CN116270989A - Application of N-acyl homoserine lactonase in preparation of salmonella typhimurium infection resisting products - Google Patents
Application of N-acyl homoserine lactonase in preparation of salmonella typhimurium infection resisting products Download PDFInfo
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- CN116270989A CN116270989A CN202310113456.8A CN202310113456A CN116270989A CN 116270989 A CN116270989 A CN 116270989A CN 202310113456 A CN202310113456 A CN 202310113456A CN 116270989 A CN116270989 A CN 116270989A
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides an application of N-acyl homoserine lactonase (AHLase) in preparing a salmonella typhimurium infection-resistant product. According to the invention, the application of AHLase to Salmonella typhimurium can inhibit not only the biofilm formation capacity but also the cluster movement capacity and the adhesion capacity to host intestinal epithelial cells, so that the AHLase can effectively reduce the virulence and/or pathogenicity of Salmonella typhimurium; the invention also applies AHLase to the salmonella typhimurium infected broiler, and discovers that the AHLase has obvious protective effect on the growth performance, organ index and intestinal health of the salmonella typhimurium infected broiler. Therefore, the invention provides the application of AHLase in preparing the salmonella typhimurium infection-resistant product, provides a new treatment scheme for salmonella typhimurium infection, and also provides a new application for AHLase.
Description
Technical Field
The invention belongs to the technical field of biological medicine. More particularly, to the use of N-acyl homoserine lactones for the preparation of a product against Salmonella typhimurium infection.
Background
Salmonella (Salmonella) belongs to gram-negative bacteria, is a very common zoonotic primordium, can infect hosts (including human beings and various animals) through contaminated foods (such as meat, eggs, milk and other animal products) and water and other materials, can cause symptoms such as vomiting, anorexia, diarrhea and even septicemia and the like of the hosts after infection, and causes serious harm to the health of animals and human bodies. Research shows that salmonella can infect poultry in a horizontal and vertical transmission mode, and the sick poultry mainly presents diseases such as white diarrhea and typhoid, and the like, and a series of clinical symptoms appear, so that intestinal damage and growth performance reduction are caused, and finally serious economic loss is caused. In addition, the poultry carrying salmonella is easy to cause food pollution in the slaughtering process, and serious threat is brought to public health safety. In all cases of infection caused by Salmonella, however, salmonella typhimurium (Salmonella typhimurium) contamination represents a significant proportion. Therefore, how to prevent and control the damage caused by salmonella typhimurium infection becomes one of the problems to be solved in the breeding industry.
Antibiotics have been widely used for the prevention and control of bacterial infections such as salmonella typhimurium in livestock and poultry farming over the past decades. However, due to a series of problems of continuous enhancement of bacterial drug resistance, antibiotic residue, ecological environment destruction and the like, the use of antibiotics in feeds is formally and comprehensively prohibited in China from 7 months and 1 day in 2020, which means that the risk of salmonella typhimurium infection in livestock and poultry cultivation is increased. Thus, the development of a green, effective antibiotic substitute for alleviating salmonella typhimurium infection in poultry is of great practical significance.
With the intensive research of microorganisms, it has been gradually discovered that bacteria regulate their virulence through Quorum Sensing (QS), thereby mediating the process of infecting a host. QS is thought to be a mechanism for information communication between and within bacterial populations that can encourage bacteria to react to changes in the external environment. By this system, the bacterium can regulate its own various physiological activities and virulence such as growth and reproduction, biofilm formation, adhesion attack, toxin secretion, and the like, thereby promoting infection of the host by the bacterium. The occurrence of QS is mediated by a variety of QS signal substances such as N-acyl homoserine lactones (N-acyl homoserine lactones, AHL) and self-inducing peptides. These signal substances accumulate gradually during the growth, propagation and metabolism of the bacteria, and when they reach a certain concentration threshold, they bind to specific receptors on the bacterial membrane, thereby activating the expression of a series of genes related to virulence and pathogenicity in the bacteria.
N-acyl homoserine lactonase (N-acyl homoserine lactonase, AHLase) is an enzyme capable of efficiently degrading lactone rings in the molecular structure of AHL, and is mainly applied to the field of aquaculture, such as: AHLase can reduce virulence (including biofilm formation and toxin secretion, etc.) of psychrophilic and pseudomonas aeruginosa; another example is: the addition of AHLase in the water body can relieve the negative influence of aeromonas hydrophila infection on the immune function of the zebra fish and the intestinal microorganism composition; and the following steps: the probiotics which can generate AHLase are added into the water body, so that the growth performance reduction and intestinal damage (including digestive enzyme activity reduction and intestinal flora structural disorder) of the weever caused by the vibrio harveyi can be relieved; and the following steps: AHLase treatment inhibits the inflammatory response and tissue damage in mice induced by Pseudomonas aeruginosa infection. The reason for this is that the pathogenic bacteria of the aquatic animals can generate AHL, so that AHLase can block the occurrence of the pathogenic bacteria QS by destroying the molecular structure of the AHL, thereby inhibiting the growth and reproduction of the pathogenic bacteria or virulence of the pathogenic bacteria.
However, because the prior art and technical studies generally consider that salmonella lacks the ability to synthesize AHL (Dai Peng et al, salmonella quorum sensing system research progress, bioprocess 2019,17 (3): 257-264), and further consider that AHLase does not inhibit salmonella typhimurium itself, although there is a great deal of application of ahase in preserving foods and preventing and resisting pathogenic bacterial infection in animals, no application of ahase in resisting pathogenic bacterial infection in domestic animals, particularly in resisting salmonella typhimurium, is currently seen.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the application of AHLase in preparing a salmonella typhimurium infection-resistant product, provides a new treatment scheme for salmonella typhimurium infection, and also provides a new application for AHLase.
