Metabolism marker related to lamb diarrhea and application thereof

Abstract

The invention relates to the technical field of sheep disease-resistant breeding molecules, in particular to a metabolic marker related to sheep lamb diarrhea and a change approach thereof. Specific metabolites related to the lamb diarrhea of the sheep are screened through related molecular means such as a metabolome sequencing technology, metabolic pathways remarkably related to the lamb diarrhea of the sheep are analyzed, and the metabolic pathways can be used as potential markers of the lamb diarrhea of the sheep. The mechanism of the lamb diarrhea is discussed from the metabolism level, so that a prevention strategy and a treatment basis are provided for the lamb diarrhea.

Description

Technical Field

[0001] The invention relates to the field of molecular technology for disease-resistant sheep breeding, and more specifically to metabolic markers of sheep lamb diarrhea and applications thereof. Background Art

[0002] With the development of intensive and large-scale aquaculture, large-scale farms are emerging, and meticulous feeding management and regular vaccination can induce bacterial and viral diarrhea. Dietary changes, weaning stress and metabolic disorders are also the main causes of diarrhea. Many studies have shown that diarrhea has multiple effects on the host, such as changing the stable state of the intestinal microbiota, pathological changes in the intestinal mucosa and reduced activity of various enzymes.

[0003] Lamb diarrhea is one of the common, multiple and high-mortality diseases of the digestive tract that seriously affects lamb production. Diarrhea can cause weight loss, growth retardation and even death of new lambs, causing huge losses to breeding companies. The intestine is the main organ for host immunity and nutrient digestion and absorption. Because the intestinal microecological system and immune system of lambs are not yet stable and mature, they are sensitive to pathogenic microorganisms and are therefore very susceptible to infection by pathogens and diarrhea. Diarrhea can cause certain damage to the lamb intestines, affect the absorption of nutrients, and then affect growth and development, causing huge economic losses to the sheep industry.

[0004] The relationship between diarrhea, intestinal microbiota and host plays a vital role in animal growth, immunity, development and nutritional metabolism. Studies have shown that enteroaggregative Escherichia coli-induced diarrhea in female calves can be predicted by changes in the intestinal microbiome and significantly change the unabsorbed carbohydrate content in feces in early life. Intestinal microorganisms can also produce metabolites, such as short-chain fatty acids associated with the production of immune proteins. Ruminococcaceae, Caldicobacteraceae, Coriobacteriaceae and Eggthellaceae are involved in the degradation of cellulose polysaccharides and the metabolism of flavonoids, and play basic functions. Studies have shown that the metabolite profile of diarrheal deer has changed, showing large-scale metabolite dysregulation, and acylcarnitines, bile acids and oxidized lipids are significantly increased in the metabolites. Although the potential effects of diarrhea on growth, immunity and metabolism have been confirmed in livestock, limited research has focused on the effects of diarrhea on the host and changes in the intestinal flora of diarrheal animals. Changes in the metabolism of diarrheal animals need to be further studied. Summary of the invention

[0005] The purpose of the present invention is to provide metabolic markers related to sheep lamb diarrhea and applications thereof.

[0006] We identified 140 metabolite structures and used PLS-DA analysis to model the relationships between metabolites and samples.

[0007] The present invention screened out statistically significant metabolites (VIP > 1 and P value < 0.05).

[0008] The invention provides a metabolic marker for sheep and lambs with diarrhea, comprising at least one of Guanosine, Hecogenin, Quinethazone, Hypotaurine, Bovinocidin, Clindamycin, Sarsasapogenin, Irbesartan, Peiminine, Taurine, Lithocholic Acid, Glyceraldehyde, Thymidine, Procymidone, Protoveratrine A, Ketoleucine, Lauric Acid, LTC4, Palmitic Acid and the like.

[0009] The metabolite markers of the embodiments of the present invention have at least the following beneficial effects: The present invention collects serum samples from lambs with diarrhea and healthy lambs and finds that there are obvious differences in 19 specific metabolites between lambs with diarrhea and healthy lambs. These metabolites with obvious differences in content can be used as biomarkers for lambs with diarrhea or as a basis for diagnosis and treatment.

