Mycobacterium tuberculosis Infection in a Closed Colony of Rhesus Macaques (Macaca mulatta)

Abstract

Mycobacterium infections in nonhuman primates can devastate the colonies and place human handlers at risk. Despite conservative measures to prevent exposure, infections occur even in closed colonies. Here we describe a recent case of M. tuberculosis within a closed colony of rhesus macaques at our Thailand facility and the procedures instituted to prevent subsequent infections. Investigation of the outbreak did not confirm the source of the infection, but even with intensive occupational safety measures in place, human contact remains the most likely possibility.

Research using nonhuman primates is crucial to help bridge gaps between small mammals (for example, mice) and humans, particularly in the development of pharmaceuticals and vaccines. Nonhuman primates in research originate from many sources, including captive colonies throughout the world. According to the Centers for Disease Control and Prevention, many macaques imported into the United States originate from China, Mauritius, and Southeast Asia¹² and derive from purpose-bred colonies, where animals routinely are transferred into and out of the colony. However, some organizations choose to maintain closed colonies of nonhuman primates for research. Nonhuman primates are quite expensive, and closed colonies are extremely useful because they reduce costs and help ensure a disease-free population of research animals. In addition, much effort is expended to keep captive nonhuman primates dedicated for research free of unintentional infections, including those of internal and external parasites, simian retroviruses (for example, SIV, simian T-cell leukemia virus), and B virus (Cercopithecine herpesvirus 1). In addition, infections of anthropozoonotic agents, including Mycobacterium tuberculosis and M. bovis, present specific animal as well as public health concerns. Nonhuman primates may be the most susceptible of all captive animals to tuberculosis.¹³ Mycobacterium infections in macaques may result in pulmonary manifestations similar to those seen in humans, including caseous granulomata and cavitary lesions^2,6,10 that allow the disease to spread through infected aerosols to other animals or animal handlers. Evidence suggesting that Old World monkeys can maintain latent infections also exists.⁴

Outbreaks of disease have previously been documented in Old World monkey colonies, including a closed colony of rhesus macaques (Macaca mulatta) ²⁰ and a semiclosed colony of rhesus and cynomolgus macaques (Macaca fascicularis), in which the outbreak occurred after the introduction of additional monkeys to boost colony census.¹⁷Here we describe a recent outbreak of M. tuberculosis within a closed colony of rhesus macaques at our Thailand facility.

Case Report

This facility conducts all animal activities in compliance with the Animal Welfare Act¹ and other federal statutes and regulations involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals.⁸ The facility where this outbreak occurred is fully accredited by AAALAC International.

Currently, the census for this closed colony of rhesus macaques (Indian origin) exceeds 600 monkeys housed singly, in pairs, or in gang community-type pens. Several different rooms exist and are separated by permanent walls. The primate areas are designed as modified outdoor housing, with natural ventilation and lighting, augmented with air handling units and misting systems. Windows are double-screened, and corridors are roofed. One wing has a double-screened roof, which allows rain and sunlight to enter, and the animals are afforded covered areas in gang cages to provide a choice of location throughout the day.

As part of the preventive maintenance program, animals are tested yearly for SIV, simian T-lymphotropic virus, and B virus. Tuberculosis-infected macaques are identified through routine tuberculin skin testing conducted semiannually at the facility.^5,7 This testing schedule exceeds recommendations from the European Primate Veterinary Association Working Group on Tuberculosis,³ which state that closed colonies of nonhuman primates with limited exposure to humans may test as infrequently as once yearly for the detection of infection. After macaques are anesthetized, skin testing is performed by administering 0.1 mL (1500 or more tuberculin units) of old mammalian tuberculin (Tuberculin Mammalian, Colorado Serum Company, Denver, CO) intradermally into the palpebra. Palpebrae are alternated for each semiannual test. The palpebra is observed at 24, 48, and 72 h after injection to determine whether a positive reaction has occurred, and reactions are graded according to standard protocol.¹⁸ In the event a borderline grade 3 reaction is recorded, a second confirmatory test is performed in the skin of the macaque's abdomen.

