A method for prevention and control of whole cycle of chicken embryo incubation period based on phage layered delivery

Abstract

The present application belongs to the technical field of poultry hatching prevention and control, and particularly relates to a chicken embryo hatching period whole cycle prevention and control method based on phage layered delivery, comprising: screening phage strains suitable for different embryo age stages; preparing a composite carrier matrix with a porous structure by using chitosan and sodium alginate; preparing a layered delivery film by using a laminating film forming process to load different phage strains in the composite carrier matrix; attaching and covering the layered delivery film on the surface of the to-be-hatched egg, and placing the coated egg in a hatching device for hatching; combining the characteristics of the outer layer of the layered delivery film degrading first and the inner layer degrading later, and cooperating with a step-by-step solidification process to ensure the interlayer pores being connected, so that the layered delivery film wrapped around the egg degrades in sequence, and each embryo age chicken embryo can obtain the suitable phage at the corresponding infection risk stage, the hatching whole process is matched with the infection prevention and control stage, and a whole cycle prevention and control system covering early prevention, middle treatment and late targeted elimination is constructed.

Description

Technical Field

[0001] This invention belongs to the field of poultry incubation and control technology, and particularly relates to a method for full-cycle control of chicken embryo incubation based on phage stratified delivery. Background Technology

[0002] Chicken embryo incubation is a crucial step in poultry farming, and the health of the embryos during this stage directly impacts the hatching rate and subsequent survival rate. Under large-scale farming conditions, *Riemerella anatipestifer*, a specific pathogen originating in chicken embryos, exhibits significant differences in host adaptability compared to *Riemerella anatipestifer* from ducks. This bacterium easily invades the embryo through pores or microcracks in the eggshell, causing infection, abnormal development, and even death, resulting in significant economic losses for poultry farms. Therefore, effective control of pathogens during the chicken embryo incubation stage is of paramount importance.

[0003] With the increasing demands for biosafety and product quality in the aquaculture industry, biocontrol technologies utilizing bacteriophages to target and lyse pathogens are gaining attention. However, existing bacteriophage control technologies primarily target the control of *Riemerella anatipestifer* from ducks, and typically employ a single bacteriophage strain or a single administration method, which is insufficient to meet the infection control needs throughout the entire chicken embryo hatching process. Chicken embryos at different embryonic stages exhibit significant differences in physiological structure, infection routes, and intraembryonic fluid environment. A single bacteriophage strain cannot be adapted to the physiological characteristics and infection risks of chicken embryos at different stages, resulting in insufficient targeting and unstable control effects.

[0004] On the other hand, existing methods of administering medication to chicken embryos mainly rely on manual injection or puncture. This requires direct contact with the egg or embryo during administration, which can easily damage the eggshell structure and potentially affect normal gas exchange through the eggshell pores, thus impacting the embryo's developmental environment. Furthermore, injection administration is typically a single-dose procedure, making it difficult to maintain its effectiveness throughout the entire incubation period and hindering dynamic control over the entire chicken embryo incubation process.

[0005] In addition, existing controlled-release vectors are mostly used in vivo or injection delivery scenarios, and can usually only release a single drug. They lack a phased delivery system suitable for the hatching process of hatching eggs, which makes it impossible to match the release sequence of bacteriophages with the infection risk at different stages of chicken embryos, making it difficult to achieve effective prevention and control throughout the entire hatching process.

[0006] Therefore, developing a technical solution that can adapt to the physiological characteristics of chicken embryos at different embryonic ages and achieve infection control throughout the entire incubation cycle by delivering bacteriophages in stages without damaging the eggshell structure or affecting gas exchange has become an urgent technical problem to be solved in this field.

[0007] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0008] To address the problems of poor targeting of infection control measures during chicken embryo incubation, easy damage to eggshells caused by drug administration, and inability to achieve continuous control throughout the entire incubation cycle in existing technologies, this invention provides a method for full-cycle control of chicken embryo incubation based on phage stratified delivery. By constructing a phage stratified delivery system adapted to different embryonic stages, precise control can be achieved throughout the entire chicken embryo incubation process without damaging the eggshell structure, thereby improving the survival rate and incubation stability of chicken embryos.

