New application of lactobacillus rhamnosus P118

Abstract

The invention relates to the technical field of prevention and control of salmonella pullorum, in particular to novel application of lactobacillus rhamnosus P118. The lactobacillus rhamnosus P118 provided by the invention has any one of the following applications: (1) the infection rate of chicks to salmonella pullorum is reduced, (2) the infection rate of the chicks to salmonella pullorum is reduced, (3) the infection rate of the chicks to salmonella pullorum is reduced, and (4) the infection rate of the chicks to salmonella pullorum is reduced; (2) the egg laying performance of hens infected with salmonella pullorum is improved; (3) reducing the bacterial load in the tissues and organs of the hens; (4) reducing horizontal propagation and vertical propagation of salmonella pullorum; and (5) reducing the risk that salmonella pullorum is vertically spread from hens to offspring through eggs. The antagonistic effect of the lactobacillus rhamnosus P118 on pullorum disease in an in-vivo and in-vitro infection model is systematically evaluated, and it is found that the P118 has a remarkable inhibiting effect on colonization infection of the pullorum disease in chick and laying hen models in the egg laying peak period.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Salmonella pullorum prevention and control, and particularly relates to a new use of Lactobacillus rhamnosus P118. BACKGROUND

[0002] Salmonella pullorum (SP) is an important serotype of Salmonella, and its infection can cause highly infectious chicken white dysentery. The wide spread of the pathogenic bacteria seriously restricts the healthy development of poultry industry. SP can infect chickens of all ages, among which the pathogenicity to 2-3 week-old chicks is the strongest and the mortality rate is the highest. Adult chickens infected show decreased egg production, dysentery, weight loss, reduced fertility and hatching rate, and long-term hidden bacterial carriage, which becomes a persistent source of transmission. There are mainly two transmission routes of SP: one is horizontal transmission, and the other is vertical transmission. After invading the intestinal epithelium, SP can be excreted outside the body with feces, polluting the environment and infecting other individuals. After being phagocytosed by macrophages and dendritic cells, SP can still survive in the cells and migrate into the lymphatic system or blood system and colonize in various organs. The SP colonized in the reproductive system will be transmitted to offspring through the eggs, forming a new infection cycle. As a facultative intracellular parasite, SP is difficult to be completely eliminated by antimicrobial drugs or immune cells and antibodies produced by the immune system during carriage or persistent infection. Therefore, it is of great significance to effectively inhibit and block the double transmission routes (horizontal transmission and vertical transmission) of SP for the prevention and control of chicken white dysentery.

[0003] At present, the main measure to inhibit chicken white dysentery is to avoid horizontal transmission through the use of antibiotics and isolation and elimination. However, in the context of "reducing, banning and replacing antibiotics", green and safe microbial preparations are considered as an important alternative solution to improve the intestinal health of livestock and poultry and promote production performance. The existing technology can prevent chicken white dysentery from horizontal transmission by using beneficial bacteria such as lactic acid bacteria to enhance intestinal barrier and immune function, but does not consider the intervention of vertical transmission route. SUMMARY

[0004] Therefore, in order to solve the above problems, the present application provides a new use of Lactobacillus rhamnosus P118.

[0005] Therefore, in order to solve the above problems, the present application provides a new use of Lactobacillus rhamnosus P118.

[0006] (1) reducing the infection rate of chicks to chicken white dysentery;

[0007] (ii) improving the egg production performance of hens infected with Salmonella pullorum;

[0008] (iii) reducing the bacterial load in the tissues and organs of hens;

[0009] (iv) reducing the horizontal and vertical transmission of Salmonella pullorum;

[0010] (v) reducing the risk of vertical transmission of Salmonella pullorum from hens to offspring via eggs.

[0011] In some embodiments, the Lactobacillus rhamnosus P118 is a live bacterium, a dead bacterium, or a culture thereof.

[0012] In some embodiments, the culture of Lactobacillus rhamnosus P118 comprises a fermentation broth of Lactobacillus rhamnosus P118.

