M. tuberculosis H37Rv Infection of Chinese Rhesus Macaques
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- Zhang, J., Ye, Y., Wang, Y. et al. J Neuroimmune Pharmacol (2011) 6: 362. doi:10.1007/s11481-010-9245-4
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Mycobacterium tuberculosis is the most common communicable infectious disease worldwide and the top killer of human immunodeficiency virus (HIV)-infected people. Because of common dual HIV and M. tuberculosis infections, the emergence of multidrug-resistant M. tuberculosis strains, the lack of effective vaccination, the morbidity, and the mortality of M. tuberculosis infection are increasing sharply. Therefore, there is an urgent need for vaccine and drug development against M. tuberculosis infection. These require appropriate animal models that closely resemble human disease. To this end, we infected Chinese rhesus macaques with the M. tuberculosis H37Rv strain. Bronchoscopy was used to inoculate nine monkeys with different doses of M. tuberculosis H37Rv strain. Regardless of the M. tuberculosis dose, all monkeys were infected successfully. This was shown by clinical, laboratory, and histopathology assessments. Among nine infected monkeys, six developed acute rapid progressive tuberculosis and the remaining animals mirrored early-stage chronic disease. These data, taken together, demonstrate that Chinese rhesus macaques are highly susceptible to M. tuberculosis infection and develop similar manifestations of disease that are seen in humans. This model affords new opportunities for studies of M. tuberculosis disease pathology and therapeutics.
KeywordsMycobacterium tuberculosisNon-human primatesChinese rhesus macaques
Animal biosafety level
Colony forming units
Erythrocyte sedimentation rate
Simian immunodeficiency virus
Tuberculin skin test
World Health Organization
Mycobacterium tuberculosis is a severe infectious disease accounting for significant morbidity and mortality globally. According to the World Health Organization (WHO) report (WHO 2009), the number of people infected with TB is reaching as many as two billion, constituting one third of the world population. In addition, M. tuberculosis is the top killer of people infected with human immunodeficiency virus (HIV). It is estimated that one third of persons living with HIV are co-infected with TB (UNAIDS 2010). In developing countries half of people with HIV infection will develop active TB. In some countries of sub-Saharan Africa, more than 70% of patients with active M. tuberculosis infection are also HIV-seropositive (UNAIDS 2010). Therefore, the need to develop new effective vaccines and drugs against TB infection is extremely urgent. However, in order to evaluate the efficacy and the safety of the candidate vaccines and drugs, appropriate animal models of M. tuberculosis infection are needed. Among the animal models, non-human primates have been extensively used for the study of the pathogen–host interactions. Because of the close evolutionary relationship between human and non-human primates as well as the similarity of the clinical and pathological manifestations of TB in them, the non-human primate is an ideal model for studies of the pathogenesis of human M. tuberculosis infection. It has been documented that the non-human primate model is particularly important and valuable for evaluating new vaccines and drugs against TB infection (Dietrich et al. 2003; Gupta and Katoch 2005; Helke et al. 2006; Langermans et al. 2001).
Several monkey models have been used for TB research for many years. However, because of cost, biocontainment, and lack of reagents for use in macaques, the monkey models for M. tuberculosis infection have been rarely used (Capuano et al. 2003). Recently, researchers from different laboratories demonstrated renewed interest in monkey models to examine TB infection using different M. tuberculosis strains and monkeys from different regions (Gormus et al. 2004; Lin et al. 2006; Sugawara et al. 2007, 2009). These studies have shown differential susceptibility of different monkey species to M. tuberculosis infection. Although it has been demonstrated that rhesus macaque is highly susceptible to TB infection, there are no reports describing M. tuberculosis infection of rhesus macaques of Chinese origin. To this end, we infected Chinese rhesus macaques with M. tuberculosis H37Rv strain. We demonstrated that Chinese rhesus macaques were highly susceptible to M. tuberculosis H37Rv infection and developed similar manifestations of disease seen in humans.
