Development and validation of a qPCR-based method for quantifying Shiga toxin-encoding and other lambdoid...

Area of Science:

• Environmental microbiology
• Virology
• Molecular biology

Background:

• Bacteriophages, particularly Shiga toxin-encoding bacteriophages (Stx phages), are crucial in microbial ecology and disease transmission.
• Conventional plaque assays may underestimate viral populations in environmental samples.

Purpose of the Study:

• To investigate seasonal variations in Stx phage abundance on a cattle farm.
• To develop and apply a culture-independent method for accurate phage enumeration.

Main Methods:

• Conventional plaque assays were used on water samples collected over 17 months.
• Quantitative PCR (qPCR) with specific primers targeting Q and stx genes was developed for culture-independent detection.
• qPCR was applied to samples negative by plaque assay.

Main Results:

• Bacteriophage numbers exhibited distinct seasonal variation, peaking from June to August.
• Cattle removal correlated with reduced phage numbers; no phages were detected in winter by plaque assay.
• qPCR detected 4.7 x 10^4 to 6.5 x 10^6 lambdoid bacteriophages/ml in plaque-assay-negative samples.
• Approximately 1 in 10^3 free lambdoid phages carried a Shiga toxin operon (stx).

Conclusions:

• Seasonal variability significantly impacts Stx phage populations in cattle farm environments.
• Culture-independent qPCR methods are essential for accurate enumeration of environmental viruses, overcoming limitations of plaque assays.
• Developed qPCR assays provide a robust tool for quantifying bacteriophages and their genetic content in situ.