Tuberculosis in Elephant
Tuberculosis (TB) is a crucial infectious disease, posing a threat to both humans and animals, especially in species like elephants that often have prolonged contact with caretakers. Mycobacterium tuberculosis complex (MTC), including M. tuberculosis, M. microti, M. bovis, and M. africanum, can lead to chronic respiratory diseases. Among these, M. tuberculosis is the primary culprit behind TB in elephants.
Traditionally, the gold standard for diagnosing TB in elephants has been bacterial culture and isolation. However, the conventional method of sample collection, known as ‘trunk wash,’ akin to human sputum culture, suffers from low sensitivity, a high false negative rate, and limited sampling capabilities. Consequently, researchers have explored alternative tools, such as serological assays like enzyme-linked immunosorbent assay (ELISA), multi-antigen print immunoassay (MAPIA), rapid serum tests (such as Elephant TB STAT-PAK® and DPP VetTB®), and interferon-gamma release assays (IGRA), to assess MTC infections in elephants. Despite these advancements, definitive confirmation of active TB in elephants still requires bacterial culture and identification. Specialized sampling techniques, particularly bronchoalveolar lavage (BAL), have become indispensable components of the diagnostic process. In the following sections, we will delve deeper into these methods.
Indirect enzyme-linked immunosorbent assay (ELISA)
Indirect enzyme-linked immunosorbent assay (ELISA) were employed for serodiagnostic screening with high sensitivity and specificity depending on technique and recombinant antigen used. In-house technique using ESAT-6, CFP-10, and MPB83 as the recombinant antigens as outlined by Angkawanish et al, 2021 exhibited a promising approach for predicting TB status in Thailand. The study, conducted on a random sample of 708 elephants across 13 provinces between 2004 and 2009, revealed a TB prevalence of 17.3% positive, 48.7% inconclusive (a combination of negative and not-negative), and 34% negative.
This research suggests that the combination of these three recombinant antigens can effectively predict positive TB status in elephants. Conversely, for predicting TB negative status, CFP-10 and MPB88 were found to be particularly useful.
Multi-antigen print immunoassay (MAPIA)
Multi-antigen print immunoassay (MAPIA) is an alternative serological examination specifically designed for early diagnosis, therapeutic monitoring, and also disease recurrent prediction with 100% sensitivity and specificity. Multiple specific antigens for TB (e.g. ESAT-6, CFP-10, Mtb8, Mtb48, MPB59, MPB64, MPB70, MPB83, Arc-1, 38-kDa) are printed on nitrocellulose membranes using specific devices. Serum samples from elephants are tested and the results are demonstrated with bands on MAPIA strips indicating the presence of antibodies to the antigens printed. A study performed by Lyashchenko, 2006 revealed that ESAT-6 was strongly recognized by elephants’ serum IgG which was a humoral immune response in TB infected elephants. The robust band in MAPIA stripe indicated the levels of antibody to certain antigens long before detected in trunk wash culture. Regarding treatment duration, antibody levels in seropositive elephants declined rather quickly down to baseline in response to antitubercular therapy. Hence, this serological assay can be applied for early detection, enabling the identification of TB-infected elephants with high accuracy and facilitating the initiation of therapeutic plans at the disease’s earliest stage. Additionally, routine monitoring during the post-treatment period has proven instrumental in predicting TB recurrence in elephants within a latent period of 2 to 6 years.
Rapid Serum Test
Various rapid serum tests have been employed for diagnosing TB in elephants, utilizing lateral flow test kits based on immunochromatography. One such tool, the Elephant TB STAT-PAK®, is a commercial diagnostic device designed specifically for elephants. It incorporates antigens ESAT-6, CFP-10, and MPB83. This method boasts an impressive sensitivity of 100% and a specificity of 95.2%. However, false positives can occur due to nontuberculous mycobacteria (NTM) infections, and limited sensitivity might lead to occasional false negatives. On the other hand, the DPP VetTB® is a field-applicable lateral flow rapid test based on MAPIA. This tool utilizes the specific antigens ESAT-6/CFP-10 fusion protein and MPB83, achieving a remarkable 100% sensitivity and specificity in its results.
Interferon gamma release assay (IGRA)
The Interferon Gamma Release Assay (IGRA) stands as one among several immunological response diagnostic tools rooted in cell-mediated immunity (CMI) for detecting TB in wildlife, akin to the tuberculin skin test. Elephants afflicted with TB release interferon-gamma (IFNγ) as a crucial cytokine in TB control. Consequently, IGRA, triggered by Mycobacterium tuberculosis complex (MTC) antigen stimulation, offers an alternative method for diagnosing TB, primarily focusing on the disease’s initial stages.