During routine testing in August 2009, one potentially infected male macaque was identified with a grade 4 reaction (Figure 1) but exhibited no signs or symptoms of active disease. This monkey was born in the facility in June 1992 and never had a positive skin reaction to old mammalian tuberculin in 47 previous tests. Facility veterinarians were notified immediately, and the monkey was euthanized without further testing. Necropsy in the Animal Biosafety Level 3 suite revealed 2 suspicious lesions in a hilar lymph node and the left lung (Figure 2). Masses measured 1.5 × 1.5 × 0.7 cm and 0.8 × 0.5 × 0.5 cm, respectively, and consisted of firm, tan, lobulated material. A lobulated pattern with necrotic cores was evident on cut surface of the lung lesion; the hilar lymph node was not opened at necropsy. Histologic examination of the lung lesion exhibited the characteristic central caseous necrosis surrounded by macrophages and multinucleated giant cells (Figure 3), and acid-fast staining revealed acid-fast bacilli (Figure 4). Culture samples were sent to The Anti-Tuberculosis Association of Thailand (Bangkok, Thailand), which confirmed the presence of M. tuberculosis. The isolated strain was susceptible to streptomycin, isoniazid, ethambutol, and rifampicin.

Figure 1.

Grade 4 tuberculin skin test at 48 h in Mycobacterium-infected macaque.

Figure 2.

Hilar (long arrow) and lung (short arrow) lymph nodes on necropsy. Masses measured 1.5 × 1.5 × 0.7 cm and 0.8 × 0.5 × 0.5 cm, respectively. The lesions consisted of firm, tan, lobulated masses.

Figure 3.

Lung: caseous necrosis surrounded by epithelioid macrophages, multinucleate giant cells, lymphocytes, and plasma cells (granuloma). Hematoxylin and eosin stain; magnification, ×100.

Figure 4.

Red acid-fast bacteria (Mycobacterium tuberculosis) within the necrotic center of a granuloma. Ziehl–Neelsen stain; magnification, ×1000.

At the time the positive skin test was confirmed, all other macaques in the room in which the infected macaque resided immediately were placed on isolation status and confined to that room. In addition, all monkeys having resided in that location at any time during the previous 6 mo were identified. Their respective new rooms were placed on isolation status, resulting in large numbers of monkeys being quarantined for further testing. According to standard operating procedure, all potentially exposed macaques in each isolation area had to undergo skin testing every 2 wk until 5 consecutive, negative tests were obtained before the quarantine was removed. Testing resulted in no additional monkeys with positive skin reactions. No other additional testing was conducted.

Discussion

This case demonstrates that Mycobacterium infections can occur in closed macaque colonies, even with stringent protection measures. Given the history of 47 previous negative PPD tests in this animal, the likelihood that the infection was latent from an earlier infection is extremely low; the infection likely was newly acquired during the previous 6 mo. Strict protocols exist at this facility for entry into nonhuman primate areas. Each person must complete an occupational health exam with a staff physician and nurse, demonstrate negative TB status (either by skin test or chest radiograph), have a respiratory fit test for an appropriate respirator, and receive safety training on the hazards of working with nonhuman primates. In the vivarium, each person must change into a dedicated scrub uniform and wear a respirator (N95 or higher, if fitted, or equivalent), gloves, rubber boots, eye protection, and head cover. Next, personnel must walk through a minimum of 3 phenol-based disinfectant trays before entering any animal housing room. Last, before entering quarantine rooms, one additional change of boots occurs, and these boots are removed when leaving the quarantine area. Even with these procedures in place, the most likely source of infection at this facility is through introduction of Mycobacterium by vivarium or construction personnel.

