Method for rapidly detecting intracellular staphylococcus aureus of dairy cow mammary gland cells

Abstract

The invention discloses a method for rapidly detecting intracellular staphylococcus aureus of dairy cow mammary gland cells, and belongs to the technical field of bacterial detection. In order to solve the technical problem that a rapid detection method for the intracellular staphylococcus aureus of the dairy cow mammary epithelial cells is lacked at present, a model for invading the dairy cow mammary epithelial cells by the staphylococcus aureus is established, and a microscopy technology based on cell perforation and Gram staining is established. The method for rapidly and accurately detecting the intracellular infection of the staphylococcus aureus verifies the accuracy of detecting the intracellular infection of the staphylococcus aureus by a'perforin-gram staining method '. The method provided by the invention has the advantages of rapidness, accuracy, simplicity, convenience, low cost and the like, can be used for carrying out specific treatment or eliminating cattle earlier in a cattle farm so as to reduce economic loss, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of bacterial detection, and particularly relates to a method for rapidly detecting intracellular Staphylococcus aureus in dairy cow mammary gland cells. Background Art

[0002] Dairy cow mastitis is a common dairy cow disease in which the mammary glands of dairy cows undergo inflammatory changes under the stimulation of external factors. This disease is widely prevalent at home and abroad, causing huge economic losses. S. aureus is one of the main pathogenic bacteria causing dairy cow mastitis and has a potential risk of zoonosis. Dairy cow mastitis caused by S. aureus is mostly subclinical and chronic, and is characterized by low cure rate and low pathogen elimination rate (Du Preez JH. Bovine mastitis therapy and why it fails. Journal of the South African Veterinary Association. 2000, 71(3): 201-208.). With the widespread use of antibacterial drugs in clinical practice and animal husbandry, the problem of S. aureus drug resistance has become increasingly serious, and multi-drug resistant (MDR) strains and superbugs - methicillin-resistant Staphylococcus aureus (MRSA) have emerged (Zhang Yue. Isolation, identification and drug resistance study of the main pathogenic bacteria of dairy cow mastitis in different regions of Hebei Province [D]. Hebei Agricultural University, 2016.). The complex S. aureus escape mechanism and drug resistance make S. aureus mastitis more difficult to cure. However, in dairy cow farms, this type of dairy cow mastitis is not taken seriously, and antibiotics are only used for a long time for dairy cows with repeated illness and no recovery. When under long-term antibiotic selection pressure, S. aureus will show drug resistance to a variety of antibacterial drugs, thus forming a vicious cycle. Therefore, the massive abuse of antibiotics has led to difficulties in treating dairy cow mastitis and low cure rates.

