CN114657093A

CN114657093A - Pigeon-derived lactobacillus crispatus BL4014 and application thereof

Info

Publication number: CN114657093A
Application number: CN202210222395.4A
Authority: CN
Inventors: 邵玉新; 王铮; 李晶; 杜少华
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2022-03-07
Filing date: 2022-03-07
Publication date: 2022-06-24
Anticipated expiration: 2042-03-07
Also published as: CN114657093B

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a pigeon source lactobacillus crispatus BL4014 and application thereof. The invention provides a strain of Lactobacillus crispatus (Lactobacillus crispatus), the preservation number of which is CGMCC No. 21213. The lactobacillus crispatus can obviously inhibit trichomonas columbae, reduce the number of trichomonas columbae in oral cavity, prevent the weight reduction of the infected pigeons and the substantial damage to the liver caused by trichomonas columbae infection, and has good prevention and treatment effects on trichomonas columbae.

Description

Pigeon-derived lactobacillus crispatus BL4014 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a pigeon source lactobacillus crispatus BL4014 and application thereof.

Background

In the process of breeding livestock and poultry, when epidemic diseases occur, livestock and poultry can cause disorder of intestinal function under the influence of pathogenic bacteria and viruses, and functional disorders caused by a plurality of epidemic diseases can be prevented and treated by using antibiotics. However, the abuse of antibiotics brings many problems to the scale breeding of animals and the safety of food, and the antibiotics forbidden in feed are inevitable trends of producing green and safe food and reducing the risk of environment drug-resistant bacteria. According to No. 194 of the ministry of rural areas in agriculture, feed production enterprises stop producing commercial feeds containing growth-promoting drug feed additives from 7/1 of 2020. The search for new green and safe feed additives to replace antibiotics is a hot spot of current research. As a novel green feed additive, the probiotics has the advantages of safety, reliability, no residue, no drug resistance, no environmental pollution and the like, and can replace antibiotics to a certain extent. Probiotics are a class of living organisms that can exert beneficial effects on the host, and lactic acid bacteria belong to the class of probiotics. The lactobacillus crispatus is a normal bacterium in intestinal tracts of humans and animals, belongs to the genus lactobacillus, can regulate the balance of intestinal flora, has a broad-spectrum antibacterial effect on common intestinal pathogenic bacteria, and also has a plurality of beneficial physiological functions of enhancing the immunity and resistance of organisms, improving the utilization rate of feed, promoting the growth performance of animals and meat quality and the like. It has been reported that desirable probiotic strains are derived from indigenous animals.

Pigeon trichomoniasis is a common pigeon disease, and its etiology is trichomonas avicularis (Trichmonas gallinae) of Trichomonas of Trichomoriaceae. The most common sign of the disease is a coarse, button-like yellow deposit of the mucous membrane of the mouth and throat; when the umbilicus is infected, a lump is formed under the skin and presents cheese-like or ulcerative lesion; when internal organs are involved, cheese-like lesions with a sharp yellow rough boundary are caused, resulting in necrosis of parenchymal organ tissues. The main route of infection of the disease is oral infection. Both sick and vermin-carrying pigeons are sources of infection. The most common site of parasitism and damage by the polypide is the digestive tract. The oral ulcer focus of the sick pigeon is the colonization point of trichomonas, and saliva also contains a large amount of live insect bodies. The polypide can be infected by the polypide through drinking water, feed, wound and unclosed umbilical ring. Sick pigeons are impaired in ingestion by oral ulcers and can cause a high mortality rate in young pigeons and growing pigeons.

In recent years, the application of probiotics in breeding pigeons and young pigeons has been reported. The screening of the pigeon source probiotics has important significance for improving the composition of intestinal flora of pigeons and improving the resistance of the pigeons to diseases so as to improve the production performance of the pigeons.

Disclosure of Invention

In order to meet the requirements of the fields, fresh pigeon milk in the crop of a young pigeon of 4 days old is collected and a strain is separated from the fresh pigeon milk, and a strain of Lactobacillus crispatus (BL 4014) with a remarkable effect of resisting trichomonas columbae is screened. Through molecular biological identification, the nucleotide sequence of the 16srDNA of the strain is shown in SEQ ID NO. 1. The strain has been patented and preserved in China general microbiological culture Collection center (CGMCC) at 11/19/2020.