Materials and methods
Nine adult Chinese rhesus macaques (five females, four males, 4–8 kg, 4–6 years old) used for this study were purchased from Sichuan Pingan Non-Human Primates Breeding and Research Center, Sichuan, China. These animals were given experimental serial numbers (WP01–WP09). All animals were strictly screened according to the National Standard (GB14922.2-2001) to ensure that they were free of underlying pathogens, including mycobacteria (M. tuberculosis and atypical mycobacteria), simian retrovirus D, simian immunodeficiency virus (SIV), simian T leukemia virus type 1, herpesvirus B, Strongyloides stercoralis, Pneumonyssus sinicola, and other microorganisms. Animals were individually housed in isolator cages of the Animal Bio-Safety Level (ABSL) III facility of Wuhan University (Wuhan, China). During the quarantine time period, the study animals received clinical evaluations, including physical exam, routine hematology testing, clinical biochemistry testing, and chest radiograph. All animals were placed in ABSL-3 facility for at least 6 weeks to allow them to adopt the environment before they were challenged with M. tuberculosis H37Rv strain. At the end of the animal adoption period, we completed a pre-inoculation workup that included measurements of weight, temperature, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and chest X-ray. ESR was measured with Random Access ESR Analyser (Linear Chemistry LENA, Spain) using anticoagulated blood collected from the study animals, in accordance with the guide provided by the manufacturer. CRP was determined by Biochemistry Analyzer (Hitachi 7080, Japan) with the reagents from Wako Pure Chemical Industries (Osaka, Japan) using animal serum isolated from coagulated blood of the study animals. Chest X-ray was performed as described below. All study protocols and procedures were approved by the Institutional Animal Care and Use Committee of Wuhan University.
M. tuberculosis strain and challenge procedures
M. tuberculosis H37Rv strain (ATCC 93009) was obtained from the Research Institute of Tuberculosis (Tokyo, Japan). M. tuberculosis H37Rv strain was prepared as described previously (Capuano et al. 2003). This strain is less virulent than another common M. tuberculosis strain (Erdman) (Gormus et al. 2004) and suitable for latent infection in the rhesus monkey. The procedure of inoculation of M. tuberculosis strain H37Rv was performed as described by others (Capuano et al. 2003). Prior to the challenge with M. tuberculosis H37Rv, atropine was intramuscularly injected into animals in order to alleviate excessive salivation and maintain heart rate. Animals anesthetized with ketamine (10 mg/kg) were placed in dorsal recumbence on the procedure table. A flexible fiber-optic bronchoscope (2.8-mm outer diameter, OLYMPUS, Japan) was inserted into the right caudal or middle lung lobes of animals. In order to determine the challenge dose of M. tuberculosis that would model human TB infection, we used four different doses of the M. tuberculosis H37Rv strain, ranging from 50 to 3,000 colony forming units (CFU). These titers of M. tuberculosis H37Rv are within the dose range (20–100,000 CFU) reported by others (Capuano et al. 2003; Gormus et al. 2004; Lin et al. 2006; Walsh et al. 1996). M. tuberculosis H37Rv bacteria suspended in 2 ml of saline were introduced into the lungs of animals through the biopsy port. Animals were then returned in a left lateral recumbent position to their cages. Inoculated animals were closely monitored for their breathing pattern and heart rate until they were recovered fully from the anesthesia. Animals were divided randomly into four groups receiving four different doses of M. tuberculosis H37Rv strain: 50 CFU (animals WP01 and WP07), 200 CFU (animals WP02 and WP08), 500 CFU (animals WP04 and WP06), and 3,000 CFU (animals WP03, WP05, and WP09). Animals were provided with full supportive care and humanely killed on the 14th week post-inoculation (PI), except those animals that died of M. tuberculosis infection prior to the 14th week PI.
After the inoculation with M. tuberculosis H37Rv, animals were observed for clinical manifestations, including appetite and coughing (daily) and weight and temperature (weekly). Blood specimens were collected via femoral venipuncture for ESR, clinical hematology, and biochemistry prior to the inoculation and biweekly PI. The clinical assessments of infected animals after M. tuberculosis inoculation were compared with the parameters measured prior to the inoculation (Capuano et al. 2003; Gormus et al. 2004; Lin et al. 2006; Walsh et al. 1996).
