Sensitivity of two methods to detect Mycoplasma agalactiae in goat milk

Abstract

BackgroundLaboratory diagnostic techniques able to detect Mycoplasma agalactiae are essential in contagious agalactia in dairy goats. This study was designed: 1) to determine the detection limits of PCR and culture in goat milk samples, 2) to examine the effects of experimental conditions including the DNA extraction method, PCR technique and storage conditions (fresh versus frozen stored milk samples) on these methods and 3), to establish agreement between PCR and culture techniques using milk samples from goats with mastitis in commercial dairy herds. The study was conducted both on artificially inoculated and field samples.ResultsOur findings indicate that culture is able to detect M. agalactiae in goat milk at lower concentrations than PCR. Qualitative detection of M.agalactiae by culture and PCR was not affected by sample freezing, though the DNA extraction method used significantly affected the results of the different PCR protocols. When clinical samples were used, both techniques showed good agreement.ConclusionsThe results from this study indicate that both culture and PCR are able to detect M. agalactiae in clinical goat mastitis samples. However, in bulk tank milk samples with presumably lower M. agalactiae concentrations, culture is recommended within the first 24 h of sample collection due to its lower limit of detection. To improve the diagnostic sensitivity of PCR in milk samples, there is a need to increase the efficiency of extracting DNA from milk samples using protocols including a previous step of enzymatic digestion.

Validation of Loop-Mediated Isothermal Amplification (LAMP) Field Tool for Rapid and Sensitive Diagnosis of Contagious Agalactia in Small Ruminants

“…[14,15,23,24]. However, it is known that performances of molecular diagnostic methods highly depend on the DNA extraction methods used and their efficiency to remove the natural inhibitors, which are present in the milk samples [25,26].…”

Milk hygiene in small ruminants: A review

“…Notwithstanding, in clinical mastitis samples the culture and PCR methods showed a good concordance due to higher M. agalactiae concentration (Tatay-Dualde et al, 2015). Contrarily to mycoplasma culture, the detection of M. agalactiae in goat milk samples by PCR resulted unaffected both by AZ or BR addition (Amores et al, 2011) and freezing of milk samples (Tatay-Dualde et al, 2015). All in all, to improve the PCR sensitivity it is recommended to extract the DNA from a specific mycoplasma culture of the milk samples, instead of from the milk sample itself (Oravcová et al, 2009).…”

“…Because of this, the culture in the first 24 hours after samples collection is recommended in BTM with low mycoplasma concentration. Notwithstanding, in clinical mastitis samples the culture and PCR methods showed a good concordance due to higher M. agalactiae concentration (Tatay-Dualde et al, 2015). Contrarily to mycoplasma culture, the detection of M. agalactiae in goat milk samples by PCR resulted unaffected both by AZ or BR addition (Amores et al, 2011) and freezing of milk samples (Tatay-Dualde et al, 2015).…”

<p>Contagious Agalactia In Sheep And Goats: Current Perspectives</p>

“…Milk (individual/pooled/tank), joint fluids, and eye swabs are relevant samples in diseased herds, and it is recommended practice to sample several animals in the same herd, due to inter-individual variability in shedding 1,107. Excretion in milk is higher in the clinical phase and might become intermittent with time,49,83,85,108 necessitating repeated samplings. Eye swabs and joint fluids tend to contain less mycoplasma (especially if lesions are not recent) and do not make the best samples in extensive surveys as they require animal containment or post-mortem sampling 43,51,84.…”