Based on the strain, the invention provides the Lactobacillus crispatus with the preservation number of CGMCC No. 21213.

The invention also provides a microbial inoculum, which comprises the lactobacillus crispatus.

The microbial inoculum can be a solid microbial inoculum or a liquid microbial inoculum. In some embodiments of the invention, the microbial inoculum is a culture solution of the lactobacillus crispatus. In some embodiments of the invention, the microbial inoculum is a lyophilized powder of the lactobacillus crispatus. In other embodiments of the present invention, the microbial inoculum further comprises an adjuvant component; the adjunct ingredient may be a biomaterial, such as corn flour or soy flour.

The invention also provides a product for preventing and/or treating trichomoniasis of pigeon, which is characterized in that: the active ingredient of the product comprises the lactobacillus crispatus.

In some embodiments of the invention, the lactobacillus crispatus of the invention is combined with other probiotics or medicaments to make a product for preventing and/or treating trichomoniasis of pigeons.

The invention also provides a product for preventing and/or treating trichomoniasis of pigeon, which is characterized in that: the active ingredient of the product is the lactobacillus crispatus.

In some embodiments of the invention, the product for the prevention and/or treatment of trichomoniasis in pigeon is a feed. In some embodiments of the invention, the feed is pigeon feed. In other embodiments of the invention, the product for the prevention and/or treatment of trichomoniasis in pigeon is a feed additive.

In some embodiments of the present invention, the formulation of the product for preventing and/or treating trichomoniasis pilaris is powder formulation, granule formulation or liquid formulation.

The invention also provides a product for inhibiting trichomonas columbae, which is characterized in that: the active ingredient of the product comprises the lactobacillus crispatus.

In some embodiments of the invention, lactobacillus crispatus according to the invention is combined with other probiotics or drugs to make a trichomonas columbae inhibiting product.

The invention also provides a product for inhibiting trichomonas columbae, which is characterized in that: the active ingredient of the product is the lactobacillus crispatus.

In some embodiments of the invention, the trichomonas columbae inhibiting product further comprises conventional adjuvant ingredients used in the preparation of probiotic preparations.

In some embodiments of the invention, the formulation of the trichomonas columbae inhibiting product is a powder formulation, a granular formulation or a liquid formulation.

The application of the lactobacillus crispatus in preparing products for preventing and/or treating trichomoniasis of pigeons also belongs to the protection scope of the invention.

The invention also provides a method for inhibiting the trichomonas columbae in vitro, which is characterized in that the lactobacillus crispatus is used for inhibiting the trichomonas columbae existing in vitro.

In some embodiments of the invention, the Lactobacillus crispatus is inoculated into MRS liquid medium and cultured anaerobically at 37 ℃ for 48h to a thallus concentration of 2 × 10⁸And (3) collecting bacterial liquid at cfu/mL (the bacterial liquid has stronger killing performance on the trichomonas columbae) and using the bacterial liquid for inhibiting the trichomonas columbae.

The Lactobacillus crispatus BL4014 provided by the invention belongs to pigeon source Lactobacillus crispatus and is an ideal indigenous probiotic. In vitro tests prove that the relative inhibition rate of lactobacillus crispatus BL4014 to trichomonas columbae is 96.30%, which is equivalent to the inhibition effect of metronidazole. In vivo experiments prove that the lactobacillus crispatus BL4014 is administrated by drenching 7 days before and 7 days after the pigeon trichomonas infection every day, or the lactobacillus crispatus BL4014 is administrated by drenching 7 days after the pigeon trichomonas infection every day, so that the weight of the infected pigeons can be effectively prevented from being reduced, the number of oral pigeon trichomonas can be obviously reduced, and the substantive damage of the pigeon trichomonas infection to the liver can be prevented. Therefore, the lactobacillus crispatus BL4014 has a good prevention and treatment effect on trichomonas columbae. The strain can be used for preparing probiotics or added into feed, improves the composition of intestinal flora of the pigeons, and promotes the metabolism of amino acid of organisms, so that the production performance of the pigeons is improved, and the strain has good application prospect.

