Mycobacterium tuberculosis var. tuberculosis infection in two captive black capuchins (Sapajus nigritus) in Southern Brazil

Abstract

Tuberculosis is a common zooanthroponosis in humans with a high incidence in Brazil, but it may also affect non-human primates (NHPs), of which Old World primates are most commonly involved. Nonetheless, its occurrence in New World primates is unknown, and therefore, this study aimed to describe the infection by Mycobacterium tuberculosis variant tuberculosis in two captive black capuchin monkeys (Sapajus nigritus) in Southern Brazil. The primates were housed in the same enclosure, wherein close contact with humans frequently occurred, and presented apathy, anorexia, and death in a clinical course of 15 days to 2 months. At the necropsy, the tracheobronchial lymph nodes were markedly enlarged and firm to hard and on the cut surface had a caseous aspect. The lungs exhibited two injury patterns: multifocal and disseminated. Microscopically, the lungs exhibited multifocal to coalescing necrotic granulomas and non-necrotic granulomas, with multiple acid-fast bacilli within the cytoplasm of epithelioid macrophages and multinucleated giant cells. Bacilli were also labeled upon immunohistochemistry (IHC) for Mycobacterium tuberculosis. Microbiological culture of lung samples from both cases yielded colonies compatible with M. tuberculosis. The isolates were identified as M. tuberculosis var. tuberculosis through polymerase chain reaction (PCR). Although tuberculosis is poorly described in New World primates, M. tuberculosis var. tuberculosis may cause a highly contagious and progressive disease with high mortality in black capuchin monkeys (S. nigritus).

Tuberculosis is a commonly identified infectious disease in humans and animals caused by Mycobacterium tuberculosis species, which comprises several variants, such as M. tuberculosis var. tuberculosis, M. tuberculosis var. africanum, M. tuberculosis var. bovis, M. tuberculosis var. canetti, and M. tuberculosis var. microti [1]. The condition has a high incidence in Brazil and is one of the leading causes of death in humans locally and globally [2]. Additionally, the disease is considered a zooanthroponosis, which may affect non-human primates (NHPs), in which it may result in progressive lung disease [3] mainly caused by M. tuberculosis var. tuberculosis, M. tuberculosis var. bovis, or M. tuberculosis var. africanum [4–7]. Although NHPs are considered more susceptible to tuberculosis infection among other captive animal species, this information is limited to records of tuberculosis in Old World primates [8], while the occurrence in Neotropical (New World) primates is uncommon [8]. The current study aimed to describe the epidemiological, pathological, microbiological, and molecular aspects of M. tuberculosis var. tuberculosis infection in two captive capuchin monkeys (Sapajus nigritus) in Southern Brazil.

Three adult, male capuchin monkeys (S. nigritus) were held captive in a public square in the city of Rio Grande, Rio Grande do Sul State, Brazil (32° 04′ 54.00″ S, 52° 09′ 48.00″ W). The primates were housed at the same enclosure for an unknown period, in poor sanitary conditions, and uncontrolled handling of the monkeys was conducted for at least 9 years prior to disease onset. Close and direct contact with humans frequently occurred at the vicinities of the enclosure, but health conditions of these humans were not assessed, as this was an open and public area. One of the capuchin monkeys presented peracute agonizing dyspnea followed by death, but necropsy was not performed. The other two capuchin monkeys (monkey 1 and monkey 2) were caught and referred for treatment at a wildlife screening center owing to clinical signs of respiratory disease. The primates had progressive inappetence, apathy, dyspnea, and died over the course of 15 days (monkey 1) to 2 months (monkey 2).

