J. Comp. Path. 2022, Vol. 199, 55e74 Available online at www.sciencedirect.

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INFECTIOUS DISEASE

Pulmonary Granuloma Is Not Always the

Tuberculosis Hallmark: Pathology of Tuberculosis
Stages in New World and Old World Monkeys
Naturally Infected with the Mycobacterium
tuberculosis Complex
Asheley H B Pereira*, Claudia A A Lopes†, Thalita A Pissinatti†,
Ana C A Pinto†, Daniel R A Oliveira‡, Gabriel M Leal†,
Luı́s C M Oliveira‡, Paulo Redner‡, Bruna E P Barbosad,
Silvia B Moreirax, Alcides Pissinattix, Fernanda H Maruyamab, Luciano
Nakazatob, Val

eria Dutrab and Daniel G Ubiali*
* Pathological Anatomy Sector, Department of Epidemiology and Public Health, Veterinary Institute, Federal University Rural
of Rio de Janeiro, Serop
edica, † Nonhuman Primate Breeding Service/Institute of Science and Technology in Biomodels,
Oswaldo Cruz Foundation, Manguinhos, Rio de Janeiro, ‡ National Laboratory for Tuberculosis and Other Mycobacterioses,
Professor H
elio Fraga Reference Center, National School of Public Health, Oswaldo Cruz Foundation, Manguinhos, Rio de
Janeiro, d Residency Training Program in Wildlife Medicine and Conservation, Veterinary Institute, Federal University Rural
of Rio de Janeiro, Serop
edica, x Rio de Janeiro Primatology Center, Guapimirim and b Microbiology and Molecular Biology
Laboratory, Federal University of Mato Grosso, Cuiab
a, Mato Grosso, Brazil

Summary
We present the pathology of monkeys naturally infected with Mycobacterium tuberculosis complex from five
different colonies in Rio de Janeiro, Brazil. On the basis of gross and histopathological findings, the lesions
were classified into chronic-active, extrapulmonary, early-activation or latent-reactivation stages. Typical
granulomatous pneumonia was seen in 46.6% of cases (six rhesus monkeys [Macaca mulatta] and one Uta Hick’s
bearded saki [Chiropotes utahickae]). The absence of pulmonary granulomas did not preclude a diagnosis of
tuberculosis (TB): classical granulomatous pneumonia was observed in the chronic-active and latent-
reactivation stages but not in the extrapulmonary and early-activation stages. The early-activation stage
was characterized by interstitial pneumonia with a predominance of foamy macrophages and molecular
and immunohistochemical evidence of M. tuberculosis complex infection. TB should be considered as a cause
of interstitial pneumonia in New World Monkeys. We recommend the use of immunohistochemistry and mo-
lecular analysis for diagnosis of TB, even when typical macroscopic or histological changes are not observed.

Ó 2022 Elsevier Ltd. All rights reserved.

Keywords: diagnosis; foamy macrophages; interstitial pneumonia; non-human primates; tuberculosis; veterinary pathology

Correspondence to: D G Ubiali (e-mail: danielubiali@ufrrj.br).