The preservation information of lactobacillus crispatus provided by the invention is as follows:

biological material (strain): BL4014

And (3) classification and naming: lactobacillus crispatus

The preservation number is as follows: CGMCC No.21213

The preservation date is as follows: year 2020, 11 and 19

The preservation unit: china general microbiological culture Collection center (CGMCC)

And (4) storage address: beijing, Chaoyang, Beicheng Xilu No. 1 Hospital No. 3, institute of microbiology, Chinese academy of sciences, zip code 100101.

Drawings

Fig. 1 shows the morphology of lactobacillus crispatus BL4014 under a 400-fold optical microscope.

Detailed Description

The present invention is described in detail below with reference to examples, it being understood that the following examples are only illustrative and illustrative of the present invention and do not limit the scope of the present invention in any way.

Animals: the 4-day-old young pigeons and 7-day-old white-feather king meat pigeons used in the following examples were provided by the pigeon breeding research institute of the animal veterinary institute of the agroforestry academy of sciences of Beijing.

Insect strain: the trichomonas columbae used in the following examples is a strain of trichomonas columbae (t. gallinae) isolated from the pigeon breeding institute of the animal husbandry veterinary institute of the agroforestry academy of sciences of beijing.

Reagent: fetal bovine serum was purchased from Biosharp (jj. kyo technologies ltd.); metronidazole water was purchased from big pharmacy of Guangdekang, Beijing; the 100 XPicillin-streptomycin solution was purchased from Biosharp (Kyoho technologies, Inc., Beijing Lankan).

TYM medium was purchased from Ku Ladbo Tech Co., Ltd, Beijing under the product number DZPM 0385. Formulation (1L): tryptose peptone 20.0g, yeast extract powder 10.0g, maltose 5.0g, L-cysteine hydrochloride 1.0g, vitamin C0.2 g, dipotassium hydrogen phosphate 0.8g, potassium dihydrogen phosphate 0.8g, agar 0.5 g. If a solid medium is prepared, 15g/L agar is added on the basis of the formula. Dissolving the above components in 900mL of distilled water, adjusting pH to 6.0 with 1N HCl, boiling to dissolve, packaging, and autoclaving at 121 deg.C for 20 min. Cooling to 45 deg.C, aseptically adding 100mL fetal calf serum inactivated at 56 deg.C for 30 min, and mixing well for use.

MRS medium was purchased from Oxoid under stock number CM 1163. Formulation (1L): casein hydrolysate 20.0g, yeast extract powder 10.0g, maltose 5.0g, L-cysteine hydrochloride 1.0g, vitamin C0.2 g, dipotassium hydrogen phosphate 0.8g, potassium dihydrogen phosphate 0.8g, and agar 0.5 g. If a solid medium is prepared, 15g/L agar is added on the basis of the formula. Dissolving the above components in 1000mL of distilled water, boiling to dissolve, packaging, autoclaving at 121 deg.C for 20 min, and cooling to room temperature.

Unless otherwise specified, the reagents used in the following examples are conventional in the art, and are either commercially available or formulated according to methods conventional in the art, and may be of laboratory pure grade. Unless otherwise specified, the experimental methods and conditions used in the following examples are all conventional in the art, and reference may be made to relevant experimental manuals, well-known literature, or manufacturer's instructions. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1 isolation and identification of strains

1. Isolation of the Strain

Collecting fresh pigeon milk in crop of 4-day-old young pigeon, weighing 5g, placing in a centrifuge tube containing 25ml sterile normal saline, and shaking up to obtain initial bacterial liquid. Then 0.1ml of the initial bacterial liquid is sucked into a test tube containing 0.9ml of sterile physiological saline, and the dilution degree is 10^-1Repeating the above processes to sequentially obtain dilution of 10^-2、10^-3、10^-4、10^-5、10^-6、10^-7The diluted bacterial liquid of (4). Selection 10^-5、10^-6、10^-7And (3) respectively sucking 0.1ml of diluted bacteria, dripping the diluted bacteria on an MRS solid culture medium, placing the MRS solid culture medium in an anaerobic jar, culturing for 24 hours at 37 ℃, and picking out typical colonies which are flat and round in appearance and are similar to the sun in shape.

