Vetnuus | March 2026 27 Moreover, current platforms do not include specific targets for M. orygis, limiting their utility in regions where this species is prevalent. The relatively high cost and infrastructure requirements further constrain their routine use in low-resource or decentralised laboratory settings (8). Whole Genome Sequencing and Targeted Next Generation Sequencing While LPAs represent a significant advance in MTBC speciation, WGS and targeted next-generation sequencing (tNGS) offer the highest resolution for species identification, antimicrobial resistance prediction, and molecular epidemiology. Given their technical complexity and specific applications, these approaches are discussed in detail in the Advanced Diagnostic Approaches and Emerging Technologies section of this review. Emerging Molecular Tools Loop-mediated isothermal amplification (LAMP) is a field-adaptable molecular method with high specificity, endorsed by the WHO for MTBC detection. However, species-level differentiation remains limited unless lineage- or species-specific primers are employed. Clustered regularly interspaced short palindromic repeats (CRISPRs)-based diagnostic platforms, including specific systems such as SHERLOCK and DETECTR, represented promising ultrasensitive nucleic acid detection technologies. Although these methods have potential for species-specific applications, they are still under development and not yet widely implemented in routine diagnostics. Spoligotyping and MTBC Lineages Spoligotyping (spacer oligonucleotide typing) is a genotyping technique that differentiates MTBC strains based on the presence or absence of unique spacer sequences within the direct repeat (DR) region of the genome. It is widely used for lineage determination, molecular epidemiology, and surveillance. Each MTBC species or lineage is associated with a characteristic spoligotype pattern. For instance, M. bovis typically lacks spacers 39 to 43, a signature feature that facilitates its identification (1,11). Mycobacterium orygis displays a unique spoligotype profile and is frequently associated with ancient or unclassified lineages. In contrast, M. caprae shares overlapping spoligotype patterns with both M. bovis and M. microti, which may complicate definitive identification when spoligotyping is used alone. Spoligotyping has contributed substantially to understanding the geographic distribution, host associations, and evolutionary relationships of MTBC species. In the context of zTB, it supports tracing infection sources and identifying potential animal reservoirs or spillover events. Although it offers lower discriminatory power than WGS, spoligotyping remains a valuable and costeffective tool for lineage assignment in settings where advanced sequencing technologies are unavailable. When combined with RD analysis or SNP-based typing, it can enhance strain differentiation and strengthen regional zTB surveillance within a One Health framework. IMMUNOLOGICAL METHODS Immunological methods detect host immune responses to MTBC antigens rather than the organisms themselves. Although widely used for TB screening and surveillance, these assays lack the ability to differentiate among MTBC species, which limits their role in the definitive diagnosis of zTB in humans. Tuberculin Skin Test The tuberculin skin test (TST), including the Mantoux test in humans and the single intradermal comparative cervical tuberculin (SICCT) test in animals, measures delayed-type hypersensitivity to purified protein derivative (PPD). These tests remain central to surveillance programs but cannot distinguish between MTBC species. In addition, they are prone to false-positive results from BCG vaccination or exposure to environmental mycobacteria (4). Interferon-Gamma Release Assays Interferon-gamma release assays (IGRAs), such as QuantiFERONTB Gold (Qiagen, Hilden, Germany) for human use and BOVIGAM™ (Thermo Fisher Scientific, Waltham, MA, USA) for animals, offer improved specificity by incorporating antigens such as early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), thereby avoiding cross-reactivity with BCG vaccination (4). Despite this advantage, IGRAs are primarily designed to detect latent TB infection and, like TSTs, do not allow differentiation between MTBC species. Enzyme-Linked Immunosorbent Assays In animals, enzyme-linked immunosorbent assays (ELISAs) are used to detect circulating antibodies against mycobacterial antigens and are particularly valuable for herd-level screening and in surveillance efforts targeting wildlife reservoirs (4). However, their diagnostic accuracy can vary widely, depending on the antigens used in the assay and the host species being tested.In humans, ELISAs play a limited role in the routine diagnosis of tuberculosis due to their relatively poor specificity and sensitivity. Nevertheless, they may serve a niche function in sero-epidemiological studies or in specific research contexts aimed at understanding immune responses to mycobacterial infection. Article Species RD1 RD4 RD9 RD12 M. tuberculosis + + + + M. bovis + − − − M. bovis BCG − − − − M. caprae + + − − M. orygis + + − − M. africanum + + − + M. canettii + + + − M. microti − + − + Table 2. Region-of-difference (RD) presence/absence signatures are commonly used to differentiate members of the M. tuberculosis complex (RD1, RD4, RD9, RD12). Presence (+) and absence (−) patterns shown are the expected signatures for typical representatives of each species; exceptions and atypical RD sizes occur (adapted from reference 1). >>>28
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