[0003] S. aureus can be surrounded by thick fibrous scar tissue in mammary gland cells, so that antibiotics cannot reach and eliminate the bacteria. Even microorganisms sensitive to the antibiotics used may not achieve the expected therapeutic effect (Linder M, Paduch JH, Grieger AS, et al. Heilungsraten chronischer subklinischer Staphylococcus aureus-Mastitiden nach antibiotischer Therapie beilaktierenden Milchkühen [Cure rates of chronic subclinical Staphylococcus aureus mastitis in lactating dairy cows after antibiotic therapy]. Berliner Und Munchener Tierarztliche Wochenschrift. 2013, 126(7-8): 291-296.). After entering the mammary gland through the teat canal and adapting to the in vivo environment, the bacteria can rapidly multiply and enter bovine mammary epithelial cells, resulting in tissue damage (Sutra L, Poutrel B. Virulence factors involved in the pathogenesis of bovine intramammary infections due to Staphylococcus aureus. Journal of Medical Microbiology. 1994, 40(2): 79-89.).The adhesion of S. aureus to epithelial cells is a key step in the infection process (Cifrian E, Guidry AJ, O'Brien CN, et al. Adherence of Staphylococcus aureus to cultured bovine mammary epithelial cells. Journal of Dairy Science. 1994, 77(4):970-983.). Although S. aureus is not generally considered an intracellular pathogen, an increasing number of studies have shown that it can parasitize within non-phagocytic cells (Ogawa SK, Yurberg ER, Hatcher VB, et al. Bacterial adherence to human endothelial cells in vitro. Infection and Immunity. 1985, 50(1):218-224.). Previous studies have shown that S. aureus has the ability to adhere to, penetrate, and divide in human and bovine aortic intimal cell lines, as well as in the bovine mammary epithelial cell line (MAC-T) (Hamill RJ, Vann JM, Proctor RA. Phagocytosis of Staphylococcus aureus by cultured bovine aortic endothelial cells: model for postadherence events in endovascular infections. Infection and Immunity. 1986, 54(3):833-836.). Intracellular parasitism is the key for S. aureus to evade host immunity and drug resistance. Therefore, clarifying whether S. aureus intracellular infection occurs in dairy cows is of great significance for the prevention and control of S. aureus mastitis in dairy cows. If it is intracellular infection, alternative antibiotic treatment strategies such as antimicrobial peptides or probiotics need to be considered. Currently, phage therapy is also in the research stage (Chang Junshuai, Zhang Qi, Cheng Lulu. Induction of lysogenic phages from Staphylococcus aureus isolated from dairy cow mastitis and analysis of their lytic ability [J]. China Dairy Cattle 2022, (2):27-31.). Such a more targeted treatment strategy can improve the cure rate of mastitis in the entire dairy cow herd.

[0004] There are currently several methods for S. aureus intracellular infectious bovine mastitis, such as PCR for detecting intracellular bacterial DNA, laser confocal microscopy, and transmission electron microscopy. The PCR method uses DNase I to remove extracellular bacterial DNA and CCK8 technology to prove the integrity of the cell membrane, and then detects whether there is S. aureus DNA in the lysed cell sample (Hébert A, Sayasith K, Sénéchal S, Dubreuil P, Lagacé J. Demonstration of intracellular Staphylococcus aureus in bovine mastitis alveolar cells and macrophages isolated from naturally infected cow milk. FEMS Microbiol Lett. 2000 Dec 1;193(1):57-62.). Its experimental steps are complicated, time-consuming, the prices of instruments and consumables are high, and it is also very inconvenient to control the dosage of DNase I. With the development of scientific instruments and equipment, more precise instruments have begun to be used to observe intracellular structures. The emergence of laser confocal microscopes can better observe the morphology and quantity of Staphylococcus aureus in bovine mammary epithelial cells. However, this method requires high-precision experimental instruments, is time-consuming (6-7 hours), the prices of antibodies and the like are high, and it is also relatively cumbersome. Transmission electron microscopy irradiation is also the most commonly used method for observing intracellular morphological structures. However, the preparation of the samples to be detected takes 3 days, the experimental instruments are expensive, and professional personnel are required for operation. None of these three methods can achieve low-cost, rapid, accurate, and point-of-care testing, which is not applicable to dairy testing stations that need to detect a large number of diseased cows. Therefore, there is an urgent need to develop a low-cost, rapid, accurate, and point-of-care testing method for judging whether S. aureus intracellular infection has occurred. Summary of the Invention

[0005] In order to solve the technical problem of the lack of a rapid detection method for Staphylococcus aureus intracellular in bovine mammary epithelial cells, based on the previous experimental results, the present invention found that Staphylococcus aureus exists in both mammary epithelial cells and macrophages in the milk of cows suffering from Staphylococcus aureus mastitis, established a model of Staphylococcus aureus invading bovine mammary epithelial cells, and established a method for rapid and accurate detection of Staphylococcus aureus intracellular infection based on cell perforation and Gram staining microscopy, and verified the accuracy of the "perforin-Gram staining method" for detecting Staphylococcus aureus intracellular infection.

