Application of ultra-micro powder of melia toosendan leaf in preparation of product for preventing rimer bacillus disease of duck

Abstract

The application relates to the field of poultry medicines and preparation of the poultry medicines, and particularly discloses application of super-micro powder of leaves of medical plant Melia toosendri in preparation of products for preventing duck Riemerella anatipestifer disease. The super-micro powder of the leaves of the medical plant Melia toosendri has the effects of clearing heat and resolving toxins, resisting bacteria and eliminating inflammation, and the like; the traditional Chinese medicine super-micro powder is simple to make, low in cost, high in safety, free of side effects, can reduce or replace the use of antibiotics, and thus can reduce drug resistance and drug residue problems; use of the super-micro powder of the leaves of the medical plant Melia toosendri can obviously prevent the duck Riemerella anatipestifer disease of meat ducks.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of poultry medicine and preparation of poultry medicine, and in particular to the application of super-micro powder of leaves of Melia toosendan in preparation of products for preventing duck Riemerella anatipestifer disease. BACKGROUND

[0002] Duck Riemerella anatipestifer disease is a chronic or acute septic infectious disease mainly attacking ducklings caused by Riemerella anatipestifer, which is susceptible to 1-8 week-old ducklings, and can infect chickens, geese and other poultry. There are many serotypes of Riemerella anatipestifer strains, and there is lack of cross immunity among different serotypes. In order to reduce the use of antibiotics and reduce bacterial drug resistance, it is necessary to start from the development of efficient, safe and inexpensive antibacterial drugs. The leaves of Melia toosendan have the effects of anti-inflammatory, anti-oxidation and immune enhancement, and become a potential choice. However, the Chinese herbal medicine faces problems such as high extraction cost and low oral absorption rate in clinical application. In order to solve this problem, high-quality leaves of Melia toosendan are selected in the present application, and the leaves are processed into super-micro powder by modern processing technology, so as to reduce the production cost and improve the absorption and utilization effect of the drug. SUMMARY

[0003] The technical problem to be solved by the present application is to provide the application of super-micro powder of leaves of Melia toosendan in preparation of products for preventing duck Riemerella anatipestifer disease, aiming at the deficiencies of the prior art.

[0004] In order to solve the above technical problem, the present application discloses the following technical scheme:

[0005] In a first aspect, the present application discloses an excellent traditional Chinese medicine for preventing and / or treating duck Riemerella anatipestifer disease and resisting Riemerella anatipestifer infection: leaves of Melia toosendan.

[0006] The Chinese herbal medicine is a plant, belonging to Magnoliophyta, Dicotyledoneae, Sapindales, Meliaceae, Melia, and Melia toosendan.

[0007] The pharmacological activity of the leaves of Melia toosendan is as follows: nature and taste and meridian: bitter and cold, and belongs to lung and liver meridians.

[0008] Indications: clearing heat and resolving toxin, benefiting the throat and reducing swelling, promoting blood circulation and relieving pain, and resisting bacteria and inflammation. It is used for treating laryngalgia, sore throat, sore and toxic sore, and sprain and pain.

[0009] In a second aspect, the present application discloses super-micro powder of leaves of Melia toosendan and a preparation method thereof.

[0010] The particle size of the super-micro powder of leaves of Melia toosendan is less than or equal to 75 μm.

[0011] The preparation method of the super-micro powder of leaves of Melia toosendan comprises the following steps:

[0012] (1) Pretreatment of medicinal materials: Purchase the leaves of Rhododendron molle, clean them, drain the water, and use a low-temperature slow drying method to prevent the destruction of active ingredients. Initial drying: 45-50℃ for 24 hours; main drying: 56-60℃ for 40-48 hours; final drying: 50℃ for 12 hours; final moisture content ≤8%, leaves are easy to break, and there is no scorching.

[0013] (2) Pulverizing medicinal materials: The dried leaves of the mountain sage were initially pulverized using a small leaf shredder, then pulverized for 2 minutes each time using an industrial universal pulverizer, for a total of 3 times; and then ultra-fine pulverization was carried out using a 200-500 mesh airflow ultra-fine powder pulverizer.