2. Screening of strains

The three typical bacterial colonies separated in the step 1 are detected by using the insect strain of the trichomonas columbae separated from the pigeon breeding research laboratory of the institute of livestock and veterinary of agriculture and forestry, Beijing City, and the bacterial strain capable of inhibiting the trichomonas columbae is screened. Three representative colonies were designated as strain A, strain B, and strain C, respectively.

Cultivating the trichomonas columbae: (1) performing aseptic operation in a super clean bench, inoculating Trichomonas Columbinis (T. gallinae) strain stored in a-80 deg.C refrigerator into TYM liquid culture medium, adding 10% sterilized fetal calf serum and appropriate amount of 100 XPcillin-streptomycin solution, and performing anaerobic culture in a 37 deg.C constant temperature incubator for 48 h. (2) Placing the cultured Pigeon trichomonad culture solution for 48h in a centrifuge tube, centrifuging at room temperature at 5000rpm/min for 3min, removing supernatant, adding TYM liquid culture medium, and suspending the precipitated insect body; (3) counting the total number of the worms and the number of the live worms, wherein the cell activity is more than 95 percent, so that the worms can be used for the test; wherein, the cell activity is the number of living polypide/total polypide multiplied by 100%; (4) the pigeon trichomonas columbae culture solution is adjusted into suspension with certain density according to the number of live larvae, and the suspension is placed at 37 ℃ for standby.

Preparing a bacterial liquid: respectively inoculating the strain A, the strain B and the strain C in MRS liquid culture medium, and anaerobically culturing at 37 deg.C for about 48 hr until the thallus concentration is 2 × 10⁸cfu/mL hourly harvestCollecting bacteria liquid for later use.

The test set is a control group, a metronidazole treatment group, a strain A treatment group, a strain B treatment group and a strain C treatment group, and each group is set with three replicates. 5X 10 was added to a 5mL sterile centrifuge tube⁵The trichomonas columbae bodies, 10% sterilized fetal calf serum and TYM liquid medium are subjected to anaerobic culture at a constant temperature of 37 ℃ until the final volume is 4 mL. After 1h of culture, 1mL of TYM liquid culture medium is added into the culture tube of the control group, 1mL of metronidazole solution with the concentration of 1mg/mL is added into the culture tube of the metronidazole treatment group, 1mL of fresh bacterial liquid of the strain A is added into the culture tube of the strain A treatment group, 1mL of fresh bacterial liquid of the strain B is added into the culture tube of the strain B treatment group, and 1mL of fresh bacterial liquid of the strain C is added into the culture tube of the strain C treatment group. The final volume of liquid in each culture tube was 5 mL. All culture tubes were placed in an anaerobic jar for anaerobic culture for 12 h. After culturing for 12h, uniformly mixing the culture solution in the culture tube by using a vortex mixer, sucking a certain suspension liquid by using a sterile gun head to drop on a blood counting plate covered with a glass slide, and counting the number of live insects of each group according to the principle of counting up and down and counting left and right. If the density of the nymphs is too high, the nymphs are diluted by sterile normal saline or preheated TYM liquid culture medium and then counted. The number of worms per group was taken as the average of the triplicates in the group. The relative inhibition rate of each group of pigeon trichomonas is (the number of the polypide in the control group-the number of the polypide in the group)/the number of the polypide in the control group is multiplied by 100%.

The results are shown in table 1, and the relative inhibition rate of metronidazole to trichomonas columbae is 99.38% in an in vitro environment; the strain A has no inhibition effect on the trichomonas columbae and promotes the growth and the propagation of the trichomonas columbae to a certain degree; the inhibition effect of the strain B on the trichomonas columbae is very small, and the relative inhibition rate is only 4.46 percent; the strain C can obviously inhibit the trichomonas columbae, has the relative inhibition rate of 96.30 percent, and has the equivalent inhibition effect with metronidazole.