0021-9975/$ - see front matter Ó 2022 Elsevier Ltd. All rights reserved.
https://doi.org/10.1016/j.jcpa.2022.09.011
56 A H B Pereira et al
Introduction
Mycobacterium tuberculosis complex (MTBC) refers to a
group of mycobacteria encompassing nine closely
related species that cause tuberculosis (TB) in ani-
mals, including humans (Kanabalan et al, 2021).
TB is considered the most impactful bacterial disease
of human populations, with 25% of all people in-
fected, and is responsible for the most significant num-
ber of infection-related deaths and long-term
disability compared with other diseases (WHO,
2020a). In 2019, TB-related health problems world-
wide reached an estimated 10 million people diagno-
ses (WHO, 2020b). TB is a chronic airborne disease
that also causes high morbidity and mortality in
non-human primates (NHPs), especially captive ma-
caque monkeys (Macaca spp) (Warit et al, 2020).
Although particular species may be more or less sus-
ceptible to the disease, all NHPs can develop TB
(M€ atz-Rensing and Lowenstine, 2018). Due to their
high genetic similarity with humans, NHPs accurately
model all aspects of TB in humans and are considered
the gold standard animal model for experimental M.
tuberculosis infection (Foreman et al, 2017). Infections
in captive NHPs by MTBC mainly occur due to direct
contact with TB-infected humans through inhalation
of aerosolized bacteria (Ghodbane and Drancourt,
2013; M€ atz-Rensing and Lowenstine, 2018; Warit
et al, 2020). Captive NHPs develop an infection clini-
cally similar to human TB (Kaushal et al, 2012;
Kanabalan et al, 2021) and demonstrate the full spec-
trum of infection and pathology seen in humans
(Scanga and Flynn, 2014).
The hallmark of TB is the formation of typical gran-
ulomas in any organ (M€atz-Rensing and Lowenstine,
2018). In macaques with active-chronic TB, classical
caseous granulomas, suppurative granulomas, non-
necrotizing granulomas, tuberculous pneumonia and
cavitary lesions have been observed (Lin et al, 2009).
These authors reported that latently infected monkeys
develop classical caseous lesions with mineralized gran-
ulomas and completely fibrotic pulmonary areas. In
cases of rapidly progressive TB, the authors reported a
pattern of lesions consistent with a primary pulmonary
disease with disseminated miliary, sometimes confluent,
small pulmonary granulomas and extrapulmonary
spread into the hepatic, splenic and mesenteric tissues.
TB is a well-known and widely reported disease in
laboratory Old World Monkeys (OWMs) and most
experimental studies have been performed on this
NHP group (Schmidt, 1956; Clarke, 1968; Walsh
et al, 1996; Okada et al, 2007; Reed et al, 2009;
Mehra et al, 2013). Natural occurrence in New World
Monkeys (NWMs) is considered uncommon (M€atz-
Rensing and Lowenstine, 2018). Reports of TB occur-
rence in NHPs in Brazil are scarce even though it is
among the 30 countries with the highest incidence
of human TB worldwide (WHO, 2019). In 2020,
Rio de Janeiro was considered the state with the sec-
ond highest number of cases of human TB (Brazil,
2020). We now describe the pathological findings of
different stages of MTBC infection in NWMs and
OWMs in captive collections in Rio de Janeiro.
Materials and Methods
Animals and Study Area
Fifteen NHPs with clinically suspected TB and positive
immunohistochemical detection of MTBC bacilli,
from five captive colonies in Rio de Janeiro, were
included in the study, which was carried out in 2020.
None of these colonies were open for public visitation.
Epidemiological data on the sex and age of each case
were acquired by the veterinarian at each colony.
Complete records on installations and management
of the five NHP colonies and routine clinical assess-
ments are presented as Supplementary Material.
Three NHP colonies (Macaca mulatta, Macaca fascicu-
laris and Saimiri spp) were located at the Nonhuman
Primate Breeding Service (SCPrim), Biomodel Science
and Technology Institute, Oswaldo Cruz Foundation
(Fiocruz), Rio de Janeiro. The NHPs were bred and
maintained according to the guidelines of the National
Council for the Control of Animal Experimentation
(CONCEA) (Brazil, 2015), the Guide for the Care
and Use of Laboratory Animals (NRC, 2003) and
the Federation of European Laboratory Animal Sci-
ence Associations (Balansard et al, 2019). Primate
handling procedures were approved by the Ethics
Committee on the Use of Animals (Fiocruz) under
licence number LW-5/16 and additive terms.
The fourth colony was at the Triage Center of Wild
Animals (CETAS), Serop
edica, Rio de Janeiro State.
CETAS is a federal environmental organization that
works with wildlife animal rescue, especially smug-
gling, and these NHPs were bred and maintained ac-
cording to CONCEA guidelines (Brazil, 2015).
The fifth colony was situated at the Rio de Janeiro
Primatological Center (CPRJ), Environmental State
Institute, Rio de Janeiro, an institution devoted to the
conservation of endangered neotropical primates.
The NHPs were bred and maintained according to
CONCEA (Brazil, 2015) and the Best Practice
Guidelines for Callithrichidae, European Association
of Zoos and Aquaria (Bairr~ao et al, 2017). The
breeding colony is authorized by the Brazilian Insti-
tute of the Environment and Renewal Natural Re-
sources, under licence number 4989806 of Instituto
Estadual do Meio Ambiente, Rio de Janeiro.
 Tuberculosis in New World and Old World Monkeys
Necropsy and Sample Collection
In cases of death or euthanasia of the studied NHPs,
complete necropsies were carried out according to
standardized protocols by two veterinary pathologists
of the Anatomy Pathology Sector, Federal University
Rural of Rio de Janeiro (SAP/UFRuralRJ) or by the
technical personnel responsible for the colony. During
the procedure, personal protective equipment was
used. A complete set for tissue samples, including
skin, skeletal muscle, tongue, thyroid gland, lymph
nodes, trachea, lungs, heart, liver, spleen, kidneys, ad-
renal glands, stomach, intestines, pancreas, brain and
nerves, were collected from each primate and fixed in
10% buffered formalin solution. Unfixed samples
from multiple organs were collected in microtubes
for bacterial culture (n ¼ 6) or in microtubes with
RNAlater Stabilization Solution (Invitrogen, www.
thermofisher.com) for molecular analyses (n ¼ 14).
Euthanasia of NHPs from the SCPrim colonies was
performed after sedation with intramuscular keta-
mine hydrochloride (10 mg kg1) and midazolam hy-
drochloride (1 mg kg1). It consisted of intravenous
injection of thiopental sodium (30e60 mg kg1)
with continuous infusion until a deep plane of anaes-
thesia was achieved, progressing to coma and death.
For euthanasia of NHPs from the CETAS colony,
sedation was performed with ketamine (40 mg kg1)
and xylazine (2 mg kg1) intramuscularly and, after
losing consciousness and protective reflexes, subse-
quent intracardiac administration of potassium chlo-
ride (19.1%). These methods followed international
best practice according to the Brazilian Guide of
Good Practices for Animal Euthanasia and CON-
CEA Normative Resolution 28/2015 (CFMV, 2012;
Brazil, 2015).
Histopathology and Immunohistochemistry
The tissue samples collected in formalin were fixed for
24e48 h for routine histological processing. Sections
were stained with haematoxylin and eosin (HE) for
optical microscopy. Histological sections with lesions
morphologically compatible with TB were submitted
to the ZiehleNeelsen (ZN) histochemical technique
for evidence of acidealcohol-resistant bacilli. To
identify the fibrous capsule of granulomas, sections
of lung tissue were submitted to Masson’s trichrome
histochemical technique.
For the immunohistochemistry (IHC) technique,
tissue blocks were cut into 3 mm slices and mounted
on silane-coated slides. Antigen retrieval was per-
formed with 0.1% trypsin for 30 min in an oven at
37 C. The sections were then incubated in metha-
nol:H2O2 solution (97%:3%) for 30 min to block
non-specific binding. The sections were incubated at
57
37 C for 2 h with primary anti-M. tuberculosis poly-
clonal antibody (GTX, 20905, 1:300 dilution; Gene-
Tex, www.genetex.com) or phosphate buffer saline
as a negative control. A bovine TB case, previously
confirmed by polymerase chain reaction (PCR),
was used as a positive control. EnVision secondary
polymer (Dako, www.agilent.com) was then applied
to the sections in an oven at 37 C for 30 min and
colour development done with 3,3-
diaminobenzidine chromogen (DAB + Substrate
Chromogen System; DakoCytomation, www.
dakocytomation.com) for 2 min. Finally, the sections
were counterstained with Harris haematoxylin and
coverslipped. Cases were considered positive where
structures morphologically compatible with Mycobac-
terium spp bacilli were seen extracellularly or within
the cytoplasm of macrophages or multinucleated gi-
ant cells.
Molecular Analysis
The tissue samples in the RNAlater solution were
submitted to DNA extraction with glass beads,
followed by phenolechloroform, according to
Sambrook and Russel (2001). The paraffin
samples were extracted by adding glass beads
according to Shi et al (2004). PCR using oligonucleo-
tides INS1(50 CGTGAGGGCATCGAGGTGGC-30 )
and INS2(50 GCGTAGGCGTCGGTGACAAA-30 ),
which detect the genomic region IS6110 (Kolk
et al, 1992), was used for the identification of
MTBC. The amplified products were separated by
electrophoresis in 1.5% agarose gel for 1 h at 60 V,
stained with GelRed (Biotium, https://biotium.
com) and visualized in ChemiDoc XRS (Bio-Rad,
www.bio-rad.com) using Image Lab Software
(Bio-Rad). For identification of mycobacteria at
the species level, oligonucleotides TB11 (50 -AC-
CAACGATGGTGTGTCCAT-30 ) and TB12 (50 -
CTTGTCGAACCGCATACCCT-30 ), which detect
one segment of the heat shock protein gene of 65 kDa
(hsp65), were used according to Telenti et al (1993).
The amplified products were separated by electro-
phoresis in 1.5% agarose gel for 1 h at 60 V, stained
with GelRed and visualized in ChemiDoc XRS us-
ing Image Lab Software. The PCR product was pu-
rified in the GFX PCR DNA and Gel Band
Purification kit (GE Healthcare, www.
gehealthcare.com) and sequenced in an automatic
ABI-PRISM 3500 Genetic Analyzer (Applied Bio-
systems, www.thermofisher.com).
For molecular diagnosis in live animals, oral swabs,
nasal swabs and rectal and gastric lavage samples
were collected (n ¼ 7). Swabs were collected without
medium, and gastric and rectal lavage was performed
with a probe and 0.9% saline solution. Samples were
58 A H B Pereira et al

