CN112080561A - Microbial marker of bovine endometritis and application thereof - Google Patents
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Abstract
The invention discloses a microbial marker of bovine endometritis, which comprises microorganisms with remarkably increased abundance: bacteroidetes, traceable bacteria, verrucomicrobia, spirillum and myxococcobacillus; also included are microorganisms with significantly increased abundance: ackermanella, Marshmania, CF231, Oscillatoria; also included are microorganisms with significantly reduced abundance: ureaplasma urealyticum, Vibrio butyricum, Dorea, Sphingomonas, Roseburia, Streptococcus. The invention discloses application of the microbial markers of bovine endometritis in preparation of tools for early screening or predicting bovine endometritis. The applicant carries out 16S rRNA gene sequencing on microbial DNA of uterine mucus samples, compares the structural difference of uterine flora of healthy and diseased cows, and has difference of microbial community composition of the uterine flora of the healthy and diseased cows. The result provides a theoretical basis for better prevention and treatment of clinical endometritis.
Description
Technical Field
The invention relates to the technical field of microbial markers, in particular to a microbial marker for bovine endometritis and application thereof.
Background
After delivery, the cervix is opened and various microorganisms "stay" in the uterus. Researches show that the healthy cow uterus also has microbial flora, and the cow uterus not only contains normal microbial flora, but also contains pathogenic bacteria and conditional pathogenic bacteria. When host immune suppression, external stress reaction and other conditions occur, the immune reaction of the cow is difficult to successfully resist the pathogenic effect of pathogenic bacteria, and the endometritis of the cow is caused.
It was found that within 3 weeks after parturition of 40% of cows developed metritis and endometritis by bacterial infection, and 15-20% of cows developed clinical endometritis within 3 weeks after parturition. Clinical endometritis is defined as: purulent uterine secretions can be detected in the vagina 21 days after delivery of the cow, or mucoid secretions can be detected in the vagina 26 days later.
Clinical endometritis has negative influence on uterus and ovary function and fertility, and can prolong luteal phase of cow after delivery, reduce fertility, and bring great economic loss to cow breeding industry. Understanding the difference of uterine flora of healthy cows and cows with endometritis has a considerable effect on preventing and treating endometritis.
The study of the uterine flora of cows with endometritis has been paid attention to by many scholars, but the study of the uterine flora of endometritis is limited due to the limitations of research means. In recent years, high throughput sequencing technology has been widely used in research in various fields as a currently advanced gene detection technology. The high-throughput sequencing can carry out deep, detailed and complete analysis on the genome, and has the advantages of rapidness, convenience, high accuracy, high throughput and the like.
Disclosure of Invention
Based on the technical problems in the background art, the invention provides a microbial marker for bovine endometritis and application thereof.
The invention provides a microbial marker of bovine endometritis, which comprises microorganisms with remarkably increased abundance: bacteroidetes, traceable bacteria, verrucomicrobia, spirillum and myxococcobacillus.
Preferably, also included are microorganisms with significantly increased abundance: ackermanella, Marshmania, CF231, Oscillatoria.
Preferably, also included are microorganisms with significantly reduced abundance: ureaplasma urealyticum, Vibrio butyricum, Dorea, Sphingomonas, Roseburia, Streptococcus.
A reagent for detecting the microbial markers of the bovine endometritis.
The use of the microbial marker of bovine endometritis or the reagent comprises the following steps: the method is used for constructing a model for predicting the risk of the bovine endometritis, preparing a diagnostic reagent for the bovine endometritis or preparing a diagnostic kit for the bovine endometritis.
The kit comprises the reagent.
An application of the microbial marker of bovine endometritis in the preparation of a tool for early screening or prediction of bovine endometritis.
An application of the microbial marker of the bovine endometritis in the preparation of a kit for early screening of the bovine endometritis, wherein the kit comprises a detection reagent of the microbial marker of the bovine endometritis.
The applicant carries out 16S rRNA gene sequencing on microbial DNA of uterine mucus samples (taken from 3 healthy cows 30-45 days after delivery and 5 cows with clinical endometritis) on a high-throughput sequencing platform, and then compares the structural difference of uterine flora of healthy cows and cows with clinical endometritis by biological analysis. The results show that the composition of the microflora of the uterine flora of healthy and clinically affected endometritis cows is different.
The relatively high abundance of the uterus flora of the cows in the healthy group is 55.58%, 15.44%, 12.57% and 10.45% of Firmicutes, Tenericutes, bacteroides and Proteobacteria; the relatively abundant uterine flora of the cow in the endometritis group are 47.88 percent, 40.92 percent, 3.05 percent and 2.01 percent of Firmicutes, Bacteroidetes, Verrucomicrobia and Proteobacteria respectively. Of these, Firmicutes (Firmicutes) are most abundant in both groups. Compared with the healthy group, the abundances of the endometritis group Bacteroidetes (bacteroides), the traceobacterium (Elusimicrobia), the Verrucomicrobia (Verrucomicrobia), the spirobacterium (Spirochaetes) and the globus mucilaginosus (Lentisphaera) are obviously increased (P < 0.05).