Using a digital X-ray unit (SIEMENS Multimobil 2.5, Mobile 810 DDR system, and PiViewSTAR WorkStation System), anterior posterior and right lateral chest X-rays were performed prior to inoculation, biweekly PI, and at the time of the euthanasia. All the thoracic radiographs were evaluated by two board-certified pulmonary radiologists.
Tuberculin skin test
Tuberculin skin test (TST) was performed on the seventh week PI. Old tuberculin (OT) (0.1 ml, purchased from Synbiotics Corporation, USA) was injected intradermally into the palpebra of the right upper eyelid. The same volume of sterile saline was injected into the left eyelid as a negative control. The eyelid was evaluated with a standard 1–5 scoring system at 48 and 72 h PI (Capuano et al. 2003).
All animals were humanely euthanized with an intravenous overdose of pentobarbital sodium. The number and size of peripheral lymph nodes were examined and documented. The entire lung–heart block was removed from the thoracic cavity and evaluated for gross pathology. All findings (the swollen peripheral and thoracic lymph nodes, the visible granulomas in the lungs, livers, spleens, and other tissues) were recorded in the autopsy report as a final pathology evaluation. Additional miscellaneous pulmonary findings associated with M. tuberculosis infection such as focal parietal pleural adhesions and other organ infection were recorded. Tissue specimens used for histopathological examination were taken randomly from lung lobes, lymph nodes, livers, and spleens of M. tuberculosis-infected animals. These tissue specimens were fixed in 10% formalin and embedded in paraffin. Standard sections were cut at 4 μm and processed for hematoxylin and eosin and Ziehl–Neelsen staining. The sections then were observed for histopathological changes and distribution of mycobacterium under a light microscope (AXIOSKOP 40, Zeiss, Germany).
Tissue specimens (the net weight ranging from 1 to 5 g) obtained from lungs and lymph nodes were weighed and then homogenized using a tissue miser homogenizer (Fisher Scientific, USA) with the addition of 1 ml PBS. The serial dilutions of homogenized tissues were plated on 7H10 agar plates and incubated at 37°C with 5% CO2. The numbers of CFU in agar plates were counted on the fourth week PI.
Clinical symptoms of M. tuberculosis infection
Clinical tests of M. tuberculosis infection
ESR and CRP
Erythrocyte sedimentation rates and C-reaction protein
Prior to inoculation
Prior to inoculation
Tuberculin skin test
TST is a standard test for the screening of M. tuberculosis infection in the human population. TST was performed on the seventh week after the M. tuberculosis inoculation. The left eyelids of animals were used as a negative control (receiving saline injection only), while the right eyelids were injected with OT. All animals receiving M. tuberculosis became positive in the TST, although the degree of positive reaction varied. Animals receiving 500 CFU or higher dose of M. tuberculosis had grade 4+ of TST, while animals in other groups had grade 3+ of TST (data not shown).
Pathological evidence of M. tuberculosis infection
Survival of experimental monkeys after challenge with different doses of TB
Challenge doses (CFU)
Cause of death
Death time (week)
Bacterial cultures demonstrated M. tuberculosis growth in all lung tissues and lymph nodes from the infected animals. The tissues from the right lungs of the infected animals contained higher numbers of bacteria than those of the left lung tissues (data not shown), which was the case for all infected animals regardless of the challenge doses of M. tuberculosis. For example, the left lung tissue from animal WP01 (the lowest dose of M. tuberculosis) had 4.9 × 105 CFU, which was half the bacterial load in the right lung tissue (data not shown). As for animal WP02 (receiving 200 CFU of M. tuberculosis), the bacterial burden of the right lung tissue was six times that in the left lung tissue (data not shown).
