Bovine tuberculosis (bTB) is a chronic inflammatory disease of cattle caused by members of the Mycobacterium tuberculosis complex, and in addition to being an important animal health problem, bTB also poses a significant threat to public health (1). It has been estimated that annual worldwide economic losses associated with bTB are ~USD 3 billion (2). The disease is well-controlled in high-income countries, however, bTB remains endemic in most low- and middle-income countries (LMICs), including India where bTB has significant impacts in terms of decreased productivity, increased mortality and zoonotic threat. While national control programs involving test-and-cull strategies have proven to be hugely successful in high-income countries, such approaches are often not feasible in LMICs for both social and economic reasons.

India has the largest livestock population in the world, including nearly 191 million cows and 109 million buffaloes (3). Livestock rearing is one of the most important activities in the rural areas of the country, and for many individuals, it is the only source of livelihood. Haryana, a state in northern India, has a large number (~4.3 million) of the Murrah breed of domestic water buffaloes (Bubalus bubalis). Indigenous buffaloes are important economically, contributing nearly 35% of the country's total milk (3). Although bTB has been well-studied in cattle generally, studies on this disease in buffalo are scarce, especially for high-producing breeds like Murrah.

A recent meta-analysis on bTB in India reported a pooled prevalence of bTB of 4.3% (95% CI: 2.7, 6.7) in buffaloes, calculated from a total of 29,037 animals tested between 1942 and 2016 (4). Without the implementation of any disease control program, this current level of endemicity is predicted to increase in the coming years, especially given the predicted intensification of dairy farming in India. Given that test-and-cull-based control programs are not implementable in India, a vaccine-based intervention strategy may be a promising solution. The Bacille Calmette-Guérin (BCG) vaccine was initially developed for control of human tuberculosis by Albert Calmette and Camille Guérin and was first used in humans in 1921 (5). While there is evidence supporting BCG-induced protection against bTB, the vaccine has not yet been licensed for use in cattle due to the presence of cross-reactive antigens that interfere with the specificity of the tuberculin skin tests recommended by the World Organisation for Animal Health (6, 7).

Recent developments in the field of bTB diagnostics have focused on fit-for-purpose tools that can reliably differentiate infected and (BCG) vaccinated animals (DIVA) in order to make implementation of BCG-based intervention strategies a possibility in LMICs. Several antigens with DIVA capability have been evaluated, of which ESAT-6, CFP-10, and Rv3615c appear to be the most promising (8, 9). These antigens have been extensively evaluated both in experimental and field conditions, and a peptide-based formulation of these antigens, henceforth referred to as the defined antigen skin test (DST), was previously evaluated for its utility in field studies in cross-bred cattle both in natural reactors and in BCG vaccinates (9, 10). Here, we assess the utility and performance of DST in buffaloes in India.

Recent developments in the field of bTB diagnostics have centered around identifying candidate antigens for DIVA in order to enable vaccine-based interventions in LMICs. Previous studies on the use of defined skin test antigens in cattle, using a combination of ESAT-6, CFP-10, MPB70, and MPB83, concluded that these antigens are promising candidates for performing the IGRA in cattle, with the ability to differentiate between vaccinated animals and those infected with M. bovis (8, 12). The current study was performed to assess the performance of a recently developed peptide-based DST cocktail in Murrah buffaloes, an economically important breed of buffaloes in India. In this study, we first performed IGRAs with DST peptides at 10 μg/ml. Although PPD-B was the most sensitive in eliciting IFN-γ responses in vitro, there was no significant difference in the responses elicited by the DST peptides and PPD(B-A). To our knowledge, this is the first study where potential of defined skin test antigens has been demonstrated in naturally tuberculin skin test reactor domestic buffaloes. Our data demonstrated that, these peptide-based antigens have promising potential for use in IGRAs as an ancillary test in concert with the skin test for distinguishing between reactor and uninfected animals. However, as this proof-of-concept study has only been performed on a small number of animals, it will be important to validate the performance of DST in a larger cohort of known infected and naïve buffaloes.