The CDC recommends “all facilities that house nonhuman primates should adhere to the Institute for Laboratory Animal Research recommendations regarding baseline and (at a minimum) annual tuberculin skin test screening of employees.”⁵ However, Thailand has an annual tuberculosis incidence of 142 cases per 100,000 people,¹⁹ and most Thais receive the Bacillus Calmette–Guerin vaccine as infants.¹⁴ All Thai vivarium staff at this facility had received the vaccination. Because of the high rates of tuberculosis exposure in the general population and potential difficulty in interpreting skin testing results after vaccination, each Thai worker at our facility receives a chest radiograph annually for surveillance purposes. However, this method of surveillance clearly may not detect every possible case of infection. Given the recent case of tuberculosis in a nonhuman primate, specific testing such as by using QuantiFERON-TB Gold (Cellestis Limited, Carnegie, Victoria, Australia) may be considered for use at this facility. Antibody-based testing to detect γ interferon released in response to M. tuberculosis proteins is more specific than skin testing and can be used in vaccinated persons, because the vaccine strain does not contain these proteins.¹¹ This approach to antibody-based testing is reported to be less sensitive than skin testing,¹¹ but given the high prevalence of infection in Thailand and the high rate of vaccination, may be a better option than skin testing for this facility.

Of particular recent historical note, this facility underwent a major renovation of the monkey facility in 2008. This project entailed hiring short-term contractors to complete the work. Rooms undergoing renovation were emptied, and areas open to the outdoors with double screening were sealed off from neighboring rooms containing animals to prevent exposure to the construction workers and equipment. Monkeys were at least 5 to 10 m away from the renovated areas. Although all construction workers were verified to have a negative chest radiograph prior to entering the areas under renovation, potential transmission from these construction workers should be considered. A single chest radiograph may not reveal asymptomatic TB or a latent infection and may not be sufficiently sensitive for screening outside personnel who will have only intermittent access to the facility.

Within several days of the described outbreak, all 32 employees who had worked in the infected macaque's room in the 6 months since its last negative skin test received an additional chest radiograph. These employees again had negative chest films, and no definitive source for the outbreak was identified.

Since 2000, 4 macaques at our facility have had positive tuberculin skin tests, which were associated with M. kansasii and M. tuberculosis infections (one each) and 2 negative cultures. With such a small number of animals affected at each time, the macaques were euthanized to protect the rest of the colony without spread of the infection. But had a much larger percent of the colony had been affected, treatment might be considered as an option in similar scenarios. This reality has led others to search for ways to preserve nonhuman primate colonies, including treatment with antituberculosis drug therapy.¹⁷ Likewise, previous tuberculosis research using rhesus macaques indicated that multidrug chemotherapy with isoniazid, rifampin, and ethambutol was a practical alternative to prevent culling of monkeys due to M. bovis infection.³ The choice to offer treatment remains at the level of the Institute's veterinary care program but generally is considered not practical in research monkey colonies, because of concerns regarding possible transmission to additional monkeys as well as animal caretakers.

Previous experimental studies using macaques have indicated that Bacillus Calmette–Guerin vaccination may protect against Mycobacterium infection. One small study using both cynomolgus and rhesus macaques found that Bacillus Calmette–Guerin significantly protected cynomolgus macaques, whereas vaccinated rhesus macaques still developed significant lung pathology after infection.⁹ In contrast, vaccination with the recombinant Tokyo (Ag85A) vaccine¹⁵ or an attenuated phoP-deficient tuberculosis vaccine protected rhesus macaques from clinical pathology including lung lesions, wasting, and systemic inflammation when challenged with a virulent M. tuberculosis strain.¹⁶

Although the source of infection in the presented case has not been resolved, further investigation may determine whether specific employees, since 2000, have been in contact with previously infected macaques. If a pattern exists, the employees may receive additional testing, such as a sputum test, to determine whether they have an undiagnosed latent Mycobacterium infection that may be responsible for these intermittent outbreaks. In addition, another possible mode of transmission exists between humans and this macaque colony. Recent construction activities at this facility may have placed potentially infected construction workers relatively close to the colony. No direct contact occurred, but due to the modified outdoor design of the facility, the macaques may have been exposed to either aerosols or sputum from construction personnel working on the screen roof above the animal enclosures.