Although Old and New World monkeys are susceptible to M. tuberculosis infection, studies of naturally acquired TB have demonstrated that the degree of susceptibility varies among the different species. It has been demonstrated that rhesus macaques are highly susceptible to TB infection (Good 1968; Gormus et al. 2004; Ribi et al. 1971). When compared with an early study showing that rhesus monkeys challenged with the M. tuberculosis strain at doses of ≤103 CFU rapidly succumbed to TB disease approximately on the 12th week PI (Chaparas et al. 1975), Walsh et al. (1996) concluded that the cynomolgus monkey is less susceptible to TB infection than the rhesus macaque. A difference in the susceptibility of the macaque subspecies to other infectious agents has also been documented. For example, differential susceptibility to SIV infection was observed between rhesus macaques of Indian and Chinese origin (Trichel et al. 2002), even though both groups of monkeys are phylogenetically found within the same species. In the present study, we provide compelling evidence demonstrating that although there was a differential susceptibility to M. tuberculosis infection, the Chinese rhesus macaque is highly susceptible to M. tuberculosis H37Rv strain infection. Regardless of the challenging doses of M. tuberculosis H37Rv strain, all animals developed clinical signs of M. tuberculosis infection, including symptoms of anorexia, weight loss, temperature changes, elevated ESR, and CRP during the course of the study. Infected animals receiving high doses (3 × 103 CFU) of M. tuberculosis demonstrated gross and microscopic changes of pathology, which are similar to those in humans. Furthermore, M. tuberculosis bacteria were found in the tissues of these animals. Due to the limited number of animals in each group, we could not conclude that there is a direct association between the doses of M. tuberculosis strain used and the severity of M. tuberculosis infection among infected animals. However, there is a trend of a positive correlation between the doses of the M. tuberculosis strain and the degrees of M. tuberculosis infection. Among nine infected animals, six receiving the doses 200 CFU or higher developed active TB disease, and five of these six animals died prior to the predetermined study time point (14th week PI). The remaining animals (receiving 200 CFU or less of M. tuberculosis) had manifestations similar to the early stage of chronic TB infection, which demonstrates no or minor lesions in chest radiographs and histopathology. Active TB disease was defined by a number of criteria, including clinical signs of disease such as temperature changes, weight loss, positive chest radiographs, and severe/extensive pathological changes in lungs. The animals with active TB died as early as on the eighth week PI. Except for one animal (WP05), all animals receiving doses of 500 or higher CFU died between the eighth and eleventh weeks PI. Interestingly, one monkey exposed to the lowest dose of M. tuberculosis H37Rv (50 CFU) developed TB disease and died on the 13th week PI. Among the clinical parameters examined during the course of M. tuberculosis infection, ESR and CRP were particularly valuable as they were consistently elevated throughout the course of infection. CRP seems to be more sensitive and reliable than ESR as all animals had a significant raise in CRP regardless of the M. tuberculosis doses used for the inoculation (Table 1). However, there was no significant association between the levels of ESR or CRP and the doses of M. tuberculosis used for the inoculation. In addition, weight loss appears to be a good indicator of M. tuberculosis infection progression as all animals with active/acute infection had a significant weight loss prior to their death (Fig. 1). These animals also developed pneumonia at the late stage of M. tuberculosis infection (sixth or tenth weeks PI; Fig. 3). Thus, these parameters may be useful for monitoring TB infection of Chinese rhesus macaques.
Given the close phylogenetic relationship of rhesus macaques to humans, the results of this study indicate that rhesus macaques of Chinese origin are highly susceptible to M. tuberculosis infection and develop disease similar to human TB clinically and pathologically. These data suggest that Chinese rhesus macaque can be used as an appropriate model for active TB infection. However, because of the limitation of this study, further characterization of M. tuberculosis infection of Chinese rhesus macaques is needed. This should include investigations of the basic host immune responses to, and pathogenesis of, M. tuberculosis infection of Chinese rhesus macaques. Since the majority of people exposed to M. tuberculosis have latent infection that can last for decades, future studies are also necessary in order to determine whether Chinese rhesus macaques are suitable as a model for latent TB infection.
The authors thank Dr. Sugawara I (the Research Institute of Tuberculosis, Tokyo) for providing the M. tuberculosis H37Rv strain. We are grateful to Junhui Wu for his kind review of the manuscript. This study was supported by the grants (2009ZX10004-402) from the Mega-Projects of Science Research for the 11th Five-Year Plan, China, 2009ZX10004-402.
Conflicts of interest
There are no any ethical or financial conflicts of interest for all authors. The corresponding authors, Drs. Wenzhe Ho and Zhi-Jiao Tang, take responsibility on behalf of all authors for the authorship, authenticity, and integrity of this manuscript.