At the necropsy, both primates exhibited poor body condition, marked enophthalmos (dehydration), and pale oral mucosa. Additionally, monkey 1 had moderate amounts of unilateral, yellow, catarrhal, nasal discharge (Fig. 1a). Pulmonary involvement occurred in two patterns: multifocal and disseminated. The first was characterized by firm, yellow, nodular areas, which measured 0.5 to 2 cm in diameter and involved multiple lung lobes (monkey 1; Fig. 1b), while in the disseminated form there was almost total parenchyma replacement by multifocal to coalescing firm, yellow, elevated or depressed irregular, nodular areas, which measured 0.3 to 3 cm in diameter. Moreover, the lungs were markedly enlarged, non-collapsed, with multifocal areas of pleural adhesions (monkey 2; Fig. 1c). The cut surface of both patterns revealed soft areas, with a caseous aspect, and slightly green central areas. The tracheobronchial and mediastinal lymph nodes of both monkeys were markedly enlarged, firm, and yellow, with a caseous and gritty aspect on the cut surface. Moreover, mesenteric lymph nodes of monkey 1 were markedly enlarged (1 cm in diameter) yellow, and firm (Fig. 1d), while the pancreas of monkey 2 presented a focal, yellow, firm nodular area, measuring 1 cm in diameter.

Fig. 1.

Gross features of Mycobacterium tuberculosis var. tuberculosis infection in Sapajus nigritus in Southern Brazil. a Unilateral, yellow, catarrhal, nasal discharge in monkey 1. b Monkey 1 lung parenchyma, multifocal yellow nodules measuring 0.5 to 2 cm in diameter (arrow), with mildly enlarged lungs. c Monkey 2 lungs are markedly enlarged, non-collapsed, with almost total parenchyma replacement by multifocal to coalescing yellow nodules measuring 0.3 to 3 cm in diameter, in addition to multifocal areas of pleural adhesion. d Monkey 1 mesenteric lymph nodes, severely enlarged, yellow, and measuring 1 cm in diameter

During the necropsy, multiple samples were collected for routine histological assessment and stained with hematoxylin and eosin (HE). In order to detect acid-fast bacilli, tissue sections of lymph nodes and lungs from both capuchin monkeys were also subjected to Ziehl–Neelsen (ZN) staining. In addition, immunohistochemistry (IHC) anti–M. tuberculosis (polyclonal antibody, 1:200 dilution, Gene Tex®) was conducted with similar tissue sections, using the Universal HRP-Polymer (MACH 4, Biocare Medical®). Reactions were revealed with 3-amino-9-ethylcarbazole chromogen (AEC, Dako®), and counterstained with Harris hematoxylin.

Fresh lung and lymph node samples from both monkeys were subjected to microbiological testing, which consisted of inoculating the tissues into 5% sheep blood agar (Kasvi®) and MacConkey agar (Kasvi®), followed by an incubation period of 72 h in aerobiosis and microaerophilia atmospheres. Moreover, considering the gross features of the lesions, the samples were submitted to a process of decontamination by the Petroff Method, followed by inoculation into Stonebrink and Löwenstein–Jensen media [9]. Samples were then incubated at 37 °C and bacterial growth was assessed every week.

Once suspicious colonies were observed in mycobacteria media, DNA extraction was performed according to a previously described method [10] with adaptations [11], followed by subsequent quantification through spectrophotometry. Mycobacterium tuberculosis variants were differentiated by polymerase chain reaction (PCR) through the presence or absence of differential 16S RNA ribosomal gene differential regions, RD1, RD4, RD9, RD12, RD1^mic, and RD2^seal [12].

Microscopically, both monkeys had partial replacement of the pulmonary parenchyma by multifocal to coalescing areas of caseous necrosis, which were characterized by intense eosinophilic granular necrotic debris surrounded by a marked inflammatory infiltrate of degenerate neutrophils, epithelioid macrophages, and rare multinucleated giant cells (Langhans type; necrotic granuloma) (Fig. 2a). Frequently, this infiltrate filled the adjacent alveolar spaces in a diffuse manner, wherein it was surrounded by lymphocytes and plasma cells in addition to mild fibrous connective tissue proliferation. The tracheobronchial and mediastinal lymph nodes of both monkeys and to the mesenteric lymph node of monkey 1 had a loss of the nodal arrangement owing to multifocal to coalescing areas of marked caseous necrosis associated with numerous epithelioid macrophages and multinucleated giant cells (Langhans type). Furthermore, multifocal areas of moderate inflammatory infiltrate composed mostly of epithelioid macrophages, lymphocytes, plasma cells, and mild amounts of neutrophils, as well as rare multinucleated giant cells (Langhans type), were observed within the lungs and lymph nodes (non-necrotic granulomas) (Fig. 2b). Regarding the focal nodular area in the pancreas of monkey 2, it consisted of an infiltrate of intact and degenerate neutrophils, plasma cells, epithelioid macrophages, and lymphocytes, as well as occasional foreign bodies and Langhans multinucleated giant cells.

