A biomarker combination for early diagnosis of latent mastitis in dairy cows and its application

Abstract

The present invention discloses a biomarker combination for early diagnosis of latent mastitis in dairy cows and its application, and relates to the field of biomedicine technology. The biomarker combination includes digalacturonic acid and N-ε-methyl-L-lysine. The present invention also provides the application of a reagent for detecting the concentration of the biomarker combination in the preparation of a product for early diagnosis of latent mastitis in dairy cows. The present invention combines metabolomics data analysis to develop a biomarker combination for early diagnosis of latent mastitis in dairy cows. The early diagnosis method constructed based on the biomarker combination can effectively identify early metabolic disorders of SCM by measuring the concentration changes of digalacturonic acid and N-ε-methyl-L-lysine, can provide accurate and efficient support for dairy cow health management, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a biomarker combination for early diagnosis of latent mastitis in dairy cows and applications thereof. Background Art

[0002] Subclinical mastitis (SCM) in dairy cows refers to a disease in which, after a pathogen infects the udder, milk quality declines, but the udder's appearance remains normal, and the cow itself exhibits no clinical symptoms. While subclinical mastitis is more difficult to detect and diagnose than clinical mastitis, its harmful effects are equally severe. Subclinical mastitis can increase the somatic cell count in milk, reduce milk quality, and thus affect the market price of milk. Furthermore, affected cows can significantly reduce milk production, impairing the overall economic profitability of the farm. Long-term subclinical mastitis affects the reproductive capacity of dairy cows, resulting in lower conception rates and prolonged gestation periods, further increasing farming costs. Subclinical mastitis can also weaken the cow's immunity, making it more susceptible to other secondary diseases, such as hoof disease and gastrointestinal disorders, making treatment more difficult and costly.

[0003] At present, the diagnosis of latent mastitis in dairy cows mainly relies on the following methods:

[0004] Somatic cell count (SCC): This method measures the number of somatic cells in milk to determine udder health. However, this method only reflects the presence or absence of inflammation and cannot identify the cause early or distinguish different types of pathogens.

[0005] Chemical detection methods: such as the detection of indicators such as lactate dehydrogenase (LDH) and β-hydroxybutyric acid (BHBA). Although these methods can help assess the degree of inflammation, their sensitivity and specificity are limited and they cannot be used as the main means of early diagnosis.

[0006] Microbial culture: Bacterial culture of milk samples is performed to identify the pathogenic bacteria. However, this method is time-consuming, usually requiring 24-48 hours to produce results, making it unsuitable for early and rapid diagnosis.

[0007] Immunological testing: Although technologies such as ELISA and PCR have high sensitivity and specificity, they require high equipment and technology, are complex to operate, and are expensive, making them difficult to be widely used in large-scale farms.

[0008] Although there are some products on the market for diagnosing cow mastitis, there is still a problem of low specificity in the early diagnosis of latent mastitis, which is prone to false negative or false positive results. In addition, many detection methods are time-consuming and cannot meet the needs of rapid screening, which is not conducive to timely prevention and control measures.

[0009] In view of the above problems, it is particularly urgent to develop an efficient, rapid and specific early diagnosis product for latent mastitis in dairy cows. Summary of the Invention

[0010] The present invention aims to provide a biomarker combination for early diagnosis of latent mastitis in dairy cows and its application, so as to solve the problems existing in the above-mentioned prior art. The biomarker combination can realize early diagnosis of latent mastitis in dairy cows with high sensitivity and high accuracy.

[0011] To achieve the above object, the present invention provides the following solutions:

[0012] The present invention provides a biomarker combination for early diagnosis of latent mastitis in dairy cows, comprising digalacturonic acid and N-ε-methyl-L-lysine.

[0013] Furthermore, when the concentration of the digalacturonic acid increases and the concentration of the N-ε-methyl-L-lysine decreases, it indicates that the risk of the dairy cow suffering from latent mastitis increases.

[0014] The present invention also provides a use of a reagent for detecting the concentration of the above-mentioned biomarker combination in the preparation of a product for early diagnosis of latent mastitis in dairy cows.

