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Updated: Dec 30, 2025
Evaluation of Microbial Safety of Dairies using Bacterial Proteomic Profiling via MALDI Approach
Published on: October 7, 2025
Propidium monoazide conventional PCR and DNA sequencing: detection of negative culture bacterial pathogens causing
M G Farhan1, M I Abd El-Hamid2, M N Hassan2
1Department of Animal Management and Treatment, Fakous Veterinary Administration, Sharkia Governorate, Fakous, Egypt.
Aims:
This study was conducted to early detect the negative culture bacterial pathogens causing subclinical mastitis for the fast diagnosis of the disease and the reduction of some milk-transmitted pathogenic bacteria to human consumers.
Methods And Results:
A total of 171 positive California mastitis test (CMT) milk samples collected from asymptomatic dairy cows in Sharkia Governorate, Egypt were examined by conventional bacteriological methods. The obtained results revealed that Streptococcus species (77·2%), followed by Staphylococcus species (48·6%) and Escherichia coli (25·7%) were the most predominant bacterial pathogens isolated from positive culture milk samples, whereas Enterobacter and Pseudomonas species were the lowest ones (1·2%, for each). Herein, 13 (7.6%) negative culture milk samples were subjected to propidium monoazide (PMA) conventional PCR assay, followed by DNA sequencing of purified PCR amplicons. Sequence analysis identified seven different types of negative culture bacterial pathogens comprising as following; 4 Enterococcus hirae, 2 Bacillus cereus, 2 Staphylococcus aureus, 1 Bacillus mycoides, 1 Bacillus subtilis, 1 Enterococcus faecium and 1 Escherichia coli.
Conclusions:
All the detected negative culture bacterial pathogens by PMA-PCR assay, followed by DNA sequencing were incriminated in causing subclinical mastitis disease and had serious implications on human public health through consumption of milk contaminated with those recovered bacterial pathogens.
Significance And Impact Of The Study:
The used methods could be useful in the routine detection of negative culture bacterial pathogens present in milk and consequently, it will help in the rapid diagnosis of subclinical mastitis disease and the reduction of many milk-transmitted diseases to human.
Insights
This study identified bacterial pathogens in milk samples that tested negative by conventional methods using propidium monoazide (PMA) PCR. This rapid detection aids in diagnosing subclinical mastitis and reducing milk-borne diseases.
Area of Science:
- Veterinary Microbiology
- Food Safety
- Molecular Diagnostics
Background:
- Subclinical mastitis poses a significant threat to dairy herds and public health.
- Early detection of causative pathogens is crucial for effective disease management.
- Conventional bacteriological methods may fail to detect all relevant pathogens in milk.
Purpose of the Study:
- To develop and validate a method for early detection of bacterial pathogens in milk samples that yield negative cultures.
- To identify specific bacterial species responsible for subclinical mastitis that are missed by standard culturing.
- To assess the public health implications of milk-transmitted pathogens.
Main Methods:
- Conventional bacteriological analysis of California Mastitis Test (CMT)-positive milk samples.
- Application of propidium monoazide (PMA) conventional PCR assay on culture-negative samples.
- DNA sequencing of purified PCR amplicons for pathogen identification.
Main Results:
- Streptococcus, Staphylococcus, and Escherichia coli were predominant in culture-positive samples.
- PMA-PCR identified seven types of bacterial pathogens in culture-negative milk, including Enterococcus hirae, Bacillus cereus, and Staphylococcus aureus.
- Several identified pathogens are known to cause subclinical mastitis and pose risks to human health.
Conclusions:
- PMA-PCR combined with DNA sequencing is effective for detecting culture-negative bacterial pathogens in milk.
- This method facilitates rapid diagnosis of subclinical mastitis.
- The findings contribute to reducing milk-transmitted diseases in consumers.
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