refrigerated for up to 2 h after collection and sent for Table 1

laboratory analysis. After treatment and purification Criteria for classification of stage of tuberculosis in non-
human primates
with N-acetyl-L-cysteine-sodium hydroxide, the sam-
ples were analyzed, using GeneXpert (Cepheid, Stage of tuberculosis Classification criteria Reference
www.cepheid.com) commercial kits Xpert MTB/
Early-activation Present: histological e
RIF or Xpert MTB/RIF Ultra, for MTBC detection interstitial
and a rifampicin resistance test. pneumonia, molecular
and
immunohistochemical
Bacterial Culture evidence of
Mycobacterium
Previously macerated tissues were cultured using
tuberculosis complex
the Kudoh method (Kudoh and Kudoh, 1974). All infection. Absent:
samples were also subjected to direct smear necrosis or granuloma
microscopy with ZN staining to identify acid- in lung parenchyma or
resistant bacilli. Bacterial culture was carried out in in any other organ
L€owensteineJensen medium and incubated for up Active-chronic Persistent evidence of Capuano et al
disease, persistent (2003)
to 72 days at 37 C in a class 3 biological safety cabi- culture positivity or
net. The MTBC species in positive cultures were iden- other signs of active
tified using an immunochromatographic assay with disease. Wide
the MPT64 marker (Silva et al, 2017). spectrum of
granulomas present in
the same pulmonary
Tuberculosis Stage Classification lobe
Extrapulmonary Typical gross or Capuano et al
The criteria established to determine the TB stages histological (2003); Lin
have been described in cynomolgus macaques as tuberculosis et al (2009)
active-chronic (Lin et al, 2009) and latent- granulomas in any
reactivation (Capuano et al, 2003). Cases in which organ without
pulmonary
typical macroscopic or microscopic TB granulomas involvement
were seen in any organ, provided there was no pulmo- Latent- Present: histological Lin et al
nary involvement, were considered as extrapulmo- reactivation pulmonary lesions (2009)
nary (Capuano et al, 2003; Lin et al, 2009). We compatible with both
classified the early-activation stage of TB according caseous and non-
necrotizing
to clinical and morphological findings. The criteria granulomas.
for the TB early-activation stage were: interstitial Multisystemic
pneumonia; molecular and immunohistochemical ev- granuloma spread
idence of MTBC infection; and absence of necrosis or Absent: respiratory
granuloma formation in the lungs or any other organ clinical signs
examined. The criteria for categorizing the TB stages
are summarized in Table 1. nine OWMs and six NWMs. The OWMs belonged to
the SCPrim rhesus monkey colony (n ¼ 7) and the
Results cynomolgus monkey colony (n ¼ 2). Of the six
NWMs, 50% (3/6) were marmosets from the CETAS
Animals colony, 33.3% (2/6) were squirrel monkeys from the
From routine pathological diagnosis at the SAP/ SCPrim colony and one (16.7%; 1/6) was a Uta
UFRuralRJ, we examined 82 NPHs in 2020, of which Hick’s bearded saki (C. utahickae) from the CPRJ col-
52.4% (43/82) were OWMs and 47.6% (39/82) were ony. The marmosets had been captured from the wild
NWMs. Of the OWMs, 88.3% (38/43) were rhesus with unknown history. Although it was impossible to
monkeys (M. mulatta) and 6.9% (3/43) cynomolgus determine their exact age, they were sexually mature.
macaques (M. fascicularis). Of the NWMs, 58.9% The sex and age of each NHP included in this study
(23/39) were squirrel monkeys (Saimiri spp), 23.1% are shown in Table 2.
(9/39) common marmosets (Callithrix spp) and one
(2.56%; 1/39) was a Uta Hick’s bearded saki (Chiro-
Clinical Assessment
potes utahickae).
Fifteen NHPs from five different colonies in Rio de Regardless of disease stage, weight loss, anorexia and
Janeiro fulfilled the criteria for inclusion in this study; lethargy were the main clinical signs in the NHPs with
 Table 2
Clinical evaluation of New World and Old World Monkeys with tuberculosis

Case no. Species Facility Sex Age** General signs Respiratory signs Natural death or Other observations
origin* euthanasia
Anorexia Weight loss Lethargy Cough Sneeze Dyspnoea Tachypnoea

Old World Monkeys 1 M. mulatta Fiocruz M 4 ++ ++ +++ ++ - + + Euthanasia None

2 M. mulatta Fiocruz F 9 + ++ +++ e e +++ +++ Death None
3 M. mulatta Fiocruz F 9 e e e e e e e Death Unknown clinical
history
4 M. mulatta Fiocruz M 3 ++ +++ +++ +++ e +++ +++ Euthanasia None
5 M. mulatta Fiocruz M 4 ++ +++ +++ +++ e e e Euthanasia None
6 M. mulatta Fiocruz M 9 ++ +++ +++ e e ++ ++ Euthanasia Severe lameness in

Tuberculosis in New World and Old World Monkeys

left pelvic limb
7 M. mulatta Fiocruz M 3 e e e e e e e Death Unknown clinical
history
8 M. fascicularis Fiocruz F 20 e e e e e e e Death Diarrhoea and severe
dehydration
9 M. fascicularis Fiocruz F 14 e e e e e e e Euthanasia Endometriosis
New World Monkeys 10 Callithrix spp CETAS F Adult +++ +++ +++ e e e e Euthanasia Proliferative
cholangitis due to
adult trematodes
11 Callithrix spp CETAS M Adult +++ +++ +++ e e e e Euthanasia Proliferative
cholangitis due to
adult trematodes
12 Callithrix spp CETAS M Adult ++ +++ +++ e e e e Death None
13 S. ustus Fiocruz M 10 +++ +++ +++ +++ +++ +++ +++ Death None
14 S. ustus Fiocruz F 3 e e e e e e e Death Unknown clinical
history
15 C. utahickae CPRJ M 10 +++ +++ +++ e e e e Death None

*Details of facility of origin for each non-human primate are presented in the Supplementary Material.
Fiocruz, Nonhuman Primate Breeding Service (SCPrim), Biomodel Science and Technology Institute (ICTB), Oswaldo Cruz Foundation; CETAS, Triage Center of Wild Animals; CPRJ, Rio de
Janeiro Primatological Center.
M, male; F, female.
**Years.
e, absent; +, mild; ++, moderate; +++, severe.