[0006] To solve the above technical problems and achieve the corresponding technical effects, the present invention specifically provides the following technical solutions:

[0007] The first object of the present invention is to provide a method for rapidly detecting intracellular Staphylococcus aureus in dairy cow mammary gland cells, and the method comprises the following steps:

[0008] S1. Establish an intracellular infection model of Staphylococcus aureus: co-incubate a dairy cow mammary epithelial cell line with Staphylococcus aureus, add lysostaphin at a concentration of 100 μg / mL and continue to incubate for 15 min to remove extracellular bacteria, and wash three times with PBS to obtain a cell sample;

[0009] S2. Permeate the cell sample with 0.3% Triton X-100 by mass for 15 min, wash 3 times with PBS, perform Gram staining, and observe Gram-positive particulate matters in the sample with an oil immersion lens of an optical microscope.

[0010] In an embodiment of the present invention, the isolation method of the dairy cow mammary epithelial cell line described in S1 is as follows: collect milk and centrifuge it. The milk is divided into three layers, the upper layer is milk fat, the middle layer is milky whey, and the lower layer is white precipitate. Discard the upper milk fat and most of the whey, leave 3 mL of whey and resuspend it with the white precipitate and transfer it to a 15 mL centrifuge tube, add triple antibiotics with a final concentration of 10% to 15 mL, mix well and centrifuge. Repeat the above steps until the supernatant after centrifugation gradually becomes clear. Discard the supernatant, add PBS to resuspend, add a macrophage scavenger, triple antibiotics, gentamicin and lysostaphin to the obtained somatic cells. After incubation, discard the supernatant and wash three times with PBS.

[0011] In an embodiment of the present invention, the dairy cow mammary epithelial cell line described in S1 is co-incubated with Staphylococcus aureus when the cell density reaches 80%.

[0012] In an embodiment of the present invention, the concentration of the Staphylococcus aureus described in S1 is 1×10 9 CFU / mL.

[0013] In an embodiment of the present invention, MOI = 8 during the co-incubation process described in S1.

[0014] In an embodiment of the present invention, the temperature of the co-incubation described in S1 is 37 °C and the time is 2 h.

[0015] In an embodiment of the present invention, the 0.3% Triton X-100 by mass described in S2 is diluted with 200 μL of PBS.

[0016] In an embodiment of the present invention, the washing time for each PBS wash described in S2 is 5 min.

[0017] In one embodiment of the present invention, the steps of the Gram staining in S2 are as follows: drop 1 drop of ammonium oxalate crystal violet staining solution and stain for 1 min; after rinsing thoroughly with distilled water, then drop another drop of Gram staining solution and stain for 1 min; rinse the culture dish with distilled water, drop ethanol with a volume fraction of 95% on the culture dish until the flowing ethanol is no longer purple, and after 20 s, rinse with distilled water; drop 1 drop of safranin staining solution and stain for 1 min, and then rinse the culture dish with distilled water.

[0018] The second object of the present invention is to provide the application of the above method in the epidemiological investigation of intracellular infectious bovine mastitis caused by Staphylococcus aureus.

[0019] The beneficial effects of the present invention:

[0020] The present invention establishes a model for Staphylococcus aureus to invade bovine mammary epithelial cells, and establishes a method based on cell perforation and Gram staining microscopy for rapid and accurate detection of intracellular infection of Staphylococcus aureus, verifying the accuracy of the "perforin-Gram staining method" for detecting intracellular infection of Staphylococcus aureus. The method provided by the present invention has the advantages of being rapid, accurate, simple, and low-cost, and has broad application prospects.

[0021] The "perforin-Gram staining method" provided by the present invention takes only 3 h from obtaining milk samples to cell extraction until the final staining, and can process a large number of samples simultaneously. It is inexpensive and does not require special instrument equipment, and farmers can conduct on-site self-examination. By comparing with the results of the gold standard, the accuracy of this detection method is also proved.

