DNA carryover in milk samples from routine milk recording used for PCR-based diagnosis of bovine Staphylococcus aureus mastitis

Abstract

Real-time PCR techniques are increasingly used to detect udder pathogens from milk samples collected non-aseptically at routine milk recording. The objectives of this study were (1) to estimate the statistical associations between cycle threshold (Ct) values for Staphylococcus aureus in non-aseptically collected composite samples taken at routine milk recording from cows milked consecutively with the same milking unit and milk meter; and (2) to formulate practical and plausible guidelines for understanding the diagnostic implications of PCR testing for Staph. aureus intramammary infection at routine milk recording. The study included 4 herds with conventional milking parlors and repeatedly low Ct-values for Staph. aureus (representing a high DNA load) in bulk tank milk. Composite milk samples were collected from all cows at all milking units during routine milk recording using the Tru-Test electronic milk meter (Tru-Test Group, Auckland, New Zealand) and analyzed using the PathoProof PCR (Thermo Fisher Scientific, Vantaa, Finland) assay. Milking clock times were retrieved at each milk meter to establish the milking order of the cows at each unit. A multinomial logistic regression was applied to estimate the association between Ct-values from cows milked consecutively with the same milking unit and milk meter. The following groups were selected based on Ct-values: (1) 0-31.3, (2) 31.4-33.9, (3) 34.0-37, (4) 37.1-39.9, and (5) 40 (negative result). The association between groups from cows milked consecutively with the same milking unit and milk meter was statistically significant. Approximately 60% of cows were in Ct group 5 if the antecedent cow was also in Ct group 5, but only 20% of cows were in Ct group 5 if the antecedent cow was in Ct group 1. The probability of cows being in Ct group 1 was not markedly influenced by the group of the antecedent cow. Statistical relationships in the intermediate range gave a plausible indication of a dose-response relationship. Carryover of bacterial DNA via the milking unit and milk meter is very likely to affect PCR results for Staph. aureus. Therefore, information about milking order must be considered in mastitis control efforts. We suggest a practical interpretation of PCR results: cows with a Ct-value <32 can be labeled "very likely to be infected with Staph. aureus," but cows with Ct-values of >37 and 32-37 can be labeled "very likely to be negative for Staph. aureus" and "uncertain Staph. aureus status," respectively.

Comparison of quantitative PCR and MALDI‐TOF mass spectrometry assays for identification of bacteria in milk samples from cows with subclinical mastitis

“…aureus and Strep. agalactiae mastitis with proposal of minor modifications, especially for diagnosis of subclinical infections (Cederlöf et al, ; Mahmmod et al, , ). This was not considered in this study since the protocol recommended by the company was followed, but more studies are clearly needed; the classical bacteriology can only characterize alive bacteria while the qPCR assays can detect the DNA of bacteria whether dead or alive (Taponen et al, ); when low bacterial loads are present in the milk samples, the number of colonies can be below the cut‐off values (100 CFU per ml in this study) and the samples classified as negative, while the qPCR assays would probably classify the same sample as positive (Hiitiö et al, ); the possible presence of antibiotic residues in the milk interfering with the bacterial growth, but not affecting the qPCR assays (Zadoks et al, ).…”

Genomic investigation of Staphylococcus aureus isolates from bulk tank milk and dairy cows with clinical mastitis

“…Furthermore, the BTM isolates were collected from farms that had shown an increased concentration of S. aureus in BTM (PCR Ct-values: 21-27). A study suggests that Ct-values < 32 very likely can be interpreted as reflecting S. aureus intra-mammary infections (Mahmmod et al, 2017).…”

Possibility of identifying plant components of the diet of Harpalus rufipes (Coleoptera, Carabidae) by visual evaluation

“…Почти 100% наличие бактерий рода Staphylococcus в пробах молочных продуктов домашнего производства можно объяснить тем, что эти молочные продукты изготавливаются из сырого термически не обработанного молока коров с нарушением санитарно-гигиенических норм и правил. Стафилококки составляют так называемую резидентную (полезную) микрофлору кожи вымени коров и почти всегда их можно выделить из сырого молока (Febler et al, 2010;Anderson et al, 2012;Mahmmod et al, 2017, Kukhtyn et al, 2017. Поэтому сапрофитные стафилококки часто выделяются из необработанных молочных продуктов домашнего производства.…”