[0010] The present invention compares and analyzes the changes of differential metabolites between lambs with diarrhea and healthy lambs, and conducts KEGG enrichment analysis to find the main metabolic pathways in which differential metabolites are enriched between lambs with diarrhea and healthy lambs.

[0011] The present invention provides a metabolic enrichment pathway for metabolite markers, including at least one of taurine, sulfinic acid metabolism, fatty acid biosynthesis, bile secretion and other metabolic pathways.

[0012] The present invention provides an application of a sheep lamb diarrhea metabolite marker, and utilizes a diarrhea risk assessment system to determine the risk of sheep lamb diarrhea or to detect whether a diarrhea drug meets a screening condition.

[0013] The present invention provides a system for evaluating the diarrhea risk of a test sheep lamb, comprising: a detection device for determining the content of the diarrhea metabolic marker described in claim 1 in a sample of the test sheep lamb; a comparison device for comparing the content with a preset threshold value, and judging the diarrhea risk or detecting whether a diarrhea drug meets the screening conditions based on the comparison result.

[0014] According to the methods of some embodiments of the present invention, if at least one of the following conditions is met, the diarrhea risk of the tested sheep lamb can be determined or whether the diarrhea drug meets the screening conditions.

[0015] (1) The content of Guanosine is lower than the threshold value.

[0016] (2) The content of Hecogenin is lower than the threshold.

[0017] (3) The content of quinethazone is lower than the threshold value.

[0018] (4) The content of hypotaurine is lower than the threshold.

[0019] (5) The content of Bovinocidin is lower than the threshold.

[0020] (6) The content of Clindamycin is lower than the threshold.

[0021] (7) The content of Sarsasapogenin is lower than the threshold.

[0022] (8) The content of Irbesartan is lower than the threshold.

[0023] (9) The content of Peiminine is higher than the threshold value.

[0024] (10) The content of taurine is higher than the threshold.

[0025] (11) The content of Lithocholic Acid is lower than the threshold value.

[0026] (12) The content of glyceraldehyde is higher than the threshold value.

[0027] (13) The Thymidine content is lower than the threshold.

[0028] (14) The content of procymidone is higher than the threshold.

[0029] (15) The content of Protoveratrine A is lower than the threshold.

[0030] (16) The content of ketolide is lower than the threshold.

[0031] (17) The content of Lauric Acid is higher than the threshold value.

[0032] (18) The level of LTC4 is lower than the threshold.

[0033] (19) The content of Palmitic Acid is lower than the threshold value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the partial least squares discriminant analysis result of the metabolic data of healthy sheep lambs and diarrhea lambs of the present invention.

[0035] Figure 2 The present invention provides the screening and analysis results of serum metabolites of healthy sheep lambs and diarrhea lambs.

[0036] Figure 3 This is the pathway enrichment result of the significantly different metabolites between the healthy sheep lambs and the diarrhea lambs of the present invention.

[0037] Figure 4 The figure is a ROC curve diagram of Guanosine according to one embodiment of the present invention.

[0038] Figure 5 The ROC curve diagram of Hecogenin according to one embodiment of the present invention.

[0039] Figure 6 The figure is a ROC curve diagram of Quinethazone according to one embodiment of the present invention.

[0040] Figure 7 The figure is a ROC curve diagram of Hypotaurine according to one embodiment of the present invention.

[0041] Figure 8 The ROC curve diagram of Bovinocidin according to one embodiment of the present invention is shown in FIG.

[0042] Fig. 9 The figure is a ROC curve diagram of Clindamycin according to one embodiment of the present invention.

[0043] Fig.10 The figure is a ROC curve diagram of Sarsasapogenin according to one embodiment of the present invention.

[0044] Fig.11 The ROC curve diagram of Irbesartan according to one embodiment of the present invention is shown in FIG.

[0045] Fig.12 It is a ROC curve diagram of Peiminine according to an embodiment of the present invention.

[0046] Fig.13 FIG. 4 is a ROC curve diagram of Taurine according to an embodiment of the present invention.

