Human Practice

Abstract

project can be found here. order to ensure that our project is relevant and applicable in the real world, we have been talking to many experts from academia and the clinic as well as people with personal experiences. In addition to that we've been doing background research through literature to understand the extent of the problem and its impact on the world.

Introduction

According to the "China Antibiotic Use in Livestock and Poultry Farming Market Research and Development Prospect Forecast Report" published by the China Report Office [1], domestic antibiotic consumption is estimated to be around 180,000 tons, with the livestock and feed sector accounting for up to 97,000 tons, or roughly 54%, or more than half of the total. This finding suggests that the primary medications used to treat animals are still antibiotics. Additionally, we think that if antibiotics are given to food animals, they will remain in the meat products and people may develop antibiotic resistance as a result of consuming them, posing a significant risk to human health. Since 2021, the state has started to advocate for non-antibiotic and low antimicrobial farming. Therefore, we also wish to find a solution to this issue and implement low-antibiotic and no-antibiotic farming.
Meanwhile, our research reveals that swine fever, chicken fever, etc., pose the greatest risks to the farming process and result in significant financial losses for farmers and the farming industry. Furthermore, according to surveys, approximately 25% to 55% of China's swine herds have died and been culled from African swine fever since the outbreak was first reported [2][3], economic losses account for about 0.78% of China's GDP in 2019 [4]. Analyzing the available data, African swine fever has resulted in direct production losses, loss of replacement breeding herds, and loss of income in China and neighboring countries in the range of approximately $55 billion to $130 billion [5]. Thus, we set out to find a possible solution. Oseltamivir, an antiviral medication, has gained in popularity as a result of the COVID-19 incidents in recent years. As its synthetic precursor, shikimic acid has caught our attention. Shikimic acid, a natural plant extract, is used as an intermediary in antiviral and anticancer medications and has anti-inflammatory and analgesic effects. It can boost immune systems in poultry, pigs, and is used in animal/pet healthcare to prevent and treat viral infections [6]. Informational searches reveal that shikimic acid has been sold as an Illicium verum Hook. f. extract for use in swine and poultry [7]. This also proves that shikimic acid can be given to pigs or poultry and that it is safe. Our initial strategy was to employ synthetic biology to produce shikimic acid as a therapeutic medicine to assist farmers in treating swine fever, poultry fever, and other illnesses in order to achieve the goal of substitution and reduction of resistance.

1 Understanding the current state of the feed additives market

• Mr. Zhao
• GUANGZHOU LEADER BIO-TECHNOLOGY CO., LTD.

Research conducted early in the study revealed that shikimic acid is used as an intermediary in antiviral and anticancer medications and has anti-inflammatory and analgesic effects. As a result, we thought it was a potential candidate to include in feed additives as a medication. Through our conversation with Mr. Zhao, he reminded us of the strict national regulations regarding GMO products and feed additives, allowing us to consider more carefully whether our products are allowed to be added into feed.
We sought out further information and spoke with specialists to address the issues. In the end, we were able to fully confirm that our products are subject to a step-by-step operational review and are permitted to be added. Details can be found in the full article. Our reasoning is supported by the following regulations:

a. Announcement No. 307 of the Ministry of Agriculture and Rural Development of the People's Republic of China
b.  Announcement of the Ministry of Agriculture of the People's Republic of China No. 2045"[9].

2 Knowing the real need for the animal husbandry

• Mr Chen, Wu Xuening Pig Farm, Sanhe Town, Taishan City
• Research field: department of animal husbandry

We requested a visit to Mr. Chen's pig farm, to understand the current practical requirements for disease control in livestock and poultry farming. Due to the regulations on food safety and disease control in farming, he declined our on-site visit but agreed to our online interview. We learned through our conversations with him that the state has severe regulations on pigs and poultry. Pigs and poultry can only be put down once they are unwell due to the danger of zoonotic diseases. Through this conversation, we changed the project's objective from therapy to prevention. It was also learned from interviews that the intestinal health is important for pig farming. Thus, anti-bacterial and anti-inflammatory properties of shikimic acid might be benefit for pig health. At the same time, it is obvious that the present pig farming industry also requires affordable and efficient preventive products.