On the genus classification level, the relatively abundant uterine flora of the healthy cow is Ureaplasma urealyticum (Ureaplasma), vibrio butyricum (Butyrivibrio), Sphingomonas (Sphingomonas), Clostridium (Clostridium), which respectively account for 13.89%, 5.44%, 4.98%, 4.38%; the relative abundance of the cow uterine flora in the endometritis group is 5-7N15, CF231, Clostridium (Clostridium) and Prevotella, which respectively account for 4.27%, 4.12%, 1.67% and 1.57%, and mycoplasma urealyticum is only found in the healthy group.
The abundances of the pathogenic group of Ackermanella, Marshmania, CF231 and Oscillatoria are obviously increased compared with the healthy group (P is less than 0.05); the abundance of mycoplasma urealyticum, vibrio butyricum, Dorea, Sphingomonas, Roseburia and Streptococcus is obviously reduced compared with that of the healthy group (P < 0.05). The research result provides a theoretical basis for better prevention and treatment of clinical endometritis.
Drawings
FIG. 1 is a graph of ven between the diseased and healthy groups.
FIG. 2 is a sparse curve between the diseased and healthy groups.
FIG. 3 is a graph of OUT Rank between the diseased and healthy groups.
FIG. 4 is a graph of PCA analysis between the diseased and healthy groups.
FIG. 5 is a graph comparing the relative abundance at the phylogenetic classification level of the diseased group and the healthy group.
FIG. 6 is a comparison of relative abundance at the genus level between the diseased group and the healthy group.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
Selecting 3-4 years old, similar body condition characteristics, 20-40 days after delivery, 3 healthy Holstein cows without drug treatment and 5 Holstein cows with endometritis in certain dairy farm in Henan province.
For determination of endometritis, reference is made to the diagnostic criteria of Sheldon: and (3) discharging purulent secretion in the vagina after 21d of the dairy cow after delivery or discharging viscous secretion in the vagina after 26d of the dairy cow after delivery, and selecting the affected group of cows by combining diagnosis of a dwelling veterinarian.
3 healthy cows were scored as H005, H095, H210, respectively, and 5 cows with clinical endometritis were scored as D012, D172, D213, D412, D420, respectively.
Example 1
By using a composition containing EDTA-K2The anticoagulant vacuum blood collection tube collects 10mL of tail vein blood of 8 cows, and the tail vein blood is stored in a refrigerator at 4 ℃.
The conventional physiological indexes of blood such as white blood cell number, neutral granulocyte percentage, neutral granulocyte number, lymphocyte percentage, lymphocyte number, monocyte percentage, monocyte number and the like in the blood of healthy and endometritis cows are measured by using a full-automatic five-classification animal blood cell analyzer.
The blood routine related indexes are as follows:
as can be seen from the above table: the percentage of neutrophils, the number of neutrophils and the percentage of lymphocytes of the cattle with endometritis are obviously different from those of the healthy control group. The percentage of neutrophils and the number of neutrophils of the cows with endometritis is obviously higher than that of a healthy control group (P <0.05), and the percentage of lymphocytes is obviously lower than that of the healthy control group (P < 0.05). The indexes of white blood cell number, monocyte percentage, lymphocyte number and the like of the diseased group and the healthy group have no significant difference.
Example 2
Before sampling, the external genitals of the dairy cows are washed by clear water, and after being wiped clean, the external genitals of the dairy cows are disinfected by 75 percent alcohol solution, so that the excrement pollution is avoided. A uterine mucus sample is collected by a sampler designed by Henan academy of agricultural sciences, and the sampler is sterilized by ethylene oxide. The sampling device is delivered into the vagina by using a rectum holding method, after the sampling device enters the uterus, the plastic catheter is pulled backwards, so that the sampling head is exposed in uterine mucus, the sampling head returns to the plastic catheter after rotating for 2-3 circles, and the sampling head is prevented from contacting the vagina. And (4) immediately flushing the sampling head with physiological saline after being taken out, collecting flushing liquid, filling the flushing liquid into a 50mL sterile centrifuge tube, and quickly putting the flushing liquid into an ice box. After all samples were collected, the samples were stored at-80 ℃.
Total bacterial DNA was extracted from cow uterine mucus samples using a DNA extraction kit. And (3) accurately and quantitatively detecting the concentration of the sample by using a Qubit-dsDNA-BR detection kit.
