White rhinoceroses (Ceratotherium simum) are classified as “Near Threatened” by the International Union for Conservation of Nature, with the majority of animals occurring in South Africa (1). Of these, a substantial number occur in the greater Kruger National Park (KNP) and the Hluhluwe-iMfolozi Park (HiP). However, because of their economic value and threatened conservation status, animals from these populations are regularly translocated to other reserves and privately owned collections. Importantly, movement of animals from these areas could present a risk of translocating Mycobacterium bovis, a major cause of tuberculosis (TB). This pathogen can infect a wide variety of domestic and wildlife hosts, including rhinoceros species (2) and has become established in both the KNP and HiP (3). The recent detection, in the KNP, of a case of severe pulmonary TB in a black rhinoceros (4) and confirmed M. bovis granulomas in lymph nodes of four white rhinoceroses (unpublished data) highlights the potential risk of movement of these species.

Tuberculosis is slowly progressive and the causative organisms may initially be contained within well circumscribed granulomas (5). For this reason, detection of the pathogen can be challenging and, as in other species, TB in rhinoceroses might only be diagnosed postmortem or once animals have developed advanced disease (2). Infection is therefore often diagnosed indirectly by measuring the host’s adaptive immune response toward M. bovis antigens. This is commonly done by quantifying either the in vivo or the in vitro immune response to purified protein derivative (PPD), a preparation containing a broad range of M. bovis antigens (6, 7). Alternatively, recombinant proteins that are more specific to M. bovis can be utilized as test antigens and these include 6 kDa early secretory antigenic target (ESAT-6), 10 kDa culture filtrate protein (CFP-10), MPB70, and MPB83 (8). Assessment of immune responses to these and other antigens might also be used to distinguish between latent infection and progressive disease or to monitor treatment in both humans and animals (8, 9).

The present study forms part of a broader project that characterized the clinical features and associated gross and histopathology of the experimental infection of three white rhinoceroses with M. bovis (10). In all cases, 20 months after infection, animals had shown no clinical signs of TB disease and had limited TB-like pathology (10). We hypothesized that quantifying the adaptive immune responses to M. bovis in these animals would provide an indirect measure of their infection and disease status. As such, we aimed to characterize (i) their immune sensitization to selected antigens following infection and (ii) the kinetics of their humoral and cell-mediated immune responses.

Following experimental endobronchial instillation of M. bovis in 3 white rhinoceroses, these developed time-dependent humoral and cell-mediated immune responses toward the antigens PPDa, PPDb, ESAT-6/CFP-10, MPB83, MPB83/MPB70, and ESAT-6/CFP-10/MPB83. Notably, cell-mediated responses toward ESAT-6/CFP-10 and PPD showed greater consistency between individuals than did humoral responses toward MPB83, MPB83/MPB70, and ESAT-6/CFP-10/MPB83. While the specific patterns of immune reactivity varied between individuals, peak responses were detected in all three animals circa 5–9 months PI, after which they gradually declined to low or undetectable levels.

In mice, the onset of adaptive immunity toward M. tuberculosis requires the active replication of the pathogen in lymph nodes draining the site of infection (13). Moreover, the progression and magnitude of this response is associated with the amount of antigen produced in these lymph nodes (13, 14). Similarly, M. bovis infection in cattle and deer invariably results in the development of granulomatous disease in lymph nodes draining the site of infection, suggesting that these tissues are the primary site of antigen presentation and immune activation (15). In the present study, during the first 5–9 months PI, the rhinoceroses displayed a consistent rise in immune reactivity toward M. bovis antigens, strongly suggesting the establishment of this infection in all three animals.

The magnitude and duration of M. bovis-specific immune reactivity appeared to reflect the disease status of each animal. PB1, which displayed the most robust and sustained responses to all test antigens was the only animal from which viable M. bovis was isolated, i.e., from a bronchoalveolar lavage sample collected 5 months following infection (10). Moreover, at postmortem examination of PB1, acid fast bacterial rods and M. bovis DNA were detected in tracheobronchial and lung lesions, respectively (10). In contrast, PB4 displayed substantially lower responses of both humoral and cell-mediated immunity (CMI) and no definitive confirmation of M. bovis infection was made in this animal by culture, histopathology, or PCR (10).

