Enterococcus faecium, microbial inoculum and application and method thereof

Abstract

The invention discloses enterococcus faecium, a microbial inoculum as well as application and a method of the enterococcus faecium, the enterococcus faecium is enterococcus faecium TC3 and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the enterococcus faecium is CCTCC NO: M 20242431. The invention also provides a microbial inoculum, wherein the microbial inoculum contains the enterococcus faecium TC3, and the enterococcus faecium TC3 can be used for preparing the microbial inoculum. Before a healthy pig is inoculated with a porcine reproductive and respiratory syndrome vaccine, the enterococcus faecium TC3 or the microbial inoculum is fed to the healthy pig, and then the healthy pig is inoculated with the porcine reproductive and respiratory syndrome vaccine, so that the effect performance of the pig inoculated with the porcine reproductive and respiratory syndrome vaccine can be improved, the antibody positive rate of the pig inoculated with the porcine reproductive and respiratory syndrome vaccine is increased, and the economic benefit of the pig is improved. The protection rate of the pigs inoculated with the porcine reproductive and respiratory syndrome vaccine is increased, the antibody dispersion of the pigs inoculated with the porcine reproductive and respiratory syndrome vaccine is reduced, and the survival rate of the pigs infected with the porcine reproductive and respiratory syndrome virus after the pigs inoculated with the porcine reproductive and respiratory syndrome vaccine is increased.

Description

Technical Field

[0001] The invention relates to the technical field of microbial products for pigs, and in particular to Enterococcus faecium, a bacterial agent and an application and method thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, is a highly contagious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). It can cause reproductive disorders such as stillbirth, mummified fetuses, weak piglets, premature births, etc. in sows. Nursery pigs have severe respiratory symptoms and are prone to secondary bacterial diseases such as Haemophilus parasuis and Streptococcus suis. If blue ear disease occurs in pigs on a pig farm, it will cause huge economic losses to the pig farming company. Vaccination of pigs is an important preventive measure for blue ear disease.

[0003] At present, although many pig farms have vaccinated their pigs against porcine reproductive and respiratory syndrome (PRRS), the effect of the vaccination is not ideal after the pigs are vaccinated. Pig farms still face great risks, making it difficult to improve breeding efficiency.

[0004] Therefore, it is very necessary to develop a product that can improve the performance of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine. Summary of the invention

[0005] The present invention provides an Enterococcus faecium, which is Enterococcus faecium TC3, which was deposited in China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China) on November 5, 2024, with a deposit number of CCTCC NO: M 20242431, and is named Enterococcus faecium TC3, Enterococcus faecium TC3.

[0006] Preferably, the Enterococcus faecium TC3 is bacterial powder.

[0007] The present invention also provides a bacterial agent, which contains the Enterococcus faecium TC3 as described above; further, the bacterial agent is a bacterial agent containing the Enterococcus faecium TC3 as described above, and the bacterial agent can also be a bacterial agent containing the Enterococcus faecium TC3 as described above and / or the metabolites of the Enterococcus faecium TC3 as described above.

[0008] Preferably, the Enterococcus faecium or the bacterial agent as described above, the Enterococcus faecium TC3 or the bacterial agent has at least one of the following characteristics:

[0009] (1) Improve the antibody positivity rate of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine;

[0010] (2) Improve the protection rate of pigs after vaccination against porcine reproductive and respiratory syndrome;

[0011] (3) Reduce the antibody dispersion of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine;

[0012] (4) Improve the survival rate of pigs infected with blue ear virus after vaccination with porcine reproductive and respiratory syndrome vaccine;

[0013] That is, the Enterococcus faecium TC3 has any one of the above characteristics or has multiple characteristics at the same time, and the bacterial agent has any one of the above characteristics or has multiple characteristics at the same time.

[0014] The present invention also provides at least one of the following uses of the Enterococcus faecium as described above:

[0015] (1) Application in the preparation of products for increasing the antibody positive rate of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine;

[0016] (2) Application in the preparation of products for improving the protection rate of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine;

[0017] (3) Application in the preparation of products that reduce antibody dispersion in pigs after vaccination with porcine reproductive and respiratory syndrome vaccine;

[0018] (4) Application in the preparation of products for improving the survival rate of pigs infected with blue ear virus after vaccination with blue ear disease vaccine;

[0019] That is, the present invention also provides any one of the above-mentioned applications of Enterococcus faecium TC3 as described above, or a plurality of applications acting simultaneously.

