Mycoplasma agalactiae p40 Gene, a novel marker for diagnosis of contagious agalactia in sheep by real-time PCR: assessment of analytical performance and in-house validation using naturally contaminated milk samples

Abstract

We evaluated the capacity of the Mycoplasma agalactiae p40 gene as a diagnostic marker for contagious agalactia in sheep by quantitative real-time PCR. The p40 gene encodes an immunodominant adhesin that plays a key role in cytoadhesion of M. agalactiae. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE), a quantification that is highly linear (R(2) > 0.992) and efficient (PCR efficiency, >0.992) over a 6-log dynamic range, down to 10 GE. We evaluated the capacity of the assay to detect Mycoplasma agalactiae in 797 milk samples (373 raw sheep milk samples from refrigerated tanks of different farms and 424 milk samples from individual sheep of a flock positive for M. agalactiae). In parallel, we also tested the samples by using microbiological isolation coupled with microscopy identification and by a PCR method recommended by the World Organization for Animal Health. While our assay was able to detect 57 (15.28%) positive samples of the 373 milk samples from different farms, identification by microbiological isolation coupled with microscopy detected only 36 (9.65%) samples, and the conventional PCR detected 31 (8.31%) samples. These findings showed that our assay based on the p40 gene is more specific and sensitive for the detection of M. agalactiae in actual natural samples and, thus, can be a promising alternative tool for diagnosis and epidemiological studies of M. agalactiae infection.

FIG. 1.

Raw sheep milk sample analysis scheme. Milk samples (797) were received, and 100 μl was cultured in 10 ml of specific microbiological media (1, MBG Mycoplasma broth base with supplement G from OXOID; and 2, MA1A medium from Mycoplasma Experience) at 37°C in 5% CO₂ chambers for 6 days. DNA was then extracted using thermolysis (3) and analyzed by PCR using the p40-IAC Q-PCR assay and the method described by Tola and coworkers (27). If the PCR tested positive, DNA was directly purified from 25 ml of milk to demonstrate direct detection by avoiding cultivation and to improve the analytical performance.