[0013] In some embodiments, the method for preparing the fermentation broth of Lactobacillus rhamnosus P118 comprises the following steps: inoculating Lactobacillus rhamnosus P118 into MRS liquid medium, and anaerobically culturing at 35-38°C for 16-24h to obtain the fermentation broth.

[0014] In some embodiments, the inoculation amount of Lactobacillus rhamnosus P118 is 1-4vol% based on the volume of MRS liquid medium.

[0015] In some embodiments, the concentration of Lactobacillus rhamnosus P118 before inoculation is 1x10 8 -1x10 9 CFU / mL.

[0016] In some embodiments, the culture of Lactobacillus rhamnosus P118 further comprises a supernatant obtained from the fermentation broth of Lactobacillus rhamnosus P118.

[0017] In some embodiments, the method for preparing the supernatant obtained from the fermentation broth of Lactobacillus rhamnosus P118 comprises the following steps: centrifuging the fermentation broth of Lactobacillus rhamnosus P118 at 4°C and a speed of 3500-4000rpm for 5-10min, filtering the supernatant obtained after centrifugation at least twice to obtain the supernatant.

[0018] In some embodiments, the step of filtering uses a sterile filter membrane with a pore size of 0.22μm.

[0019] In some embodiments, the Lactobacillus rhamnosus P118 is deposited with the China Center for Type Culture Collection (CCTCC) and has the accession number CCTCC NO:M20221065.

[0020] In some embodiments, the amount of Lactobacillus rhamnosus P118 added to the feed is 0.5-1 wt%.

[0021] The technical scheme of the present application has the following advantages:

[0022] The Lactobacillus rhamnosus P118 provided by the present application has any of the following uses: (1) reducing the infection rate of chicks by Salmonella pullorum; (2) improving the egg production performance of hens infected with Salmonella pullorum; (3) reducing the bacterial load in the tissues and organs of hens; (4) reducing the horizontal and vertical transmission of Salmonella pullorum; (5) reducing the risk of vertical transmission of Salmonella pullorum from hens to offspring through eggs. The present application evaluates the antagonistic effect of Lactobacillus rhamnosus P118 on chicken pullorum in in vivo and in vitro infection models, and finds that P118 has a significant inhibitory effect on the colonization and infection of chicken pullorum in chick and laying hen models at the peak of egg production, providing a reliable solution to the difficult problem of chicken pullorum epidemic prevention in poultry farming. It is also found that Lactobacillus rhamnosus P118 has a synergistic blocking strategy for both horizontal and vertical transmission. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and those skilled in the art can also obtain other drawings based on these drawings without creative labor.

[0024] Figure 1 is the detection result of the antibacterial ability of Lactobacillus rhamnosus P118 on Salmonella pullorum in experimental example 1 of the present application, wherein, Figure 1 GEN in the above formula represents the antibacterial ability of gentamicin on Salmonella pullorum;

[0025] Figure 2 is the detection result of the influence of Lactobacillus rhamnosus P118 on the biofilm formation ability of Salmonella pullorum in experimental example 1 of the present application;

[0026] Figure 3 is the interaction between Lactobacillus rhamnosus P118 and Salmonella pullorum in experimental example 1 of the present application, wherein, Figure 3 A in the above formula represents the inhibitory effect of Lactobacillus rhamnosus P118 on the growth of Salmonella pullorum, Figure 3 B in the above formula represents the inhibitory effect of Salmonella pullorum on the growth of Lactobacillus rhamnosus P118;

[0027] Figure 4is the influence of Lactobacillus rhamnosus P118 on the survival rate of chicks infected with Salmonella pullorum in Experimental Example 2 of the present application, wherein, Figure 4 C in the above formula (1) represents the change in the survival rate of chicks one week after infection, Figure 4 D in the above formula (1) represents the survival rate of chicks infected at the age of 7 days after infection;

[0028] Figure 5 is the influence of the experimental group and the control group on the tissue bacterial load of chicks infected with Salmonella pullorum in Experimental Example 2 of the present application;