[0009] This invention proposes a method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery, comprising the following steps: (1) Screening and preparation of bacteriophage lines Chicken-derived Riemerella anatipestifer was isolated from chicken embryo infection foci as host bacteria. Specific phage strains suitable for early, middle and late embryonic stages were screened, and phage stock solutions for different embryonic stages were prepared by culturing and purification. (2) Preparation of composite carrier matrix A composite carrier matrix with a porous structure was prepared by mixing and dissolving chitosan and sodium alginate, and adjusting the ratio and pH. (3) Preparation of layered delivery membrane Using a composite carrier matrix as the film-forming material, a step-by-step curing lamination film-forming process is adopted to load phages adapted to early, middle and late embryonic stages into different layers to form a multilayered delivery membrane with a multi-layered structure. (4) Egg incubation with covering The layered delivery film is applied to the surface of the eggs to be incubated, and the coated eggs are placed in an incubation device for incubation. (5) Time-sequenced release and hierarchical delivery During incubation, the layered delivery membrane degrades and releases bacteriophages in sequence. The bacteriophages enter the chicken embryo and target and lyse pathogens in the early, middle, and late embryonic stages, thus achieving infection control throughout the entire chicken embryo incubation process.

[0010] Preferably, the phage and the carrier formed by the degradation of the composite carrier matrix in step (5) constitute a phage complex, which permeates to the eggshell surface through the membrane structure and then permeates into the embryo through the natural gas pores of the eggshell, thereby targeting and lysing pathogenic bacteria at different embryonic stages and achieving infection control throughout the entire process of chicken embryo hatching.

[0011] Preferably, the different embryo ages mentioned in step (1) are 7-day embryo age, 14-day embryo age and 18-day embryo age, and the specific phage strains are used for infection control in the early, middle and late stages of chicken embryo hatching.

[0012] Preferably, the mass ratio of chitosan to sodium alginate in step (2) is 1:1.5 to 1:2.5, the mass concentration of the mixed raw materials after dissolving in water is 2% to 5%, the pH is 6.5 to 7.0, and the mixture is stirred continuously at 35 to 40°C until completely dissolved. After stirring, the mixture is allowed to stand for 10 to 30 minutes to degas, thus obtaining a porous composite carrier matrix.

[0013] Preferably, the porous structure in step (2) combined with the interlayer pores formed by the stepwise curing process in step (3) provides a channel for the penetration of the phage complex.

[0014] Preferably, the layered delivery membrane in step (3) is based on the eggshell of the seed egg. The first layer of composite carrier matrix is ​​used as the inner layer to load a phage strain adapted to 18 days of embryonic age. After initial curing, the second layer of composite carrier matrix is ​​coated as the middle layer and loaded with a phage strain adapted to 14 days of embryonic age. After further curing, the third layer of composite carrier matrix is ​​coated as the outer layer and loaded with a phage strain adapted to 7 days of embryonic age.

[0015] Preferably, in step (3), the coating thickness of each layer of the layered delivery membrane is 0.05~0.15 mm, and the phage loading is 10. 8 ~10 9 PFU / cm 2 .

[0016] Preferably, the curing environment in step (3) is a temperature of 20~25℃, a relative humidity of 40%~60%, and a curing time of 4~8 h. The resulting film is a flexible and bendable structure, without high-temperature curing or hardening processes, so that the coating film can closely adhere to the surface of the hatching egg and adapt to the curved shape of the eggshell, and does not affect the pore structure and gas exchange of the eggshell during incubation.

[0017] Preferably, the hatching eggs in step (4) meet the requirements of having no surface damage, no stains, and uniform weight; the covering meets the requirements of completely covering the eggshell without wrinkles or air bubbles; and the incubation conditions are a temperature of 37.5~38.5℃, a relative humidity of 50~65%, and a ventilation rate of 0.3~0.8 m³ / h. 3 / h, turn the eggs once every 2~4 hours.