TABLE 1 relative inhibition of each group against Pigeon trichomonas

Group of	Number of worms after 12h of culture (number/mL)	Relative inhibition rate
			Control group	21.07×10⁵	0.00％
Metronidazole treatment group	0.13×10⁵	99.38％
			Strain A treatment group	21.60×10⁵	-2.52％
Strain B treatment group	20.13×10⁵	4.46％
			Strain C treatment group	0.78×10⁵	96.30％

3. Identification of strains

And (3) sending the screened strain C capable of obviously inhibiting the trichomonas columbae to a China Industrial microbial culture Collection management center (CICC) for strain identification. The results of morphological observation are shown in FIG. 1, and the strain C is in the form of a rod under an optical microscope of 400X. Through molecular biological identification, the strain C is determined to be Lactobacillus crispatus (Lactobacillus crispatus). The strain C is delivered to China general microbiological culture Collection center (CGMCC) for preservation, the preservation name is BL4014, the classification name is Lactobacillus crispatus, the preservation number is CGMCC No.21213, and the preservation date is 2020 years, 11 months and 19 days.

The sequence of 16srDNA of Lactobacillus crispatus BL4014 is as follows:

CTTTGGGCATTGCAGACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCAGCTTCGTGCAGTCGAGTTGCAGACTGCAGTCCGAACTGAGAACAGCTTTCAGAGATTCGCTTGCCTTCGCAGGCTCGCTTCTCGTTGTACTGCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCATTAGAGTGCCCAACTTAATGCTGGCAACTAATAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCTTAGCGTCCCCGAAGGGAACTTTGTATCTCTACAAATGGCACTAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAGAGGCGGAAACCTCCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTCTCCTCTTCTGCACTCAAGAAAAACAGTTTCCGATGCAGTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTATTCTTCCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTGATTACCGTCAAATAAAGGCCAGTTACTACCTCTATCCTTCTTCACCAACAACAGAGCTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAAGCCTTTACCTTACCAACTAGCTAATGCACCGCGGGGCCATCCCATAGCGACAGCTTACGCCGCCTTTTAAAAGCTGATCATGCGATCTGCTTTCTTATCCGGTATTAGCACCTGTTTCCAAGTGGTATCCCAGACTATGGGGCAGGTTCCCCACGTGTTACTCACCCATCCGCCGCTCGCTTTCCTAACGTCATTACCGAAGTAAATCT(SEQ ID NO:1)。

example 2 detection of Effect of Lactobacillus crispatus BL4014 on in vivo inhibition of Trichomonas Columba

1. Animal testing

Selecting 100 white-feather king meat pigeons which are artificially fed for 7 days old, and breeding the pigeons in cages in a strictly-sterilized environment without pigeon trichomonas contamination. The method comprises the steps of wearing foot rings on 100 7-day-old white-feather king meat pigeons which are not infected with trichomonas, weighing, calculating average weight, randomly dividing the meat pigeons into five groups according to the average weight, wherein each group comprises 20 meat pigeons, each group is fed in 3 cages, and the vertical direction, the middle layer and the lower layer form one group, so that the total weight of each group is basically consistent. Five groups of meat pigeons are respectively set as A group (negative control group), B group (positive control group), C group (metronidazole treatment group), D group (BL4014 control group) and E group (BL4014 treatment group). The effect of each group on the inhibition of the trichomonas columbae was tested using a strain of trichomonas columbae (t. gallinae) isolated from the pigeon breeding research institute of the institute of zootechnics and veterinarian of the agroforestrial academy of beijing. The treatment methods for each group are shown in Table 2.

Preparing BL4014 bacterial liquid: inoculating Lactobacillus crispatus BL4014 into MRS liquid culture medium, and anaerobically culturing at 37 deg.C for about 48 hr until thallus concentration is 2 × 10⁸And collecting bacterial liquid for later use when cfu/mL is obtained.