[0029] Figure 6 is the influence of the experimental group, the control group and the blank control group on the histopathological changes of chicks infected with Salmonella pullorum in Experimental Example 2 of the present application;

[0030] Figure 7 is the regulatory effect of the experimental group, the control group and the blank control group on the intestinal barrier function of chicks infected with Salmonella pullorum in Experimental Example 2 of the present application;

[0031] Figure 8 is the influence of the experimental group, the control group and the blank control group on the egg production performance of SP-infected laying hens in Experimental Example 3 of the present application;

[0032] Figure 9 is the influence of the experimental group, the control group and the blank control group on the spleen infection graph of SP-infected laying hens in Experimental Example 3 of the present application;

[0033] Figure 10 is the influence of the experimental group, the control group and the blank control group on the spleen infection index of SP-infected laying hens in Experimental Example 3 of the present application;

[0034] Figure 11 is the influence of the experimental group, the control group and the blank control group on the organ infection graph of SP-infected laying hens in Experimental Example 3 of the present application;

[0035] Figure 12 is the detection rate of SP in the liver, spleen, intestine and reproductive tract of SP-infected laying hens in Experimental Example 3 of the present application;

[0036] Figure 13 is the bacterial load of SP in the spleen of SP-infected laying hens in Experimental Example 3 of the present application;

[0037] Figure 14 is the bacterial load of SP in the liver of SP-infected laying hens in Experimental Example 3 of the present application;

[0038] Figure 15 is the bacterial load of SP in the jejunum of SP-infected laying hens in Experimental Example 3 of the present application;

[0039] Figure 16SP load in cecum of the experimental group and the control group in Example 3 of the present application;

[0040] Figure 17 SP load in ovary of the experimental group and the control group in Example 3 of the present application;

[0041] Figure 18 SP load in bursa of the experimental group and the control group in Example 3 of the present application;

[0042] Figure 19 SP load in vaginal sample of the experimental group and the control group in Example 3 of the present application;

[0043] Figure 20 SP load in egg of the experimental group and the control group in Example 3 of the present application. DETAILED DESCRIPTION

[0044] The following examples are provided to better further understand the present application, and are not limited to the best mode, and do not constitute a limitation on the content and scope of protection of the present application, and any person under the inspiration of the present application or the combination of the present application with other prior art features, any product identical or similar to the present application falls within the scope of protection of the present application.

[0045] The specific experimental steps or conditions are not indicated in the examples, and can be performed according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not indicated by the manufacturer, and are conventional reagent products that can be obtained by purchase.

[0046] In the practice of poultry breeding industry, Salmonella Pullorum (SP) infection causes high mortality rate in chicks, and shows persistent carrier state in adult chicken flocks. More notably, the pathogen has high efficient horizontal transmission (via fecal-oral route) and vertical transmission (via reproductive system) ability, which poses multiple prevention and control challenges to poultry healthy breeding. Most of the existing technical researches are focused on blocking the horizontal transmission of Salmonella Pullorum, and the examples of the present application take Lacticaseibacillus rhamnosus P118 as probiotics, through systematic evaluation of the comprehensive antagonistic effect of Lacticaseibacillus rhamnosus P118 on horizontal and vertical transmission of Salmonella Pullorum in in vivo and in vitro infection models, in-depth study of the antagonistic effect of Salmonella infection in the peak egg production model of laying hens, to solve the key epidemic prevention problems in poultry breeding.

[0047] The Lacticaseibacillus rhamnosus P118 in the embodiment of the application is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20221065.

[0048] The Salmonella pullorum (SP) in the embodiment of the application is preserved in the laboratory germ bank of the Hangzhou Institute for Advanced Study, University of Science and Technology of China.

[0049] The preparation method of the Salmonella pullorum bacterial suspension is as follows: the frozen strain is inoculated on TSB agar medium, and cultured at 37 DEG C overnight. A single colony is picked from the streak plate, and cultured at 37 DEG C on a shaking table at 180 r / min for 12-16 h. The bacterial liquid is adjusted to 1x10 8 CFU / mL.