In this technique, in-vitro peripheral blood mononuclear cells (PBMCs) generated by T cells are stimulated by TB-specific antigens like ESAT-6 and CFP-10, prompting the release of elephant-IFNγ (eIFNγ). The quantity of eIFNγ is gauged through a developed sandwich ELISA, using mitogens against elephant PBMCs such as ConA or PWM. Adapted from the human IGRA test, this method yields results indicating negative, MTC-suspected, or MTC-infected statuses. Additionally, positive results can distinguish between MTC and non-tuberculous mycobacteria (NTM) infections. Notably, a recent study (Pongma et al, 2023) employing elephant whole blood culture, as opposed to PBMCs, reduces blood volume collection, shortens diagnosis time, and enhances user-friendliness of the technique.
Bacterial culture and Polymerase chain reaction (PCR)
The identification of the MTC serves as a definitive method for tuberculosis diagnosis, achievable through direct microscopy, bacterial culture, and molecular techniques. In elephants, MTC has been isolated from various sources such as respiratory secretions, feces, vaginal discharge, and trunk washes. However, limitations existed, particularly in trunk washes, due to their low sensitivity, leading to failure in isolation, susceptibility to contamination, and prolonged reporting times.
To address these challenges, a novel technique called bronchoalveolar lavage (BAL) has been developed for elephants. This method involves endoscopy for collecting bronchoalveolar fluid and exploring the respiratory tract. Culturing and isolating MTC requires a biosafety level 3 laboratory, specific media, and the results depend on the disease stage and sample quality.
Molecular identification methods involve extracting DNA directly from clinical samples, tissues, or growing colonies with high bacterial loads. Polymerase chain reaction (PCR) and genotyping have proven advantageous for MTC identification due to their rapidity, high sensitivity, and usefulness in epidemiological studies. In Thailand, common primers used for TB detection include 16S rRNA and 16S–23S-rDNA internal transcribed spacers (ITS) to confirm M. tuberculosis complex. Additionally, gyrase B (gyrB) is employed for specific strains such as the M. tuberculosis strain American Type Culture Collection (ATCC) 27294 and others.
Figure 3 shows a PCR analysis targeting Mycobacterium spp. Lane 4 contains a 100 bp DNA ladder, which serves as a molecular weight marker for accurate sizing of the PCR product. Lane 5 represents the positive sample of Mycobacterium spp. with a band size of 506 bp. Lane 6 includes a positive control and Lane 7 is the blank control.
As has been shown above, the laboratory procedures employed for the diagnosis of elephant tuberculosis can be effectively conducted in Thailand, albeit necessitating the application of specialized techniques and equipment. Nevertheless, MoZWE has the capability to conduct tuberculosis DNA detection through the polymerase chain reaction (PCR) technique.
References
Angkwanish T, Vernooij HJCM, Sirimalaisuwan A, Charernpan P, Nielen M, Rutten VPMG. Prevalence and demographic risk factors of Mycobacterium tuberculosis infections in captive Asian elephants (Elephas maximus) based on serological assays. Front Vet Sci. 2021;8.
Lyashchenko KP, Greenwald R, Esfandiari J, Olsen JH, Ball R, Dumonceaux G, et al. Tuberculosis in elephants: Antibody responses to defined antigens of Mycobacterium tuberculosis, potential for early diagnosis, and monitoring of treatment. Clin Vaccine Immunol. 2006;13(7):722–32.
Pongma C, Songthammanuphap S, Puthong S, Buakeaw A, Prammananan T, Warit S, et al. Using whole blood cultures in interferon gamma release assays to detect Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus). PLoS One. 2023;18(7):e0288161.
Songthammanuphap S, Puthong S, Pongma C, Buakeaw A, Prammananan T, Warit S, et al. Detection of Mycobacterium tuberculosis complex infection in Asian elephants (Elephas maximus) using an interferon gamma release assay in a captive elephant herd. Sci Rep . 2020;10(1).
Thomas J, Balseiro A, Gortázar C, Risalde MA. Diagnosis of tuberculosis in wildlife: a systematic review. Vet Res. 2021;52(1).
Verma-Kumar S, Abraham D, Dendukuri N, Cheeran JV, Sukumar R, Balaji KN. Serodiagnosis of tuberculosis in Asian elephants (Elephas maximus) in southern India: A latent class analysis. PLoS One. 2012;7(11):e49548.
Choenkwan Pabutta
Veterinarian, The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals (MoZWE)