Cultivation of eggs of Pigeon trichomonad (T.gallinae): (1) performing aseptic operation in a super clean bench, inoculating Pigeon trichomonas in a refrigerator at-80 deg.C in TYM liquid culture medium, adding 10% sterilized fetal calf serum and appropriate amount of 100 XPcillin-streptomycin solution, and performing anaerobic culture in a constant temperature incubator at 37 deg.C for 48 h. (2) Placing the trichomonas columbae culture solution cultured for 48h into a centrifuge tube, centrifuging at room temperature at 5000rpm/min for 3min, removing supernatant, adding TYM liquid culture medium, and suspending precipitated insect body; (3) counting the total number of the worms and the number of the live worms, wherein the cell activity is more than 95 percent, so that the method can be used for tests; wherein, the cell activity is the number of living polypide/total polypide multiplied by 100%; (4) the pigeon trichomonas columbae culture solution is adjusted into suspension with certain density according to the number of live larvae, and the suspension is placed at 37 ℃ for standby.

The operating method of drenching comprises the following steps: the corresponding solution was orally drenched using a pipette.

TABLE 2 treatment methods for each group

2. Influence of Lactobacillus crispatus BL4014 before and after trichomonas columbae infection on average weight gain of young pigeons

The weights of the young pigeons in each group were measured at 7-day-old, 14-day-old and 21-day-old, respectively, and the average weight was calculated. The results are shown in table 3, the average weights of the young pigeons in each group have no significant difference (P >0.05) at the age of 7 days, which indicates that the test grouping is correct; the average body weight of each group has no significant difference at the age of 14 days, and the average body weight of the BL4014 control group is higher than that of the other four groups; at the age of 21 days, the average body weight of the young pigeon infected with the trichomonas columbae in the positive control group is significantly lower than that of the young pigeon not infected with the trichomonas columbae in the negative control group (P <0.05), but the average body weight of the metronidazole treatment group, the BL4014 control group and the BL4014 treatment group is not significantly different from that of the negative control group (P > 0.05). The result shows that the young pigeon weight is reduced due to trichomonas infection of the pigeon, but the young pigeon weight is not reduced due to the metronidazole treatment and the drenching of the lactobacillus crispatus BL4014, which indicates that the lactobacillus crispatus BL4014 has a weight increasing effect on the young pigeon to a certain extent.

TABLE 3 weight gain and relative weight gain of each group before and after insect attack

Note: data are presented as mean ± sd, with the same column of shoulder-marked lower case letters indicating significant difference (P <0.05), upper case letters indicating very significant difference (P <0.01), and the following.

3. Number of trichomonas Columba in each group

And (3) detecting the number of trichomonas stomachis of the young pigeons of each group 7 days after the attack infection, namely when the young pigeons are 21 days old. The method comprises the following steps: collecting oral cotton swab samples of pigeons in 1.5mL centrifuge tubes containing 500 mu L sterile physiological saline, repeatedly collecting each pigeon for 3 times, oscillating for 3min, and counting the number of pigeon trichomonas in each sample by using a blood cell counting plate, wherein the average value is the number of pigeon trichomonas carried in the oral cavity of each pigeon.

As a result, as shown in Table 4, the number of trichomonas oralis in the negative control group was 0, and the number of trichomonas oralis in the positive control group reached 1.38X 10⁶individual/mL, oral cavity pigeon trichomonas of other attacking groupsNumber is only 10⁵one/mL. The quantity of the oral cavity pigeon trichomonas mellifera of the metronidazole treatment group and the BL4014 prevention group is obviously less than that of the positive control group, and the difference is obvious (P)<0.05). The number of the oral pigeon trichomonas columbae in the BL4014 treatment group is less than that of the positive control group, but the difference is not significant. The test result shows that the continuous irrigation of the lactobacillus crispatus BL4014 for 7 days before and after the infection of the pigeon trichomonas columbae can obviously reduce the number of the pigeon trichomonas columbae in the oral cavity of the young pigeons, thereby effectively preventing and treating the pigeon trichomonas columbae, and the effect is similar to that of the metronidazole for treating the pigeon trichomonas columbae.

