Respiratory Burst Evaluation Using SECM in Milk from Mastitis Bovine and Healthy Bovine
Authors: Kumagai Ryoma , Prasad Ankush , Kasai Shigenobu 
Published: December 2020

1. Introduction

A large number of somatic cells are found in milk from mastitis bovine than one from healthy bovine. About 70% of the somatic cells are neutrophils and monocytes1). We monitored respiratory burst and respiration activity of human immune cells using scanning electrochemical microscopy (SECM) 2). In this study, we have described the results on the respiratory burst of neutrophils and monocytes in bovine milk using SECM.

2. Methods

2.1 Sample and cell chip preparation

We centrifuged the milk sample from mastitis bovine to remove fat. The cell number of the cell suspension obtained by centrifugation was 4.2×106 cells/mL. We then mixed the cell suspension and collagen in a 9:1 ratio. We transferred 1.5 μL aliquots of the aggregated cells to collagen-embedded and pyramidal wells that had been anisotropically etched with silicon.
Collagen embedded cells in pyramid well are called the cell chip. For measurement of normal cellular respiration, somatic cells in bovine milk were added just prior to measurement. During measurement of respiratory burst, PMA was added under the same experimental conditions to the somatic cells at a final concentration of 100 nM. We prepared the cell chip of the milk sample from healthy bovine by the same method. The cell number of the cell suspension obtained from the milk of health bovine was 2.1×105 cells/mL.

2.2 SECM evaluation of respiratory burst using cell chip

The respiration activity was measured by scanning using a Pt microelectrode in the Z direction between 30 μm (the cell suspension surface) and 530 μm (the bulk) from the cell suspension. The measurement solution was PBS buffer containing 11.4 mM glucose. The scanning distance, scanning speed and sampling time are 500 μm, 50 μm/s and 100 msec, respectively. In this study, we conducted experiments on two milk samples, one of which was diagnosed with mastitis and the other of which was control (non-mastitis), and we compared the results.

3. Results and discussion

Fig. 1 (A) shows the result of mastitis sample, and (B) shows the result from a healthy sample. The working electrode was located 30 μm above the well top (N) at t = 0, 20, 40, and 60 s. The electrode was located 530 μm above the well top (Bulk) at t = 10, 30, and 50 s. From Fig. 1, in all measurement results, the reduction current value is lower at Point N than at Bulk. This indicates that the dissolved oxygen concentration in the vicinity is decreased due to the respiration of somatic cells. We calculated the average ΔI as the electrode moved up and down during N-bulk each of the three scans. The change in the oxygen concentration (ΔC) at 25 °C was calculated assuming a dissolved oxygen content of 248 μM and an oxygen reduction current of -1.7 nA (summarized in Table.1). From the PMA (-) experimental results, the magnitude relationship between the number of somatic cells and the oxygen consumption rate agree. Moreover, the values at PMA (+) are higher in both samples than PMA (-). This result suggests that PMA induced respiratory burst and increased respiratory volume of neutrophils and monocytes. The value of F in the mastitis sample is about 5 times that during normal respiration and about the value of F in the health sample is about 3 times during normal respiration. This suggests that the mastitis bovine has a higher proportion of neutrophils and monocytes in the somatic cells than healthy bovine. This result is consistent with the literature1). This result suggests that SECM respiratory burst assessment can distinguish mastitis bovine from healthy bovine. In addition, we suggest we can discover early stage of mastitis by measuring the increase in neutrophils.

Reference

M. Damm, C. Holm, M. Blaabjerg, M. N. Bro,D. Schwarz: J.Dairy Sci.100, 4926-4940 (2017).

H. Kikuchi, A. Prasad, R. Matsuoka, S. Aoyagi, T. Matsue, S. Kasai, Frontiers in Physiology 7, 25, 1-6 (2016)

Figure 1