[0022] Through large-scale epidemiological investigations of dairy cows in dairy farms, it is also clear that the proportion of dairy cows with intracellular infectious bovine mastitis caused by Staphylococcus aureus among dairy cows with Staphylococcus aureus bovine mastitis is 82.9%, indicating that intracellular infectious bovine mastitis caused by Staphylococcus aureus is a highly prevalent type of mastitis worthy of attention. The "perforin-Gram staining method" provided by the present invention enables dairy farms to carry out specific treatment or cull cows earlier to reduce economic losses. Description of the Drawings

[0023] Figure 1 It is a result diagram for detecting the infection of Staphylococcus aureus in bovine mammary gland cells without using Triton X-100 permeabilization based on the perforin-Gram staining method; Figure 1 The magnification is 1000 times;

[0024] Figure 2 It is a result diagram for detecting the infection of Staphylococcus aureus in bovine mammary gland cells by only using Triton X-100 permeabilization without adding lysostaphin incubation based on the perforin-Gram staining method; Figure 2 The magnification is 1000 times;

[0025] Figure 3 The figure shows the result of detecting the infection of Staphylococcus aureus in dairy cow mammary gland cells by perforin-Gram staining method without adding lysostaphin incubation and without permeabilizing with Triton X-100; Figure 3 The magnification is 1000 times;

[0026] Figure 4 The figure shows the result of detecting the infection of Staphylococcus aureus in dairy cow mammary gland cells by perforin-Gram staining method; Figure 4 The magnification is 1000 times;

[0027] Figure 5 The figure shows the result of observing Staphylococcus aureus in dairy cow mammary gland cells by transmission electron microscope; Figure 5 The scale bar of A in is 2μm, Figure 5 The scale bar of B in is 1μm, → points to Staphylococcus aureus, * points to autophagosome;

[0028] Figure 6 The figure shows the result of detecting Staphylococcus aureus in dairy cow mammary gland cells by laser confocal microscope; among them, the high-magnification image of the contour area is shown on the right, → points to Staphylococcus aureus;

[0029] Figure 7 The figure shows the result of PCR detection of Staphylococcus aureus in dairy cow mammary gland cells; among them, 1 is Staphylococcus aureus, 2 is Staphylococcus aureus, 3 is negative control, 4 is Staphylococcus aureus. Detailed implementation manners

[0030] The following further elaborates on the present invention in conjunction with specific embodiments and the accompanying drawings. The following embodiments facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The drugs, reagents, and materials used in the following embodiments can all be obtained through commercial channels unless otherwise specified.

[0031] Sources of bacteria and clinical samples:

[0032] The dairy cow mammary epithelial cell line (MAC-T) and the S. aureus standard strain USA300 (ATCC BAA-1560) were both provided by Heilongjiang Bayi Agricultural University (Provincial Key Laboratory for the Prevention and Control of Bovine Diseases), and the milk samples were collected from a dairy farm in Daqing City, Heilongjiang Province.

[0033] The method for isolating mammary epithelial cells from milk is as follows:

[0034] Collect the milk of dairy cows and store it at 4°C for later use. Put 200 mL of milk into a centrifuge tube, centrifuge it at 4°C and 1000 r / min for 10 min. After centrifugation, the milk is divided into three layers: the upper layer is milk fat, the middle layer is milky whey, and the lower layer is white precipitate. Discard the upper milk fat and most of the whey, leave 3 mL of whey, resuspend it with the white precipitate, transfer it to a 15 mL centrifuge tube, and add 10% triple antibody diluted with PBS to 15 mL. After mixing, centrifuge at 1000 r / min. Then repeat this step until the supernatant after centrifugation gradually becomes clear. Discard the supernatant, add 1 mL of PBS to resuspend, transfer the obtained somatic cells to a 24-well plate with coverslips, add macrophage scavenger at 100 μg / mL, triple antibody at 500 μg / mL, gentamicin at 500 μg / mL, and lysostaphin at 100 μg / mL to each well. Incubate at 37°C for 30 min, then discard the supernatant, and wash three times with PBS for later detection.