[0050] The MRS culture solution has the following formula: casein peptone 10.0 g, beef extract 10.0 g, yeast powder 5.0 g, glucose 5.0 g, sodium acetate 5.0 g, diammonium citrate 2.0 g, Tween 80 1.0 g, K2HPO4 2.0 g, MgSO4·7H2O 0.2 g, MnSO4·H2O 0.05 g, CaCO3 20.0 g, agar 15.0 g, ultrapure water 1.0 L, pH = 6.8. If a liquid culture medium is prepared, the agar in the formula is removed.

[0051] The formula of the XLD culture medium is as follows: yeast powder 3.0 g, L-lysine 5.0 g, xylose 3.75 g, lactose 7.5 g, sucrose 7.5 g, sodium chloride 5.0 g, sodium thiosulfate 6.8 g, ferric ammonium citrate 0.8 g, phenol red 0.08 g, agar 15.0 g, ultrapure water 1.0 L, pH = 7.4±0.2. If a liquid culture medium is prepared, the agar in the formula is removed.

[0052] The formula of the TSB culture medium is as follows: tryptone 17.0 g, soybean peptone 3.0 g, sodium chloride 5.0 g, potassium phosphate dibasic 2.5 g, glucose 2.5 g, ultrapure water 1.0 L. pH = 7.3±0.2.

[0053] The formula of the BPW culture medium is as follows: peptone 5.0 g, bovine bile salt 1.0 g, calcium carbonate 10.0 g, sodium thiosulfate 4.5 g, L-cystine 0.01 g, brilliant green 0.0025 g, ultrapure water 1.0 L, pH = 8.4±0.2.

[0054] The formula of the TTB culture medium is as follows: peptone 10.0 g, sodium chloride 5.0 g, sodium phosphate dibasic 3.57 g, potassium phosphate monobasic 1.5 g, ultrapure water 1.0 L.

[0055] Embodiment

[0056] The embodiment provides a preparation method of lactobacillus rhamnosus P118 fermentation liquor, and specific steps and parameters are as follows:

[0057] (1) inoculate the activated lactobacillus rhamnosus P118 into MRS liquid medium at an inoculation amount of 1 vol%, and anaerobically culture at 37 DEG C for 24 hours to obtain a fermentation liquor;

[0058] The step of activating the lactobacillus rhamnosus P118 is as follows: after diluting the lactobacillus rhamnosus P118 with deionized water, 100 μL of the diluent is spread on an MRS solid culture medium plate, a sterilized glass rod is used for spreading, and then the plate is inverted and placed in a 37 DEG C constant-temperature box for culture for 36 hours, single colonies appear, the single colonies are taken and transferred into 50 mL of MRS liquid medium for culture, and the initial inoculation concentration is 1×10 6 CFU / mL, and after 37 DEG C culture for 24 hours, the bacterial liquid is adjusted to 1×10 8 CFU / mL for standby.

[0059] (2) centrifuge the fermentation liquor obtained in step (1) at 4 DEG C and 4000 rpm for 5 minutes, collect the supernatant after centrifugation, filter twice through a 0.22 μm sterile filter membrane to obtain sterile supernatant, wash the precipitate after centrifugation with pre-cooled PBS buffer three times, adjust the bacterial concentration to the original culture volume to obtain a live bacterial suspension, and adjust the bacterial liquid to 1×10 8 CFU / mL for standby.

[0060] Experimental example 1

[0061] 1.1, using the Oxford cup method to detect the bacteriostatic ability of the lactobacillus rhamnosus P118 fermentation liquor:

[0062] After sterilizing the LB medium by high pressure, the medium is cooled to 50 DEG C, chicken white diarrhea salmonella (the final concentration of chicken white diarrhea salmonella in the LB medium is 1×10 6 CFU / mL), pour the plate (20 mL / dish), and after the medium solidifies, sterile Oxford cups are uniformly placed in the medium, 200 μL of the fermentation liquor in step (1) of example 1, the sterile supernatant of step (2), 500 μg / mL of gentamicin and PBS buffer are added into the Oxford cups respectively, wherein the gentamicin treatment group is used as a positive control, and after 37 DEG C culture for 24 hours, the bacteriostatic ring diameter is recorded, and the results are shown in Figure 1 .