59
60 A H B Pereira et al

Table 3
Gross lesions of tuberculosis in New World and Old World Monkeys

Gross lesions Old World Monkeys New World Monkeys

M. mulatta (n ¼ 7) M. fascicularis (n ¼ 2) Callithrix spp (n ¼ 3) S. ustus (n ¼ 2) C. utahickae

No./severity No./severity No./severity No./severity (n ¼ 1)
No./severity

Lung oedema 6/+++ 2/+ 3/+++ 1/++ 1/+++

Lung consolidation 6/+++ e 2/+ 1/++ 1/+++
Granulomatous
pneumonia
Cranial lobes 6/+++ e e e 1/+++
Caudal lobes 6/++ e e e 1/++
Granulomatous pleuritis 4/+++ e 1/+++ e e
Granulomatous
lymphadenitis
Mediastinal lymph 6/+++ e e e 1/+++
node
Mesenteric lymph 3/+++ e e e 1/+++
node
Others lymph nodes 5/++ 1/+++ e e 1/+++
Granulomatous 1/+++ e e e 1/+++
tracheitis
Granulomatous 1/+++ e e e e
oesophagitis
Granulomatous gastritis 1/+++ e e e e
Granulomatous 2/+++ e e e e
pancreatitis
Granulomatous 1/+++ e e e e
duodenitis
Granulomatous hepatitis 6/+++ e e 1/++ 1/+++
Granulomatous splenitis 4/+++ e e e e
Granulomatous nephritis 1/+++ e e e e
Granulomatous e 1/++ e e 1/+++
epicarditis
Granulomatous 1/+++ e e e e
encephalitis
Granulomatous neuritis 1/+++ e e e e
Granulomatous arthritis 1/+++ e e e e
Granulomatous myositis 3/+++ e 1/++ e e
Other lesions
Endometriosis 1/+++ 1/+++ e e e
Chronic proliferative e e 2/+++ e e
cholangitis due to
trematodes

e, absent; +, mild; ++, moderate; +++, severe.

TB. Clinical respiratory signs were observed in 55.5%
(5/9) of OWM cases and in 16.7% (1/6) of NWM
cases. The clinical findings and other relevant clinical
considerations are summarized in Table 2.
Gross Findings
The changes and severity of macroscopic alterations
of all nonhuman primates (NHP) with tuberculosis
are available in Table 3. Within the OWM group,
66.7% (6/9), all rhesus monkeys, had severe granulo-
matous pneumonia with well-demarcated, sometimes
elevated, firm yellowewhite or greyish nodules of
different sizes, typical of pulmonary granulomas
(Fig. 1). In all cases, single or coalescing nodules
were seen in all pulmonary lobes, but the cranial lobes
were most severely affected. The lungs were diffusely
firm, enlarged and reddish with multiple consolidated
areas. Multiple cavitations of variable sizes were
sometimes seen in the airways (Fig. 1). All six cases
with granulomatous pneumonia had variable degrees
of mediastinal lymph node enlargement.
In all TB cases in OWMs, typical granulomas were
seen in at least one organ. The gastrointestinal,
lymphatic, musculoskeletal, urinary and nervous sys-
tems had multiple granulomas of variable size and
severity, and were the systems most affected in multi-
systemic TB. Of these animals, granulomatous
 Tuberculosis in New World and Old World Monkeys
Figs. 1–4. Tuberculosis, rhesus monkeys, chronic-active stage tuberculosis. (1) NHP 4. Multifocal to coalescent granulomatous pneumonia with well-demarcated, sometimes elevated, firm
yellow–white or greyish nodules. Lungs diffusely firm, enlarged and reddish with multiple consolidated areas. Inset: multiple cavitations of airways. (2) NHP 4. Multifocal gran-
ulomatous hepatitis. Inset: multifocal granulomas throughout parenchyma. (3) NHP 4. Multifocal granulomatous splenitis. (4) NHP 6. Multifocal granulomatous neuritis. Mul-
tiple granulomas adherent to median nerve (black arrow) and musculocutaneous nerve (white arrow).