[0015] Furthermore, the product is a kit.

[0016] The present invention also provides a product for early diagnosis of latent mastitis in dairy cows, comprising a reagent for detecting the concentration of the above-mentioned biomarker combination.

[0017] Furthermore, the product is a kit.

[0018] Furthermore, the diagnostic criteria of the product are: when the concentration of digalacturonic acid exceeds 10 μmol / L and the concentration of N-ε-methyl-L-lysine is lower than 5 μmol / L, the cow is diagnosed with latent mastitis.

[0019] The present invention discloses the following technical effects:

[0020] The present invention combines metabolomics data analysis to develop a biomarker combination for the early diagnosis of latent mastitis in dairy cows, including digalacturonic acid and N-ε-methyl-L-lysine. The early diagnosis method constructed based on this biomarker combination can effectively identify early metabolic disorders of SCM by measuring the concentration changes of digalacturonic acid and N-ε-methyl-L-lysine. This early diagnosis method has significant diagnostic advantages, can provide accurate and efficient support for dairy cow health management, and has broad application prospects, especially in the fields of dairy product quality assurance and dairy cow health monitoring. By comparison with traditional methods, it is shown that the early diagnosis method of the present invention not only has high sensitivity and high accuracy, but also has a low misdiagnosis rate and high economic benefits, and is suitable for large-scale, economical and efficient clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Statistical graph of the concentrations of digalacturonic acid and N-ε-methyl-L-lysine in serum samples of dairy cows in the healthy group and the mastitis group;

[0023] Figure 2 This is a receiver operating characteristic curve analysis diagram using digalacturonic acid and N-ε-methyl-L-lysine as markers. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The structural formula of digalacturonic acid referred to in the present invention is as follows:

[0030]

[0031] The structural formula of N-ε-methyl-L-lysine is as follows:

[0032]

[0033] Example 1

[0034] Omics analysis showed that the metabolic disturbances of digalacturonic acid and N-ε-methyl-L-lysine in subclinical mastitis in dairy cows showed consistent patterns:

[0035] Digalacturonic acid: The significant increase in serum, milk, feces and rumen fluid samples (VIP (Variable Importance in the Projection) = 1.86, P = 0.005) is consistent with the results of the intestinal inflammatory response study, indicating that it may become a key biomarker for intestinal health and SCM diagnosis.

[0036] N-ε-methyl-L-lysine: The concentration of this metabolite is generally reduced in serum, milk, feces, and rumen fluid, indicating its important role in systemic metabolic responses. Its reduction can serve as an early indicator of systemic metabolic disorders and inflammatory responses.

[0037] The details are as follows:

[0038] 1. Sample Collection and Processing

[0039] Serum, milk, feces, and rumen fluid samples were collected from 100 dairy cows (50 SCM cows and 50 healthy cows). Metabolites were extracted using standardized methods to remove proteins and impurities to ensure sample purity.

[0040] 2.HPLC separation and mass spectrometry analysis

[0041] The concentrations of digalacturonic acid and N-ε-methyl-L-lysine were quantified using HPLC-MS.

[0042] HPLC-MS detection conditions include:

[0043] The HPLC separation column was a C18 reverse phase column, and the mobile phase was a gradient system of acetonitrile and water. The acidity was adjusted to 0.1% formic acid or acetic acid to improve the analytical sensitivity.

[0044] Mass spectrometry analysis used the electrospray ionization (ESI) mode to detect the parent ion (m / z) signals of digalacturonic acid and N-ε-methyl-L-lysine, analyze the peak intensities in their mass spectra, and calculate the metabolite concentrations;

[0045] Internal standards are used for quantitative calibration to ensure the accuracy and reliability of the test results.

[0046] 3. Quantitative Analysis

[0047] The concentrations of digalacturonic acid and N-ε-methyl-L-lysine in each sample were obtained by comparison with the standard and calibration with the internal standard.

[0048] 4. Test results

[0049] The HPLC-MS results of digalacturonic acid and N-ε-methyl-L-lysine in serum samples are shown in Figure 1 and Table 1. The test results showed that digalacturonic acid and N-ε-methyl-L-lysine had significant concentration differences in cows with subclinical mastitis.