[0063] According to Figure 1 , the lactobacillus rhamnosus P118 fermentation liquor and the supernatant both present significant bacteriostatic rings compared with the positive control group gentamicin.

[0064] 1.2, determining the influence of lactobacillus rhamnosus P118 on the SP biofilm formation ability:

[0065] According to the volume ratio of 1:1, the supernatant of Lactobacillus rhamnosus P118 and the suspension of Salmonella pullorum (1×10 6 CFU / mL) were mixed as the experimental group, and the same volume of the suspension of Salmonella pullorum was added as the control group, and the same volume of PBS buffer was added as the blank control group. The bacterial liquid of the experimental group, the control group and the PBS buffer of the blank control group were added to the 96-well plate (96-well plate was set with MRS culture medium), and the temperature conditions of 30°C and 37°C were set respectively, and the culture was static for 5 days. 0.1% crystal violet staining was used, and the dye was absorbed in the hole after static staining at room temperature for 18 min. ddH2O was used to wash the plate until no fading occurred. 200μL of decolorizing solution (ethanol: acetone volume ratio of 3:1) was added to each well, and the dye was completely dissolved and decolorized at 25°C for 20 min. The value of OD550nm was measured by enzyme-labeled instrument. The biofilm formation rate = (experimental hole OD550nm-blank control hole OD550nm) / blank control hole OD550nm×100%, and the results are shown in Figure 2 .

[0066] According to Figure 2 , whether at 30°C or 37°C, the supernatant of Lactobacillus rhamnosus P118 can significantly inhibit the biofilm formation ability of SP.

[0067] 1.3, evaluate the co-growth of Lactobacillus rhamnosus P118 and SP:

[0068] The Lactobacillus rhamnosus P118 live bacteria suspension formed in step (2) of Example 1 and Salmonella pullorum were diluted according to the gradient concentration, and the concentration was 1×10 4 ~ 10 9 CFU / mL. The chessboard method was used to design the co-culture system, 200μL of mixed bacterial liquid (the volume ratio of Lactobacillus rhamnosus and SP in the mixed bacterial liquid was 1:1) was added to the 96-well plate, and the mixed fermentation liquid was obtained after static culture at 37°C for 20h. The fermentation liquid was inoculated on MRS agar plate (only Lactobacillus rhamnosus P118 can grow) and XLD agar plate (only SP can grow), and cultured in a constant temperature incubator at 37°C. According to the growth of colonies, the plate count was carried out at 18-24h of culture, and the results are shown in Figure 3 .

[0069] According to Figure 3 , P118 live bacteria can significantly inhibit the proliferation of SP, and SP has no significant effect on the proliferation of P118.

[0070] Experimental Example 2

[0071] 2.1, the influence of Lactobacillus rhamnosus P118 on the survival rate of chicks infected with SP:

[0072] Clean-skinned chicken embryos (SPF embryos, purchased from Ningbo Chunpai Co., Ltd.) from a specific pathogen-free flock were incubated until 1 day old. Chicks were then orally inoculated with a Salmonella pullorum suspension (concentration 1×10⁻⁶). 8 (CFU / mL, 100 μL orally administered to each chick, once orally throughout the experimental period) SP was administered to 1-day-old chicks, with an SP inoculation dose of 1 × 10⁻⁶ per chick. 7 CFU;

[0073] After infection, the experimental group was given 50 μL of Lactobacillus rhamnosus P118 fermentation broth orally every other day. The control group was given an equal volume of sterile PBS, and a blank control group was set up. That is, clean chicken embryos were given PBS buffer orally, and 50 μL of PBS buffer was given to the chicks orally every other day during the experiment.

[0074] Observe continuously for 7 days, record daily clinical symptoms and mortality, and see the results below. Figure 4 .

[0075] according to Figure 4 It can be seen that the cumulative mortality rate of the control group reached 55% on the 7th day, while the mortality rate of the experimental group decreased to 35% during the same period, and the survival rate increased by 20%.