The above object of the present invention is achieved by the following technical scheme:
according to the invention, the AHLase is acted on the salmonella typhimurium for the first time, so that the biomass of a biofilm of the salmonella typhimurium can be reduced, the inhibition rate of the biofilm formation can be increased, the secretion of extracellular DNA (eDNA) can be inhibited, the expression of genes related to the biofilm formation can be reduced, the biofilm formation capacity can be further inhibited, the cluster movement capacity and the adhesion capacity to host intestinal epithelial cells can be inhibited, and the AHLase can be used for effectively reducing the virulence and/or pathogenicity of the salmonella typhimurium; the invention also applies AHLase to the salmonella typhimurium infected broiler, and discovers that the AHLase has obvious protective effect on the growth performance, organ index and intestinal health of the salmonella typhimurium infected broiler. The above shows that AHLase is suitable for preparing salmonella typhimurium infection resisting products, and therefore, the application of AHLase in preparing salmonella typhimurium infection resisting products is within the protection scope of the invention.
Preferably, the product is a feed, a feed additive, an enzyme preparation, a microecological preparation or a pharmaceutical product.
Preferably, the addition amount of the AHLase in the feed, the feed additive, the enzyme preparation and the microecological preparation is not less than 10000U/kg daily ration.
Preferably, the anti-salmonella typhimurium infection is a reduction in virulence and/or pathogenicity of salmonella typhimurium.
Preferably, the reduction in virulence and/or pathogenicity of salmonella typhimurium is inhibition of biofilm formation, exercise and/or adhesion capacity of salmonella typhimurium.
Preferably, the ability to inhibit the formation of a biofilm of salmonella typhimurium is one or more of reducing the biomass of the biofilm, increasing the inhibition rate of the formation of the biofilm, inhibiting the secretion of extracellular DNA, and down-regulating the expression of genes associated with the formation of the biofilm.
Preferably, the ability to inhibit the motility of salmonella typhimurium is the ability to inhibit the aggregation of salmonella typhimurium.
Preferably, the ability to inhibit adhesion of salmonella typhimurium is to inhibit adhesion of salmonella typhimurium to host intestinal epithelial cells.
Preferably, the anti-salmonella typhimurium infection is to protect the growth performance, organ index and/or intestinal health of an animal infected with salmonella typhimurium.
Preferably, the protection of the intestinal health of the salmonella typhimurium infected animal is one or more of protecting the morphological structure of the intestinal tract, improving the barrier function of the intestinal tract, and inhibiting the inflammatory reaction of the intestinal tract.
Preferably, the improvement of intestinal barrier function is inhibition of expression of tight junction proteins, such as mRNA expression of occludin, ZO-1.
Preferably, the inhibition of the inflammatory response in the gut is inhibition of inflammatory factor expression, such as inhibition of IL-8 expression.
Preferably, the animal is a broiler chicken.
The invention has the following beneficial effects:
according to the invention, the application of AHLase to Salmonella typhimurium can inhibit not only the biofilm formation capacity but also the cluster movement capacity and the adhesion capacity to host intestinal epithelial cells, so that the AHLase can effectively reduce the virulence and/or pathogenicity of Salmonella typhimurium; the invention also applies AHLase to the salmonella typhimurium infected broiler, and discovers that the AHLase has obvious protective effect on the growth performance, organ index and intestinal health of the salmonella typhimurium infected broiler. Therefore, the invention provides the application of AHLase in preparing the salmonella typhimurium infection-resistant product, provides a new treatment scheme for salmonella typhimurium infection, and also provides a new application for AHLase.
Drawings
Fig. 1A is a statistical result of biofilm biomass, and fig. 1B is a statistical result of biofilm formation inhibition rate, wherein the results are expressed as mean ± standard deviation, P <0.05, P <0.01, and P <0.001.
Fig. 2 is a statistical graph of the effect of AHLase on the amount of eDNA secretion of salmonella typhimurium, wherein the results are expressed as mean ± standard error, and P <0.01.
FIG. 3 is a graph showing the statistical result of the influence of AHLase on the expression level of genes involved in the formation of a biofilm of Salmonella typhimurium, wherein the result is expressed as mean value.+ -. Standard error, and P <0.05.
Fig. 4 is a graph showing the results of salmonella typhimurium cluster exercise expressed as mean ± standard error, P <0.05, red arrow pointing to the circle of salmonella typhimurium.
Fig. 5 is a graph showing the effect of AHLase on adhesion ability of salmonella typhimurium expressed as mean ± standard error, P <0.05, P <0.01, yellow arrow pointing to salmonella typhimurium adhered to IPEC-J2 cells.
FIG. 6A is a result of the effect of AHLase on the mRNA expression level of a tight junction protein of a broiler infected with Salmonella typhimurium at test period 11D, FIG. 6B is a result of the effect of AHLase on the mRNA expression level of a tight junction protein of a broiler infected with Salmonella typhimurium at test period 21D, FIG. 6C is a result of the effect of AHLase on the serum diamine oxidase activity of a broiler infected with Salmonella typhimurium at test period 11D, FIG. 6D is a result of the effect of AHLase on the serum diamine oxidase activity of a broiler infected with Salmonella typhimurium at test period 21D, wherein the results are expressed as mean.+ -. Standard error, no letter or the same letter above the histogram indicates that the difference is not significant (P > 0.05), and the different letters indicate that the difference is significant (P < 0.05).
