An alert of Mycobacterium tuberculosis infection of rhesus macaques in a wild zoo in China

Abstract

Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), is becoming increasingly recognized as an important cause of fatal chronic illnesses in China. In this study, we report an infectious disease among 84 rhesus macaques at a Chinese zoo. Their clinical signs and symptoms were very similar with the manifestations of TB in humans. To determine the potential pathogens of this outbreak, many methods were used. First, tuberculin skin tests showed that none of the monkeys displayed significant skin reactions. Subsequently, the sera were tested for specific antibody IgG; 29 (34.5%) and 39 (46.4%) blood samples tested positive by TB-IgG and TB-DOT, respectively. Radiographic examination showed characteristic imageology changes in 14 (16.7%) monkeys. One individual determined as positive by the above three methods was euthanized, and histopathological analysis demonstrated typical granulomas and caseous necrosis in the lung, liver, spleen, and intestine. Furthermore, the pathogenic mycobacteria were isolated from lung lobe, cultured on acidic Lowenstein-Jensen culture medium, and identified as M. tuberculosis by real-time PCR and DNA sequencing. Nevertheless, the origin of the infection remained unknown. These findings emphasize the need to strengthen the management and training of staff, especially those working at animal shelters.

Keywords: Mycobacterium tuberculosis; infectious disease; outbreak; rhesus macaque.

Fig. 1.

Tuberculin skin test in M. tuberculosis-infected representative rhesus macaque. The rhesus macaques were intradermally injected in the left eyelid with 0.1 ml of 5 IU TB-PPD and in the right eyelid with 1 µg recombinant CFP10/ESAT6 fusion protein, respectively. No significant swelling and nodules were observed on the left or right eyelid of experimental animals at 24, 48, and 72 h after injection.

Fig. 2.

Chest X-ray of representative rhesus macaques in each group. Eight representative rhesus macaques (No. 2, 5, 11, 31, 77, 47, 70 and 53) in D⁻I⁻X⁻, D⁺I⁻X⁻, D⁻I⁺X⁻, D⁺I⁺X⁻, D⁻I⁻X⁺, D⁺I⁻X⁺, D⁻I⁺X⁺, and D⁺I⁺X⁺ group were chosen to evaluate the condition of lung, respectively. Their radiographs are indicated with A, B, C, D, E, F, G, and H in this figure. No obvious radiographic lesion was observed in the lung of rhesus macaques from D^+/−I^+/−X⁻ groups. However, kinds of radiographic lesions were observed in the lung of rhesus macaques from D^+/−I^+/−X⁺ groups, including flocculent shadows, tuberculoma, calcifications, and uneven distribution of tubercles (indicated by arrow in E to H).

Fig. 3.

Gross pathological changes of organs collected from rhesus macaques infected with M. tuberculosis. One rhesus macaque each in D⁺I⁺X⁺ and D⁻I⁻X⁻ (as a negative control) group was euthanized via intravenous injection with overdose of pentobarbital sodium. Vivisection and tissue dissection revealed no obvious pathological changes in the main visceral organs of the rhesus macaque in D⁻I⁻X⁻ group (A). In contrast, numerous miliary tubercles and extensive caseous necrosis were observed on the surface of the lung (B), spleen (C), and liver (D) collected from the rhesus macaque in D⁺I⁺X⁺ group.

Fig. 4.

Histopathological analyses. The organs collected from the rhesus macaque in D⁺I⁺X⁺ group were subjected to histopathological examination. Large caseous granuloma and infiltration of inflammatory cells in lung (A), localized calcification, proliferation of connective tissue, and caseous necrosis in spleen (B), large caseous granuloma with central necrosis surrounded by a narrow mantle zone in the liver (C), inflammatory cell infiltration, caseous necrosis, and proliferation of connective tissue in intestines (D) were observed by H&E stain using light microscopy at a magnification of 100×. Additionally, acid-fast bacilli were observed in a tissue section of lung by acid-fast stain (E) under light microscope at original magnification times 1,000×.

Fig. 5.

qRT-PCR analysis. Genomic DNA was extracted from the colony cultured from the lung tissue of the rhesus macaque in D⁺I⁺X⁺ group and amplified using qRT-PCR kit according to the manufacturer’s instructions. The fluorescence intensity of the sample (gray), positive control (red), and negative control (green) was detected with 7,300 Real Time PCR System (Applied Biosystems).