[0076] 2.2 Effect of Lactobacillus rhamnosus P118 on bacterial load in SP-infected chick tissues:

[0077] Chicks from both the experimental and control groups were euthanized on day 7 post-infection (as described in section 2.1). Liver, spleen, cecum, and fecal samples were collected under aseptic conditions. Tissue samples were placed in sterile PBS, and mechanical homogenization was performed using a tissue homogenizer. The homogenate was diluted with PBS and inoculated onto solid XLD selective medium for colony counting. Results are shown in [Figure 1]. Figure 5 .

[0078] according to Figure 5 It can be seen that the SP load in the experimental group treated with Lactobacillus rhamnosus P118 was significantly reduced in the liver, cecum and fecal samples (P<0.001, P<0.05, P<0.01), and there was also a decreasing trend in the spleen.

[0079] 2.3 Effects of Lactobacillus rhamnosus P118 on histopathological changes in SP-infected chicks:

[0080] Histopathological examination of tissue sections can reveal the microscopic effects of SP infection on the liver, spleen, and intestines, allowing for a direct observation of whether Lactobacillus rhamnosus can alleviate SP infection in chicks.

[0081] Specifically, the collected tissue blocks of liver, spleen and cecum were soaked in 4% (w / v) paraformaldehyde buffer for 24 h for fixation, washed with PBS and embedded in paraffin. The tissues were cut into 5 μm thick sections, stained with hematoxylin-eosin (H&E), and subjected to histopathological analysis, and the results are shown in Figure 6 .

[0082] According to Figure 6 , in the control group sections: the spleen tissue was severely damaged, the red and white pulp boundary was unclear (gray arrow), a large number of neutrophil infiltration appeared (green arrow), and cell necrosis, nuclear fragmentation and pyknosis appeared (orange arrow) Figure 6 E); the liver cells were arranged in disorder and disorder (white arrow), the cell membrane was dissolved and the structure was incomplete (black arrow), and a large number of inflammatory cell infiltration appeared (green arrow) Figure 6 F); the cecum was severely damaged, and local mucosal epithelial cell shedding, unclear intestinal villi and exposed lamina propria appeared (red arrow) Figure 6 G). The experimental group significantly improved the pathological damage: the spleen tissue damage was relieved Figure 6 H); the liver cells were arranged in order, the structure was complete (white arrow), and there was basically no inflammatory cell infiltration Figure 6 I); the cecum was normal in shape, the tissue structure was complete, and the crypt structure was complete (blue arrow) Figure 6 J).

[0083] 2.4, Regulation of Lactobacillus rhamnosus P118 on the intestinal barrier function of SP-infected chicks:

[0084] After embedding the intestinal tissue samples fixed in 4% paraformaldehyde in paraffin, sectioning, dehydration and blocking, immunofluorescence staining was performed, and the tight junction proteins Occludin and ZO-1 proteins were labeled on the same section. Finally, the intestinal tissue section was visualized by immunofluorescence using a microscope slide scanner Pannoramic MIDI (3DHISTECH), and the results are shown in Figure 7 .

[0085] According to Figure 7 , the results showed that the expression of Occludin and ZO-1 proteins in the control group was significantly reduced, indicating that the intestinal barrier function of chicks was severely damaged after SP infection. Compared with the control group, the expression levels of the two proteins in the experimental group were significantly reversed, and returned to the same level as the blank control group.

[0086] Experimental Example 3

[0087] 3.1, Effect of Lactobacillus rhamnosus P118 on the egg production performance of SP-infected laying hens:

[0088] The live bacteria suspension of Lactobacillus rhamnosus P118 in Example Step (2) was inoculated into MRS liquid medium at a ratio of 1% (v / v), and after 24 hours of culture at 37°C, centrifugation was performed at 4°C and 4000 rpm / min, the bacterial precipitate was taken, and the bacterial powder was prepared by mixing with corn starch at a ratio of 4:1 (w:w) after drying at 50°C for 6-8 hours, with 1 x 10 8 CFU / g of bacteria powder. The dry bacteria powder was added to the feed at a ratio of 0.8 wt% to form the feed.