[0014] (3) Packaging of medicinal materials: Weigh 1 kg of ultrafine powder and package it using a vacuum packaging machine.

[0015] Thirdly, the present invention discloses the application of the *Syzygium aromaticum* leaf described in the first aspect and the ultrafine powder of *Syzygium aromaticum* leaf described in the second aspect.

[0016] The application is in the preparation of products for the prevention and / or treatment of duck plague Riemerella infection.

[0017] The aforementioned Riemerella anatipestifer infection is caused by Riemerella anatipestifer.

[0018] The prevention and / or treatment of duck riberi infection includes any of the following:

[0019] i. To alleviate any one or a combination of serositis, liver swelling, and effusion in ducks caused by Riemerella anatipestifer infection; said serositis includes fibrinous pericarditis and / or fibrinous perihepatitis;

[0020] ii. Reduce mortality caused by duck riberi infection;

[0021] iii. Reduce any one or a combination of white diarrhea, depression and neurological symptoms caused by duck plague Riemerella infection;

[0022] iv. Reduce weight loss caused by duck riberi infection;

[0023] v. Reduces any one or a combination of bacterial loads in the heart, liver, and brain tissue caused by duck plague Riemerella infection.

[0024] When the application is for prevention, the feed is made by directly feeding the feed with ultrafine powder of Rhododendron simsii leaves at a dose of 2-2.5 g / kg body weight, preferably 2.3 g / kg body weight, which can be taken in divided doses or once; or the feed can be made by adding 1-10 wt% of Rhododendron simsii leaves ultrafine powder to the feed.

[0025] The application can also be in the preparation of products for treating duck plague Riemerella infection.

[0026] The anti-duck Riemerella infection includes any of the following:

[0027] 1) Relieves any one or a combination of serositis, liver swelling, and effusion in ducks caused by Riemerella anatipestifer infection; said serositis includes fibrinous pericarditis and / or fibrinous perihepatitis;

[0028] 2) Reduce mortality caused by Riemerella anatipestifer infection;

[0029] 3) Reduce any one or a combination of white diarrhea, depression, and neurological symptoms caused by Riemerella anatipestifer infection;

[0030] 4) Reduce weight loss caused by Riemerella anatipestifer infection;

[0031] 5) Reduces any one or a combination of bacterial loads in the heart, liver, and brain tissue caused by Riemerella anatipestifer infection.

[0032] Fourthly, the present invention discloses a product comprising the *Syzygium aromaticum* leaf as described in the first aspect or the *Syzygium aromaticum* leaf ultrafine powder as described in the second aspect.

[0033] The product in question is for the prevention and / or treatment of duck riberi infection.

[0034] The aforementioned Riemerella anatipestifer infection is caused by Riemerella anatipestifer.

[0035] The prevention and / or treatment of duck riberi infection includes any of the following:

[0036] i. To alleviate any one or a combination of serositis, liver swelling, and effusion in ducks caused by Riemerella anatipestifer infection; said serositis includes fibrinous pericarditis and / or fibrinous perihepatitis;

[0037] ii. Reduce mortality caused by duck riberi infection;

[0038] iii. Reduce any one or a combination of white diarrhea, depression and neurological symptoms caused by duck plague Riemerella infection;

[0039] iv. Reduce weight loss caused by duck riberi infection;

[0040] v. Reduces any one or a combination of bacterial loads in the heart, liver, and brain tissue caused by duck plague Riemerella infection.

[0041] The product may also be a product for treating duck plague Riemerella infection.

[0042] The anti-duck Riemerella infection includes any of the following:

[0043] 1) Relieves any one or a combination of serositis, liver swelling, and effusion in ducks caused by Riemerella anatipestifer infection; said serositis includes fibrinous pericarditis and / or fibrinous perihepatitis;

[0044] 2) Reduce mortality caused by Riemerella anatipestifer infection;

[0045] 3) Reduce any one or a combination of white diarrhea, depression, and neurological symptoms caused by Riemerella anatipestifer infection;

[0046] 4) Reduce weight loss caused by Riemerella anatipestifer infection;

[0047] 5) Reduces any one or a combination of bacterial loads in the heart, liver, and brain tissue caused by Riemerella anatipestifer infection.