[0020] The present invention also provides at least one of the following applications of the above-mentioned bacterial agent:

[0021] (1) Application in the preparation of products for increasing the antibody positive rate of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine;

[0022] (2) Application in the preparation of products for improving the protection rate of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine;

[0023] (3) Application in the preparation of products that reduce antibody dispersion in pigs after vaccination with porcine reproductive and respiratory syndrome vaccine;

[0024] (4) Application in the preparation of products for improving the survival rate of pigs infected with blue ear virus after vaccination with blue ear disease vaccine;

[0025] That is, the present invention also provides any one of the above-mentioned applications of the above-mentioned bacterial agent or a plurality of applications acting simultaneously.

[0026] Preferably, in the bacterial agent as described above, the viable bacterial count of Enterococcus faecium TC3 is (1.0-9.0)×10 9 CFU / g.

[0027] The present invention also provides a method for improving the effect of pigs after vaccination with a porcine reproductive and respiratory syndrome vaccine, wherein the faecal enterococcus or the bacterial agent as described above is added to the daily diet of the pigs, mixed evenly and provided to healthy pigs for feeding, and after at least 7 days of feeding, the healthy pigs are vaccinated with the porcine reproductive and respiratory syndrome vaccine, and after 21 days of feeding, the healthy pigs are vaccinated with the porcine reproductive and respiratory syndrome vaccine again.

[0028] Preferably, in the above method for improving the effect of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine, the effect is manifested as at least one of the following:

[0029] (1) Antibody positivity rate of porcine reproductive and respiratory syndrome vaccine;

[0030] (2) Protection rate of porcine reproductive and respiratory syndrome vaccine;

[0031] (3) Antibody dispersion of porcine reproductive and respiratory syndrome vaccine;

[0032] (4) survival rate after infection with PRRS virus;

[0033] That is, according to the above method in the present invention, by feeding pigs with the Enterococcus faecium or the bacterial agent as described above in the present invention, the effect of the pigs after being vaccinated with the porcine reproductive and respiratory syndrome vaccine is any one of the above-mentioned effects or multiple effects that work simultaneously.

[0034] Preferably, in the above method for improving the effect performance of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine, the added amount of the Enterococcus faecium or the bacterial agent is 200g / t.

[0035] That is, the Enterococcus faecium or the bacterial agent as described above is added to the basic diet of pigs, and the added amount of the Enterococcus faecium or the bacterial agent is 200g / t (gram / ton), that is, 200 grams of the Enterococcus faecium or the bacterial agent is added to the basic diet (complete feed) of 1 ton of pigs, and mixed well to make a mixture for healthy pigs to eat. After at least 7 days of feeding, that is, after the healthy pigs have been freely eating the mixture for at least 7 days, the healthy pigs are vaccinated with porcine reproductive and respiratory syndrome vaccine. As above, the healthy pigs are still allowed to freely eat the mixture. After 21 days of feeding, the healthy pigs are vaccinated with porcine reproductive and respiratory syndrome vaccine again; if necessary, the healthy pigs can continue to freely eat the mixture.

[0036] The Enterococcus faecium TC3 provided by the present invention is separated from the intestinal contents of healthy pigs.

[0037] Beneficial effects of the present invention:

[0038] Before healthy pigs are vaccinated with the blue ear disease vaccine, the Enterococcus TC3 or the bacterial agent described in the present invention is fed to healthy pigs (mixed with feed for feeding), and then the healthy pigs are vaccinated with the blue ear disease vaccine. This can improve the effect performance of pigs after being vaccinated with the blue ear disease vaccine, increase the antibody positivity rate of pigs after being vaccinated with the blue ear disease vaccine, improve the immune system level of the pig herd, and increase the sensitivity of the pig herd to produce immune response when attacked by the blue ear virus. If the pig herd is attacked by the blue ear virus, more pigs in the pig herd can quickly produce antibodies and produce immune defense in time, and extend the protection period of the blue ear disease vaccine; improve the protection rate of pigs after being vaccinated with the blue ear disease vaccine, and increase the pigs' The titer of antibodies produced can better protect the health of pigs; the dispersion of antibodies after pigs are vaccinated with porcine reproductive and respiratory syndrome vaccine is reduced, and the antibody titer produced by each individual pig in the herd tends to the average level of the antibody titer of the herd, with high uniformity. The antibodies produced by the herd are stable and balanced, eliminating the fluctuation of the overall antibody level of the herd in routine immunization, the overall antibody level of the herd is stable, and the risk of blue ear disease is greatly reduced; the survival rate of pigs infected with blue ear virus after vaccination with porcine reproductive and respiratory syndrome vaccine is increased, the physical health status and immunity of pigs are improved, and after pigs are subsequently infected with blue ear virus, more pigs can survive through their own immune regulation, thereby reducing the occurrence of pig deaths. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below.