[0035] Sources of the main reagents involved in the present invention:

[0036] Lysostaphin was purchased from Shanghai Macklin Biochemical Co., Ltd.; macrophage scavenger was purchased from Target Technology (Beijing) Co., Ltd.; Triton X-100 was purchased from BioFroxx Biotechnology Co., Ltd. in Germany; mouse anti-tubulin α antibody was purchased from Beijing Protein Innovation Co., Ltd.; 10×PCR buffer, TaKaRa Taq, dNTPs, and DNA Marker DL2000 were all purchased from TaKaRa Biotechnology (Dalian) Co., Ltd.

[0037] Example 1: A method for rapid detection of Staphylococcus aureus in dairy cow mammary gland cells

[0038] 1. Establishment of an intracellular infection model of Staphylococcus aureus

[0039] Culture Staphylococcus aureus USA 300 in Luria-Bertani (LB) broth at 37°C for 8 h, wash it three times with phosphate buffer (PBS) and set aside. Inoculate the dairy cow mammary epithelial cell line (MAC-T) into a six-well plate with coverslips for culture. When the cell density reaches 80%, incubate it with Staphylococcus aureus USA 300 at a concentration of 1×10 9 CFU / mL (MOI = 8) in a 37°C incubator for 2 h. Then add lysostaphin (100 μg / mL) and continue to incubate for 15 min to remove extracellular bacteria, and finally wash three times with PBS.

[0040] 2. Detection of Staphylococcus aureus infection in dairy cow mammary gland cells by perforin-Gram staining

[0041] The cell samples were permeabilized with 0.3% Triton X-100 diluted with 200 μL of PBS for 15 min, washed 3 times with PBS for 5 min each for subsequent Gram staining; first, 1 drop of ammonium oxalate crystal violet staining solution was added and stained for 1 min; after rinsing thoroughly with distilled water, 1 drop of Gram staining solution was added again and stained for 1 min; the culture dish was rinsed with distilled water, 95% ethanol by volume was added dropwise to the culture dish until the flowing ethanol was not purple, and after 20 s, it was rinsed with distilled water; 1 drop of safranin staining solution was added and stained for 1 min, and the culture dish was rinsed with distilled water; the Gram-positive particulate matter in the sample was observed with an oil immersion objective of an optical microscope.

[0042] Triton X-100 is a commonly used non-ionic surfactant and emulsifier that can permeabilize the cell membrane of living cells. Lysostaphin can cleave the polyglycine cross-linking bonds in the peptidoglycan layer of the cell wall of Staphylococcus spp. and was used in this study to eliminate extracellular Staphylococcus aureus.

[0043] As Figure 1 shown, in the model group without permeabilization with Triton X-100, no Staphylococcus aureus was seen in MAC-T, indicating that without perforin, Gram staining could not stain Staphylococcus aureus in MAC-T; in the treatment group incubated only with Triton X-100 without adding lysostaphin, a large number of Staphylococcus aureus were observed in the field of view, and it was impossible to distinguish intracellular bacteria from extracellular bacteria ( Figure 2 ); in the treatment group incubated without adding lysostaphin and perforin, extracellular Staphylococcus aureus was observed in the field of view, but intracellular bacteria could not be observed ( Figure 3 ); in the treatment group of the perforin-Gram staining method incubated with lysostaphin and Triton X-100, Staphylococcus aureus in MAC-T could be clearly observed ( Figure 4 ).

[0044] 3. Comparison of the detection effects of the perforin-Gram staining method provided by the present invention with transmission electron microscopy, laser confocal microscopy and PCR

[0045] (1) Method for observing Staphylococcus aureus in dairy cow mammary gland cells by transmission electron microscopy

[0046] The samples were fixed with 2.5% glutaraldehyde and 5% formaldehyde by mass for 2 h, washed 3 times with PBS, and then fixed with 1% osmium tetroxide by mass for 2 h. All the above operations were carried out at 4 °C. Subsequently, the samples were dehydrated with acetone at 50%, 70%, 80%, 90% and 100% by mass for 15 min respectively. The samples were immersed in a mixed solution of embedding solution and propylene oxide with a volume ratio of 1:1 and 3:1 at room temperature for 5 h, and then immersed in the embedding solution for 5 h. Finally, they were left standing at 37 °C for 12 h, at 45 °C for 24 h, and at 60 °C for 24 h. Then, ultra-thin sections were prepared and stained, and transmission electron microscopy was used to observe Staphylococcus aureus in cells.