[0018] Furthermore, under the incubation conditions, the outer layer of the layered delivery membrane degrades within 1 to 7 days, the middle layer degrades within 8 to 14 days, and the inner layer degrades within 15 to 21 days. The phages released from each layer correspond to the suitable 7-day, 14-day, and 18-day embryonic ages, respectively.

[0019] Furthermore, the phage complex released from the outer layer permeates into the embryo through the pores of the middle layer, the pores of the inner layer, the pores of the eggshell, and the air cell membrane of the embryo. The phage complex released from the middle layer is delivered to the target tissue of the chorioallantoic membrane through the pores of the inner layer, the pores of the eggshell, and the circulation of body fluids within the embryo. The phage complex released from the inner layer is delivered to the target tissue of the yolk sac through the pores of the eggshell and the air cell membrane of the embryo, thus achieving targeted delivery of the phage.

[0020] Based on the same inventive concept, the present invention also provides a layered delivery membrane for the whole-cycle prevention and control of chicken embryo incubation, comprising a multi-layer structure formed of biodegradable polymer material, wherein each layer is loaded with a phage strain adapted to different embryonic stages, and the multi-layer structure degrades sequentially in the incubation environment according to a preset time sequence.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention achieves timed release and targeted delivery of chicken embryos in the early, middle and late stages of hatching by screening chicken phage strains in stages and combining them with layered delivery membranes. This can effectively inhibit or eliminate the infection of Riemerella anatipestifer in chicken embryos at different embryonic stages and achieve full-cycle prevention and control during the hatching period of chicken embryos. (2) The control method uses a flexible polymer coating membrane material (chitosan-sodium alginate), the drying process is mild (20~25℃, relative humidity 40%-60%), the membrane is flexible and bendable, does not damage the eggshell structure and the normal development environment of the embryo, and significantly improves biosafety; (3) By constructing a multi-layer controlled-release vector, the time-sequential and targeted release of bacteriophages is realized, and the release of bacteriophages is precisely matched with the risk of infection at the embryonic age, avoiding the problems of low efficiency of single-dose administration and difficulty in continuous prevention and control, and improving the overall prevention and control efficiency and resource utilization. Attached Figure Description

[0022] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the following description is only a part of the embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a flowchart of a chicken embryo incubation period control method based on phage stratified delivery.

[0024] Figure 2This is a flowchart of the preparation process of the composite carrier matrix in a chicken embryo incubation cycle control method based on phage stratified delivery.

[0025] Figure 3 This is a flowchart of the preparation of the layered delivery membrane in a chicken embryo incubation period control method based on phage layered delivery. Detailed Implementation

[0026] This invention proposes a method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery. To facilitate understanding of this invention by those skilled in the art, the specific embodiments of this invention are described below with reference to the accompanying drawings.

[0027] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the art. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] Example 1: Infection Control During Spring Embryo Hatching in Large-Scale Chicken Farms In this embodiment, the chicken farm is located in a humid southern region where Riemerella anatipestifer infection is prevalent in spring. The operating procedure is as follows: Figure 1 As shown, the details are as follows.

[0029] 1.1 Phage line screening Infected lesion tissue was extracted from dead chicken embryos sent from the hatchery of a poultry farm. *Riemerella anatipestifer*, a bacteriogen, was isolated and purified under aseptic conditions and used as the host bacterium. The appropriate phage strains for 7-day, 14-day, and 18-day-old chicken embryos were screened using the double-layer agar plate method in a biosafety cabinet. The screening criteria were: using *Riemerella anatipestifer* isolated from the infected lesions of the corresponding embryonic age as the target, and determining the phage lysis rate using the double-layer agar plate method. The phages were inoculated with a resolution rate of ≥90% to ensure that they had specific infection capabilities against pathogens at the corresponding embryonic stages. The primary screening phages were then inoculated into the body fluid environment simulating different embryonic ages (7-day, 14-day, and 18-day) for secondary screening. The retention rate of the primary screening phages was measured, and those with a retention rate of ≥85% were selected to ensure stable survival in the embryonic environment at the corresponding embryonic age. Finally, after inoculating the corresponding embryonic age embryos with pathogens, the secondary screening phages were administered. The change in the number of pathogens in the embryonic cavity was measured after 24 hours. The screening requirements of ≥90% infection inhibition rate at the early 7-day embryonic age, ≥88% infection clearance rate at the mid-stage 14-day embryonic age, and ≥92% infection clearance rate at the late 18-day embryonic age were determined to obtain phages for three-stage screening, ensuring that the selected phage strains were suitable for infection control needs at different embryonic ages.