[0040] Embodiment 1:

[0041] The intestinal contents of healthy Tongcheng piglets were collected, mixed with sterile saline at a ratio of 1:50, and 1 mL of liquid was inoculated on the selective medium for Enterococcus faecium (Qingdao Haibo Biological), cultured in an anaerobic incubator at 37°C for 48±2h, and dark red or pink colonies with smooth and raised surfaces and neat peripheries were selected. After purification 3 times, a single colony was transferred to the Enterococcus broth medium and cultured at 37°C for 4h. Gram staining was positive, the colony morphology was oval, and there were no spores. Biochemical identification showed that glucose, sucrose, lactose, mannitol, arabinose, and arginine dihydrolase were positive, and xylose, raffinose, sorbitol, and 0.04% potassium tellurite were negative.

[0042] Biochemical identification showed that glucose, sucrose, lactose, mannitol, arabinose, and arginine dihydrolase were positive, and xylose, raffinose, sorbitol, and potassium tellurite were negative.

[0043] The strain was sent to Aoke (Wuhan) Biotechnology Co., Ltd. for gene sequencing. The comparison results of the NCBI database showed that the isolated strain had a homology of up to 99% with the sequences of Enterococcus faecium strain gp117, Enterococcus faecium strain gp34, Enterococcus faecium strain HBUR51181, etc. The 16s rRNA sequence of the strain is shown in SEQ NO.1. The strain belongs to Enterococcus faecium and is named Enterococcus faecium TC3.

[0044] SEQ NO.1

[0045]

[0046] Embodiment 2:

[0047] A total of 96 healthy three-way hybrid piglets aged 5 weeks with no symptoms of blue ear disease and no blue ear disease vaccine were selected from a large pig farm in Hubei. Before the experiment, serum and nasal swabs were collected for PRRSV ELISA antibody and RT-PCR antigen testing, and both were confirmed to be negative.

[0048] Blank group: fed with basic diet.

[0049] Experimental group 1: fed with basic diet + 200g / t Enterococcus faecium TC3 powder (the viable count of Enterococcus faecium TC3 was 1.0×10 9 CFU / g).

[0050] Experimental group 2: Feeding the basic diet + 200g / t Enterococcus faecium TC3 powder (the viable count of Enterococcus faecium TC3 was 5.0×10 9 CFU / g).

[0051] Experimental group 3: fed with basic diet + 200g / t Enterococcus faecium TC3 powder (the viable count of Enterococcus faecium TC3 was 9.0×10 9 CFU / g).

[0052] Each group had 2 replicates, with 12 weaned piglets in each replicate. They were raised in isolation and vaccinated with the highly pathogenic porcine reproductive and respiratory syndrome live vaccine JXA1-R strain (porcine blue ear disease vaccine) (Wuhan Keqian Biological Co., Ltd.) after 7 days of feeding.

[0053] After 21 days of continued feeding, the healthy pigs were vaccinated again with the same porcine reproductive and respiratory syndrome vaccine as mentioned above.

[0054] After 21 days of feeding, blood was drawn to detect antibody levels, and the antibody positive rate (S / P>0.4) and dispersion were calculated based on the test results. Dispersion is an important indicator to measure the uniformity of the antibody value of the pig herd, which can reflect the uniformity of the antibody level of the pig farm and provide a reference for the overall disease prevention and control of the pig farm. After the blood was drawn, the JXA1-R strain was used for virus attack. The clinical symptoms of the experimental pigs in each immunization group and the control group after vaccination and the lung lesions after slaughter were observed. At the same time, the results of body temperature measurement were referred to, and the antibody positive rate, dispersion and morbidity were comprehensively evaluated to evaluate the effect of Enterococcus faecium.

[0055] The calculation and judgment criteria of each indicator are as follows:

[0056] If S / P≥0.4, it is judged as PRRSV antibody positive; otherwise it is judged as negative. S / P=(OD 650 - Negative control OD 650 Average value) / (positive control OD 650 Mean - negative control OD 650 average value).

[0057] Dispersion = (standard deviation / sample mean) × 100%.