[0047] Fig.14 The ROC curve diagram of Lithocholic Acid according to one embodiment of the present invention is shown in FIG.

[0048] Fig.15 The ROC curve diagram of Glyceraldehyde according to one embodiment of the present invention is shown in FIG.

[0049] Fig.16 The figure is a ROC curve diagram of Thymidine according to one embodiment of the present invention.

[0050] Fig.17 The figure is a ROC curve diagram of Procymidone according to one embodiment of the present invention.

[0051] Fig.18 The figure is a ROC curve diagram of Protoveratrine A according to one embodiment of the present invention.

[0052] Fig.19 The ROC curve diagram of Ketoleucine according to one embodiment of the present invention is shown in FIG.

[0053] Fig. 20 The ROC curve diagram of Lauric Acid according to one embodiment of the present invention is shown in FIG.

[0054] Fig.21 It is a ROC curve diagram of LTC4 according to an embodiment of the present invention.

[0055] Fig. 22 FIG. 4 is a ROC curve diagram of Palmitic Acid according to an embodiment of the present invention.

[0056] Fig.23 A schematic diagram of the components of the system for assessing diarrhea risk in test sheep lambs according to the present invention. DETAILED DESCRIPTION

[0057] Example 1 Collection of samples from sheep diarrhea lambs and healthy lambs.

[0058] Samples were collected from 11 lambs with diarrhea and 17 healthy lambs at the Huyang Intensive Farm. Serum was extracted from the jugular vein for metabolomics analysis. All samples were stored at -80 °C before further analysis.

[0059] Example 2 Extraction and LC-MS / MS analysis of metabolites.

[0060] The sample was thawed at 4 °C, 50 mg of the sample was weighed into an EP tube, 3 steel beads were added, 300 μL of methanol-water solution (4:1, v / v) was added, the sample was broken in a tissue crusher, and 700 μL of methanol-water solution (4:1, v / v) was added and vortexed, ultrasonicated in an ice bath for 20 min, allowed to stand at -20 °C for 1 h, centrifuged at 16000×g at 4 °C for 20 min, and the supernatant was taken. The supernatant was evaporated to dryness in a high-speed vacuum concentrator centrifuge. For mass spectrometry detection, 100 μL of methanol-water solution (1:1, v / v) was added for re-dissolution, centrifuged at 20000×g at 4 °C for 20 min, and the supernatant was taken for sample analysis. During the entire analysis process, the samples were placed in an autosampler at 4 °C, and the samples were analyzed using a SHIMADZU-LC30 ultra-high performance liquid chromatography system (UHPLC) using an ACQUITY UPLC® HSS T3 (2.1×100 mm, 1.8 µm) (Waters, Milford, MA, USA) column.

[0061] Example 3 Data preprocessing.

[0062] The raw data were aligned, corrected for retention time, and extracted for peak area using MSDIAL software. Metabolite structures were identified using accurate mass matching (mass tolerance < 20 ppm) and secondary spectrum matching (mass tolerance < 0.02 Da). For the extracted data, ion peaks with > 50% missing values ​​within the group were deleted and excluded from subsequent statistical analysis; the total peak area of ​​positive and negative ion data was normalized, the positive and negative ion peaks were integrated, and R software was used for pattern recognition. The data were preprocessed by Unit variance scaling (UV) before subsequent data analysis.

[0063] Example 4 Data modeling analysis.

[0064] Partial least squares discriminant analysis (PLS-DA) was used to model the relationship between metabolites and samples, and to establish a relationship model between metabolite expression and sample category to predict sample category.

[0065] Example 5 Screening of differential metabolites and performing differential comparison and pathway enrichment.

[0066] By comparing with the metabolite database, statistically significant metabolites (VIP > 1 and P <0.05), and compared the differential metabolites between sheep diarrhea lambs and healthy lambs to analyze their changes. In addition, based on the KEGG database, the differential metabolites were enriched and the main metabolic pathways of differential metabolite enrichment between sheep diarrhea lambs and healthy lambs were found.

[0067] Example 6 ROC curve analysis of differential metabolites.

