A PCR detection method for discerning Serratia marcescens in extended boar semen

Serratia marcescens is a bacterial contaminant that can be spermicidal when present in extended boar semen that is typically stored prior to breeding use at 15 to 18 °C for several days. This particular contaminant appears to originate from carrier boars, where it resides in the preputial cavity, but has also been shown to then easily contaminate the semen-processing laboratory. Screening for carrier boars to date has been performed through detection of S.marcescens in ejaculates using traditional agar plate culture techniques. These agar growth techniques are labor and time consuming due to the need for sample °titration and temporal growth followed by isolation, leading to delays in identification. The aim of this study was to develop a rapid, sensitive traditional PCR technique that can detect the presence of S.marcescens in extended boar semen. Primers for the detection of S. marcescens 16S rRNA were designed and specificity tested. After PCR optimization, assay sensitivity was evaluated using extended boar semen that was inoculated with various physiological ratios of spermatozoa: S.marcescens (100:1, 50:1, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1. 1:1 and 1:10). Samples, held at 16 °C, were tested every 24 h over a 96 h period, with bacterial DNA extraction performed at each time point using a commercial kit. As a final step, the developed technique was used to screen random samples of extended boar semen for S. marcescens contamination. Results showed that this PCR technique had a sensitivity (90%) and specificity (100%) at detecting S.marcescens in the different inoculated ratios as well as in random, naturally contaminated samples of extended boar semen. In conclusion, this study reports a traditional PCR technique that is effective at rapidly and accurately detecting the presence of S.marcescens in boar extended semen.