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.

Evaluation of Extraction Methods for Efficient Detection of Enteric Viruses in Pork Meat Products

“…The performance of the in-house method was similar to that achieved with 0.25 g when 1 g of pig liver was analyzed (Table 5). It is consistent with results obtained in other microbial pathogens and food products (Rodriguez-Lazaro et al 2004a, b, c, 2005a, b, c, 2006Lopez-Enriquez et al 2007;Oravcova et al 2009). Additionally the method allowed nonquantitative viral RNA detection (down to 500 pfu/reaction) in 2.5 g of spiked pig liver samples, and adequate quantitative detection of viral DNA down to 500 pfu/ sample (Table 5).…”

A survey of Mycoplasma agalactiaein dairy sheep farms in Spain

“…Consequently, in the present study we aimed to assess for the first time the presence and geographic distribution of the four Mycoplasma species causing CA, by analyzing raw milk samples from Spanish dairy sheep farms by classical microbiological methods, and PCR-based methods which have been demonstrated to be specific and sensitive [11-13]. The knowledge acquired will allow the implementation of appropriate control programs for those pathogens.…”

Molecular Characterization of Mycoplasma agalactiae Reveals the Presence of an Endemic Clone in Spain

“…All the information regarding the sampling and the isolation procedure is detailed in the work of Ariza-Miguel et al (11). The species designation of the isolates was confirmed by real-time PCR targeting the p40 gene (12,13). All isolates were analyzed by PFGE with the restriction enzyme SmaI and by MLVA at 4 highly variable VNTR loci (i.e., MagaI VNTR 5, MagaI VNTR 14, MagaI VNTR 17, and MagaI VNTR 19) as previously described (3).…”