FIG. 7A shows the results of the effect of AHLase on the ileal inflammatory response of Salmonella typhimurium-infected broilers at test stage 11d, and FIG. 7B shows the results of the effect of AHLase on the ileal inflammatory response of Salmonella typhimurium-infected broilers at test stage 21d, where the results are expressed as mean.+ -. Standard error, no letter or the same letter above the histogram indicates that the difference is insignificant (P > 0.05), and the different letters indicate that the difference is significant (P < 0.05).
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
N-acyl homoserine lactonase (AHLase): purchased from beijing challenge biotechnology limited.
Salmonella typhimurium: strain number ATCC14028, available from the food safety engineering research and development center in guangdong province.
EXAMPLE 1 inhibition of the biofilm formation ability of Salmonella typhimurium by AHLase
1) Influence of AHLase on biofilm biomass and biofilm formation inhibition rate of Salmonella typhimurium
Selecting Salmonella typhimurium single colony, placing in LB broth culture medium, shake culturing in a constant temperature shaker at 180 rpm (37deg.C) for 12 hr, and adjusting bacterial liquid to 10 9 After CFU/mL, the bacterial solution is mixed with AHLase solution to prepare enzyme bacterial mixed solution with final concentration of AHLase of 0, 0.2, 0.4, 0.6, 0.8 and 1.0U/mL (6 repeats of each group, the group with final concentration of 0 is blank group, the rest is treatment group, and the bacteria are in the enzyme bacterial mixed solution)The concentration is 5 multiplied by 10 8 CFU/mL), transferred to 96-well plates, incubated at 37 ℃ for 24 hours, the medium was discarded, rinsed with distilled water, and 200 μl of 0.1% crystal violet aqueous solution was added to each well to dye for 30min, and the plates were rinsed. 200. Mu.L of acetic acid was added to each well, dissolved by shaking, and the absorbance at 590nm (OD 590 I.e., the biomass of the biofilm), according to the formula "(blank OD 590 Treatment group OD 590 ) Blank OD 590 Calculating inhibition rate of biofilm formation by x 100% "and then carrying out statistical analysis on the data result by adopting a single factor analysis of variance method, and carrying out multiple comparison by Duncan's method when the difference is obvious, wherein P is the same as that of the biological film<0.05 is significant difference, P<0.01 is very significant.
The results are shown in FIG. 1, wherein FIG. 1A shows the statistics of biofilm biomass, and FIG. 1B shows the statistics of biofilm formation inhibition rate. It can be seen that with increasing AHLase addition, biofilm biomass produced by salmonella typhimurium decreased very significantly (P < 0.01); compared with a blank group, the treatment group added with 1.0U/mL AHLase has the best inhibition effect on the salmonella typhimurium biofilm, and the inhibition rate reaches 86.37%. The AHLase can obviously inhibit the formation of a biological film of salmonella typhimurium, reduce the generation amount of the biological film, inhibit the resistance and continuous infection capacity of salmonella typhimurium, and further reduce the infectivity of salmonella typhimurium.
2) Effect of AHLase on extracellular DNA (eDNA) secretion of Salmonella typhimurium
Selecting Salmonella typhimurium single colony, shake culturing in LB broth medium at 180 rpm (37deg.C) in a constant temperature shaker for 12 hr, and adjusting bacterial liquid to 10 9 After CFU/mL, the bacterial solution is mixed with AHLase solution to prepare an enzyme bacterial mixed solution with the final concentration of AHLase of 0 and 1.0U/mL (6 repeats each group, the group with the final concentration of 0 is blank group, and the concentration of bacteria in the enzyme bacterial mixed solution is 5 multiplied by 10) 8 CFU/mL), removing planktonic bacteria and cleaning biofilm after aerobic static culture in 1.5mL centrifuge tube at 37deg.C for 24h, adding 5 μL EDTA (0.5M) to each well, standing at 4deg.C for 1h, adding 700 μL 1mmol/L TEN (Tris-EDTA-NaCl) buffer to each well to resuspend biofilm, and thenCentrifuge at 12000g for 5min at 4deg.C, transfer 100. Mu.L of the supernatant to a centrifuge tube containing 300. Mu.L of 5mmol/L TE (Tris-EDTA) Buffer, then add the above mixture with an equal volume of DNA extract (Beijing QINGSHOCUK) to a column Spin Columns T1 (Beijing QINGSHOCUK), centrifuge at 12000g for 60s at 4deg.C, wash the column with washing Buffer BL (Beijing QINGSHOCUK), and centrifuge repeatedly for 30s at 12000 g. Finally adding sterile water, centrifuging to collect eDNA, measuring the concentration of the eDNA by using an ultra-micro ultraviolet spectrophotometer, and carrying out statistical analysis on the data result by adopting a t-test method, and P<0.05 is significant difference, P<0.01 is very significant.
The results are shown in FIG. 2. Compared with a blank group (NC), the concentration of the eDNA secreted by the salmonella typhimurium can be remarkably reduced by 1.0U/mL AHLase (P < 0.05), and because the eDNA is a main component of a bacterial biofilm, inhibition of the eDNA secretion represents inhibition of the formation of the bacterial biofilm, so that the AHLase can inhibit the formation of the salmonella typhimurium biofilm, and further reduce the infectivity of the salmonella typhimurium.