[0030] The screening requirements for the third screening mentioned above are set according to the physiological characteristics and infection features of chicken embryos at different embryonic ages, as follows: 7-day-old chicken embryos: In the early stage of embryonic development, the embryo is fragile and the body fluid environment is mainly composed of egg white. After infection, pathogens can easily proliferate rapidly. Therefore, the core is to effectively inhibit the spread of infection, and the infection inhibition rate is set at ≥90% to ensure that the infection process is blocked in the early stage of pathogen proliferation and avoid damage to the embryo. 14-day-old chicken embryos: In the mid-stage of development, the embryo is gradually maturing. The body fluid environment consists of egg white and allantoic fluid. Infections are mostly localized colonization infections. Pathogens have already reached a certain scale of proliferation. It is necessary to eliminate existing pathogens while avoiding damage to the embryo. Therefore, the infection clearance rate is set at ≥88% to effectively kill bacteria while reducing the potential impact of excessive bacteriophages on the embryo. 18-day-old chicken embryos: Approaching the hatching period, the embryos have a certain level of immunity. The body fluid environment is mainly composed of yolk sac fluid and allantoic fluid. The goal is to eliminate pathogens, with an infection clearance rate of ≥92%.

[0031] Each strain after three screenings was inoculated into pre-sterilized LB liquid medium containing the corresponding host bacteria and cultured in an intelligent constant temperature shaking incubator at 37℃ with shaking at 220 r / min for 13 h. After the culture, the bacterial solution was transferred to a high-speed refrigerated centrifuge and centrifuged at 10000 r / min for 22 min to remove bacterial precipitate. The supernatant was filtered through a 0.22 μm sterile filter membrane in a negative pressure filtration device for sterilization. PEG6000 powder was added to the sterilized supernatant until completely dissolved, and the solution was placed in a 4℃ refrigerator for static precipitation for 12 h. Subsequently, it was purified by dialysis with phosphate buffer for 5 h to finally obtain high-purity bacteriophage stock solution, which was aliquoted and temporarily stored in a -20℃ refrigerator for later use.

[0032] 1.2 Preparation of composite carrier matrix like Figure 2 As shown, the raw materials were accurately weighed using an electronic balance at a mass ratio of chitosan to sodium alginate of 1:2.2. Deionized water was added to bring the mass concentration of the mixed raw materials to 3.5%. The mixture was poured into a container of a digital display constant temperature water bath stirrer, and the temperature was set to 38℃ with a stirring speed of 250 r / min. The stirring was continued for 3 h until the raw materials were completely dissolved. During the stirring process, 0.3 mol / L hydrochloric acid solution was slowly added dropwise using a pipette to adjust the pH of the mixture to 6.7 to avoid local pH changes affecting the matrix performance. After stirring, the mixture was transferred to a vacuum degassing tank, the tank door was closed, and the tank was evacuated and allowed to stand for 25 min to degas. After the bubbles were completely eliminated, the mixture was removed to obtain a uniform, transparent, and porous composite carrier matrix with a uniform pore distribution. The matrix was then sealed and stored at room temperature for later use.