[0058] Onset criteria: (1) body temperature rises above 41°C for more than 3 days; (2) decreased spirit and appetite, conjunctivitis, cough, wheezing and other respiratory symptoms; (3) pathological changes in the lungs such as pneumonia and consolidation. Patients who meet the above 3 criteria can be diagnosed with the disease.

[0059] Incidence rate = (number of cases / total number of samples) × 100%.

[0060] Protection rate = (number of healthy individuals / total number of samples) × 100%.

[0061] Survival rate = (total number of samples - number of dead pigs) / total number of samples.

[0062] Table 1 Effect of Enterococcus faecium on vaccination against blue ear disease

[0063]

[0064]

[0065] The results are shown above. Before healthy pigs are vaccinated with the blue ear disease vaccine, the faecal Enterococcus TC3 or the bacterial agent described in the present invention is fed to healthy pigs (mixed with feed for feeding), and then the healthy pigs are vaccinated with the blue ear disease vaccine. This can improve the performance of pigs after being vaccinated with the blue ear disease vaccine, increase the antibody positivity rate of pigs after being vaccinated with the blue ear disease vaccine, increase the protection rate of pigs after being vaccinated with the blue ear disease vaccine, reduce the antibody dispersion of pigs after being vaccinated with the blue ear disease vaccine, and increase the survival rate of pigs infected with blue ear virus after being vaccinated with the blue ear disease vaccine.

Claims

1. An Enterococcus faecium, characterized in that The Enterococcus faecium is Enterococcus faecium TC3, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20242431.

2. A bacterial agent, characterized in that The bacterial agent contains the Enterococcus faecium TC3 according to claim 1.

3. The Enterococcus faecium according to claim 1 or the bacterial agent according to claim 2, characterized in that: The Enterococcus faecium TC3 or the bacterial agent has at least one of the following characteristics: (1) Improve the antibody positivity rate of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine; (2) Improve the protection rate of pigs after vaccination against porcine reproductive and respiratory syndrome; (3) Reduce the antibody dispersion of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine; (4) Improve the survival rate of pigs infected with porcine reproductive and respiratory syndrome virus after being vaccinated with porcine reproductive and respiratory syndrome vaccine.

4. At least one of the following uses of the Enterococcus faecium according to claim 1: (1) Application in the preparation of products for increasing the antibody positive rate of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine; (2) Application in the preparation of products for improving the protection rate of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine; (3) Application in the preparation of products that reduce antibody dispersion in pigs after vaccination with porcine reproductive and respiratory syndrome vaccine; (4) Application in the preparation of products that improve the survival rate of pigs infected with blue ear virus after being vaccinated with porcine blue ear disease vaccine.

5. At least one of the following uses of the bacterial agent according to claim 2: (1) Application in the preparation of products for increasing the antibody positive rate of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine; (2) Application in the preparation of products for improving the protection rate of pigs after vaccination with porcine reproductive and respiratory syndrome vaccine; (3) Application in the preparation of products that reduce antibody dispersion in pigs after vaccination with porcine reproductive and respiratory syndrome vaccine; (4) Application in the preparation of products that improve the survival rate of pigs infected with blue ear virus after being vaccinated with porcine blue ear disease vaccine.

6. The bacterial agent according to claim 2, characterized in that In the bacterial agent, the viable bacterial count of Enterococcus faecium TC3 is (1.0-9.0)×10 9 CFU / g.

7. A method for improving the effect of pigs after vaccination with a porcine reproductive and respiratory syndrome vaccine, characterized in that: Add the Enterococcus faecium described in claim 1 or the bacterial agent described in claim 2 to the daily diet of pigs, mix well and feed to healthy pigs. After at least 7 days of feeding, start to vaccinate the healthy pigs with porcine reproductive and respiratory syndrome vaccine. After 21 days of feeding, vaccinate the healthy pigs with porcine reproductive and respiratory syndrome vaccine again.

8. The method according to claim 7, characterized in that The effect is manifested as at least one of the following: (1) Antibody positivity rate of porcine reproductive and respiratory syndrome vaccine; (2) Protection rate of porcine reproductive and respiratory syndrome vaccine; (3) Antibody dispersion of porcine reproductive and respiratory syndrome vaccine; (4) Survival rate after infection with PRRS virus.

9. The method according to claim 7 or 8, characterized in that: The added amount of the Enterococcus faecium or the bacterial agent is 200g / t.

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