3) Influence of AHLase on expression level of genes related to formation of biofilm of Salmonella typhimurium
RNA sample extraction and purity detection: selecting Salmonella typhimurium single colony, shake culturing in LB broth medium at 180 rpm (37deg.C) in a constant temperature shaker for 12 hr, and adjusting bacterial liquid to 10 9 After CFU/mL, the bacterial solution is mixed with AHLase solution to prepare an enzyme bacterial mixed solution with the final concentration of AHLase of 0 and 1.0U/mL (6 repeats of each group, the group with the final concentration of 0 is blank group, and the concentration of bacteria in the enzyme bacterial mixed solution is 5 multiplied by 10) 8 CFU/mL), cells were collected by centrifugation at 12000g after incubation at 37 ℃ for 6h in a 50mL centrifuge tube. Extracting total RNA from bacteria by using BIOG accurate bacterial RNA extraction kit of hundred-generation biological company, dissolving in RNase-free double distilled water, measuring concentration of RNA by using Nanodrop-2000 ultra-micro spectrophotometer (2.5 mu L of RNase-free double distilled water is taken before measurement to wash sample measuring hole, filter paper is used for sucking clean, and RNA concentration of all samples accords with subsequent operation requirement and is uniformly regulated to 500 ng/. Mu.L), and measuring light absorption at 260nmValue (OD) 260nm ) With absorbance at 280nm (OD 280nm ) To evaluate the purity, OD of RNA 260nm /OD 280nm The value between 1.8 and 2.2 indicates that the purity of the RNA sample is higher.
RNA integrity detection: weighing 0.5g of agarose, placing into a conical flask, adding 40mL of distilled water, heating in a microwave oven to thoroughly and uniformly dissolve the agarose, adding 0.5 mu L of nucleic acid dye (ethidium bromide) when the temperature is reduced to 60 ℃, pouring into a gel-making mold, transferring gel to an electrophoresis tank after solidification molding, and adding 1 xTAE buffer to the submerged gel surface. 1. Mu.L of RNA sample with higher purity, 1. Mu.L of 6×loading buffer and 4. Mu.L of RNase-free double distilled water were mixed and loaded, and electrophoresis was performed at 130V for 25 min. After the end, a photo is taken by a full-automatic digital gel imaging system, and the appearance of 28S and 18S bands on the gel, wherein the brightness of the 28S band is twice that of the 18S band, indicates that the RNA integrity is good.
RNA reverse transcription: after confirming good purity and integrity, reverse transcription is carried out on the RNA sample by using a reverse transcription kit HiScript II qRT SuperMiH of Novain biological company, the reaction system is shown in Table 1, and the reaction procedure is as follows: the reaction was carried out at 50℃for 40min and at 85℃for 5s. After the completion of the RNA reverse transcription reaction, cDNA was diluted 1-fold with RNase-free double distilled water, and gradient PCR was performed using the Novamat Bio Inc. kit 2 XTaq Master MiH, the reaction system is shown in Table 2, and the reaction procedure is as follows: (1) pre-denaturation: 95 ℃ for 3min; (2) and (3) a circulation stage: denaturation: 95 ℃ for 15s; annealing: 60.1 ℃ for 15s; extension: 72 ℃,30s;30 cycles. (3) Extending thoroughly: 72℃for 5min.
TABLE 1 reverse transcription reaction system
TABLE 2cDNA reaction System
Using cDNA sample as template, and Novozan biological companyThe fluorescent quantitation kit ChamQ Universal SYBR qPCR Master MiH of (2) was performed on an ABI 7500Real Time PCR instrument system for Real Time fluorescent quantitation. The DNA gyrase A subunit (gyrA) is used as an internal reference gene, the target gene is a Curli bacterium Mao Bianma gene (curli subunit gene D, csgD), and the lengths of the upstream primer and the downstream primer and the product are shown in Table 3. By 2 -ΔΔCt Measuring the relative expression quantity of mRNA of target gene by using a method, using a t-test method to make statistical analysis on the data result, * P<0.05 is a significant difference.
TABLE 3 fluorescent quantitative PCR primer sequences
The results are shown in FIG. 3. Compared with a blank group (NC), 1.0U/mL AHLase can obviously reduce the relative expression quantity (P < 0.05) of the CsgD gene of the salmonella typhimurium, and because CsgD is a core gene for encoding Curli pilus, the Curli pilus of the salmonella typhimurium is a key component of a bacterial biofilm, can promote bacteria to adhere to the surfaces of various substances (such as animal intestinal tracts, plants and the like) and adhere to each other to finally form the biofilm, and can inhibit the expression of the CsgD gene to represent that the formation of the biofilm can be inhibited, so that the AHLase can inhibit the formation of the salmonella typhimurium biofilm, thereby reducing the infectivity of the salmonella typhimurium.
Example 2 inhibition of Salmonella typhimurium motility by AHLase
Selecting Salmonella typhimurium single colony, shake culturing in LB broth medium at 180 rpm (37deg.C) in a constant temperature shaker for 12 hr, and adjusting bacterial liquid to 10 9 After CFU/mL, 1 mu L of bacterial liquid is sucked up, and the bacterial liquid is gently spotted at the center of a cluster motility flat plate culture medium (the group with the final concentration of 0 is a blank group) with the final concentration of 0 and 1.0U/mL, and the formula of the culture medium is as follows: 25g/L LB, 5g/L agar powder and 0.5g/L glucose. The flat plate is kept stand and incubated for 24 hours at 37 ℃, the size of a bacterial moving coil is observed and measured, then a t-test method is adopted to carry out statistical analysis on the data result, * P<0.05 is a significant difference.