[0033] 1.3 Preparation of Layered Delivery Membrane Based on the requirement of layered delivery, where the outer membrane degrades first and the inner membrane degrades later, phages of different embryonic ages are loaded, such as... Figure 3 As shown, the eggshell of the hatching egg was used as the bonding reference in a fully automatic lamination film forming machine to coat the composite carrier matrix on the stainless steel carrier plate of the machine. The first coating process was initiated, uniformly coating the composite carrier matrix as the inner layer, with a coating thickness controlled at 0.12 mm. Subsequently, an automatic spraying device was used to uniformly load the phage stock solution adapted to 18-day-old embryos, with a loading rate controlled at 10. 9 PFU / cm 2 The substrate is then initially cured at 23°C and 55% relative humidity for 1.8 h. After curing, the second coating process is automatically started, coating the middle layer with a composite carrier matrix of the same thickness and loading a phage stock solution adapted to 14-day embryos. After maintaining the same loading amount, it is cured again for 1.8 h. Finally, the third coating process is started, coating the outer layer with the composite carrier matrix and loading a phage stock solution adapted to 7-day embryos. After curing, the carrier plate is transferred to a constant temperature and humidity drying oven and cured continuously for 6.5 h under the same temperature and humidity conditions, finally producing a layered delivery membrane.

[0034] 1.4 Coating and Incubation of Hatching Eggs In the sterile incubation preparation workshop, hatching eggs undergo pretreatment. Each egg is manually screened and weighed using an electronic scale, selecting uniform eggs with smooth surfaces, no damage or stains, and a weight between 58 and 62g. A layered delivery membrane is then applied to the egg surface with the inner layer facing inwards, ensuring complete coverage of the eggshell without wrinkles or air bubbles. The wrapped eggs are then evenly placed on incubation trays and pushed into the intelligent incubation chamber, where the incubation temperature is set to 38.2℃, relative humidity to 60%, and ventilation to 0.6 m³ / h. 3 / h, the automatic egg-turning component of the intelligent linkage incubator is set to turn the eggs once every 3 hours, and the incubation process is carried out from 0 to 21 days. During this period, the incubation status can be viewed in real time through the remote monitoring unit.

[0035] 1.5 Timing Release and Layered Delivery During incubation, the temperature and humidity sensors and membrane degradation observation window built into the intelligent incubator provide real-time monitoring. The outer membrane gradually degrades within 1-7 days due to the temperature and humidity of the incubation environment. The released bacteriophages and the nanocarriers formed by the degradation of the composite carrier matrix constitute a bacteriophage complex. This complex slowly permeates to the eggshell surface through the interconnected pores of the middle and inner layers, and then penetrates into the embryo through the eggshell gaps and the air cell membrane, forming early protection. After the outer layer is completely degraded, the middle layer is exposed to the incubation environment and degrades simultaneously within 8-14 days. The released bacteriophage complex continues to permeate to the eggshell surface through the pores of the inner layer and is precisely delivered to the chorioallantoic membrane target tissue through the intraembryonic fluid circulation. After the middle layer is degraded, the inner layer finally degrades within 15-21 days. The released bacteriophage complex directly contacts the eggshell surface and is delivered to the yolk sac target tissue through the eggshell gaps and the air cell membrane. The entire process achieves the time-sequential release and targeted delivery of bacteriophages.

[0036] Example 2: Infection Control During Autumn Chicken Embryo Hatching in Small and Medium-Sized Breeding Cooperatives In this embodiment, the breeding cooperative is relatively small in scale. Previously, due to a lack of effective prevention and control measures, the infection rate of chicken embryos fluctuated greatly, resulting in poor hatching stability. This prevention and control measure was carried out in the autumn, and the operation procedure is as follows: Figure 1 As shown.

[0037] 1.1 Phage line screening Liver and chorioallantoic membrane tissues were collected from diseased chicken embryos at the cooperative's own breeding base. These tissues were processed under sterile conditions to isolate and purify *Riemerella anatipestifer*, a bacteriophage derived from duck plague, as the host bacterium. The double-layer agar plate method was used to screen suitable phage strains for 7-day, 14-day, and 18-day-old chicken embryos in a constant-temperature incubator. Each strain was inoculated into sterilized LB broth containing the corresponding host bacterium and cultured in a conventional constant-temperature shaking incubator at 36°C with shaking at 180 rpm for 15 h. After culture, the bacterial culture was centrifuged at 9000 rpm for 25 min to remove bacterial precipitate. The supernatant was filtered through a 0.45 μm sterile filter for sterilization. PEG6000 was added to the supernatant to a concentration of 10% and stirred to dissolve. The mixture was allowed to settle in the refrigerator for 10 h, followed by purification by dialyzing with physiological saline for 4 h, yielding a bacterium with an activity of 10. 8 The PFU / mL phage stock solution was placed in sterile test tubes, sealed, and refrigerated for later use.

