Vet World   Vol.18   October-2025  Article - 27 

Development and evaluation of a formaldehyde-stabilized tuberculin as a safe and potent alternative to phenol-based purified protein derivative for the diagnosis of animal tuberculosis

Background and Aim: Bovine tuberculosis (bTB), caused by Mycobacterium bovis, remains a significant zoonotic threat to livestock and public health, resulting in major economic losses. The diagnostic accuracy of purified protein derivative (PPD) tuberculin, the cornerstone of in vivo screening, is influenced by the allergen’s composition and stabilizer. Conventional phenol-stabilized tuberculin raises toxicity and safety concerns, prompting calls for safer alternatives. This study aimed to enhance the diagnostic value of tuberculin by replacing phenol with formaldehyde as a preservative and stabilizer.

Materials and Methods: Tuberculin was prepared from M. bovis cultures according to the Kazakhstan national stan­dard (Standard Republic of Kazakhstan 1130) and Government standard (16739). Experimental formulations containing 0.1%–10% formaldehyde were produced, and their physicochemical, biological, and allergenic properties were assessed. Diagnostic performance was evaluated in guinea pigs sensitized with M. bovis and atypical mycobacteria (Mycobacterium kansasii, Mycobacterium scrofulaceum, Mycobacterium avium, Mycobacterium phlei), in calves experimentally infected with M. bovis Bacillus Calmette-Guérin, and in naturally infected cattle herds. Results were compared with commercial PPD tuberculin (KazBioPharm, Kazakhstan; Kursk Biofactory, Russia). Statistical analysis was performed using Student’s t-test and analysis of variance (p < 0.05).

Results: Formaldehyde at 3% yielded the highest biological activity and stability without local irritation. In M. bovis-sensitized guinea pigs, 3% formaldehyde-stabilized tuberculin exceeded commercial PPD by 19.9% in reaction intensity. In calves, mean skin-fold thickness increased by 13.1% compared to control PPD. No cross-reactions were observed in animals sensitized to atyp­ical mycobacteria. In a tuberculosis (TB)-affected cattle herd (n = 87), the new formulation induced a mean skin-fold thickness of 5.23 mm, approximately 10% higher than the commercial controls, and identified 21% more infected animals. Receiver Operating Characteristic (ROC) analysis confirmed superior diagnostic accuracy (area under the ROC curve = 0.928, Youden Index = 0.80).

Conclusion: Replacing phenol with 3% formaldehyde significantly enhanced the sensitivity, stability, and biosafety of tuber­culin without compromising specificity. The new formulation eliminates phenol toxicity while improving diagnostic yield in animal TB screening. These findings support the integration of formaldehyde-stabilized tuberculin into national and inter­national diagnostic standards as a reliable and safer alternative for large-scale veterinary applications.

Keywords: biosafety, bovine tuberculosis, diagnostic sensitivity, formaldehyde stabilization, Mycobacterium bovis, purified protein derivative, tuberculin, veterinary diagnostics.

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How to cite this article: Turgenbayev K, Borsynbayeva A, Ozatbekuly A, Dyusenov S, Tlepov A, and Turgenbayev R (2025) Development and evaluation of a formaldehyde-stabilized tuberculin as a safe and potent alternative to phenol-based purified protein derivative for the diagnosis of animal tuberculosis, Veterinary World, 18(10): 3268-3287.

Received: 23-08-2025   Accepted: 17-10-2025   Published online: 31-10-2025

Corresponding author: Assiya Borsynbayeva    E-mail: asiajan@mail.ru

DOI: 10.14202/vetworld.2025.3268-3287

Copyright: Turgenbayev, et al. This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.