[0048] The product is in the form of an oral medication, including oral liquid, granules, powder, pills, tablets or capsules.

[0049] The *Syzygium aromaticum* leaf or *Syzygium aromaticum* leaf ultrafine powder provided by this invention is an excellent traditional Chinese veterinary medicine that can be used to replace or reduce the use of antibiotics in the prevention of duck plague Riemerella vaginalis infection. The *Syzygium aromaticum* leaf ultrafine powder provided by this invention can also improve the absorption and efficacy of traditional Chinese medicine.

[0050] The products described in this invention include feed, feed additives, or veterinary drugs; for example, the *Syzygium aromaticum* leaf or *Syzygium aromaticum* leaf ultrafine powder can be added to feed at a dosage of 1-10 wt%, such as 2 wt%, 3 wt%, 4 wt%, 5 wt%, or 8 wt%, thereby reducing or replacing antibiotics and significantly preventing infection with *Riemerella anatipestifer*.

[0051] The *Syzygium aromaticum* leaf or *Syzygium aromaticum* leaf ultrafine powder described in this invention can be administered to ducklings aged 4 to 18 days.

[0052] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0053] (1) The ultrafine powder of Rhododendron simsii leaves provided by the present invention has the effects of clearing heat and detoxifying, antibacterial and anti-inflammatory, and is not prone to drug resistance.

[0054] (2) The *Syzygium aromaticum* leaf ultrafine powder provided by this invention is safe, has significant therapeutic effects, and is easy to use. It is a traditional Chinese medicine ultrafine powder used to prevent duck plague Riemerella vaginalis infection.

[0055] (3) The ultrafine powder of Rhododendron simsii leaves provided by the present invention has significant preventive effects, does not require injection or oral antibiotics, and can reduce or replace antibiotics in clinical disease prevention.

[0056] (4) The mountain jasmine round leaf provided by the present invention is an ultrafine powder with a high cell wall breakage rate, which improves the dissolution rate and bioavailability of the effective ingredients.

[0057] (5) Ultrafine powder can be directly absorbed through the intestinal mucosa, increasing bioavailability, reducing damage to medicinal materials, lowering medication costs, and facilitating storage and transportation. Attached Figure Description

[0058] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0059] Figure 1 This is a anatomical image of the challenge group in Example 1.

[0060] Figure 2 This is a anatomical image of the *Syzygium stenoptera* round-leaf prevention group in Example 1.

[0061] Figure 3 This is a comparison chart of feces from each group in Example 2.

[0062] Figure 4 This is a comparison chart of weight changes in each group in Example 2.

[0063] Figure 5 This is a comparative anatomical image of the liver at 1 dpi in Example 2.

[0064] Figure 6 This is a comparative anatomical diagram of the heart at 1 dpi in Example 2.

[0065] Figure 7 This is a comparative anatomical diagram of each group dissected at 4 dpi in Example 2.

[0066] Figure 8 This is a comparative anatomical diagram of each group dissected at 7 dpi in Example 2.

[0067] Figure 9 This is a comparison chart of bacterial load in target organs in Example 2.

[0068] Figure 10 This is an anatomical diagram illustrating the clinical preventive application of *Syzygium serratum* leaf ultrafine powder in Example 3.

[0069] Figure 11 This is an anatomical diagram of clinical production without preventative treatment in Example 3. Detailed Implementation

[0070] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0071] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0072] The particle size of the *Syzygium aromaticum* leaf ultrafine powder described in the following examples is below 75 μm.

[0073] In this invention, dpi refers to the number of days after the poison attack, such as 1 dpi means 1 day after the poison attack.

[0074] Example 1

[0075] 1. Materials and Methods

[0076] 1.1 Experimental Animals

[0077] From June to July 2024, 60 one-day-old Muscovy ducks were purchased from Nanchang, Jiangxi Province. All ducklings were healthy and tested negative for Riemerella anatipestifer.

[0078] 1.2 Test Drugs

[0079] Mountain fragrant round leaf ultrafine powder.