TABLE 4 number of oral Pigeon trichomonads for each group

Group of	Number of trichomonas of 21-day-old young pigeon (10)⁵one/mL)
		Negative control group (group A)	0^c
Positive control group (group B)	13.80±8.77^a
		Metronidazole therapy group (group C)	2.98±1.31^b
BL4014 prevention and cure group (group D)	3.47±3.62^b
		BL4014 treatment group (E group)	8.44±1.27^ab
P value	0.046

4. Blood general index

And (4) carrying out blood routine detection on each group of young pigeons 7 days after the attack of the pests, namely 21 days old of the young pigeons. The method comprises the following steps: after hunger treatment of each group of pigeons, collecting whole blood samples by using disposable vacuum blood collection tube wings containing anticoagulant EDTA-K2 with soft needles to collect blood samples through veins, slightly shaking the test tube for 4-5 times to fully mix the blood samples with the anticoagulant, and collecting 2 tubes of each pigeon, wherein each tube is 3 mL. The collected whole blood was subjected to blood routine testing using a fully automatic veterinary blood cell analyzer (K4500, josmen, japan) within 4 hours.

The results are shown in table 5, and the red blood cell content and the hemoglobin content of each group of young pigeons are not significantly different; the leukocyte content of the positive control group is obviously increased and has obvious difference (P <0.05) with that of the negative control group, and the leukocyte content of the other three test groups has no obvious difference (P >0.05) with that of the negative control group; the number of mononuclear cells in the positive control group is remarkably increased, and is remarkably different from that in other four test groups (P <0.05), and compared with the negative control group, the BL4014 control group and the BL4014 treatment group are not remarkably different (P > 0.05); compared with the negative control group, the positive control group had a tendency to increase the numbers of eosinophils and basophils, and the other three test groups had no tendency to increase the numbers of eosinophils and basophils.

By combining the blood conventional index changes of each group, the significant changes of the blood conventional indexes related to the parasitic diseases of the BL4014 prevention group and the BL4014 treatment group are not caused by the trichomonas columbae infection, and are similar to the metronidazole treatment group. The result shows that the lactobacillus crispatus BL4014 is continuously administrated 7 days before and after the young pigeon is infected with the trichomonas columni or the lactobacillus crispatus BL4014 is continuously administrated 7 days after the young pigeon is infected with the trichomonas columni has good prevention and treatment effects on the trichomonas columni infection, and the effect of the lactobacillus crispatus BL4014 administrated 7 days before and after the infection is better and is similar to the effect of metronidazole treatment.

Blood routine index of different test groups at day 521

TABLE 5 (CONTINUOUS) blood general indices of 21-day-old different test groups

5. Measurement of blood biochemical indices

After 7 days of insect attack infection, namely when the young pigeons are 21 days old, the enzyme linked immunosorbent assay kit is used for detecting the content of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), total serum protein and albumin in the serum of each group of young pigeons, and the detection method refers to the use instruction of each kit. The enzyme linked immunosorbent assay kit is used as follows: alanine aminotransferase test kit (ALT), Nanjing, cat # A009-2-1; aspartate aminotransferase test kit (AST), manufactured by Nanjing, cat # C010-2-1; a total protein determination kit, which is built by Nanjing and has the product number A045-4-2; albumin assay kit, Nanjing Kangkui, cat # A028-2-1.

The detection results are shown in table 6, after young pigeons are infected with trichomonas pigeon, the ALT expression level and the AST expression level of the positive control group are both obviously increased and have obvious difference (P <0.05) with the negative control group, while the ALT expression level and the AST expression level in the serum of other three test groups are not obviously increased after the young pigeons are infected with trichomonas pigeon, and have no obvious difference (P >0.05) with the negative control group. Test results show that the lactobacillus crispatus BL4014 is administrated 7 days before and after the pigeon trichomonas infection, so that the ALT and AST content in serum of the young pigeon can be kept at a normal level, and the effect is the same as that generated by metronidazole treatment, therefore, the lactobacillus crispatus BL4014 is administrated, the substantive damage of the pigeon trichomonas infection to the liver can be effectively prevented, and the prevention and treatment effect on the pigeon trichomonas is good.

TABLE 6 Biochemical index of blood of each group

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make various modifications and decorations without departing from the technical principle of the present invention, and the modifications and decorations also fall within the protection scope of the present invention.