[0038] 1.2 Preparation of composite carrier matrix Weigh the raw materials using an electronic balance at a mass ratio of chitosan to sodium alginate of 1:1.7. Add deionized water to achieve a mass concentration of 2.8% for the mixed raw materials. Pour the mixture into a container in a constant temperature water bath, set the temperature to 36℃, and continuously stir with an electric stirrer at a speed of 180 r / min for 3.5 h until the raw materials are completely dissolved. During stirring, use a dropper to add 0.2 mol / L sodium hydroxide solution dropwise to adjust the pH of the mixture to 6.6, ensuring that the pH value remains stable within the set range. After stirring, pour the mixture into a clean glass container and let it stand at room temperature for 18 min to degas. After the bubbles in the solution dissipate, a uniform and porous composite carrier matrix is ​​obtained. Seal and store in a cool place.

[0039] 1.3 Preparation of Layered Delivery Membrane Using a lamination film-forming equipment in conjunction with manual operation, layer-by-layer coating is performed on the carrier plate of the lamination film-forming equipment, with the eggshell of the hatching egg as the bonding reference. Figure 3 As shown, a composite carrier matrix was first uniformly coated as an inner layer using a coater, with a coating thickness controlled at 0.09 mm. Then, phage stock solution adapted for 18-day-old embryos was uniformly loaded using a sprayer, with a loading capacity of 10... 8 PFU / cm 2 The carrier plate was placed in a constant temperature and humidity chamber and initially cured for 1.2 h at 21℃ and 48% relative humidity. After initial curing, a second layer of composite carrier matrix was coated as the middle layer with a thickness of 0.09 mm. After loading with phage stock solution adapted to 14-day embryos, it was cured again for 1.2 h. Finally, a third layer of composite carrier matrix was coated as the outer layer with a thickness of 0.09 mm. After loading with phage stock solution adapted to 7-day embryos, the carrier plate was left in the constant temperature and humidity chamber to continue drying for 5 h, thus obtaining a layered delivery membrane with interconnected pores in each layer.

[0040] 1.4 Coating and Incubation of Hatching Eggs Before incubation, the eggs are screened, ensuring the surface is free of damage and stains. They are weighed using an electronic scale to ensure the weight is between 53 and 57 g and evenly distributed. The inner layer of the layered delivery membrane is then tightly wrapped around the egg, ensuring complete coverage of the shell without wrinkles or air bubbles. If necessary, a small amount of biodegradable adhesive is used to secure the edges. The wrapped eggs are then placed on the incubation rack in the incubator, with the incubation temperature set to 37.7℃, relative humidity 53%, and ventilation rate 0.4 m³ / h. 3 / h, and manually turn the eggs every 2.5 hours, and carry out the entire incubation process according to the incubation cycle of 0 to 21 days. Record the temperature, humidity and status of the eggs in the incubator at regular intervals every day.

[0041] 1.5 Timing Release and Layered Delivery During incubation, the incubator is opened regularly each day to observe the degradation of the outer membrane. The outer membrane gradually absorbs moisture from the incubation environment and slowly degrades within 1 to 7 days. The released bacteriophages and the nanocarriers formed by the degradation of the composite carrier matrix constitute a bacteriophage complex. This complex gradually penetrates to the eggshell surface through the pores of the middle and inner layers, and then penetrates into the embryo through the eggshell gaps and the air cell membrane to play an early role in infection control. After the outer layer is completely degraded, the middle layer begins to degrade within 8 to 14 days. The released bacteriophage complex penetrates to the eggshell surface through the pores of the inner layer and is delivered to the chorioallantoic membrane target tissue with the help of the intraembryonic fluid circulation. After the middle layer is degraded, the inner layer degrades within 15 to 21 days. The released bacteriophage complex comes into direct contact with the eggshell surface and is delivered to the yolk sac target tissue through the eggshell gaps and the air cell membrane, thus achieving infection control throughout the entire incubation cycle of chicken embryos.