[0050] Table 1 HPLC-MS detection results of digalacturonic acid and N-ε-methyl-L-lysine in serum samples

[0051]

[0052] From this, we can derive the diagnostic criteria for latent mastitis in dairy cows:

[0053] When the serum digalacturonic acid concentration exceeds 10 μmol / L and the N-ε-methyl-L-lysine concentration is less than 5 μmol / L, the cow can be diagnosed with latent mastitis. In addition, the severity of the disease can be determined based on the magnitude of the change in concentration (e.g., ≥2-fold) to help guide subsequent treatment and management.

[0054] The receiver operating characteristic curves using digalacturonic acid and N-ε-methyl-L-lysine as markers are shown in Figure 2 , the AUC value reached 1.0, indicating that digalacturonic acid and N-ε-methyl-L-lysine had high diagnostic value.

[0055] Example 2

[0056] In addition, 100 dairy cows (50 SCM cows and 50 healthy cows) were selected and diagnosed for subclinical mastitis using both the traditional milk cell count (SCC) method and the HPLC-MS method of Example 1. The results are shown in Table 2. The results demonstrated that the HPLC-MS method of the present invention significantly outperformed the traditional SCC detection method in terms of sensitivity and accuracy in diagnosing SCM and had a lower misdiagnosis rate.

[0057] Table 2 Diagnostic results of SCC and HPLC-MS methods

[0058]

[0059] Note: False positive rate (FPR) = false positives / [false positives + true negatives], where: false positives (FP) are the number of healthy samples incorrectly classified as positive; true negatives (TN) are the number of healthy samples correctly classified as negative.

[0060] Economic benefit analysis:

[0061] 1. Reduce the risk of misdiagnosis and missed diagnosis

[0062] Traditional SCC methods have a high misdiagnosis rate. Missed and misdiagnosed cases not only lead to health problems for dairy cows, but also result in unnecessary treatment and production losses. By using the HPLC-MS method of the present invention, the misdiagnosis rate has been reduced from 22% using traditional methods to 2%, significantly improving detection accuracy. Reducing misdiagnosis not only improves dairy cow health but also reduces the resulting treatment and management costs.

[0063] 2. Improve the efficiency of early diagnosis

[0064] The HPLC-MS method of this invention offers significant advantages for the early diagnosis of latent mastitis. By accurately detecting changes in metabolite concentrations, it can identify affected cows in the early stages of the disease, enabling timely intervention and treatment. This prevents the decline in production performance caused by disease progression, reduces long-term treatment costs for dairy cows, and reduces dairy product quality issues.

[0065] 3. Reduce overall breeding costs

[0066] Compared to traditional methods, the HPLC-MS method of this invention allows for early screening and diagnosis of latent mastitis in dairy cows, reducing unnecessary medication and treatment costs. Furthermore, early intervention effectively prevents further deterioration of the cow's health, ensuring cow production performance and the quality of dairy products, thereby improving the economic benefits of the farm.

[0067] On a medium-sized dairy farm (500 cows), the average cost of diagnosing and treating SCC using traditional methods is RMB 250 per cow (including testing, medication, and treatment costs). If this method can diagnose SCC earlier and reduce misdiagnosis rates, it is expected to reduce treatment costs by 10% annually, or reduce the treatment costs of 50 cows, saving approximately RMB 12,500.

[0068] By adopting the HPLC-MS method of the present invention, the misdiagnosis rate is reduced to 2%, while the efficiency of early screening is improved. It is expected that the farm can save about 20% of the mastitis-related treatment costs each year, with a total cost saving of more than RMB 25,000.

[0069] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of a reagent for detecting the concentration of a biomarker combination in the preparation of a kit for early diagnosis of latent mastitis in dairy cows, characterized in that: The biomarker combination includes digalacturonic acid and N-ε-methyl-L-lysine.

2. The use according to claim 1, characterized in that When the concentration of digalacturonic acid increases and the concentration of N-ε-methyl-L-lysine decreases, it indicates that the risk of dairy cows suffering from subclinical mastitis increases.

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