Fig. 2.

Microscopic features of Mycobacterium tuberculosis var. tuberculosis infection in Sapajus nigritus in Southern Brazil. a Lung, caseous granuloma, severe caseous necrosis surrounded by an inflammatory infiltrate of epithelioid macrophages, degenerate neutrophils, and rare multinucleated giant cells. Hematoxylin and eosin (HE), × 100. b Lung, noncaseous granuloma, severe inflammatory infiltrate of epithelioid macrophages, lymphocytes, plasma cells, and neutrophils. HE, × 200. c Large amounts of acid-fast bacilli within the cytoplasm of macrophages and Langhans multinucleated giant cells. Ziehl–Neelsen staining, × 1000. d Marked immunolabeling is observed within the cytoplasm of epithelioid macrophages upon immunohistochemistry anti–M. tuberculosis. 3-amino-9-ethylcarbazole chromogen (AEC), × 1000

ZN histochemical staining revealed multiple acid-fast bacilli within the cytoplasm of epithelioid macrophages and multinucleated giant cells, which were minimal in monkey 1 but numerous in monkey 2 (Fig. 2c). The same bacilli were markedly immunolabeled for M. tuberculosis upon IHC (Fig. 2d).

Colonies compatible with M. tuberculosis were obtained in both samples through Stonebrink culture. Moreover, the isolates were identified as M. tuberculosis var. tuberculosis based on the presence of RD1, RD4, RD9, and RD12 regions through PCR. In contrast, no bacterial growth was observed in blood agar or MacConkey agar at both aerobic and anaerobic conditions.

The diagnosis of tuberculosis in both S. nigritus of this study was obtained by an association of the epidemiological, pathological, immunohistochemical, microbiological, and molecular features. Tuberculosis is an endemic condition in Brazil, which is listed among the 30 countries with the highest index of disease in humans [2]. The occurrence of tuberculosis in free-ranging NHPs is uncommon; however, it is one of the main infectious diseases diagnosed in captive NHPs, as close contact with humans favors disease occurrence [4, 7, 13–15]. Likewise, higher frequencies of tuberculosis have been detected in NHPs maintained in areas with a concomitant higher occurrence of tuberculosis in humans [3, 13, 15], which includes the region of the present work.

NHPs are considered more susceptible to infection by M. tuberculosis var. tuberculosis among several other captive species [3, 4, 8]. Although the frequency of tuberculosis in Old World primates is well known, the condition is considered uncommon in South American primates [8, 16]; however, this may be based on limited investigations and reports within this area, such as previous molecular studies that identified M. tuberculosis in New World primates in Colombia and Peru, including primates from the Cebidae family [5, 17]. Furthermore, recent reports described cases of active tuberculosis in Neotropical primates involving Ateles spp. and Aotus spp. [7, 14]; in the present work, both S. nigritus succumbed to the condition and died, demonstrating the occurrence of active tuberculosis in NHPs in South America.

M. tuberculosis var. tuberculosis mainly affects humans; however, as it is easily transmitted to NHPs, it is considered a zooanthroponosis [3]. In this study, we suggest humans were the source of transmission of M. tuberculosis var. tuberculosis to capuchin monkeys, as there was close contact between species. The transmission of M. tuberculosis var. tuberculosis occurs mainly through inhalation of aerosols; however, although rare, it may also occur by ingestion [3]. Airborne transmission occurs through contact with humans or animals with active infection. We suggest that airway transmission occurred in both monkeys of this investigation, because, grossly and microscopically, most of the lesions involved the lungs and tracheobronchial lymph nodes [4].