[0047] It can be seen from Figure 5 that there are black spherical substances with a diameter of about 1 μm in MAC-T, which are Staphylococcus aureus. There are many autophagosomes in the cells, and autophagosome membrane structures are presented around Staphylococcus aureus.

[0048] (2) Method for detecting Staphylococcus aureus in dairy cow mammary gland cells by laser confocal microscopy

[0049] The cell slides were taken out and fixed with 4% paraformaldehyde by mass for 15 min, permeated with 0.3% Triton X-100 for 15 min, and blocked with 5% skim milk at room temperature for 1 h. Incubated with anti-tubulin α antibody (1:200) at 37 °C for 2 h, and the cells were washed 3 times with PBS, 10 min each time. Then incubated with goat anti-mouse peroxidase-conjugated AffiniPure (1:100) secondary antibody for 1 h, and the cells were washed three times with PBS again, 10 min each time. After staining with 100 μL of 4,6-diamidino-2'-phenylindole dihydrochloride (DAPI), the cell culture dish was sealed with an anti-quenching mounting medium. The DNA staining outside the cell nucleus was observed with a laser confocal microscope ×63 (1.3 numerical aperture) oil immersion objective lens.

[0050] As Figure 6 shown, the Blank group was the negative control group of MAC-T cells not co-cultured with Staphylococcus aureus; the Test group was the positive group of MAC-T cells co-cultured with Staphylococcus aureus. It could be observed that the cytoskeleton labeled with α-tubulin wrapped the DNA substance outside the cell nucleus labeled with DAPI, which was Staphylococcus aureus; the Without lysostaphin group was the group without adding lysostaphin. It could be observed that there were unkilled Staphylococcus aureus outside the cytoskeleton, which verified that lysostaphin (100 μg / mL) could completely and effectively remove extracellular bacteria by treating the cells at 37 °C for 15 min.

[0051] (3) Method for detecting Staphylococcus aureus in dairy cow mammary gland cells by PCR

[0052] The obtained samples were incubated with DNase I (concentration 0.5 U / μL) and MgCl 2 (10 mM) at 37 °C for 30 min, centrifuged at 1500 r / min for 5 min, the supernatant was discarded, and then washed three times with PBS; 150 μL of PBS was added to each tube, and then the samples were boiled in boiling water at 100 °C for 10 min, centrifuged at 12000 r / min, and the supernatant was taken for subsequent identification of the staphylococcal nuclease gene (nuc). According to the nuc gene sequence, primers were designed and synthesized, and the primer sequences were as follows: SF: 5'-GCGATTGATGGTGATACGGTT-3' (SEQ ID NO.1); SR: 5'-AGCCAAGCCTTGACGAACTAAAGC-3' (SEQ ID NO.2). PCR reaction system: ddH 2 O 9.5 μL, DNA template 1 μL, 2×Taq PCR Master Mix 12.5 μL, upstream primer 1 μL, downstream primer 1 μL. PCR reaction program: preheat at 94.0 °C for 5 min; denature at 94.0 °C for 30 s, anneal at 54.0 °C for 40 s, extend at 72.0 °C for 40 s, 35 cycles; extend at 72.0 °C for 7 min, store at 4 °C. Take 5 μL of the PCR product and perform electrophoresis on a 2% agarose gel, and observe the results using a gel imaging system. The method for identifying the genotype of the nuc gene of Staphylococcus aureus in the sample by PCR technology refers to the following literature: Yang Yang, Zhang Wei, Yuan Yaowu, etc. Detection of Staphylococcus aureus in dairy products by PCR [J]. Scientia Agricultura Sinica, 2006, (05): 990-996.

[0053] It can be seen from Figure 7 that using the staphylococcal nuc gene as the target gene, a 279 bp product was obtained after PCR amplification. Since the extracellular DNA substances were all cleared by DNA lyase, this result indicates that Staphylococcus aureus invaded into MAC-T cells.