61
 62
A H B Pereira et al
Figs. 5–8. Tuberculosis, New World Monkeys. (5) NHP 12. Extrapulmonary tuberculosis in a common marmoset. Multifocal granulomas on parietal pleura and intercostal muscles. (6)
NHP 15. Latent-reactivation stage tuberculosis in a Uta Hick’s bearded saki. Multifocal well-demarcated yellowish areas in airways. Bar, 1 cm. (7) NHP 15. Systemic granulo-
matous spread in latent-reactivation stage tuberculosis in a Uta Hick’s bearded saki. Granulomatous hepatitis and mesenteric lymphadenitis. Bar, 1 cm. (8) NHP 15. Latent-
reactivation stage tuberculosis in a Uta Hick’s bearded saki. Multifocal granulomatous nephritis. Bar, 1 cm.
 Tuberculosis in New World and Old World Monkeys
lymphadenitis and granulomatous hepatitis (Fig. 2)
were seen in 66.7% (6/9) of cases, followed by granu-
lomatous splenitis (Fig. 3) in 44.44% (4/9) and gran-
ulomatous myositis in 33.3% (3/9) of cases. One case
(NHP 6, a rhesus monkey) had multiple granulomas
of variable size in nerves of the brachial plexus
(Fig. 4).
A female rhesus monkey (NHP 2) with no pulmo-
nary involvement had multifocal ulcers in the respira-
tory epithelium and many granulomas in lymph
nodes, mesentery, duodenum, pancreas, liver, spleen
and submucosa and muscular layer of the trachea.
In addition to the mesenteric granulomas, this animal
had multifocal variably sized dark-red cystic masses,
which extended from the uterus to the abdominal
wall and serosa of multiple intestinal segments. These
structures contained a large amount of dark-red fluid
and were also found on the surface of multiple abdom-
inal organs of a cynomolgus monkey (NHP 9) that
had considerable enlargement of the mediastinal
lymph nodes with diffuse replacement of tissue archi-
tecture by amorphous multifocal to coalescent
yellowish granulomas.
In NWMs, the typical pulmonary lesion with
granulomas was seen only in the Uta Hick’s bearded
saki, representing 16.7% (1/6) of cases. The main
gross change observed in the NWMs was a moderate
increase in lung volume with marked oedema in
83.3% (5/6) of the cases, followed by different de-
grees and severity of pulmonary consolidation with
irregular multifocal red-dark areas in 66.7% (4/6)
of cases. In two marmosets (NHPs 10 and 11), an
enhanced hepatic lobular pattern with an increased
Fig. 9. Tuberculosis, chronic-active stage, rhesus monkey (NHP
4). Fibrin and many neutrophils surrounded by epithelioid
macrophages, lymphocytes and multinucleated giant cell
in suppurative granuloma. HE. 40.
63
lumen diameter of biliary ducts and gallbladder
was seen.
Granulomatous changes were seen in 50% (3/6) of
the NWM cases. A marmoset (NHP 12) presented
with granulomatous myositis, periostitis and pleuritis
(Fig. 5). A squirrel monkey (NHP 14) had granulo-
matous hepatitis, with many granulomas in the liver
parenchyma. Multisystemic granulomatous change
was observed in the Uta Hick’s bearded saki (NHP
15). In this case, the pulmonary granulomas were
multifocal and well-delimited, sometimes encapsu-
lated and variably sized in all pulmonary lobes.
Some areas of pulmonary lobes were gritty on cut sur-
face (Fig. 6). Furthermore, this NHP had evidence of
extrapulmonary spread to lymph nodes, liver (Fig. 7),
spleen and kidney (Fig. 8).
Histopathological and Immunohistochemical Findings
In this study, 66.7% (6/9) of OWMs and 83.3% (5/
6) of NWMs showed pulmonary changes at histol-
ogy. A severe multifocal to coalescent granulomatous
pneumonia was seen in all six rhesus monkeys with
pulmonary changes and 16.7% (1/6) of NWM TB
cases. In 66.7% (4/6) of NWM TB cases, different
degrees of diffuse foamy interstitial pneumonia
were observed with no evidence of necrosis or pulmo-
nary granuloma formation. The pulmonary histo-
logical changes of all the NHPs with TB are
summarized in Table 4.
On histological examination, the lung lesions in
OWMs were characterized by multiple areas with
typical caseous granulomas, solid-cellular granu-
lomas (non-necrotizing) and suppurative granulomas
in each of the six rhesus monkeys. The caseous granu-
lomas had an acellular centre with a large amount of
amorphous hypereosinophilic material and pyknotic
debris, surrounded mainly by epithelioid macro-
phages, multinucleated giant cells (Langhans-type
and foreign body giant cells) and a variable number
of lymphocytes, neutrophils and rare foam cells. In
the periphery of the caseous granulomas, solid-
cellular granulomas composed of epithelioid macro-
phages and multinucleated giant cells were often
seen. Solid-cellular granulomas were characterized
by macrophage aggregates, epithelioid macrophages,
foamy cells and lymphocytes without necrosis. Sup-
purative granulomas were characterized by a central
area with degenerated neutrophils without caseous
necrosis, surrounded by epithelioid macrophages,
lymphocytes and multinucleated giant cells (Fig. 9).
In 66.7% (4/6) of cases, ulceration of the bronchial
epithelium and neutrophil infiltration (NHPs 1, 4, 5
and 7) and the formation of multiple cavities in the
major airways (Fig. 10) were observed.
 64
A H B Pereira et al
Figs. 10–13. Tuberculosis, chronic-active stage, rhesus monkeys. (10) NHP 4. Severe granulomatous pneumonia. Airways (asterisk) replaced by epithelioid macrophages, Langhans- and
foreign body-type giant cells, variable numbers of lymphocytes, neutrophils and rare foam cells, focal ulceration of bronchial epithelium and cavitation of airways (arrow).
Multifocal areas of necrosis with pyknotic cell debris and amorphous eosinophilic material. HE. 2.5. (11) NHP 5. Granulomatous hepatitis with large central caseous gran-
uloma. HE.  5. (12) NHP 4. Few acid-fast bacilli in cytoplasm of multinucleated giant cell (arrow) and in extracellular spaces. ZN.  40. (13) NHP 2. Intense intracytoplasmic
immunolabelling of intact (arrow) and apparently degenerate mycobacteria in multinucleated giant cells and macrophages. IHC. 63.
 Tuberculosis in New World and Old World Monkeys
Figs. 14–17. Tuberculosis, New World Monkeys. (14) NHP 10. Interstitial pneumonia with predominance of foamy macrophages in a common marmoset with early-activation stage tuber-
culosis. Alveoli distended by many foamy macrophages and lymphocytes. HE. 40. Inset: intense intracytoplasmic immunolabelling of intact and apparently degenerate my-
cobacteria in foamy macrophages. IHC.  40. (15) NHP 15. Multifocal solid-cellular mineralized granuloma with areas of caseous necrosis in lung of a Uta Hick’s bearded saki
with latent-reactivation stage tuberculosis. HE.  2.5. (16) NHP 15. Acid-fast bacilli in extracellular matrix (arrows) of caseous granuloma in lung of a Uta Hick’s bearded saki
with latent-reactivation stage tuberculosis. ZN. 40. (17) NHP 15. Intense intracytoplasmic immunolabelling of intact and apparently degenerate mycobacterial (black ar-
rows) in intrabronchial foamy macrophages. Numerous bacilli in intra- and extracellular matrix in bronchial submucosa (white arrows). IHC. 40.

65
 66
Table 4
Pulmonary histopathological tuberculosis lesions in New World and Old World Monkeys

Case no. Species Distribution of Granulomas Leucocyte profile in lung lesions

pneumonia/
severity Caseous Mineralization Fibrous Cavitation Giant Epithelioid Foamy Lymphocytes Neutrophils
necrosis capsule cells macrophages macrophages

Old World 1 M. mulatta MTC/+++ +++ e + + + ++ + ++ +++

Monkeys 2 M. mulatta e e e e e e e e e e
3 M. mulatta MTC/+++ +++ e +++ e ++ ++ + ++ e
4 M. mulatta MTC/+++ +++ e +++ +++ +++ +++ e +++ +
5 M. mulatta MTC/+++ +++ e ++ + + ++ e +++ ++
6 M. mulatta MTC/+++ +++ e ++ e + ++ + +++ +
7 M. mulatta MTC/+++ +++ e ++ ++ +++ ++ e ++ +++
8 M. fascicularis e e e e e e e e e e
9 M. fascicularis e e e e e e e e e e
New World 10 Callithrix spp ID/+++ e e e e + e +++ +++ e
Monkeys 11 Callithrix spp ID/+++ e e e e e e ++ ++ e
12 Callithrix spp ID/+ e e e e e e e +++ e
13 S. ustus ID/++ e e e e e e ++ + e

A H B Pereira et al
14 S. ustus e e e e e e e e e e
15 C. utahickae MTC/+++ +++ +++ + e + + +++ +++ +

MTC, multifocal to coalescent; ID, interstitial diffuse; e, absent; +, mild; ++, moderate; +++, severe.
 Table 5
Methods applied to diagnosis of tuberculosis in New World and Old World Monkeys

Evaluation of bacilli in histological sections Molecular analysis MTBC# culture

ZiehleNielsen IHC PCR Xpert*

Case no. Species Intracellular Extracellular Intracellular Extracellular INS1/2 HSP IS1081/IS16110 Lung Lymph node Liver Spleen

Old World Monkeys 1 M. mulatta +a ++a +++a +a Pa N Pa P P P P

2 M. mulatta +e +e +++e ++e Pa N Pa P P NP P
3 M. mulatta +a +a ++a e Pa N Pa P NP NP N
4 M. mulatta ++a +a ++a +++a Pa Pa Pa P P N P
5 M. mulatta e e +a +a Pa N Pa N NP N P