SEQUENCE LISTING

<110> agriculture and forestry academy of sciences of Beijing City

<120> pigeon source lactobacillus crispatus BL4014 and application thereof

<130> P220074-NLK

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1377

<212> DNA

<213> Lactobacillus crispatus

<400> 1

ctttgggcat tgcagactcc catggtgtga cgggcggtgt gtacaaggcc cgggaacgta 60

ttcaccgcgg cgtgctgatc cgcgattact agcgattcca gcttcgtgca gtcgagttgc 120

agactgcagt ccgaactgag aacagctttc agagattcgc ttgccttcgc aggctcgctt 180

ctcgttgtac tgcccattgt agcacgtgtg tagcccaggt cataaggggc atgatgactt 240

gacgtcatcc ccaccttcct ccggtttgtc accggcagtc tcattagagt gcccaactta 300

atgctggcaa ctaataacaa gggttgcgct cgttgcggga cttaacccaa catctcacga 360

cacgagctga cgacagccat gcaccacctg tcttagcgtc cccgaaggga actttgtatc 420

tctacaaatg gcactagatg tcaagacctg gtaaggttct tcgcgttgct tcgaattaaa 480

ccacatgctc caccgcttgt gcgggccccc gtcaattcct ttgagtttca accttgcggt 540

cgtactcccc aggcggagtg cttaatgcgt tagctgcagc actgagaggc ggaaacctcc 600

caacacttag cactcatcgt ttacggcatg gactaccagg gtatctaatc ctgttcgcta 660

cccatgcttt cgagcctcag cgtcagttgc agaccagaga gccgccttcg ccactggtgt 720

tcttccatat atctacgcat tccaccgcta cacatggagt tccactctcc tcttctgcac 780

tcaagaaaaa cagtttccga tgcagttcct cggttaagcc gagggctttc acatcagact 840

tattcttccg cctgcgctcg ctttacgccc aataaatccg gacaacgctt gccacctacg 900

tattaccgcg gctgctggca cgtagttagc cgtgactttc tggttgatta ccgtcaaata 960

aaggccagtt actacctcta tccttcttca ccaacaacag agctttacga tccgaaaacc 1020

ttcttcactc acgcggcgtt gctccatcag acttgcgtcc attgtggaag attccctact 1080

gctgcctccc gtaggagttt gggccgtgtc tcagtcccaa tgtggccgat cagtctctca 1140

actcggctat gcatcatcgc cttggtaagc ctttacctta ccaactagct aatgcaccgc 1200

ggggccatcc catagcgaca gcttacgccg ccttttaaaa gctgatcatg cgatctgctt 1260

tcttatccgg tattagcacc tgtttccaag tggtatccca gactatgggg caggttcccc 1320

acgtgttact cacccatccg ccgctcgctt tcctaacgtc attaccgaag taaatct 1377

Claims

1. Lactobacillus crispatus (Lactobacillus crispatus) with the preservation number of CGMCC No. 21213.

2. A microbial inoculant comprising lactobacillus crispatus as claimed in claim 1.

3. A product for preventing and/or treating trichomoniasis of pigeon is characterized in that: the active ingredient of the product comprises lactobacillus crispatus as claimed in claim 1.

4. A product for preventing and/or treating trichomoniasis of pigeon is characterized in that: the active ingredient of the product is lactobacillus crispatus as claimed in claim 1.

5. The product for the prevention and/or treatment of trichomoniasis of pigeon according to claim 3 or 4, characterized in that: the product is a feed or a feed additive.

6. A product for inhibiting trichomonas columbae is characterized in that: the active ingredient of the product comprises lactobacillus crispatus as claimed in claim 1.

7. A product for inhibiting trichomonas columbae is characterized in that: the active ingredient of the product is lactobacillus crispatus as claimed in claim 1.

8. A product for the inhibition of trichomonas columbae according to claim 6 or 7, wherein: the product also contains conventional adjuvant ingredients for preparing probiotic preparations.

9. Use of lactobacillus crispatus according to claim 1 for the preparation of a product for the prevention and/or treatment of trichomoniasis of pigeon.

10. A method for inhibiting Pigeon trichomonas in vitro, which comprises inhibiting Pigeon trichomonas in vitro using the Lactobacillus crispatus of claim 1.