[0089] P118 was inoculated into MRS liquid medium at a ratio of 1% (v / v), and after 24 hours of culture at 37°C, drinking water was added at a ratio of 1% (v / v) to form drinking water containing 1 x 10 7 CFU / mL of live bacteria.

[0090] SP-infected 180-day-old hens were infected by oral inoculation of SP, with 1 x 10 9 CFU of SP bacteria per hen, and the infection was performed once. From 14 days before infection, the SP-infected hens were given Lactobacillus rhamnosus P118 of Example Step (2) by feed addition and drinking water addition, with daily addition of feed and every two-day addition of drinking water containing Lactobacillus rhamnosus P118. The control group was normally fed with feed and drinking water, and the blank control group was normally fed with feed and drinking water. The number of eggs laid per day was recorded to calculate the egg laying rate = number of eggs laid / number of individuals x 100%, and the results are shown in Figure 8 .

[0091] As shown in Figure 8 , the egg laying rate of the control group decreased in a time-dependent manner, and decreased to 63.96% on the 14th day, which was significantly lower than that of the blank control group (P < 0.001), indicating that the hen was in the peak period of SP infection at this time. Compared with the control group, the egg laying rate of the experimental group was significantly improved (P < 0.05), indicating that P118 treatment significantly improved the egg laying performance of the infected hens.

[0092] 3.2, Improvement of Lactobacillus rhamnosus P118 on the pathological damage of SP-infected hens:

[0093] In order to understand the macroscopic pathological changes of each organ of the hen after infection with SP, in the experiment of 3.1, the hens were serially dissected at 1, 2, 3, 4, 7 and 11 weeks after infection, the external tissue damage was observed and recorded, the spleen weight was weighed to obtain the organ infection index (organ infection index = organ weight / body weight), and the tissue samples (jejunum, cecum, liver, spleen, ovary, bursa of Fabricius, vagina) were collected for subsequent detection.

[0094] According to Figure 9It can be seen that the organ lesions of the control group after SP infection are serious, the spleen is swollen and congested, multiple lesions appear in the liver, the liver capsule ruptures leading to hemorrhage, and the overall texture is fragile and easy to break; the ovary appears obvious deformation and discoloration, and part of the follicle content is caseous degeneration. Compared with the control group, the experimental group has slight tissue lesions, showing the same appearance as the blank control group, according to Figure 10 It can be seen that the spleen index of the chickens is significantly reduced in the second and third weeks (P<0.001, P<0.05).

[0095] The collected tissue samples (jejunum, cecum, liver, spleen, ovary, bursa of Fabricius, vagina) were subjected to histopathological section detection using the method of 2.3, which can reflect the microscopic effects of SP infection on the liver, spleen, intestinal tract and reproductive tract, and directly observe whether P118 can reduce the infection of SP in chickens, and the results are shown in Figure 11 .

[0096] As shown in Figure 11 , in the control group, the spleen tissue damage is serious, a large number of neutrophil infiltration (green arrow) and a large number of hemosiderin deposition (yellow arrow) appear in the red pulp; the liver cells are arranged in disorder (white arrow), the cell membrane is dissolved, the structure is incomplete (black arrow), and a large number of inflammatory cell infiltration (green arrow) appears; the cecal tissue damage is serious, the local mucosal epithelial cells fall off, the intestinal villi are not clear, the lamina propria is exposed (red arrow), the tissue structure is abnormal, the crypt number is reduced, and the local crypt structure disappears (gray arrow), a large number of neutrophil infiltration (green arrow) appears, the cells appear necrosis, nuclear fragmentation and pyknosis (orange arrow); in the ovary tissue, the number of primary follicles is reduced, follicle degeneration occurs (blue arrow), and inflammatory cell infiltration (green arrow) appears. In the experimental group, the spleen tissue damage is slight, the red and white pulp boundary is clear; the liver cells are arranged in order, the structure is complete (white arrow), and there is basically no inflammatory cell infiltration; the cecum shape is normal, the tissue structure is complete, and the crypt structure is complete; the follicle density and number in the ovary tissue increase (purple arrow), and the follicle shape is complete.