30ng of qualified genome DNA sample is used as a template, the V4 region of the bacteria 16Sr RNA is sequenced through an Illumina HiSeq platform, amplification primers are 515F (GTGCCAGCMGCCCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT), an Agencour AMPure XP magnetic bead is used for purifying a PCR amplification product and dissolving the PCR amplification product in an output Buffer, Agilent 2100Bioanalyzer is used for detecting the range and the concentration of fragments, and the qualified fragments are sequenced on the HiSeq platform. Sequencing data analysis includes OTU clustering and analysis, species composition analysis, single sample diversity analysis, inter-sample diversity analysis, and the like. Sequencing is carried out by Shenzhen Hua Dagen science and technology service Limited.
Example 3
The applicant carries out 16S rRNA sequencing by carrying out high-throughput sequencing on V4 regions of 16SrRNA genes of 8 vaginal mucus samples through an Illumina HiSeq platform, 601017 high-quality sequences are obtained after quality trimming and chimera examination are carried out, and the effective length of the sequences of the samples is 252bp on average. From the above results, a ven plot between the diseased group and the healthy group was established, as shown in fig. 1.
FIG. 1 shows: of 2281 OTUs, 627 were shared by two groups, and these 627 OTUs accounted for 80.07% and 41.85% of the total OTUs of the healthy and diseased cows, respectively, indicating that the level of microbial diversity of the uterine flora of the diseased cows was partially similar to that of the healthy cows, sharing a portion of the bacterial species, but the bacterial diversity of the diseased group was more complex.
The sparse curve and the OUT Rank curve are respectively plotted according to the above results, as shown in fig. 2 and 3. Fig. 2 and 3 show that: this sequencing depth was sufficient to cover the overall bacterial diversity and the good Coverage index of each sample was greater than 99%, indicating that the sequencing method can represent the true composition of the uterine flora.
The Shannon index is a diversity measure based on species abundance and uniformity, and t-tests were performed on Shannon index values of the two groups of samples, as shown in the table below, to show that the Shannon index values have statistical significance (P <0.01), indicating that the two groups have significantly different flora diversity. The PCA analysis was then performed, as shown in FIG. 4, which FIG. 4 shows: the sample points of the diseased group and the healthy group are clearly separated on the coordinates, which further illustrates the significant difference in the flora structures of the two groups.
Example 4
The study contained 8 samples in total and all sample populations contained 19 phyla in total. On the phylum classification level, Firmicutes, Bacteroidetes, Tenericutes, Proteobacteria, Actinobacteria and Verrucomicrobia are the most abundant phyla among all samples, accounting for more than 95% of the total. The relative abundance at the phylogenetic classification level of the diseased and healthy groups was plotted as shown in figure 5.
The most abundant uterine flora of cows in the healthy group are Firmicutes (Firmicutes), Tenericutes (mollicutes), Bacteroidetes (Bacteroidetes), Proteobacteria (Proteobacteria), which account for 55.58%, 15.44%, 12.57%, 10.45%, respectively.
The vaginal flora of the affected group of cows with the highest relative abundance was Firmicutes (Firmicutes), Bacteroidetes (Bacteroidetes), Verrucomicrobia (Verrucomicrobia), Proteobacteria (Proteobacteria), accounting for 47.88%, 40.92%, 3.05%, 2.01%, respectively.
The relative abundance at the phylum level was analyzed by Kruskal-Wallis rank sum test comparison, as shown in the following table. The following table shows: compared with the healthy group, the abundance of the diseased group Elusiicrobia (the phylum of Trachelospermum), Verrucomicrobia (the phylum of Microbacteroides), Bacteroides (the phylum of Bacteroides), Spirochaetes (the phylum of Spirochaetes) and Lentiispharmare (the phylum of Gliocladium mucilaginosum) is obviously increased, and the difference has statistical significance (P is less than 0.05); the abundance of mollicutes (Tenericultes), actinomycetes (Actinobacillus) and Proteobacteria (Proteobacteria) in the diseased group is obviously reduced compared with that in the healthy group.
Example 5
The two groups of samples were compared at the genus level and analyzed to obtain 156 different species of bacteria. At the genus taxonomic level, urealasma (Ureaplasma urealyticum), 5-7N15, Butyrivibrio (vibrio butyrate), sphingamonas (Sphingomonas sp.), CF231, Clostridium (Clostridium sp.) are the most abundant genera in all samples. The relative abundance at the genus level of the diseased and healthy groups was plotted as shown in fig. 6.
5-7N15 was found to be more abundant in the diseased and healthy groups, accounting for 4.27% and 2.39%, respectively. The relative abundance of mycoplasma urealyticum in the diseased group and the healthy group is 0.00% and 13.89%, respectively; the relative abundance of the butyric acid vibrio in the diseased group and the healthy group is 0.56% and 5.44% respectively; the relative abundance of sphingomonas in the diseased group and healthy group was 0.24% and 4.98%, respectively; the relative abundance of clostridium in the diseased group and the healthy group is 1.67% and 4.38% respectively; the relative abundance of CF231 in the diseased and healthy groups was 4.12% and 0.90%, respectively.