Tuberculosis in New World and Old World Monkeys

6 M. mulatta e e +a e Pa Pa Pa P P N N
7 M. mulatta e e ++a ++a N N Pa NP NP NP NP
8 M. fascicularis e e +b e Pb N NP NP NP NP NP
9 M. fascicularis + e +++e +e NP NP NP NP NP NP NP
New World Monkeys 10 Callithrix spp +a +a +++a +a Pa N Pa NP NP NP NP
11 Callithrix spp +a +a +a e NP NP Pa NP NP NP NP
12 Callithrix spp +a +a +++a +a Pc N Pa NP NP NP NP
13 S. ustus e e +a e NP NP Pa NP NP NP NP
14 S. ustus +d e ++d e NP NP Pd NP NP NP NP
15 C. utahickae +a ++a +++a ++a NP NP NP NP NP NP NP

*, GeneXpert, Cepheid; HSP, heat shock protein gene; e, absent; +, mild; ++, moderate; +++, severe; P, positive; N, negative; NP, not performed; PCR, polymerase chain reaction; IHC, immu-
nohistochemistry using a polyclonal anti-Mycobacterium tuberculosis antibody.
#
, Mycobacterium tuberculosis complex.
a
, lung; b, heart; c, pleura and striated muscle; d, liver; e, lymph node.

67
68 A H B Pereira et al

Table 6
Stage of tuberculosis in New World and Old World Monkeys

Case no. Species Stage of tuberculosis Respiratory signs Typical lung granulomas Extrapulmonary involvement

Gross Microscopic

Old World Monkeys 1 M. mulatta Active-chronic + + + Multisystemic

2 M. mulatta Extrapulmonary + e e Multisystemic
3 M. mulatta Active-chronic e + + Multisystemic
4 M. mulatta Active-chronic + + + Multisystemic
5 M. mulatta Active-chronic + + + Multisystemic
6 M. mulatta Active-chronic + + + Multisystemic
7 M. mulatta Active-chronic e + + Multisystemic
8 M. fascicularis Extrapulmonary e e e Heart
9 M. fascicularis Extrapulmonary e e e Lymph node
New World Monkeys 10 Callithrix spp Early activation e e e e
11 Callithrix spp Early activation e e e e
12 Callithrix spp Extrapulmonary e e e Multisystemic
13 S. ustus Early activation + e e e
14 S. ustus Extrapulmonary e e e Liver
15 C. utahickae Latent-reactivation e + + Multisystemic

e, absent; +, present.