[0054] It can be seen that the test results by the above four methods show that the transmission electron microscopy irradiation method, laser confocal irradiation method, PCR method, and perforin-Gram staining method can all accurately detect Staphylococcus aureus in bovine mammary epithelial cells, but the perforin-Gram staining method provided by the present invention is the most convenient and economical.

[0055] Example 2: Clinical detection of intracellular Staphylococcus aureus infection in bovine mammary epithelial cells

[0056] 70 cows reported with Staphylococcus aureus mastitis were selected from a dairy farm, and their milk was taken to isolate mammary epithelial cells. The epidemiological investigation of Staphylococcus aureus intracellular infectious mastitis in cows was carried out on the cell samples by perforin-Gram staining method. The test results showed that the positive rate was 82.9% and the negative rate was 17.1% (see Table 1), which was consistent with the PCR test results.

[0057] Table 1 Results of the epidemiological investigation of Staphylococcus aureus intracellular infectious mastitis in cows by perforin-Gram staining method

[0058]

[0059] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for rapid detection of intracellular Staphylococcus aureus in bovine mammary cells, characterized in that: The steps include: S1. Establishment of Staphylococcus aureus intracellular infection model: Cow mammary epithelial cell line was co-incubated with Staphylococcus aureus, 100 μg / mL lysostaphin was added and incubated for 15 min to remove extracellular bacteria, and cell samples were obtained by washing three times with PBS; S2. The cell samples were permeated with 0.3% Triton X-100 for 15 min, washed 3 times with PBS, and Gram-stained. Gram-positive particles in the samples were observed using an optical microscope with an oil immersion lens.

2. The method according to claim 1, characterized in that S1 The isolation method of the bovine mammary epithelial cell line is as follows: collect bovine milk and centrifuge it. The bovine milk is divided into three layers, the upper layer is cream, the middle layer is milky white whey, and the lower layer is white precipitate. The upper layer of cream and most of the whey are discarded, leaving 3mL of whey and the white precipitate, resuspended and transferred to a 15mL centrifuge tube, and a third antibody with a final concentration of 10% is added to 15mL. After mixing, centrifugation is performed, and the above steps are repeated until the supernatant after centrifugation gradually becomes clear, the supernatant is discarded, and PBS is added to resuspend, and macrophage scavenger, third antibody, gentamicin and lysostaphin are added to the obtained somatic cells. After incubation, the supernatant is discarded and the cells are washed three times with PBS.

3. The method according to claim 1, characterized in that The bovine mammary epithelial cell line described in S1 is cultured to a cell density of 80% and then co-incubated with Staphylococcus aureus.

4. The method according to claim 1, characterized in that: The concentration of Staphylococcus aureus in S1 is 1×10 9 CFU / mL.

5. The method according to claim 1, characterized in that During the co-incubation described in S1, MOI=8.

6. The method according to claim 1, characterized in that The co-incubation temperature in S1 is 37° C. and the time is 2 h.

7. The method according to claim 1, characterized in that S2 The Triton X-100 with a mass fraction of 0.3% was diluted with 200 μL PBS.

8. The method according to claim 1, characterized in that The washing time of each PBS washing in S2 is 5 minutes.

9. The method according to claim 1, characterized in that: The steps of Gram staining described in S2 are as follows: adding 1 drop of ammonium oxalate crystal violet stain solution, staining for 1 minute; after rinsing with distilled water, adding another drop of Gram stain solution, staining for 1 minute; rinsing the culture dish with distilled water, adding 95% ethanol by volume on the culture dish until the ethanol flowing down is no longer purple, and rinsing with distilled water after 20 seconds; adding 1 drop of safflower yellow stain solution, staining for 1 minute, and rinsing the culture dish with distilled water.

10. Use of the method according to any one of claims 1 to 9 in epidemiological investigation of dairy cow mastitis caused by intracellular Staphylococcus aureus infection.

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