[0080] 1.3 Test Methods

[0081] Ducklings were randomly divided into two groups: the *Rhizoctonia solani* prevention group (TF+RA) and the *Rhizoctonia solani* challenge group (RA), with 30 ducklings in each group.

[0082] After acclimatizing one-day-old ducks in the *Syzygium aromaticum* leaf prevention group for three days, 1% *Syzygium aromaticum* leaf ultrafine powder was added to their feed starting on the fourth day of age until the end of the experiment. During the experiment, ducks had free access to water and feed. On the eleventh day of age, ducks were challenged with a viral load of 2.15 × 10⁻⁶ bacteria. 10 CFU / mL, after bacterial resuscitation, centrifuge at 3500r / min for 10min, discard the culture medium, add physiological saline and resuspend. Challenge method is intraperitoneal injection of 1mL.

[0083] The group challenged with Riemerella anatipestifer was fed a basal diet until the end of the experiment. During the experiment, they had free access to food and water. They were challenged on day 11 with a bacterial concentration of 2.15 × 10⁻⁶. 10CFU / mL, after bacterial resuscitation, centrifuge at 3500r / min for 10min, discard the culture medium, add physiological saline and resuspend. Challenge method is intraperitoneal injection of 1mL.

[0084] Seven days after the viral load was introduced, the mortality rate was tallied, and autopsies were performed to observe changes in fibrinous inflammation in the heart and liver.

[0085] 2. Test Results

[0086] 2.1 Mortality rate

[0087] As shown in Table 1, 12 birds died in the challenge group, with a mortality rate of 40%; 3 birds died in the prevention group of Rhizoctonia solani, with a mortality rate of 10%. Adding the Rhizoctonia solani ultrafine powder of this invention to the feed can significantly reduce the mortality rate of Rhizoctonia solani infection in ducks.

[0088] Table 1. Mortality rates of ducks infected with Riemerella anatipestifer in the challenge group and the prevention group.

[0089]

[0090] 2.2 Post-mortem Changes

[0091] Eight days after the viral invasion, ten ducks were randomly selected for dissection. Figure 1 and Figure 2 Red circles indicate fibrinous pericarditis, and yellow circles indicate fibrinous perihepatitis. In the challenge group, 9 birds developed fibrinous pericarditis, and 5 developed fibrinous perihepatitis; in the prevention group using *Rhizoctonia solani* leaf ultrafine powder, 3 birds developed fibrinous pericarditis, and 1 developed fibrinous perihepatitis. Adding *Rhizoctonia solani* leaf ultrafine powder to the feed significantly reduced fibrinous pericarditis and fibrinous perihepatitis caused by *Rhizoctonia solani* infection.

[0092] Example 2

[0093] 1. Materials and Methods

[0094] 1.1 Experimental Animals

[0095] From August to September 2024, 200 one-day-old Muscovy ducks were purchased from Nanchang, Jiangxi Province. All ducklings were healthy and tested negative for Riemerella anatipestifer.

[0096] 1.2 Test Drugs

[0097] Mountain fragrant round leaf ultrafine powder.

[0098] 1.3 Test Methods

[0099] Two hundred one-day-old Muscovy ducks were purchased and tested for Riemerella anatipestifer pathogen. After testing negative, they were acclimatized to a basal diet for three days. On the fourth day, they were divided into a basal diet group and a 1 wt% *Syzygium aromaticum* leaf feed supplement group, with 100 ducks in each group. On the tenth day, the basal diet group was divided into a control group (C group, n=50) and a challenge group (RA group, n=50), while the 1 wt% *Syzygium aromaticum* leaf feed supplement group was divided into a control group (C+TF group, n=50) and a prevention group (RA+TF group, n=50).

[0100] The control group (Group C) was fed a basal diet until the end of the experiment. During the experiment, the animals had free access to food and water. On the 11th day of age, 1 mL of physiological saline was injected into their peritoneum.

[0101] The challenge group (RA group) was fed a basal diet until the end of the experiment, with free access to food and water during the experiment. Challenge was performed on day 11 of age, with a challenge concentration of 2.15 × 10⁻⁶ based on previous animal pre-experiments. 10 CFU / mL, after bacterial resuscitation, centrifuge at 3500 r / min for 10 min, discard the culture medium, add physiological saline to resuspend, and inject 1 mL of bacterial solution into the peritoneum.

