced tuberculosis in subhuman primates and the course of the disease after intravenous BCG vaccination. Infect Immun. 1970;2:574-582.

8. Walsh GP, Tan EV, dela Cruz EC, et al. The Philippine cynomolgus monkey (Macaca fasicularis) provides a new nonhuman primate model of tuberculosis that resembles human disease. Nat Med. 1996;2(4):430-436.

9. Alfonso R, Romero RE, Diaz A, et al. Isolation and identification of mycobacterium in New World primates maintained in captivity. Veterinary Microbiology. 2004;98:285-295.

10. Vervenne RA, Jones SL, van Soolingen D, et al. TB Diagnosis in Non-Human Primates: Comparison of 2 interferon-gamma assays and the skin test for identification of Mycobacterium tuberculosis infection. Veterinary Immunology and Immunopathology. 2004;100:61-71.

11. Sirois, M. Laboratory Animal Medicine: Principle and Procedures. Philadelphia, PA: Elsevier;2005:195-211.

Acknowledgements

Japanese Macaque - Portrait © by Larry Merrill is from the September 2004 web page in his Year of the Monkey collection and is used with permission.

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