[0042] Example 3: Verification of Prevention and Control Effectiveness This embodiment uses two sets of experiments to illustrate the effectiveness of the prevention and control method of the present invention and the advantages of the layered design.

[0043] 3.1 Feasibility and efficacy comparison of phage delivery Three hundred healthy hatching eggs were selected and randomly divided into three groups of 100 eggs each: experimental group A, control group B, and control group C. The eggs in each group were from the same source, had the same embryonic age, and similar weight, and were confirmed to be fertilized and viable embryos by candling. The chicken-derived Riemerella anatipestifer was isolated from dead chicken embryos and had a titer of 10. 9 PFU / mL. Group A hatching eggs were wrapped with a layered delivery membrane as described in Example 1; Group B received no preventative treatment, no membrane wrapping, and no drug injection; Group C received conventional injection, without membrane wrapping, and the injected drug was a chicken-derived duck plague Riemerella phage, with an injection dose of 10 PFU / mL. 9 PFU / egg. All three groups were artificially inoculated through the eggshell pores on day 1 of incubation. 5 Chicken-derived Riemerella anatipestifer (CFU / piece) was used to simulate natural infection, followed by incubation for 21 days under the same incubation conditions as in Example 1. Infection rate and hatchability were recorded for the three groups.

[0044] After 21 days, the infection rate of group A was ≤5% and the hatching rate was ≥92%; the infection rate of group B was ≥30% and the hatching rate was ≤70%; and the infection rate of group C was ≤8% and the hatching rate was ≥88%. Through data comparison, it was indirectly proved that the bacteriophage successfully entered the embryo and exerted an anti-infection effect. Its anti-infection effect was not inferior to the traditional injection method, and it avoided the physical damage to the eggs and potential contamination risks caused by the injection operation.

[0045] 3.2 Comparative Experiment of Control Effects of Layered and Monolayer Loaded Phage Membranes Two hundred healthy hatching eggs were randomly divided into two groups of 100 eggs each: an experimental group and a control group. All eggs in the groups were from the same source, had the same embryonic age, and similar weight, and were confirmed to be fertilized and viable embryos by candling. The experimental group eggs were wrapped with a layered delivery membrane with a total thickness of 0.36 mm, and each layer was loaded with one of the three bacteriophages isolated in Example 1. The control group eggs were wrapped with a single-layer membrane loaded with only one type of bacteriophage, with a thickness of 0.36 mm. The bacteriophage used was the 7-day embryo-appropriate bacteriophage isolated in Example 1. Both groups were inoculated with pathogenic bacteria on day 1 of incubation, manually inoculated through the eggshell pores. 5 Chicken-derived Riemerella anatipestifer (CFU / egg) was used to simulate natural infection, followed by incubation for 21 days under the same incubation conditions as in Example 1. The infection rate, hatchability, and pathogen clearance efficiency at different embryonic stages were then tested.

[0046] The experimental group achieved pathogen clearance rates of ≥90%, ≥88%, and ≥92% at 7, 14, and 21 days, respectively, with a hatching rate of ≥92% and a final infection rate of ≤4%. In contrast, the control group only achieved a pathogen clearance rate of ≥85% in the early stage, ≤60% in the middle and late stages, a hatching rate of ≤75%, and a final infection rate of ≥18%. These results demonstrate that the stratified design can cover the infection risk throughout the entire embryonic stage, while a single-layer membrane cannot meet the prevention and control needs in the middle and late stages.