In addition to the lungs, caseous granulomas and
solid-cellular granulomas of variable severity were
found in multiple organs of the rhesus monkeys. In
NHPs 1, 3, 4, 5, 6 and 7, there was marked replace-
ment of lymph node architecture by numerous
caseous and solid-cellular granulomas. The liver
(Fig. 11) (NHPs 1, 3, 4, 5 and 7), spleen (NHPs 3,
4, 5 and 7), pleura (NHPs 1, 3, 5 and 7), skeletal mus-
cle (NHPs 4, 5 and 6), oesophagus (NHP 1), stomach
(NHP 4), pancreas (NHP 4), duodenum (NHP 4),
kidney (NHP 1), one adrenal gland (NHP 1), joints
(NHP 6), brain (NHP 1) and nerves (NHP 6) also
contained multiple granulomas of variable severity.
In 50% (3/6) of OWM cases with granulomatous
pneumonia, straight or slightly curved bacilli
(1e5 mm long and 0.2e0.8 mm wide) were seen in
the areas of necrosis or within the cytoplasm of mac-
rophages, epithelioid macrophages or multinucleated
giant cells using the ZN technique (Fig. 12). In all six
cases of granulomatous pneumonia (Fig. 13), IHC
with anti-M. tuberculosis antibody revealed intact
and degenerated bacilli in necrotic areas and in the
cytoplasm of multinucleated giant cells, macrophages
and epithelioid macrophages.
A rhesus monkey (NHP 2) with no pulmonary
changes had multifocal to coalescent caseous granu-
lomas in the trachea, lymph nodes, mesentery, duo-
denum, pancreas, liver and spleen. Dark-red cystic
masses of the abdominal wall (Supplementary
Fig. S1) and mesenteric nodules, in addition to the
TB granulomas, were seen. At histological evaluation,
a moderately cellular proliferation, composed of
columnar and ciliated endometrial cells with a mod-
erate amount of clear eosinophilic cytoplasm and
round nuclei, was observed. Multifocally, there were
areas with moderate haemorrhage and haemosider-
ophages containing abundant intracytoplasmic
brown pigment. The lesions in this animal were
compatible with endometriosis (Supplementary
Fig. S2).
A cynomolgus monkey (NHP 9) with no pulmo-
nary lesions also had endometriosis. This monkey pre-
sented with a multifocal to coalescent granulomatous
lymphadenitis with replacement of normal lymph
node architecture by severe caseous granuloma for-
mation. In a rhesus monkey (NHP 8), histological
changes were observed in the cardiac ganglia, which
were expanded or replaced by a few multinucleated
foreign body giant cells. Adjacent well-demarcated
multifocal areas with a necrotic centre and dystrophic
mineralization expanded the epicardium.
Out of three NWMs with histological pulmonary
changes, two (66.7%) marmosets and one squirrel
monkey (NHP 13) had variable degrees of prolifera-
tion of foamy macrophages that thickened alveolar
septa and infiltrated the air spaces (Fig. 14). These
macrophages had abundant foamy cytoplasm and
often concentric and peripheral nuclei. Although a
few giant cells were seen in one marmoset (NHP
10), no evidence of necrosis or granuloma formation
was seen in lung sections or in any other organ in
this NWM.
A squirrel monkey (NHP 14) had multifocal gran-
ulomatous hepatitis with many solid-cellular granu-
lomas and a few caseous granulomas surrounded
mainly by some epithelioid macrophages, multinucle-
ated giant cells and lymphocytes. A marmoset (NHP
12) had granulomatous pleuritis with a large caseous
 Tuberculosis in New World and Old World Monkeys
granuloma in the intercostal muscles that extended to
the adjacent rib with associated periostitis. A mild
diffuse interstitial pneumonia with a predominance
of lymphocytes was also seen in this marmoset, which
was the only case without pneumonia in this group.
In the single case of granulomatous multifocal to
coalescent bronchopneumonia in a NWM, the Uta
Hick’s bearded saki had many well-demarcated and
encapsulated mineralized granulomas of variable
size in the pulmonary parenchyma (Fig. 15). These
granulomas were often surrounded by many foamy
macrophages with abundant cytoplasm, epithelioid
macrophages, lymphocytes and a few multinucleated
giant cells and neutrophils. Adjacent to these lesions,
the alveolar spaces contained a large amount of amor-
phous eosinophilic oedema fluid. Sometimes, multi-
focal fibroblastic proliferation in the alveolar wall
was seen. Many foamy macrophages infiltrated the
airways multifocally as well as the bronchial and
bronchiolar lumen. Many solid-cellular granulomas
were observed in the lung tissue.
ZN staining revealed a few bacilli, predominantly
intracellularly in foamy cells of the marmosets. The
liver and lungs sections of the Uta Hick’s bearded
saki contained a few extracellular bacilli in necrotic
areas (Fig. 16). Intracellular bacilli were seen in all
cases, mainly in the airways and intrabronchial foamy
macrophages (Fig. 17). The ZN staining and immu-
nohistochemical results are presented in Table 5.
Molecular Findings
Molecular analysis was performed on 14 of the 15
NHPs (Table 5). Molecular detection with oligonu-
cleotides INS1 and INS2 was positive in 64.28% (9/
14) of cases and with oligonucleotides TB11 and
TB12 was positive in 14.28% (2/14) of cases. Gene
sequencing confirmed the diagnosis of MTBC. The
GeneXpert system detected the presence of MTBC
species DNA in all analysed samples. All samples
were sensitive to rifampicin, with amplification of
the genes rpoB1, rpoB2, rpoB3 and rpoB4.
Bacterial Culture
Bacterial culture was performed on six of the 15
NHPs. In all cases, growth of MTBC bacteria was
observed (Table 5).
Classification of Stage of Tuberculosis
Based on the clinical, morphological, immunohisto-
chemical, molecular and bacteriological results, the
NHPs were classified into different stages of TB
(Table 6). Of the OWMs, 66.7% (6/9) of cases were
classified as the active-chronic stage. Typical pulmo-
69
nary granulomas were seen in all cases in this group
and clinical respiratory signs in 83.3% (5/6) of cases.
In 33.3% (3/9) of OWM cases, the stage was classified
as extrapulmonary due to the absence of gross or his-
tological lesions of granulomatous pneumonia. Ex-
trapulmonary TB was seen in two cynomolgus
monkeys and one rhesus monkey. In this group, respi-
ratory signs were observed in 33.3% (1/3) of cases.
In the NWMs, 50% (3/6) of cases were classified as
the early-activation stage, 33.3% (2/6) as the extrap-
ulmonary stage and 16.7% (1/6) as the latent-
reactivation stage. In the early-activation group,
66.7% (2/3) of the primates were marmosets and
one (33.3%, 1/3) was a squirrel monkey. Pulmonary
granulomas were not observed in any of the early-
activation cases and only the squirrel monkey had
clinical respiratory signs. The extrapulmonary stage
group comprised one marmoset and one squirrel
monkey. No clinical respiratory signs were observed
in the NWMs with the early-activation stage. The
latent-reactivation stage was identified in the Uta
Hick’s bearded saki. This primate had caseous and
non-necrotizing pulmonary granulomas (typical of
latent pulmonary granulomas) but no clinical respi-
ratory signs.
Discussion
In humans, pulmonary granuloma formation is
considered the pathological hallmark of TB (Wadee
and Wadee, 2021). Studies of experimental MTCB
infections in NHPs (Capuano et al, 2003; Lin et al,
2014) and humans (Cadena et al, 2017) have
described the morphological heterogeneity of TB. In
cynomolgus macaques infected with MTBC, a wide
spectrum of lesions can be seen within and between
monkeys of the same stage classification (Capuano
et al, 2003; Lin et al, 2014), as described in humans
(Flynn and Klein, 2011).
TB diagnosis in the NHPs in this study was based
on the association of pathological, immunohisto-
chemical, molecular and, when possible, bacteriolog-
ical culture findings. We found different lesion
patterns of the diverse stages of TB in the NWMs
and OWMs kept under human care. Although the
typical TB presentation was observed in some cases,
absence of pulmonary granuloma did not preclude a
TB diagnosis, as indicated by the TB cases in
NWMs that had interstitial pneumonia. Extrapulmo-
nary or early pulmonary changes in NWMs and
OWMs naturally infected with MTBC also represent
an important pattern of TB lesions. The pathological
heterogeneity of TB lesions probably was due to the
variable clinical evolution of the disease at the time
of death or euthanasia of the NHPs.
70 A H B Pereira et al
We identified the chronic-active TB stage predom-
inately in OWMs, especially rhesus monkeys,
compared with cynomolgus macaques. Presumably,
this was because rhesus monkeys are more susceptible
to M. tuberculosis infection than cynomolgus macaques
(Langermans et al, 2001; Maiello et al, 2018). This
relatively higher susceptibility to M. tuberculosis
infection may have favoured the activation stage of
the disease. In the cases of naturally occurring
chronic-active TB identified in rhesus monkeys in
this study, the lesions closely resembled those induced
experimentally in this species (Zhang et al, 2011,
2014). In our study, all rhesus monkeys with
chronic-active TB had a wide spectrum of histological
changes with different granulomas types present in
the same individual, as in humans (Capuano et al,
2003).
Extrapulmonary TB refers to TB involving poten-
