Tuberculosis in elephants: antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment

Abstract

Tuberculosis (TB) in elephants is a re-emerging zoonotic disease caused primarily by Mycobacterium tuberculosis. Current diagnosis relies on trunk wash culture, the only officially recognized test, which has serious limitations. Innovative and efficient diagnostic methods are urgently needed. Rapid identification of infected animals is a crucial prerequisite for more effective control of TB, as early diagnosis allows timely initiation of chemotherapy. Serology has diagnostic potential, although key antigens have not been identified and optimal immunoassay formats are not established. To characterize the humoral responses in elephant TB, we tested 143 serum samples collected from 15 elephants over time. These included 48 samples from five culture-confirmed TB cases, of which four were in Asian elephants infected with M. tuberculosis and one was in an African elephant with Mycobacterium bovis. Multiantigen print immunoassay (MAPIA) employing a panel of 12 defined antigens was used to identify serologic correlates of active disease. ESAT-6 was the immunodominant antigen recognized in elephant TB. Serum immunoglobulin G antibodies to ESAT-6 and other proteins were detected up to 3.5 years prior to culture of M. tuberculosis from trunk washes. Antibody levels to certain antigens gradually decreased in response to antitubercular therapy, suggesting the possibility of treatment monitoring. In addition to MAPIA, serum samples were evaluated with a recently developed rapid test (RT) based on lateral flow technology (ElephantTB STAT-PAK). Similarly to MAPIA, infected elephants were identified using the RT up to 4 years prior to positive culture. These findings demonstrate the potential for TB surveillance and treatment monitoring using the RT and MAPIA, respectively.

FIG. 1.

Serum IgG antibody responses in culture-positive elephants to individual antigens of M. tuberculosis. MAPIA was performed as described in Materials and Methods. Each panel of strips represents a set of serum samples collected over time from one elephant, as shown (K, I, C, L, and N). Each strip represents one serum sample collected on the date indicated. Bands on MAPIA strips show the presence of antibodies to antigens printed (listed on the right). Time of diagnosis by positive culture is shown by arrows, and treatment duration is indicated for each elephant.

FIG. 2.

Rapid detection of serum antibodies by the ElephantTB STAT-PAK kit. The RT was performed as described in Materials and Methods. In the test window, the upper line is the control band and the lower line is the test band. Results were obtained with selected serum samples from elephant K (1, November 1995; 2, April 2000), elephant L (3, October 1996), and elephant N (4, August 2000; 5, January 1999). RT images 1 and 5 represent nonreactive samples collected from elephants K and N, respectively, before seroconversions. RT images 2 and 4 represent reactive samples from the same two elephants collected at the time of diagnosis by positive cultures of M. tuberculosis and M. bovis, respectively. Semiquantitative results obtained with an optical reader (intensity of test band measured as peak area) were as follows: 1, 0 units; 2, 714 units; 3, 458 units; 4, 257 units; and 5, 0 units.

FIG. 3.

Detection of serum antibodies by ElephantTB STAT-PAK kit in elephant TB before, during, and after treatment. The RT was performed as described in Materials and Methods. Results were measured with an optical reader and expressed in reflectance units (peak area). Each panel shows kinetics of antibody responses found by the RT with a set of serum samples collected over time from one elephant, as indicated (K, I, C, and L). The time of diagnosis by positive culture is shown by arrows, and treatment duration is indicated for each elephant.

FIG. 4.

Effect of antitubercular treatment on IgG antibody responses to selected antigens of M. tuberculosis in culture-positive elephants. MAPIA was performed as described in Materials and Methods. Each panel represents a set of serum samples collected over time from one elephant, as indicated (K, I, C, and L). Results are expressed in arbitrary units of optical density obtained from measuring intensities of MAPIA bands by scanning and densitometry using Scion Image (version Beta 4.0.2). The time of diagnosis by positive culture is shown by arrows, and treatment duration is indicated for each elephant.