The relative abundance at the genus level was analyzed by Kruskal-Wallis rank sum test comparison, as shown in the following Table. The following table shows: the relative abundance of the diseased group CF231, the oscillatoria, the akkermansia and the swamp bacillus is obviously increased compared with that of the healthy group (P is less than 0.05); the relative abundance of the diseased groups such as ureaplasma urealyticum, vibrio butyricum, Dorea, Sphingomonas, Roseburia and Streptococcus is obviously reduced compared with that of the healthy groups, and the difference has statistical significance (P is less than 0.05).
In summary, the applicant studied the difference in uterine flora between healthy cows and cows with endometritis in the same feeding environment based on Illumina HiSeq high throughput sequencing technology. And (3) carrying OUT data processing such as data filtering, Tags splicing, OUT clustering and the like on the original sequencing data to obtain 601017 final optimized sequences. After OTU analysis and Alpha diversity analysis among samples are carried out on the optimized sequence, the sequencing quantity and the sequencing depth of the experimental sample are proved to be enough to cover all groups of flora in the sample, and the richness and the uniformity of each group of samples are better. In order to obtain the species classification information corresponding to each OTU, the OTU representative sequence is subjected to taxonomic analysis by using an RDP classificator Bayesian algorithm in the test, and the community composition of each sample is counted on each level of the phylum compendium.
The species diversity of the two groups of samples is analyzed at each level, and the diversity of the uterine flora in the endometritis group is found to be obviously higher than that in the healthy group; on the genus classification level, the relative abundance of CF231, Oscillospira, Akkermansia and Paludibacter in the diseased group is remarkably higher than that in the healthy group (P < 0.05).
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (9)
1. A microbial marker of bovine endometritis, which is characterized by comprising a remarkably increased abundance of microorganisms: bacteroidetes, traceable bacteria, verrucomicrobia, spirillum and myxococcobacillus.
2. The microbial markers of bovine endometritis of claim 1, further comprising a significant increase in abundance of a microorganism: ackermanella, Marshmania, CF231, Oscillatoria.
3. The microbial markers of bovine endometritis of claim 1, further comprising a significantly reduced abundance of a microorganism: ureaplasma urealyticum, Vibrio butyricum, Dorea, Sphingomonas, Roseburia, Streptococcus.
4. A reagent for detecting a microbial marker of bovine endometritis according to any one of claims 1-3.
5. Use of a microbial marker of bovine endometritis according to any one of claims 1-3 or an agent according to claim 4, comprising: the method is used for constructing a model for predicting the risk of the bovine endometritis, preparing a diagnostic reagent for the bovine endometritis or preparing a diagnostic kit for the bovine endometritis.
6. A kit comprising the reagent of claim 4.
7. Use of a microbial marker of bovine endometritis according to any one of claims 1-3 in the preparation of a tool for early screening or prediction of bovine endometritis.
8. Use of a microbial marker of bovine endometritis according to any one of claims 1-3 in the preparation of a kit for early screening of bovine endometritis.
9. The use according to claim 8, wherein the kit comprises the reagent according to claim 4.
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| CN113897446A (en) * | 2021-09-18 | 2022-01-07 | 深圳临研医学有限公司 | Diagnosis and prognosis marker for rheumatoid arthritis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101906478A (en) * | 2010-08-11 | 2010-12-08 | 吉林大学 | Method for quickly diagnosing bovine endometritis pathogen |
| CN108030814A (en) * | 2017-12-06 | 2018-05-15 | 成都点石创想科技有限公司 | A kind of Chinese medicinal preparation method for the mucus or purulent inflammation that can treat cow uteri mucous membrane |
| CN109706257A (en) * | 2019-03-12 | 2019-05-03 | 西北农林科技大学 | A kind of method of pathogen in quick detection cow endometritis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906478A (en) * | 2010-08-11 | 2010-12-08 | 吉林大学 | Method for quickly diagnosing bovine endometritis pathogen |
| CN108030814A (en) * | 2017-12-06 | 2018-05-15 | 成都点石创想科技有限公司 | A kind of Chinese medicinal preparation method for the mucus or purulent inflammation that can treat cow uteri mucous membrane |
| CN109706257A (en) * | 2019-03-12 | 2019-05-03 | 西北农林科技大学 | A kind of method of pathogen in quick detection cow endometritis |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113897446A (en) * | 2021-09-18 | 2022-01-07 | 深圳临研医学有限公司 | Diagnosis and prognosis marker for rheumatoid arthritis |
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