[0047] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery, characterized in that, Includes the following steps: (1) Screening and preparation of bacteriophage lines Chicken-derived Riemerella anatipestifer was isolated from chicken embryo infection foci as host bacteria. Specific phage strains suitable for early, middle and late embryonic stages were screened, and phage stock solutions for different embryonic stages were prepared by culturing and purification. (2) Preparation of composite carrier matrix A composite carrier matrix with a porous structure was prepared by mixing and dissolving chitosan and sodium alginate, and adjusting the ratio and pH. (3) Preparation of layered delivery membrane Using a composite carrier matrix as the film-forming material, a step-by-step curing lamination film-forming process is adopted to load phages adapted to early, middle and late embryonic stages into different layers to form a multilayered delivery membrane with a multi-layered structure. (4) Egg incubation with covering The layered delivery film is applied to the surface of the eggs to be incubated, and the coated eggs are placed in an incubation device for incubation. (5) Time-sequenced release and hierarchical delivery During incubation, the layered delivery membrane degrades and releases bacteriophages in sequence. The bacteriophages enter the chicken embryo and target and lyse pathogens in the early, middle, and late embryonic stages, thus achieving infection control throughout the entire chicken embryo incubation process.

2. The method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery according to claim 1, characterized in that: The different embryo ages mentioned in step (1) are 7-day embryo age, 14-day embryo age and 18-day embryo age, and the specific phage strains are used for infection control in the early, middle and late stages of chicken embryo hatching.

3. The method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery according to claim 1, characterized in that: The mass ratio of chitosan and sodium alginate in step (2) is 1:1.5 to 1:2.

5. After dissolving in water, the mass concentration of the mixed raw materials is 2% to 5%, and the pH is 6.5 to 7.

0. The mixture is stirred continuously at 35 to 40°C until completely dissolved. After stirring, the mixture is allowed to stand for 10 to 30 minutes to remove bubbles.

4. The method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery according to claim 1, characterized in that: The phage and the carrier formed by the degradation of the composite carrier matrix in step (5) constitute the phage complex. The porous structure in step (2) combined with the interlayer pores formed by the stepwise solidification process in step (3) provides a channel for the penetration of the phage complex.

5. The method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery according to claim 1, characterized in that: In step (3), the coating thickness of each layer of the layered delivery membrane is 0.05~0.15 mm, and the phage loading is 10. 8 ~10 9 PFU / cm 2 .

6. The method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery according to claim 5, characterized in that: The layered delivery membrane described in step (3) is based on the eggshell of the seed egg. The first layer of composite carrier matrix is ​​used as the inner layer to load a phage strain adapted to 18-day embryos. After initial curing, the second layer of composite carrier matrix is ​​coated as the middle layer and loaded with a phage strain adapted to 14-day embryos. After further curing, the third layer of composite carrier matrix is ​​coated as the outer layer and loaded with a phage strain adapted to 7-day embryos.

7. The method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery according to claim 6, characterized in that: The curing environment described in step (3) is a temperature of 20~25℃, a relative humidity of 40%~60%, and a curing time of 4~8 h.

8. The method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery according to claim 6, characterized in that: The incubation conditions described in step (4) are a temperature of 37.5~38.5℃, a relative humidity of 50~65%, and a ventilation rate of 0.3~0.8 m³ / s. 3 / h, turn the eggs once every 2~4 hours.

9. The method for full-cycle prevention and control of chicken embryo incubation based on phage stratified delivery according to claim 8, characterized in that: Under the incubation conditions, the outer layer of the layered delivery membrane degrades within 1 to 7 days, the middle layer degrades within 8 to 14 days, and the inner layer degrades within 15 to 21 days. The phages released from each layer correspond to the suitable 7-day, 14-day, and 18-day embryonic ages, respectively.

10. A layered delivery membrane for whole-cycle prevention and control during chicken embryo incubation, characterized in that: The layered delivery membrane comprises a multilayer structure formed of a biodegradable polymer material, with each layer loaded with a phage strain adapted to different embryonic stages, and the multilayer structure degrades sequentially in the incubation environment according to a preset time sequence.

Images