Salmonella typhimurium has exercise capacity, can promote the contact and adhesion of thalli and host cells, and the exercise capacity is positively related to the contact and adhesion capacity of the salmonella typhimurium and host cells, and can enter cells after the salmonella typhimurium is closely contacted with the host, so that systemic infection is initiated. The clustering movement means that many bacteria progress in the same direction by virtue of the rotational movement of the flagellum.
The results are shown in FIG. 4. Compared with a blank group (NC), the diameter of a salmonella typhimurium group movement ring can be obviously reduced by 1.0U/mL AHLase treatment, which shows that the AHLase can reduce the ability of salmonella typhimurium group movement, is beneficial to inhibiting the contact and adhesion of the salmonella typhimurium group movement to host cells, and further reduces the infectivity of salmonella typhimurium.
EXAMPLE 3 inhibition of the ability of AHLase to adhere to Salmonella typhimurium
1) Culture of intestinal epithelial cells IPEC-J2 cells (from agricultural university of south China)
(1) Cell resuscitation: IPEC-J2 cells were removed from liquid nitrogen, thawed in a 37℃water bath, centrifuged at 800r/min for 5min, the supernatant was discarded, 4mL of complete medium (10% fetal bovine serum FBS+1% penicillin/streptomycin+high sugar DMEM) was added, thoroughly blown and mixed, and then the cell suspension was transferred to a sterile 10cm cell culture dish, 5% CO 2 Culturing in an incubator at 37 ℃.
(2) Cell digestion: when the cells grow to 80% -90% of the area of the culture dish, discarding the culture medium, washing twice with sterile PBS, adding 1mL pancreatin containing EDTA, reacting for 5min at 37 ℃, observing the degree of cell shedding, stopping digestion when the cells are loosened, adding an equivalent amount of complete culture medium, and blowing the cells until the cells are completely shed from the bottom of the culture dish.
(3) Cell passage: the cell suspension was transferred to a sterile 15mL centrifuge tube and centrifuged at 800r/min at 4℃for 5min. Adding 1mL of complete culture medium, blowing cells, mixing thoroughly, inoculating into new sterile culture dish at a ratio of one transmission to two transmission, adding 5% CO 2 Culturing in a constant temperature incubator at 37 ℃ for 24 hours.
(4) Cell plating: 1mL of cells were digested according to step (2), and each well was performed in a 12-well cell culture plate1mL of the cell suspension was added at 5% CO 2 Culturing in a 37 ℃ incubator, and treating when the cell grows to 80% -90%.
2) Determination of adhesion Rate of Salmonella typhimurium to intestinal epithelial cells
Selecting Salmonella typhimurium single colony, shake culturing in LB broth medium at 180 rpm (37deg.C) in a constant temperature shaker for 12 hr, and adjusting bacterial liquid to 10 9 After CFU/mL, the bacterial solution was mixed with AHLase solution to prepare an enzyme-bacteria mixture having an AHLase final concentration of 0 and 1.0U/mL (the group having a final concentration of 0 is a blank group, and the concentration of bacteria in the enzyme-bacteria mixture is 5X 10) 8 CFU/mL), streaking (Z-type) the enzyme bacteria mixture on an LB agar plate, and culturing the plate in a constant temperature incubator at 37 ℃ for 12 hours. Salmonella typhimurium single colonies were picked from the plates and cultured in fresh LB broth medium at 37℃with an air shaker 180r/min for 12h. According to 1:100 volume ratio of the bacterial liquid is added into a new LB broth culture medium, and the bacterial liquid is cultured to OD in an air shaking table at 37 ℃ at 220r/min 600 =1.0. Centrifuging at 4000r/min for 15min, washing thallus twice with sterile PBS, and adjusting bacterial liquid concentration to 10 9 CFU/mL, ready for use.
Inoculating intestinal epithelial cells IPEC-J2 into 12-well culture plate, washing with sterile PBS for 2 times after cells grow to 90%, adding complete medium without double antibody, adding prepared bacterial solution into cells according to infection complex (MOI) of about 100, adding into 5% CO 2 Culturing in a constant temperature incubator at 37 ℃ for 1h. Cells were washed 3 times with sterile PBS and lysed by adding 1mL of PBS containing 0.1% Triton-100 for 30min, and thoroughly blown. After crushing and mixing evenly, the cell lysate is diluted by a ten-fold gradient ratio, coated in an LB culture plate, cultured for 12 hours at a constant temperature of 37 ℃, and the colony number of the flat plate is recorded. Bacterial adhesion calculation formula: bacterial adhesion (%) = (number of colonies of plate x dilution)/number of added bacteria x 100%. Statistical analysis is carried out on the data result by adopting a t-test method, * P<0.05 is the difference is significant and, ** P<0.01 is very significant.
Bacterial adhesion to intestinal epithelial cells is a prerequisite and critical step for its infection of the host. After Salmonella typhimurium enters the intestinal tract, it must adhere to intestinal epithelial cells to infect the host.
As shown in fig. 5, the 1.0U/mL ahase treatment significantly reduced the amount of salmonella typhimurium that adhered to the host intestinal epithelial cells (IPEC-J2) compared to the blank (NC), indicating that ahase can reduce the ability of salmonella typhimurium to adhere, inhibit infection of the host, and thus reduce the infectivity of salmonella typhimurium.