[0102] The control group of *Syzygium aromaticum* (C+TF group) was fed with 1 wt% *Syzygium aromaticum* ultrafine powder added to the basal diet starting on the fourth day until the end of the experiment. During the experiment, the animals had free access to food and water, and 1 mL of physiological saline was injected into the peritoneum on the 11th day of age.

[0103] The *Syzygium aromaticum* leaf prevention group (RA+TF) was fed with 1 wt% *Syzygium aromaticum* leaf ultrafine powder added to their basal diet starting on day four until the end of the experiment. During the experiment, they had free access to food and water. On day 11, they were challenged with the virus at a concentration of 2.15 × 10⁻⁶, based on previous animal pre-experiments. 10 CFU / mL, after bacterial resuscitation, centrifuge at 3500 r / min for 10 min, discard the culture medium, add physiological saline to resuspend, and inject 1 mL of bacterial solution into the peritoneum.

[0104] Weight data were recorded before challenge (day 10), and defecation was observed 10 hours after challenge. Animals were sacrificed and sampled at 1 dpi, 4 dpi, and 7 dpi. Eight animals were randomly selected for each sampling. Animals were fasted for 12 hours before sacrifice, and their weight data were recorded. The bacterial load of Riemerella anatipestifer, a target organ, was measured in the heart, liver, and brain tissue.

[0105] 2. Test Results

[0106] 2.1 Changes in fecal excretion

[0107] like Figure 3 As shown, white diarrhea refers to white, watery stools. The yellow arrow represents white diarrhea. The changes in defecation 10 hours after the virus challenge are shown in the figure. It can be seen from the figure that the *Syzygium aromaticum* leaf group can significantly alleviate white diarrhea caused by *Rimerella anatipestifer* infection.

[0108] 2.2 Weight Changes

[0109] like Figure 4 As shown, at 4 dpi and 7 dpi, *Syzygium aromaticum* leaf can significantly increase body weight and reduce the impact of *Rimerella anatipestifer* infection on weight loss, and the addition of *Syzygium aromaticum* leaf ultrafine powder feed has no effect on the weight of ducklings.

[0110] 2.3 Anatomical Changes

[0111] 1dpi liver anatomy diagram as shown Figure 5 As shown, compared with group C ( Figure 5 Compared to group A), group C+TF ( Figure 5 In group B), there was no significant change; in group RA ( Figure 5 In group D), the liver was significantly enlarged, and in the RA+TF group ( Figure 5 C) in the formula can alleviate liver swelling caused by RA infection. A cardiac anatomy diagram at 1 dpi is shown below. Figure 6 As shown, compared with group C ( Figure 6 Compared to group A), group C+TF ( Figure 6 In group B), there was no significant change; in group RA ( Figure 6 In group D), significant pericardial effusion was observed in the RA+TF group. Figure 6 In case C), pericardial effusion will not occur.

[0112] 4dpi anatomical diagram as shown Figure 7 As shown, compared with group C ( Figure 7 Compared to group A), group C+TF ( Figure 7 In group B), there was no significant change; in group RA ( Figure 7 In group D), significant fibrinous pericarditis and perihepatitis were observed. In the RA+TF group ( Figure 7 In case C), there was no obvious fibrinous pericarditis or perihepatitis.

[0113] 7dpi anatomical diagram as shown Figure 8 As shown, compared with group C ( Figure 8 Compared to group A), group C+TF ( Figure 8 In group B), there was no significant change; in group RA ( Figure 8 In group D), significant fibrinous pericarditis and perihepatitis were observed. In the RA+TF group ( Figure 8 In case C), there was no obvious fibrinous perihepatitis, but mild fibrinous pericarditis was observed.