[0068] Figure 4 to Figure 22 : is the ROC curve of the metabolite marker of one embodiment of the present invention. As can be seen from the figure, the AUC values ​​of the 19 metabolites are all greater than 0.7, which indicates that the diagnostic value of these 19 metabolites is moderately or highly reliable and has a high application value.

[0069] Example 6 Diarrhea risk assessment in sheep lambs.

[0070] Fig.23 : This is a schematic diagram of the composition of a system for assessing the diarrhea risk of a test sheep lamb according to another embodiment of the present invention. The system includes: a metabolite separation device for separating a metabolite sample from a sample; and a comparison device for comparing the content of a marker with a preset threshold value, and judging the risk according to the relative size of the detected content and the threshold value.

[0071] Example 7 Method for implementing risk assessment of diarrhea in sheep and lambs.

[0072] If at least one of the following conditions is met, the lambs being tested can be judged to be at risk of diarrhea or the drug does not meet the screening criteria.

[0073] (1) The content of Guanosine is lower than the threshold value.

[0074] (2) The content of Hecogenin is lower than the threshold.

[0075] (3) The content of quinethazone is lower than the threshold value.

[0076] (4) The content of hypotaurine is lower than the threshold.

[0077] (5) The content of Bovinocidin is lower than the threshold.

[0078] (6) The content of Clindamycin is lower than the threshold.

[0079] (7) The content of Sarsasapogenin is lower than the threshold.

[0080] (8) The content of Irbesartan is lower than the threshold.

[0081] (9) The content of Peiminine is higher than the threshold value.

[0082] (10) The content of taurine is higher than the threshold.

[0083] (11) The content of Lithocholic Acid is lower than the threshold value.

[0084] (12) The content of glyceraldehyde is higher than the threshold value.

[0085] (13) The Thymidine content is lower than the threshold.

[0086] (14) The content of procymidone is higher than the threshold.

[0087] (15) The content of Protoveratrine A is lower than the threshold.

[0088] (16) The content of ketolide is lower than the threshold.

[0089] (17) The content of Lauric Acid is higher than the threshold value.

[0090] (18) The level of LTC4 is lower than the threshold.

[0091] (19) The content of Palmitic Acid is lower than the threshold value.

Claims

1. Compared with healthy lambs, diarrhea lambs have changes in metabolism and different metabolic markers.

2. According to claim 1, the metabolic markers of diarrhea lambs include at least one of 19 types including Guanosine, Hecogenin, Quinethazone, Hypotaurine, Bovinocidin, Clindamycin, Sarsasapogenin, Irbesartan, Peiminine, Taurine, Lithocholic Acid, Glyceraldehyde, Thymidine, Procymidone, Protoveratrine A, Ketoleucine, Lauric Acid, LTC4, and Palmitic Acid, as shown in Figure 2.

3. The differential metabolites between diarrhea lambs and healthy lambs were mainly enriched in metabolic pathways such as taurine, sulfite metabolism, fatty acid biosynthesis, and bile secretion, as shown in Figure 3.

4. Use of the metabolite described in claim 2 or the metabolic pathway described in claim 3 in preventing and / or treating lamb diarrhea.

5. A kit, characterized in that: The invention comprises a reagent for detecting the content of a marker, wherein the marker is the sheep diarrhea metabolic marker according to claim 1.

6. According to claims 1 and 3, the method for detecting metabolic changes between diarrhea lambs and healthy lambs is as follows: (1) collecting serum samples from healthy lambs and diarrhea lambs, extracting and detecting metabolites; (2) filtering the original data, screening differential metabolites, and comparing their changes; (3) comparing and analyzing the differential metabolites with the KEGG database to find the significantly enriched pathways of differential metabolites between diarrhea lambs and healthy lambs.

7. A system for assessing the risk of diarrhea in a sheep subject, characterized in that: include: A detection device for determining the content of the diarrhea metabolic marker according to claim 1 in a test sheep sample; The comparison device is used to compare the content with a preset threshold value and determine the diarrhea risk based on the comparison result.

Images