Bovine mastitis is the most costly disease for dairy farmers, hence, control measures to prevent it are crucial for dairy farm sustainability. Staphylococcus aureus is considered a major mastitis pathogen because of its impact on milk quality and low cure rates. Prevention of S. aureus mastitis includes segregation of infected animals, whilst treatment of such animals should be performed for a longer time to improve cure rates. This makes identification of S. aureus infected quarters and animals of significant importance. The experiments reported in this research paper aimed to develop and validate a sensitive method for magnetic detection of S. aureus and of the Staphylococcus genus in raw milk samples. Mastitic milk samples were collected aseptically from 47 cows with subclinical mastitis, from 12 Portuguese dairy farms. Forty nine quarter milk samples were selected based on bacteriological results. All samples were submitted to PCR analysis. In parallel, these milk samples were mixed with a solution combining specific antibodies and magnetic nanoparticles, to be analysed using a lab-on-a-chip magnetoresistive cytometer, with microfluidic sample handling. The antibodies used in this work were a rabbit polyclonal IgG anti-S. aureus ScpA protein and a mouse monoclonal IgM anti-S. aureus ATCC 29740. This paper describes the methodology used for magnetic detection of bacteria, including analysis of false positive/negative results. This immunological recognition was able to detect bacterial presence above 100 cfu/ml, independently of antibody and targeted bacteria used in this work. Comparison with PCR results showed sensitivities of 57·1 and 79·3%, specificity values of 75 and 50%, and PPV values of 40 and 95·8% for magnetic identification of Staphylococci species with an anti-S. aureus antibody and an anti-Staphylococcus spp. antibody, respectively. Some constraints are described as well as the method's limitations in bacterial quantification. Sensitivities and specificities require to be improved, nevertheless, the methodology described may form the basis for a means of identifying S. aureus infected cows at the point of care.

Geographically related Staphylococcus epidermidis isolates from human patients (n=30), dairy farms (farmers and individual raw milk from cattle, n=36) and a dairy plant (n=55) were examined for epidemiological relatedness by pulsed-field gel electrophoresis and, using in vitro methods, for the ability to produce biofilm and antimicrobial resistance. Methicillin-resistant isolates (MRSE) were also identified and characterized. Isolates from farmers and dairy cattle were found to be genetically related, while isolates from human patients were highly diverse. Some dairy plant isolates (18·2%) were closely related to those from dairy farms. Biofilm production and resistance to antimicrobial agents were most typical for isolates from human patients, of which 76·7% were MRSE. Methicillin resistance was also widespread in farm-related isolates (61·1%). This study indicates the possible transmission of S. epidermidis between cattle and farmers. Dairy products were not proven to be an important source of either human infections or methicillin-resistant strains.

This article presents a study on the influence of the protocol used for immobilization of bacterial cells onto surfaces by mechanically trapping them into a filter. In this sense, the surface and structure of trapped cells are analyzed. Bacteria can be present solely or with extracellular polymeric substances (EPS). To test the behavior of the EPS layer duing the filtering process, different strains of a well-known EPS-producer bacteria (Staphylococcus epidermidis), which produce an extracellular matrix clearly visible in AFM images, have been used. Results show that this immobilization method can cause severe structural and mechanical deformation to the cell membrane. This altered mechanical state may possibly influence the parameters derived from AFM force curves (which are micro/nano-mechanical tests). Also, our results suggest that the EPS layer might move during the filtering process and could accumulate at the upper part of the cell, thus favoring distorted data of adhesion/pull-off forces as measured by an AFM tip, especially in the case of submicron-sized microbial cells such as bacteria.

The responses of five lactating East Friesian milk ewes to experimental mammary infection with Staphylococcus epidermidis and of five control ewes were examined over a period of 10 weeks. Infection caused an influx of neutrophils into milk, the numbers of which started to rise 4 h post infection and peaked 24 h after infection. The initial response was accompanied by mild fever and mild leucopaenia in blood (8 h after infection). No other signs of systemic infection were observed. Milk appeared normal at all times, although the milk yield of infected ewes tended to decline. Staphylococci were absent in milk from four ewes at 2 d and at 3 d after infection, but re-emerged intermittently in four of five ewes at subsequent samplings. Cytokines in milk were measured by ELISA. IL-8 was elevated in infected glands at 2 h and peaked at 8 h. In the four ewes intermittently shedding bacteria, IL-8 remained elevated until the final sampling at 10 weeks. IL-1β was transiently elevated at 1 d and 2 d and showed a pronounced peak in one sheep. Milk samples from this ewe were bacteriologically negative, somatic cell count (SCC) was within the normal range and the concentrations of IL-1β, as well as IL-8, were similiar to the control group (n=5) from 1 week after infection until the final sampling. Histological examination revealed leucocytic infiltrates in the four glands remaining infected at the end of the experiment, and a high level of CD5+ lymphocytes in three ewes. The results suggest that the relationship between the initial neutrophil influx and the proinflammatory cytokines may be responsible for determining the course of infection. Subclinical mastitis due to coagulase-negative staphylococci leads to minor changes in milk yield and milk constituents.