EXAMPLE 4 protection of Salmonella typhimurium infected broilers by AHLase
1) Breeding of broiler chickens
240 large Huang Yurou roosters (1 day old (d) of Ling nan Huang Qing Kuai) are selected and divided into 3 groups according to the weight balance principle: control group (feeding basal diet), infected group (feeding basal diet and infecting Salmonella typhimurium), AHLase group (10000U AHLase/kg diet added on the basis of infected group). Each group had 8 replicates (initial weights of broiler chickens were similar) with 10 chickens per replicate for a 21 day trial period. All broilers were kept in metal cages with 16 hours of daily light, the temperature in the house was maintained at 34 ℃ for the first week, and then gradually lowered to 24 ℃ (third week). All broilers were free to eat and drink. The basic diet formulation and nutritional composition are shown in table 4.
TABLE 4 basal diet composition and nutrient levels (air-dried basis,%)
The premix in table 4 provides for each kg of diet: vitamin A5 000IU, vitamin B 1 2.5mg, vitamin VB 6 4mg, vitamin B 12 0.015mg, vitamin D 3 80.75mg, 31mg of vitamin E, 1.6mg of vitamin K, 0.05mg of biotin, 1.5mg of folic acid, 60mg of pantothenic acid, 15mg of nicotinic acid, 450mg of choline chloride and 15mg of riboflavin; copper 9.5mg, iron 70mg, manganese 121mg,60mg of zinc, 1.40mg of iodine and 0.45mg of selenium.
2) Infection with Salmonella typhimurium
Salmonella typhimurium liquid is streaked (Z-shaped) on an LB agar plate, and the plate is placed in a constant temperature incubator at 37 ℃ for culturing for 12 hours. Salmonella typhimurium single colonies were picked from the plates and cultured in fresh LB broth medium at 37℃with an air shaker 180r/min for 12h. According to 1:100 volume ratio of the bacterial liquid is added into a new LB broth culture medium, and the bacterial liquid is cultured to OD in an air shaking table at 37 ℃ at 220r/min 600 After=1.0, the bacterial liquid concentration was adjusted to 5×10 9 CFU/mL, ready for use.
At the 8 th to 10 th days of the test period, 2mL of bacteria liquid is infused into the oral cavity of the broiler chickens in the infection group and the AHLase group, and the same amount of LB broth is infused into the oral cavity of the broiler chickens in the control group.
3) Index measurement
a. Growth performance: each repeat broiler of each group was weighed and the corresponding feed intake recorded at trial 11d and 21d, and the final weight, average daily gain, average daily feed intake and feed weight ratio of the three phases 1 to 11d, 12 to 21d and 1 to 21d were calculated.
b. Organ index: at trial 11d and 21d, one chicken from each repetition of each group was randomly selected for weighing and slaughter, viscera (liver, spleen, thymus and bursa) were isolated, weighed and organ index (i.e. organ weight (g)/living weight (kg)) was calculated. Statistical analysis is carried out on the data results by adopting a single-factor analysis of variance method, multiple comparison is carried out by adopting a Duncan's method when the difference is obvious, P <0.05 is obvious, and P <0.01 is extremely obvious.
4) Analysis of results
a. The results of the growth performance measurement are shown in Table 5. It can be seen that the end weight, average daily gain and average daily feed intake during the 1 to 11d and 12 to 21d period and the average daily gain and average daily feed intake during the 1 to 21d period were significantly reduced (P < 0.05) and the feed weight ratio during the 1 to 11d period was significantly increased (P < 0.05) as compared to the control group. The end weight, average daily gain and average daily feed intake during the AHLase groups 1 to 11d and 12 to 21d, and the average daily gain and average daily feed intake during the 1 to 21d were significantly higher (P < 0.05) than the infected group and were not significantly different (P > 0.05) from the control group. In addition, the feed weight ratio during AHLase groups 1 to 11d was significantly lower (P < 0.05) than in the infected group and there was no significant difference (P > 0.05) from the control group.
The growth performance status of livestock and poultry can directly reflect the use effect of the additive. The results show that the addition of AHLase in the diet has an obvious protective effect on the growth performance of the broiler chicken infected by salmonella typhimurium, can completely counteract the reduction of the growth performance of the broiler chicken, and is beneficial to reducing the reduction of the culture benefit caused by salmonella typhimurium infection.
TABLE 5 protection of growth performance of Salmonella typhimurium infected broilers by AHLase
In table 5, the same row of data shoulder marks have no letter or the same letter indicates that the difference is not significant (P > 0.05), and the different letter indicates that the difference is significant (P < 0.05).
b. The results of organ index measurement are shown in Table 5. It can be seen that the spleen index, liver index and bursa index of the broiler 11d were all significantly elevated (P < 0.05) in the infected group compared to the control group, while the spleen index, liver index and bursa index of the AHLase group broiler were all significantly lower (P < 0.05) than in the infected group and were not significantly different (P > 0.05) from the control group. There were no significant differences in thymus index, spleen index, liver index, and bursa index between groups at 21d (P > 0.05).
The most important immune organs of poultry are thymus, spleen and bursa of Fabricius. The thymus and the french capsule belong to central immune organs, and are main sites for T, B lymphocyte development, differentiation and maturation respectively. The spleen (the largest peripheral lymphoid immune organ in the body) and liver (the largest solid organ in the body) both contain a large number of immune cells, which are important parts of cellular and humoral immune responses. In the infected state, an increase in organ index (i.e., organ relative weight) means that the pathogenic bacteria overstimulate the immune system of the body, resulting in a large number of immune cells and immune factors entering the tissue organ, resulting in compensatory swelling of the organ. The test result shows that the addition of AHLase in the diet has obvious protection effect on organ indexes of broilers infected by salmonella typhimurium, and can completely counteract organ swelling caused by salmonella typhimurium infection.
