Evaluation of Respiration Activity for Diagnosis of Mastitis Using SECM

1. Introduction Mastitis is a generic term for biological reactions leading to damage of mammary gland tissue, which occurs when intestinal microbes invade into mammary glands. Currently, Somatic cell count (SCC) 1) is widely used as mastitis diagnosis method utilizing the fact that a large number of somatic cells are present in the milk of mastitis cattle. However, early diagnosis is difficult with this method. We have evaluated respiratory activity and respiratory burst of immune cells by Scanning electrochemical microscopy (SECM) 2) . In this study, attention was paid to the fact that neutrophils are particularly likely to increase among somatic cells, and evaluation of respiratory activity can be used as a marker for the diagnosis of mastitis. 2. Experiment 2.1 Type of milk sample used Two milk samples were obtained from Holstein cows. The somatic cell count of each of the two samples was 211 × 105 cells / mL and 31 × 105 cells / mL. 2.2 Evaluation of respiration activity for the somatic cell in milk method 2μL of the sample was put into the inverted conical well and was incubated at 37 °C for 15 minutes. Following this step, 10 mL of phosphate buffered saline (PBS) containing 11.4 mM glucose was added into the well. An Ag/AgCl electrode and a Pt electrode (d=20μm) were installed. The Pt electrode was scanned in the Z direction at the center of the well with the sample (Fig.1). Oxygen concentration profile was monitored by applying -0.5 Vvs.Ag/AgCl using SECM and scanning 500 μm in the vicinity of the sample. The scanning speed was 10 μm/s, the number of scans was kept at 3 with a sampling interval was 100 msec. 3.Results and Discussion As shown in Fig. 2, differences in reduction current value were obtained. The average reduction current (where n=3) for ΔIr was observed to be -630pA while for ΔIb, it was found to be lower, reflecting a change in reduction current value of -400 pA. The difference observed in two different densities of cell lines shows a strong correlation between the number of somatic cells and its relation to changes in reduction current. It is thus proposed that the monitoring of respiratory burst can serve as a method for early diagnosis and detection of mastitis. Acknowledgement We would like to thank Kakuda farm that provided us with milk samples and for their excellent cooperation during the course of study. References 1) M. Damm, et al., J.Dairy Sci.100:4926-4940,2017 2) H. Kikuchi, et al.,frontiers vol.7 Article25 2016 Figure 1