Following an initial peak circa 5–9 months PI, all rhinoceroses displayed a gradual decline in the magnitude of their immune responses, and antigen-induced IFN-γ release reached low levels within 12–16 months after infection. In cattle, the severity of TB disease correlates with both the magnitude of ESAT-6-specific CMI (5) and the humoral response to M. bovis (6). Similarly, in rabbits experimentally infected with M. tuberculosis, progressive disease is associated with strongly rising titers of anti-PPD antibodies (16), while control of the infection is characterized by low seroreactivity to PPD that declines over time (17). A similar pattern of waning sensitization to ESAT-6/CFP-10 has been documented in naturally infected black rhinoceroses during treatment for M. tuberculosis infection (8). Such patterns are associated with a decrease in pathogen burden and antigenic load (17). Notably, for PB1, the peak in CMI responses at 5 months PI occurred at the only time point that viable M. bovis was isolated from the respiratory tract. Hereafter, the gradual decline in M. bovis-specific immune responses, as for the other two experimentally infected animals, was associated with limited TB-like pathology at postmortem examination and a failure to isolate viable organisms from multiple tissues. Together, these immune response patterns appear to reflect the control of M. bovis infection in these animals following the development of the adaptive immune response.

Humoral responses broadly mirrored those of CMI, with PB1 showing strong seroreactivity to all test antigens. However, PB2 and PB4 showed limited detectable sensitization to the M. bovis-specific antigens MPB83, MPB83/MPB70, and ESAT-6/CFP-10. Similar differences in seroreactivity of individual animals are seen following experimental infection in other species, including cattle and badgers (18, 19). As for these hosts, this phenomenon in rhinoceroses could be related to the infection dose administered to each animal or reflect a natural animal-to-animal diversity in antigen recognition (18, 19). Nonetheless, following infection, PB2 and PB4 did display a transient elevation in seroreactivity to PPDb. In part, this may reflect the far greater range of antigenic epitopes present in PPDb compared to the more specific antigens such as MPB83 and ESAT-6. Alternatively, because PPD includes antigens that are shared between various mycobacterial species (20), the detection of immunological sensitization to PPDb might in part reflect the boosting of immune responses to cross-reactive antigens. Evidence of such cross-reactivity is indicated by the distinct CMI response of PB1 to PPDa following M. bovis infection.

At 20 months PI, all rhinoceroses displayed a rapid increase in IFN-γ release in response to the antigens PPDb, PPDa, and ESAT-6/CFP-10/TB7.7. This boosting of the immune response might have reflected acute progression of TB disease in these animals (5); however, this is unlikely given the paucity of M. bovis-associated pathology at postmortem examination shortly after this sampling time point (10). Alternatively, this observation may be related to viral pneumonia observed in PB2 and PB4 (10). The immune response associated with acute viral infections can induce non-specific “bystander” activation and proliferation of lymphocytes (21), giving rise to IFN-γ production (22). Notably, at this time, PB2 and PB4 displayed a greater response to PPDa than to PPDb. This may be further evidence that this phenomenon resulted from a non-specific mycobacterial or viral infection rather than an M. bovis-specific event.

Results from the present study provide insight into the temporal patterns of immunological responses to M. bovis infection in white rhinoceroses. However, limitations in the study design must be considered when extrapolating findings to animals under natural conditions. First, the nature of the study precluded statistical analysis of data, partly because of the limited sample size and partly because of the substantial variation in immune responses between individuals. Unlike studies using inbred species such as experimental mice (13), this limitation is commonly experienced in studies investigating outbred animals, especially wildlife (19). Second, differences in the infective doses administered to each animal may have accounted for the differences in pathological outcomes and immune response profiles and these may not reflect natural infection. Nonetheless, it is notable that while the magnitude of these responses differed between individuals, their kinetics were comparable. Lastly, the three experimentally infected individuals were all relatively young, had been treated for ectoparasites, were generally free from severe stressors and were well fed (10). As such, the immunological profiles described in this study may reflect an optimal outcome of infection and not necessarily a scenario in which these and other factors might affect either disease progression or resolution.

In summary, this study has characterized antigen-specific immune response patterns of white rhinoceroses that appear to reflect the effective control of M. bovis infection by this host. Importantly, the measurement of these responses might prove useful for assessing the disease status or response to treatment of naturally infected animals. Moreover, the immunological markers identified in this study might be used for the detection of infection; however, further studies investigating these markers in M. bovis-uninfected animals are required to confirm their diagnostic utility.

This study was carried out with the approval of the Animal Ethics Committees of the South African National Parks (SANParks) and Stellenbosch University. Animal care was in accordance with the SANParks Standard Operating Procedures for the Capture, Transportation and Maintenance in Holding Facilities of Wildlife.

AM, MM, VR, and PH designed the study; MM, PB, and JH provided animal care and performed chemical immobilization and blood collection; SP, DM-L, JH, RM, and MM performed laboratory assays; data analysis was done by SP, DM-L, VR, PH, MM, and AM; the first draft of the manuscript was written by SP and all authors contributed to editorial revision.

The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.