Estimation of Performance Characteristics of Analytical Methods for Mycobacterium avium subsp. paratuberculosis Detection in Dairy Products

Paratuberculosis is a chronic enteric infection, caused by Mycobacterium avium subsp. paratuberculosis (MAP), affecting virtually all ruminants as well as other animals. MAP is also suspected to be involved in the etiology of some human diseases, like Crohn’s disease and others. In surveillance studies, different analytical methodologies were employed to detect MAP, showing different results and incidence in dairy products. The aim of this study was to evaluate the performance characteristics of three analytical methods [culture, quantitative PCR (qPCR) and peptide-mediated magnetic separation (PMS) phage-based assay] for MAP detection in raw, heat-treated and powdered milk. The methods were evaluated according to performance characteristics defined for qualitative methods in ISO 16140-2:2016. To estimate sensitivity (including trueness) and LOD, 720, and 900 test portions, respectively, were blind tested by two laboratories. Considering all matrices, different sensitivities, expressed as the percentage of positives from the total of true positive test portions, were obtained for IS900 qPCR (94%), f57 qPCR (76%), culture (83%), and PMS-phage (40%). Trueness, expressed as results correctly assigned (including positive and negative) to the reference value, was 93% for the IS900 qPCR method, 89% for culture and 49% for the PMS-phage. The LODs obtained in this study were similar to the LODs previously published for cultural and qPCR methods. However, for the PMS-phage method, the obtained results showed higher LOD values compared to the limited data available in the scientific literature. Our results highlight that while the PMS-phage assay is workable in pure liquid culture for estimation of MAP counts, its usage for surveillance of dairy matrices should be treated with a lot of caution as performance characteristics obtained were lower than for the two other methods tested. qPCR and culture are the most appropriate methods to detect MAP in milk-based matrices according to ISO 16140 methodology. Cultural techniques are considered the gold standard for detection of viable MAP, but qPCR, which is widely used in analytical and surveillance studies, can be considered a suitable and recommendable alternative to cultural methods for screening, if confirmation of MAP’s viability is not requested

Comparaison de différentes méthodes de concentration et de détection des virus entériques dans des boues

Deux méthodes appliquées à la recherche des virus entériques dans les boues de station d’épuration ont été étudiées. Dans un premier temps, les deux méthodes, comprenant chacune une étape de concentration et une étape de détection par culture cellulaire, ont été comparées entre elles. Dans un second temps, seules les techniques de concentration ont été comparées en analysant les concentrats par RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). Trois types de virus entériques (entérovirus, rotavirus, virus de l’hépatite A) ont été recherchés dans différentes boues (stabilisées ou non et chaulées ou non). Les résultats obtenus avec les deux méthodes sont cohérents. Toutefois, l’une d’entre elles, bien que légèrement moins sensible, offre un certain nombre d’avantages, notamment en termes de facilité de mise en œuvre et de délai de réponse. L’utilisation de la RT-PCR pour cette comparaison a permis de confirmer l’intérêt de cette technique en termes de coût et de rapidité. Elle constitue également une alternative intéressante pour les virus qui se multiplient mal en culture cellulaire. Enfin, les résultats des différentes analyses réalisées au cours de cette étude, et notamment les résultats de dénombrement, montrent que le procédé d’hygiénisation des boues permet de diminuer l’infectiosité des entérovirus

Inactivation of viruses and bacteria on strawberries using a levulinic acid plus sodium dodecyl sulfate based sanitizer, taking sensorial and chemical food safety aspects into account

The efficacy of levulinic acid (LVA) in combination with sodium dodecyl sulfate (SDS) in removal of foodbome viruses, enteric bacterial pathogens and their surrogates on fresh strawberries was investigated. Inoculated strawberries were treated with potable water, sodium hypochlorite solution (50 ppm), 0.5% LVA plus 0.5% SDS solution, and 5% LVA plus 2% SDS solution respectively for 2 min, followed by spray-rinsing with potable water. Water washing removed at least 1.0-log of the tested viral and bacterial strains from the strawberries' surfaces. The 50 ppm chlorine wash induced 3.4, 1.5 and 2.1-log reductions for hepatitis A virus (HAV), murine norovirus-1 (MNV-1) and MS2 bacteriophage, respectively. In comparison, the tested bacterial strains showed uniform reductions around 1.6-log CFU/ml. The 0.5% LVA plus 0.5% SDS wash induced 2.7, 1.4 and 2.4-log reductions for HAV, MNV-1 and MS2, which were comparable with the reductions induced by chlorine (P > 0.05). For bacteria, over 2.0-log reductions were obtained for Enterococcus faeciurn, Listeria monocytogenes and Salmonella, while Escherichia coli 0157:H7 and Escherichia coli P1 showed reductions of 1.9 and 1.8-log CFU/ml. Higher concentration of LVA plus SDS showed no significantly higher reductions (P > 0.05). Sensory tests of washed strawberries and chemical residue analysis of LVA on strawberries after washing were also performed. In conclusion, this study demonstrates good performance of 0.5% LVA plus 0.5% SDS to reduce the levels of enteric pathogens if present on strawberries without altering taste and introducing chemical safety issues