TABLE 6 protection of organ index of Salmonella typhimurium infected broilers by AHLase
In table 6, the same row of data shoulder marks have no letter or the same letter indicates that the difference is not significant (P > 0.05), and the different letter indicates that the difference is significant (P < 0.05).
EXAMPLE 5 protection of the intestinal health of Salmonella typhimurium infected broilers by AHLase
1) Feeding of broiler chickens, infection with Salmonella typhimurium was the same as in example 4.
2) Sample collection: at 11d and 21d of the test period, one chicken from each repetition of each group was randomly selected for fin vein blood collection, centrifuged at 3000rpm at 4℃for 10min, and serum was isolated and stored at-30 ℃. Then slaughtering the chickens, separating intestinal tracts, shearing back intestinal middle section tissues, dividing the intestinal middle section tissues into two small blocks, and fixing one small block in 4% paraformaldehyde solution for detecting the morphological structure of the intestinal tracts; another small block of liquid nitrogen is frozen and stored at-80 ℃ for measuring the ileum gene expression.
3) And (3) measuring indexes:
a. intestinal morphology and structure analysis: ileal tissues stored in 4% paraformaldehyde solution were sectioned for paraffin embedding and hematoxylin-eosin staining. The flat and morphologically intact intestinal villi was observed and measured under a microscope in each field of view in each sample, its Villi Height (VH) and Crypt Depth (CD) were measured, and the ratio (VH/CD) of the two was calculated. Where VH refers to the distance between the tip of the villus to the junction of the villus and the crypt, and CD refers to the depth of the tubular gland formed by the depression of the villus root into the lamina propria.
b. Serum diamine oxidase (DAO) activity assay: the activity of serum DAO was determined spectrophotometrically (kit purchased from beijing solebao biosystems) at a wavelength of 500nm on an enzyme-labeled instrument. The measurement principle is as follows: DAO catalyzes cadaverine to produce aldehyde and hydrogen peroxide, excessive horseradish peroxidase is exogenously added to catalyze hydrogen peroxide to oxidize o-dianisidine to produce colored matter, absorption peak is present at 500nm, and DAO activity can be calculated by measuring the increasing rate of absorbance of the wavelength.
c. Measurement of relative expression amounts of mRNA of intestinal claudin and cytokine:
total RNA of ileal tissue was extracted using the RNA extraction kit FastPure Cell/Tissue Total RNA Isolation Kit V2 from Nanjinouzan biosystems and the obtained RNA was stored at-80 ℃.
The concentration of RNA samples was determined as in example 1 (all samples were RNA concentrations meeting the requirements of the subsequent procedure and were uniformly adjusted to 500 ng/. Mu.L) and tested for purity and integrity, after which reverse transcription and real-time fluorescent quantitative PCR were performed after confirming good purity and integrity of the RNA samples. With glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal gene, the target gene includes various kinds of closely linked proteins, namely, claudin-1 (claudin-1), occlusal protein (occludin) and zonules (ZO-1), and various kinds of inflammatory factors including interleukin 1 beta (IL-1 beta), interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha). Primer information for the reference gene and the target gene is shown in Table 7. By 2 -ΔΔCt The relative expression level of mRNA of the target gene is measured by the method.
TABLE 7 primer information for genes
4) Test results:
a. the results of the intestinal morphology analysis are shown in Table 8. It can be seen that both the ileal VH and VH/CD were significantly reduced (P < 0.05) in the broilers 11d and 21d of the infected group compared to the control group. Whereas the ileal VH of the ahlay group broiler 11d and the ileal VH and VH/CD of 21d were significantly higher (P < 0.05) than the infected group and were not significantly different (P > 0.05) from the control group; furthermore the ileal VH/CD of ahlay broiler 11d was even significantly higher (P < 0.05) than control.
The morphological structure of the intestinal canal can reflect the quality of the digestive absorption function of the intestinal canal. The larger intestinal VH and VH/CD means that the larger the intestinal villus surface area is, the more mature the intestinal epithelial cells are, and the more favorable the digestive absorption function of the intestinal tract is. The test result shows that the addition of AHLase in the diet can completely counteract intestinal morphology and structure damage caused by salmonella typhimurium infection, thereby being beneficial to the digestion, absorption and growth performance improvement of intestinal tracts.
TABLE 8 influence of AHLase on the ileum morphology and structure of Salmonella typhimurium infected broilers
In table 8, the same row of data shoulder marks have no letter or the same letter indicates that the difference is not significant (P > 0.05), and the different letter indicates that the difference is significant (P < 0.05).
b. The results of the intestinal barrier function test are shown in fig. 6, wherein fig. 6A shows the effect of AHLase on the expression level of the closely connected protein mRNA of the salmonella typhimurium-infected broiler at the 11D of the test period, fig. 6B shows the effect of AHLase on the expression level of the closely connected protein mRNA of the salmonella typhimurium-infected broiler at the 21D of the test period, fig. 6C shows the effect of AHLase on the activity of serum diamine oxidase (DAO) of the salmonella typhimurium-infected broiler at the 11D of the test period, and fig. 6D shows the effect of AHLase on the activity of serum DAO of the salmonella typhimurium-infected broiler at the 21D of the test period.
