Establishment of a neonatal rhesus macaque model to study Mycobacterium tuberculosis infection

Abstract

Mycobacterium tuberculosis (Mtb) is the causative agent of human tuberculosis (TB) with an estimated 8.8 million new TB cases and 1.4 million deaths annually. Tuberculosis is the leading cause of death in AIDS patients worldwide but very little is known about early TB infection or TB/HIV co-infection in infants. A clinically relevant newborn animal model to study TB infection is urgently needed. We have successfully established an aerosol newborn/infant model in neonatal nonhuman primates (NHPs) that mimics clinical and bacteriological characteristics of Mtb infection as seen in human newborns/infants. Further, this model will allow the establishment of a TB coinfection model of pediatric AIDS. Aerosol versus intra broncho-alveolar Mtb infection was studied. Interestingly, 42 days post infection specific lesions were detected suggestive of the classic Ghon focus in human children. Concurrently, specific cellular immune responses developed 4-6 weeks after Mtb infection. Using the enzyme-linked immunospot (ELISPOT) assays, we found that IL-12 production correlated with early Mtb infection lesions seen by routine thoracic radiographs. Overall, this work represents the first example of early Mtb infection of newborn macaques. This study gives us a unique opportunity to further characterize immunopathogenesis and establish a TB/SIV co-infection model for pediatric AIDS.

Keywords: ELISPOT IL-12; Ghon focus; TB; newborn; rhesus macaque.

Figure 1

(A) Each inoculum dose was confirmed by plating onto 7H10 agar plates. (B) An ultrasonic nebulizer was used for aerosol Mtb infection. The flow rate of the ultrasonic nebulizer was at 0.5 ml/minute. (C) Experimental design, animal procedures and assays performed

Figure 2

Weight and Temperature charts with relevance to Erdman infected newborns. The growth curves of all Redman-infected newborns are comparable to that of naïve animal. Weight gain was gradual and temperature remained at a normal range for all animals. Each graph represents the weight in kilograms (Kg) and the temperature in Fahrenheit (F).

Figure 3

Absolute frequency (number) of lymphocyte subsets in Mtb-infected newborn/infant macaques. Those included CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocytes as well as CD3⁻CD8⁺ and CD3⁻CD8⁺CD16⁺ NK cells isolated from freshly collected blood samples over the 12 weeks study.

Figure 4

Chest radiograph of neonatal rhesus macaques infected with M. tuberculosis. Parenchymal lesions are circle.

Figure 5

Representative microscopic examination of lung and lymph node tissues from a newborn macaque showing typical lesions consistent with M. tuberculosis infection. (A) Lymph node: epithelioid macrophages with early granuloma formation (arrow) 31242; and, (B) Lung infiltrates with interstitial epithelioid macrophages are shown. (C-D) Lymph node tissues from a neonatal macaque infected via the aerosol route with low dose M. tuberculosis. Panel C shows a low magnification (2× magnification) view of the lymph node with numerous active germinal centers. Panel D shows a normal lymph node tissue for comparison.

Figure 6

Immune responses to Mycobacterium tuberculosis infection in newborn macaques. (A) Frequency of PPD and ESAT-6 specific IFN-γ and IL-12 secreting cells measured by ELISPOT assay. The secretion of IFN-γ and IL-12 was measured longitudinally in PBMC isolated from two newborn macaques (animals: 31338 and 31669) infected with aerosol Mtb Erdman at 10 and 50 CFU, respectively. (B) Frequency of PPD and ESAT-6 specific IFN-γ and IL-12 secreting cells isolated from multiple tissues/organs postmortem.