[0097] 3.3, the effect of Lactobacillus rhamnosus P118 on the bacterial load of SP infected chickens:

[0098] The jejunum, cecum, blood, liver, spleen, ovary, bursa of Fabricius and vaginal samples collected in each week were placed in sterile PBS, the tissue samples were mechanically broken using a tissue homogenizer, the tissue homogenate was diluted with PBS and inoculated into XLD selective medium for bacterial counting, and the SP detection rate was calculated (SP detection rate % = the number of chickens detected with SP infection / the total number of chickens x 100%).

[0099] After the infection of laying hens in 3.1, the eggs were continuously collected, and the SP in the eggs was qualitatively detected according to the GB4789.4-2024 National Food Safety Standard, BPW medium pre-enrichment, TTB medium selective enrichment and XLD medium separation bacteria.

[0100] According to Figure 12 It can be seen that the experimental group can reduce the detection rate of SP in the liver and spleen, intestinal tract and reproductive tract as a whole.

[0101] Specifically, according to Figures 13-19 It can be seen that the SP load in the spleen, liver, jejunum, cecum, ovary, dilatation and vaginal samples of the experimental group was significantly reduced (P<0.001 or P<0.01 or P<0.05) in the corresponding weeks.

[0102] As Figure 20 shown, the detection of SP in the eggs of the control group continued to increase within four weeks after infection, and the highest detection rate was 14%, while the experimental group could reduce the detection rate of SP in the eggs throughout the whole process.

[0103] Obviously, the above examples are only examples for the sake of clarity, and are not limited to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. Lactobacillus rhamnosus P118 has any of the following uses: (i) Reduce the infection rate of Salmonella pullorum in chicks; (ii) Improve the egg production performance of hens infected with Salmonella pullorum; (iii) Reduce the bacterial load in the tissues and organs of hens; (iv) Reduce the horizontal and vertical transmission of Salmonella pullorum in chickens; (v) Reduce the risk of Salmonella pullorum being transmitted vertically from the hen to the offspring via the eggs.

2. The use according to claim 1, characterized in that, The Lactobacillus rhamnosus P118 is its live bacterial cells, its dead bacterial cells, or its culture.

3. The use according to claim 2, characterized in that, The culture of Lactobacillus rhamnosus P118 includes the fermentation broth of Lactobacillus rhamnosus P118.

4. The use according to claim 3, characterized in that, The preparation method of fermentation broth of Lactobacillus rhamnosus P118 includes the following steps: Lactobacillus rhamnosus P118 was inoculated into MRS liquid medium and cultured anaerobically at 35–38°C for 16–24 h to obtain the fermentation broth.

5. The use according to claim 4, characterized in that, The inoculum size of *Lactobacillus rhamnosus* P118 was 1–4 vol% based on the volume of MRS liquid culture medium.

6. The use according to claim 4, characterized in that, Before inoculation, the concentration of Lactobacillus rhamnosus P118 was 1×10⁻⁶. 8 ~1×10 9 CFU / mL.

7. The use according to claim 3, characterized in that, The culture of Lactobacillus rhamnosus P118 also includes the supernatant obtained from the fermentation broth of Lactobacillus rhamnosus P118.

8. The use according to claim 7, characterized in that, The method for preparing the supernatant obtained from the fermentation broth of Lactobacillus rhamnosus P118 includes the following steps: The fermentation broth of Lactobacillus rhamnosus P118 was centrifuged at 4℃ and 3500-4000 rpm for 5-10 min. The supernatant was then filtered at least twice to obtain the supernatant.

9. The use according to claim 8, characterized in that, The filtration step uses a 0.22 μm sterile filter membrane.

10. The use according to any one of claims 1-9, characterized in that, The Lactobacillus rhamnosus P118 strain is deposited at the China Center for Type Culture Collection (CCTCC) under accession number CCTCCNO:M20221065.

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