As can be seen from fig. 6A and 6B, mRNA expression levels of the infected broilers 11d and 21d ileocecal cludins and 21d ileo ZO-1 were significantly up-regulated (P < 0.05) compared to the control group. Whereas the mRNA expression level of 11d ileal occludins was significantly down-regulated in the ahlay group compared to the infected group (P < 0.05) and was not significantly different from the control group (P > 0.05). In addition, the mRNA expression level of 21d ileal occludins was also restored to no significant difference (P > 0.05) from the control group in the AHLase group broiler chickens. As can be seen from fig. 6C and 6D, there was no significant difference in serum DAO activity (P > 0.05) for each group of broilers 11D and 21D.
Intestinal tight junctions are composed of a variety of tight junction proteins including transmembrane proteins (e.g., claudin family and occludin) and intramembrane proteins (e.g., ZO family). The tight junction proteins maintain the permeability barrier of the intestine, preventing the passage of harmful macromolecular substances in the intestinal lumen through the intestinal wall into the blood circulation. DAO is a highly active intracellular enzyme that is present in the intestinal mucosa and is released into the blood when the intestinal mucosa is damaged, so that DAO activity in the blood may reflect intestinal damage and barrier function conditions. The results of this experiment found that Salmonella typhimurium infection had no significant disruption to the intestinal barrier function of broilers (because serum DAO activity did not change significantly), whereas mRNA expression levels of ileum occludin in infected groups were significantly up-regulated, which is thought to be because Salmonella typhimurium infection caused self-protection of the body and feedback caused the up-regulation of intestinal occludin expression levels to combat Salmonella typhimurium invasion of the intestinal barrier. The addition of AHLase reduces the virulence of Salmonella typhimurium, relieves the stimulation of Salmonella typhimurium to the intestinal barrier of broilers and the burden of intestinal self-protection, and further causes the expression quantity of intestinal occludins to fall back, but does not destroy the intestinal barrier function of broilers (because the serum DAO activity has no obvious change).
c. The results of the intestinal inflammatory reaction are shown in fig. 7, wherein fig. 7A shows the effect of AHLase on the inflammatory reaction of the ileum of the salmonella typhimurium infected with the same, and fig. 7B shows the effect of ahase on the inflammatory reaction of the ileum of the salmonella typhimurium infected with the same, at the time of the test period 21 d.
It can be seen that the mRNA expression level of the ileal pro-inflammatory factor IL-8 of the broiler 11d in the infected group is significantly up-regulated (P < 0.05) compared with the control group, which indicates that Salmonella typhimurium infection causes a certain inflammatory reaction in the intestinal tract of the broiler, and may aggravate intestinal damage (such as intestinal morphological structure damage). And the mRNA expression level of the IL-8 in the 11d ileum of the broiler chicken in the AHLase group is restored to be not significantly different from that in the control group (P > 0.05), which proves that the AHLase can relieve the inflammatory reaction of the intestinal tract of the broiler chicken caused by salmonella typhimurium infection, thereby being beneficial to the alleviation of intestinal damage, which is thought to be because the AHLase reduces the virulence of the salmonella typhimurium.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
- Use of an n-acyl homoserine lactonase for the preparation of a product against salmonella typhimurium infection.
- 2. The use according to claim 1, wherein the product is a feed, a feed additive, an enzyme preparation, a microecological preparation or a pharmaceutical product.
- 3. The use according to claim 1, wherein the salmonella typhimurium infection is a reduction in virulence and/or pathogenicity of salmonella typhimurium.
- 4. The use according to claim 3, wherein the reduction of virulence and/or pathogenicity of salmonella typhimurium is inhibition of biofilm formation, motility and/or adhesion of salmonella typhimurium.
- 5. The use according to claim 4, wherein the ability to inhibit the formation of a biofilm of salmonella typhimurium is one or more of decreasing the biomass of the biofilm, increasing the inhibition rate of the formation of the biofilm, inhibiting the secretion of extracellular DNA, and down-regulating the expression of genes associated with the formation of the biofilm.
- 6. The use according to claim 4, wherein the ability to inhibit the motility of salmonella typhimurium is the ability to inhibit the colonisation of salmonella typhimurium.
- 7. The use according to claim 4, wherein the ability to inhibit adhesion of salmonella typhimurium is to inhibit adhesion of salmonella typhimurium to host intestinal epithelial cells.
- 8. The use according to claim 1, wherein the salmonella typhimurium infection is to protect the growth performance, organ index and/or intestinal health of an animal infected with salmonella typhimurium.
- 9. The use according to claim 8, wherein the protection of the intestinal health of the salmonella typhimurium infected animal is one or more of protection of the morphological structure of the intestinal tract, improvement of the intestinal barrier function, inhibition of the inflammatory response of the intestinal tract.
- 10. The use according to claim 8, wherein the animal is a broiler chicken.
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| CN114424800A (en) * | 2022-04-06 | 2022-05-03 | 北京挑战生物技术有限公司 | Feed additive for improving intestinal health index of broiler chickens and application thereof |
| CN115197888A (en) * | 2021-09-28 | 2022-10-18 | 华南农业大学 | Salmonella typhimurium htpG and construction method and application thereof |
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| CN115197888A (en) * | 2021-09-28 | 2022-10-18 | 华南农业大学 | Salmonella typhimurium htpG and construction method and application thereof |
| CN114424800A (en) * | 2022-04-06 | 2022-05-03 | 北京挑战生物技术有限公司 | Feed additive for improving intestinal health index of broiler chickens and application thereof |
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