We also assessed the utility of the DST peptide cocktail as a skin test reagent in buffaloes. Of the three different doses (5, 10, and 20 μg per peptide) of DST that were compared, 20 μg/ml appeared to have the highest sensitivity, although higher concentrations may need to be tested to achieve an optimal balance between sensitivity and specificity. Most importantly, the peptide cocktail also proved to be highly specific, as no measurable skin induration response was observed following injection into naïve uninfected buffaloes. Both SIT and the highest dose of DST were found to be 100% sensitive, identifying all 11 naturally-infected buffaloes as reactors. In this context, it is important to note that, given the high burden of environmental mycobacteria in India, PPD-B can induce non-specific responses, often leading to high rates of false-positivity with SIT. The CCT with the simultaneous injection of PPD-A helps overcome this loss of specificity associated with SIT, but only at the expense of sensitivity. The results of this study showed that the peptide-based DST provides sensitivity and specificity equivalent to that of the OIE-recommended CCT, without the need for a second injection. Moreover, DST at its highest tested dose (20 μg/ml) elicits a lower amplitude of skin test response in infected animals (6.18 ± 2.60 mm) as compared with PPD-B (13.81 ± 4.68 mm) without hindering the specificity estimate. Such exuberant non-specific PPD-B responses tend to raise concerns and limit acceptability of the SIT among farmers. In reactor buffaloes, we found that 20 μg DST produced a skin response greater than the cut-off of 2 mm in all of the animals tested. In this context, it is crucial to highlight the difference in antigen dosage that may be necessary for accurate bTB diagnosis in cattle and buffaloes. In a previous study with cross-bred cattle, pilot dose titration experiments showed that DST@10 may be the optimal dose in cross-bred cattle (9). However, here the data show that DST@20 may be better suited for bTB diagnosis in buffaloes as it identified all reactors as test-positive without compromising test Specificity. Future studies that are adequately statistically powered are required to accurately determine the antigen dosage required for diagnosis in buffaloes.

In 2019, the World Health Organization reported that over nine million people developed TB, of which ~1.5 million died (13). Only eight countries account for about two-thirds of the total reported cases of TB, with India leading the count. Moreover, some recent estimates suggest that ~9% of all human TB cases in India may be of zoonotic origin (14). Hence, it is increasingly recognized that controlling TB in the livestock population in these settings could be a major step toward attaining the ambitious End TB goal of 2035 (15). As part of these control efforts, effective methods of identifying infected animals are needed. However, there are several major limitations to the OIE-recommended tuberculin-based skin tests, including the fact that the active components in the reagent are undefined and their interference with BCG-based vaccination. In contrast, the peptide-based DST offers superior quality control and potential for DIVA has previously been demonstrated in cattle (10), and the current study indicates that the DST antigens are promising candidates for differentiating between reactor and uninfected buffaloes. Limitations of the study include the relatively small number of animals and their origin from a single dairy farm. Hence, these results will need to be verified in larger cohorts of naturally infected and naïve animals to establish more refined estimates of sensitivity and specificity of the DST in buffaloes prior to consideration for replacement of the traditional tuberculin standard. Moreover, while the DIVA capability of DST enables implementation of BCG vaccination as an intervention, the efficacy of BCG vaccination in buffaloes remains unknown. Field trials are in process to evaluate the performance of DST in larger cohorts of infected, BCG-vaccinated and naïve buffaloes, and the efficacy of BCG in buffaloes in natural transmission settings in India is being planned.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by Institutional Animal Ethics Committee, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar, Haryana, India.

SS, VK, MV, DBa, and NJ conceptualized the study. TK, MS, BJ, DA, DBi, and DY conducted the animal experiments. MS, SS, and MV conducted the lab experiments. TK, MS, and SS analyzed the data. TK prepared the first draft. All authors contributed to the article and approved the submitted version.

MV was employed by company Cisgen Biotech Discoveries Pvt. Ltd. SS and VK have filed intellectual property protection for the DST. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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