Tuberculosis is a slowly progressing disease that may show nonspecific clinical signs, which may be absent in the early stages of the condition [6]. Sometimes, anorexia and weight loss are the first clinical signs observed in these cases [14, 16, 18], similarly to that noted in this study. Although cough is often reported when the lung lobes are affected [6, 16], dyspnea was the most consistent respiratory clinical sign observed in the current work, which probably occurred owing to pulmonary impairment.

Tuberculosis is a highly contagious and progressive disease with high mortality rates in NHP [16], similarly to that observed in this study, in which two out of three S. nigritus specimens died because of this condition. The gross and microscopic findings of both cases with predominantly pulmonary involvement were similar to those previously described in Old World primates [13, 15, 18]. However, two patterns of macroscopic lesions were observed, which possibly occurred due to a longer clinical course in monkey 2, as the involvement of more than one lung lobe and dissemination to other tissues is mostly related to chronic active disease [19]. Aside from the lungs, the tracheobronchial and mediastinal lymph nodes are frequently affected by the condition [15], similarly to the observations of this investigation, in which granulomatous infiltrate obliterated the nodal parenchyma. Moreover, there was the dissemination of granulomatous lesions to the pancreas and mesenteric lymph nodes, which was probably related to the advanced stage of the condition [15].

In primates, a variety of granulomatous responses directed towards Mycobacterium spp. may be observed at a microscopic examination, which are mostly related to the length and extent of the disease [13, 19]. In this study, the lungs exhibited multifocal to coalescing areas with necrotizing granulomas, which are hallmark lesions of tuberculosis [19]. Furthermore, non-necrotizing granulomas with infiltrate of epithelioid macrophages, lymphocytes, rare degenerate neutrophils, and multinucleated giant cells were observed in the lungs and lymph nodes; this type of presentation occurs exclusively in cases of active tuberculosis [19]. Some of the granulomas located in the lungs were surrounded by mild proliferation of fibrous connective tissue, and, although fibrous capsule formation is infrequent in granulomatous lesions of NHPs, these may be associated with the latent form of tuberculosis [18, 20]. However, there are situations in which the active and latent form of tuberculosis may occur concomitantly, which would represent a chronic active process of the disease [18], similarly to that evidenced in the capuchin monkeys in this work. Acid-fast bacilli were observed predominantly within the cytoplasm of epithelioid macrophages and Langhans multinucleated giant cells in this study. Although acid-fast bacilli can be observed either intra- or extracellularly, the amount of these may vary considerably [13, 20], as observed in this study, wherein monkey 1 had mild gross lesions associated with lower amounts of acid-fast bacilli, while monkey 2 showed severe and diffuse lung lesions and higher amounts of bacilli.

Albeit less frequent, Mycobacterium tuberculosis var. bovis may cause tuberculosis in NHPs [15, 16]. Similarly to humans, diseases caused by M. tuberculosis var. bovis or M. tuberculosis var. tuberculosis have indistinguishable clinical and pathological features [21]; thus, microbiological culture and molecular methods are essential to obtain a differential diagnosis [16]. In this investigation, the bacterial culture and PCR were major features to determine the specific variant of Mycobacterium tuberculosis involved. Moreover, the association of these diagnostic methods with the epidemiological and pathological findings was essential in order to establish an accurate diagnosis of the condition.

Although the occurrence of tuberculosis in New World primates is poorly described in South America, it may occur as a highly contagious and progressive disease with a high mortality rate in captive Sapajus nigritus. Lesions are mainly restricted to the lungs and mediastinal/tracheobronchial lymph nodes, but spread to other organs may occur.

Funding information

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

We, authors of this study, declare, for all due purposes, that this study was approved by the Veterinary Animal Research Committee of the Faculdade de Veterinária of the Universidade Federal do Rio Grande do Sul (no. 33660). No ethical approval was needed, according to the guidelines of the institutional Animal Research Ethics Committee, as this investigation was conducted only with primate cadavers, and thus, it did not involve any experimental or clinical trial in wild animals.