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.