Development of a Rapid PCR Test for Identification of Streptococcus agalactiae in Milk Samples Collected on Filter Paper Disks

Abstract

Streptococcus (Strep.) agalactiae is one of the major pathogens of bovine mastitis and is the main cause of subclinical infection. This study attempted to develop a rapid PCR diagnosis procedure using milk samples collected on filter paper disks. Chromatographic filter paper was employed as the preservation media and kept at room temperature for one to four weeks. The revival rate of Strep. agalactiae kept on dried filter paper disks was affected by the pretreatment preservation time. The revival test suggested that not all the bacteria in artificially contaminated milk samples on the filter paper disks could be recovered. After that, a PCR based on the 16-23S intergenic spacer region of Strep agalactiae was performed. The results distinguished the strep. agalactiae from major pathogens of bovine mastitis at a 2×10 2 colony forming units (CFU)/ml level, which showed similar sensitivity to the results from liquid milk samples. The results also showed that milk samples collected on filter paper disks could be kept at room temperature for one to four weeks with little negative effect on sensitivity and specificity. The field test showed that the diagnostic sensitivity and specificity was 96.15% and 98.60%, respectively. In conclusion, the protocol will provide a rapid and economic procedure for the detection of bovine mastitis.

Extraction of Mastitis Pathogen DNA from Sample Collecting Cards: Practical Consequences

“…Specificity and sensitivity were estimated on a small batch of samples. They are however quite close to those calculated by Wu et al (2008) [7].…”

“…In studies performed in the early 2000's, where DNA was extracted from liquid milk, S. aureus could be detected at a concentration of 5x10 3 cfu/mL [12] and S. agalactiae at a concentration of 10 3 to 10 4 cfu/mL [18]; an enrichment process was needed in order to lower the limit of detection (LOD). However, things are improving fast and LODs for mammary pathogens in milk by PCR without enrichment are as low as 40 cfu/mL for S. aureus [19], 2x10 2 cfu/mL for S. agalactiae [7], and 10 cfu/mL for M. bovis [15]. In the present study S. aureus was inconsistently detected in some samples at concentrations close to 20 cfu/mL.…”

“…Lampel et al (2000) [20] demonstrated that various aliquots from concentrated food washes subsequently spotted onto sample collecting cards and assayed by PCR, yielded consistently positive results and LOD similar to those observed with pure-culture dilutions. A few studies have investigated DNA extraction from milk spotted onto cards [7,8,21]. DNA identification is supposed to be carried out with very small parts of the card (2 punches of Ø1.2 mm), and quantification of the bacterial load in the initial milk sample with such a limited matrix is not feasible.…”

“…These adaptations allow the use of larger amounts of milk, up to 250 µL. LODs for these techniques would be 2x10 2 cfu/mL for S. agalactiae, 10 to 10 2 for S. aureus, 10 2 to 10 3 for E. coli and CNS [7].…”

“…Whereas detection of mastitis pathogens by PCR from liquid milk has been carefully substantiated [5] and is now routinely used for large scale udder health surveys [6], extraction from filter paper has not been extensively tested [7,8] and some details of the technique deserve to be improved.…”