tially any organ other than the lungs. Reports of
this presentation in macaques are scarce (Stockinger
et al, 2011; Lee, 2015). Extrapulmonary spread was
variable among the NHPs in our study, and when
the disease is advanced they can exhibit extrapulmo-
nary dissemination (Capuano et al, 2003). The macro-
scopic and histopathological granulomatous lesions
found in several organs of the rhesus monkeys, lymph
nodes and heart of the cynomolgus macaques, liver of
the squirrel monkeys and skeletal muscle and parietal
pleura of the common marmosets demonstrate the
ability of MTBC to cause various changes in multiple
organs even when pulmonary changes are absent. On
the basis of these findings, we suspect that systemic
involvement had resulted from haematogenous
spread of MTBC bacilli after initial infection.
Haematogenous dissemination within organs can
occur in TB infections. Despite widespread dissemina-
tion of M. tuberculosis during primary infection, most
infected but otherwise healthy individuals resolve
these lesions without becoming symptomatic
(Sakamoto, 2012). Exposure of NHPs to M. tubercu-
losis results in a wide range of outcomes (Scanga
and Flynn, 2014). Unlike the granulomatous changes
in many organs, the extrapulmonary lesions in our
study suggest possible pulmonary resolution associ-
ated with an absence of clinical respiratory signs in
most of the NHPs with this stage presentation.
MTBC species infect primarily macrophages in the
lungs (Agarwal et al, 2021). Often, these macrophages
contain an intracellular accumulation of MTBC lipid
(foam cells) (Guerrini et al, 2018). Studies have shown
that this lipid-laden macrophage formation is
induced by oxygenated forms of mycolic acid, such
as oxygenated ketomycolic and hydroxyl-mycolic
acids synthesized by MTBC species (Peyron et al,
2008). Although the occurrence of foam cells is typi-
cally associated with necrotic granulomas (Peyron
et al, 2008), the absence of necrosis, granulomatous
changes or interstitial fibrosis in the early-activation
TB stage can indicate that foamy macrophages are
a key component in the initial pathogenesis of TB in
NHPs.
Lesions of early pulmonary TB can develop along
several pathways (Hunter, 2011). In the early-
activation stage, we observed diffuse interstitial foamy
pneumonia. Although lipidic pneumonia was associ-
ated with post-primary TB (Hunter et al, 2007), no
evidence of endobronchial TB producing bronchial
obstruction, necrosis or cavitation was found in the
NHPs during the early-activation stage in this study.
In one marmoset in the early-activation stage, we
observed many interstitial foamy macrophages and
a few multinucleated giant cells without necrosis.
The TB granuloma formation can occur due to the
activation of these interstitial macrophages. The
mechanism of initial granuloma formation, especially
in NWMs, requires further investigation.
It has been proposed that intracellular bacilli in
macrophages overproduce M. tuberculosis lipids,
which accumulate in the internal vesicles in the multi-
vesicular body and are subsequently exocytosed into
the extracellular matrix (Beatty et al, 2000, 2001). It
has been demonstrated that foam cell formation is
induced by oxygenated forms of mycolic acid, such
as oxygenated ketomycolic and hydroxyl-mycolic
acids synthesized by pathogenic mycobacterial spe-
cies such as M. tuberculosis (Peyron et al, 2008;
Russell et al, 2009). In our study, the presence of
bacilli in foamy macrophages was confirmed by
acid-fast staining and IHC.
As in human TB, infection in NHPs can be stably
maintained, spontaneously reactivate or reactivate
in response to immunosuppression (Lin et al, 2009;
Lin and Flynn, 2010). Based on the clinical history,
it was not possible to determine a potential immuno-
suppression factor associated with the latent-
reactivation stage in the Uta Hick’s bearded saki in
this study, which appears to have spontaneously reac-
tivated a latent infection. We observed intense intra-
cytoplasmic immunolabelling to anti-M. tuberculosis in
foamy macrophages in the latent-reactivation stage,
indicating that foam cell formation is due to active
M. tuberculosis replication. In addition to the necro-
tizing granulomas, this monkey had non-necrotizing
granulomas in lung tissue, which has not been
observed in latent infection in NHPs (Lin et al, 2009).
This study shows that clinical respiratory signs
should not be the considered as the only cause for
suspicion of TB in NHPs. Coughing is infrequent
in NHPs with TB but may occur (Mansfield and
Fox, 2019), as in three rhesus monkeys and one
 Tuberculosis in New World and Old World Monkeys
squirrel monkey in this study. As reported by
Simons and Gibson (2012) and Via et al (2013),
weight loss, lethargy and anorexia were the most
frequent clinical signs in NHPs with TB in our
study.
Mycobacterial antigens and changes in tissue
morphology can be evaluated using IHC (Karimi
et al, 2014). IHC is generally considered more sensi-
tive than acid-fast staining for identification of
MTBC bacilli and has been investigated as an
improved method for diagnosis of TB (Mustafa et al,
2006). Due to the high sensitivity of IHC compared
with acid-fast staining, we recommend its use for
routine diagnosis even in cases without typical TB
microscopic changes. In cattle, cross-reactivity to
polyclonal antibodies may result in low specificity in
distinguishing MTBC microorganisms from M. tuber-
culosis and Mycobacterium bovis (Konradt et al, 2016).
Our molecular analysis demonstrated microorgan-
isms belonging to the MTBC. Genetic sequencing
failed to discriminate Mycobacterium spp due to the
high genetic homology among different species within
the complex (Neshani et al, 2018). Although rifam-
picin resistance was not identified in any case in the
present study, we suggest using GeneXpert as a sur-
veillance tool for bacterial resistance to antibiotics.
All NHPs in this study were naturally infected with
MTBC, although there are reports that OWMs are
more susceptible than NWMs (Brack, 1987;
Montali et al, 2001; Blanchard and Russell-
Lodrigue, 2012). No predisposing factors were
observed in the OWMs compared with the NWMs.
TB in free-ranging NHPs is uncommon (Rocha et al,
2011). However, we believe that all NHPs with direct
contact with humans are susceptible to M. tuberculosis
infection. As NHPs with naturally occurring TB were
included in this study, it was not possible to determine
the route, strain, infectious dose or time of evolution of
the TB infection.
We believe that complete and specific ante-mortem
examinations for the diagnosis of TB must be carried
out frequently in NHP colonies, even when clinical
respiratory signs are not evident. Due to the possible
retransmission from monkeys to man (M€atz-
Rensing and Lowenstine, 2018), TB in NHPs offers
a potential health risk for staff members. Because of
the inherent risk a NHP infected with TB poses to
the colony and staff, it is recommended that monkeys
infected with or suspected of being infected with TB
are euthanized (Bushmitz et al, 2009).
In conclusion, we found a wide spectrum of lesions
in NHPs with different stages of TB. As the early-
activation stage was characterized by foamy intersti-
tial pneumonia without typical TB granulomas, TB
71
should be included in the differential diagnosis of
foamy interstitial pneumonia in NHPs, especially in
the New World species. We recommend the use of
IHC and molecular analysis for the diagnosis of TB,
even when typical macroscopic or histological
changes are not evident.
Acknowledgments
We thank Edson Moleta Colodel for his contributions
and revision of the draft paper.
Funding
We thank the Conselho Nacional de Desenvolvi-
mento Cientı́fico e Tecnol
ogico (CNPq) and Coor-
denaç~ao de Aperfeiçoamento de Pessoal de Nı́vel
Superior (CAPES) for financial support for this study.
CRediT Author Statement
A H B Pereira, D G Ubiali: Manuscript conceptu-
alization, Data curation, Investigation. A H B Per-
eira: Writing - original draft preparation. T A
Pissinatti, A C A Pinto, D R A Oliveira, G M
Leal, B E P Barbosa, S B Moreira, A Pissinattti:
Clinical evaluation of non-human primates. A H B
Pereira, C A A Lopes, S B Moreira, A Pissinattti,
D G Ubiali: Non-human primates necropsy. L C M
Oliveira, P Redner, F H Maruyama, L Naka-
zato, V Dutra: Molecular analysis. A H B Pereira,
C A A Lopes, T A Pissinatti, A C A Pinto, D R A
Oliveira, G M Leal, L C M Oliveira, P Redner,
B E P Barbosa, S B Moreira, A Pissinattti, F H
Maruyama, L Nakazato, V Dutra, D G Ubiali:
Methodology, Data curation, Writing - reviewing
and editing, Formal analysis.
Conflict of Interest Statement
The authors declared no potential conflicts of interest
with respect to the research, authorship or publica-
tion of this article.
Supplementary Data
Supplementary data to this article can be found on-
line at https://doi.org/10.1016/j.jcpa.2022.09.011.
Data Availability
The datasets used and analysed during this study are
available from the corresponding author on request.
72 A H B Pereira et al
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½ Received,
Accepted, September 23rd, 2022 
March 10th, 2022