[0114] 2.4 Changes in bacterial load on target organs

[0115] like Figure 9 As shown, cardiac bacterial load ( Figure 9In the A group, compared with the RA group, the cardiac bacterial load in the RA+TF group was significantly lower at 1 dpi, and extremely significantly lower at 4 dpi and 7 dpi; the liver bacterial load ( Figure 9 In the B group, compared with the RA group, the bacterial load in the liver of the RA+TF group was significantly lower at 1 dpi and 4 dpi, and extremely significantly lower at 7 dpi; the bacterial load in the brain tissue ( Figure 9 In the C group, compared with the RA group, the bacterial load in the brain tissue of the RA+TF group showed a significant decreasing trend at 1 dpi and 7 dpi, but a decreasing trend at 4 dpi, which was not significant. This indicates that *Rhizoctonia solani* leaf can reduce the bacterial load of *Riemerella anatipestifer* in the target organ tissues of ducklings.

[0116] Example 3

[0117] 1. Materials and Methods

[0118] 1.1 Experimental Animals

[0119] From October to November 2024, a clinical trial was conducted at a duck farm in Nanchang, Jiangxi Province, that tested positive for Riemerella duck plague.

[0120] 1.2 Test Drugs

[0121] Mountain fragrant round leaf ultrafine powder.

[0122] 1.3 Test Methods

[0123] 800 one-day-old ducklings were randomly divided into group A and group B, with 400 ducklings in each group. Group A was not treated in any way (fed according to the normal feeding process of the farm). Group B started to have 1 wt% of *Syzygium aromaticum* leaf ultrafine powder added to their feed from the fourth day of age, and continued to be added for 18 days.

[0124] The initial symptom of Riemerella anatipestifer infection in ducks is the excretion of white, watery feces. As the disease progresses, lethargy, neurological symptoms, mortality, and the number of healthy ducks are recorded. Finally, 15 ducks from each group are randomly selected for dissection to observe lesions. The criteria for determining white, watery feces are the presence of white, diarrheal material around the anus; lethargy is defined as listlessness and huddling together; and neurological symptoms include head and neck tremors or a stargazing posture, and reluctance to move. Healthy ducks are defined as those without any of the above symptoms (i.e., ducks not exhibiting the initial symptoms of the disease, such as white, watery feces).

[0125] 2. Test Results

[0126] As shown in Table 2, in duck farms positive for Riemerella anatipestifer infection, the *Syzygium aromaticum* leaflet prevention group significantly reduced white watery feces, lethargy, neurological symptoms, and duck mortality. Anatomical dissection of Group A is shown below. Figure 11 As shown, the anatomy of group B is as follows: Figure 10As shown, red circles represent fibrinous pericarditis, and yellow circles represent fibrinous perihepatitis. Figure 10 As shown, the ultrafine powder of Rhizoma Cymbidium can significantly alleviate fibrinous pericarditis and perihepatitis caused by Riemerella anatipestifer.

[0127] Table 2. Effects of clinical application of *Syzygium aromaticum* leaf ultrafine powder on duckling infection with Riemerella anatipestifer.

[0128]

[0129] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of Rhizoma Cynanchi Amaranthus leaf in the preparation of products for the prevention and / or treatment of duck plague Riemerella vaginalis infection.

2. Application of Rhizoma Cynanchi Amaranthus leaf in the preparation of products for treating Rhizoma Riemereum infection in ducks.

3. The application according to claim 1 or 2, characterized in that, The *Syzygium aromaticum* leaf is *Syzygium aromaticum* leaf ultrafine powder, and the particle size of the *Syzygium aromaticum* leaf ultrafine powder is less than 75 μm.

4. The application according to claim 2, characterized in that, The anti-Duck Riemerella infection includes any of the following: Relieves any one or a combination of serositis, liver swelling, and pericardial effusion in ducks caused by Riemerella anatipestifer infection; Reduce mortality caused by Riemereum anatipestifer infection in ducks; Reduce any one or a combination of white diarrhea and neurological symptoms caused by Riemerella anatipestifer infection; Reduce weight loss caused by Riemereum anatipestifer infection in ducks; Reduce any one or a combination of bacterial loads in the heart, liver and brain tissue caused by Riemerella anatipestifer infection; The serositis includes fibrinous pericarditis and / or fibrinous perihepatitis.

5. The application according to claim 4, characterized in that, The neurological symptoms mentioned include depression.

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