Development and Validation of a Test System to Detect Brucella abortus in Whole Milk, Soft Cheese and Leafy Greens

Introduction: Brucellosis is a zoonotic disease that affects both humans and animals. In the United States, brucellosis in humans frequently occurs after ingestion of unpasteurized milk or dairy products, or after being in contact with infected meat or placenta of infected animals.  The causative agents, Brucella spp., are fastidious, slow-growing organisms that do not grow in conventional laboratory media. 

Purpose: To develop a test system for the detection of Brucella spp. from food matrices, and to validate its performance on artificially-inoculated whole milk, soft cheese and leafy greens.

Methods: Brucella abortus S19 (vaccine strain) was artificially inoculated into home-made soft cheese (25 g), leafy greens (25 g) or whole milk (25 ml), stored overnight at 4 °C, and enriched with Brucella media supplemented with polymyxin B, cycloheximide, vancomycin, and nalidixic acid. Twenty of 25 samples for each food matrix were inoculated with 1 to 80 CFU, and 5 served as uninoculated controls. Sample aliquots were taken daily for 5 days and analyzed using a test system that included multiplex PCR assay, lateral flow immunoassay (LFI) and immunomagnetic particles (IMP). 

Results: A test system for screening and confirming the presence of Brucella in food samples was developed. The test system was validated in house using artificially inoculated whole milk, soft cheese and leafy greens at a level of 1 to 80 CFU/25 g or ml of food matrices. Using a combination of IMP-multiplex PCR, B. abortus S19 was detected in food samples as early as after 2 days of enrichment, and all confirmed Brucella­-positive samples were identified when incubation was extended up to 3 to 4 days. LFI detected all PCR-positive samples after 4-days of enrichment. The PCR-negative and uninoculated enrichment samples did not produce any positive signal from IMP-multiplex PCR and LFI even after 7 days of enrichment. These results suggest that the multiplex PCR and LFI methods exhibited 100% sensitivity (ability to detect positives when positives are present) and 100% specificity (ability to declare a negative when sample is truly negative). The limit of analytical sensitivity of the multiplex PCR assay was 10⁴ CFU/ml, while that of the LFI was 10⁵ CFU/ml, which explains the additional 24 hr enrichment required to detect positive signals using LFI from samples that had already tested positive using multiplex PCR.

Significance: This test system provides a rapid and robust method for detection and isolation of Brucella spp. from some contaminated foods.