a. To stop the disease from spreading, the primary therapy for contagious animal diseases is culling, but farms may suffer significant financial losses as a result. From this, we realized that there was little possibility to turn the product into a therapeutic drug.
b. They typically immunize their pigs and closely regulate the surroundings and access of humans in their farms to limit the possibility that pigs will become ill to prevent financial losses. Our request to visit the site was thus denied. These actions do not, however, deal with the underlying cause of pig disease. Farmers have therefore been exploring solutions to the issue.
c. There are several flaws in current traditional Chinese medicine feed additives, including adding too much and taking too long to work will result in too high an economic investment; adding synthetic peptides has obvious effects, but the price is on the high side; adding probiotics needs to be in a humid environment to work better, and it is complicated to operate in the process of raising animals. There are no satisfactory products in the market at present. As a result, it is currently of greater urgency to develop efficient and reasonably priced products.
d. Mr. Chen made the argument that the pig intestine serves an important immunological function in addition to being a digesting organ. We can only assure the orderly and sustainable development of the pig farming business by ensuring the healthy development of the intestinal system. As a result, a product that can sterilize and have anti-inflammatory effects on the pig digestive tract is required. Thankfully, shikimic acid possesses anti-inflammatory and antibacterial effects.

We proceed with a pertinent document search in relation to the techniques of preventing and controlling African swine flu.

To further strengthen the prevention and control of African swine fever and comprehensively improve the capacity to prevent and control animal diseases, State Office Development [2019] No. 31[10] proposed to strengthen the prevention and control of African swine fever by strengthening the regionalization and entry/exit management, strengthening the prevention of epidemic prevention in pig farms. The aforementioned documents provide evidence that pig farms are quite tight and thorough about maintaining a sterile environment and managing people during the breeding season.

3 Identify the possiblity for shikimic acid as a preventative medicine and application object

• Mr Zhang, Doctor of Veterinary Surgeon
• Research field: Former Lecturer, College of Animal Husbandry and Veterinary Medicine, Shenyang Agricultural University

Our original plan was to employ shikimic acid to treat illnesses that affect livestock, poultry, and pets with the flu virus. Therefore, to learn more about the potential for shikimic acid as a medication, we spoke with Mr. Zhang, a veterinarian. We changed our application goals to focus on livestock and poultry control after speaking with Mr. Zhang, and we identified that the state permits the addition of shikimic acid as a natural plant ingredient.These are the main ideas:

a. China has severe regulations regulating the use of pharmaceuticals in food animals due to zoonotic concerns. As a natural plant ingredient, shikimic acid can be used in pigs or poultry.
b. He thinks there is little chance for influenza to infect pets. The pet market, unlike livestock and poultry farming, has more mature and convenient prevention and control strategies, such as symptomatic care and vaccination, so the demand for such drugs is relatively low.
c. Livestock and poultry require a product to boost resistance and decrease illness risk more than domestic pets do, according to the market and demand.

And we further search the related regulations and ensured the possibility to use shikimic acid as the feed additives.

i.According to the Announcement No. 307 of the Ministry of Agriculture and Rural Development of the People's Republic of China [8]: Farmers must adhere to Ministry regulations on restricted feed ingredients and additives, using only those listed in the Catalogue of Feed Ingredients and Catalogue of Varieties, with exceptions for local plant raw materials  (excluding medicinal plants) and agricultural by-products. This statute clarifies that shikimic acid can be used to add.
ii. According to the Ministry of Agriculture and Rural Affairs' document Agricultural and Animal Husbandry Development [2021] No. 37[11], which can be found at the development of the Chinese veterinary medicine industry should be accelerated, and additives for Chinese veterinary medicines should be strengthened in terms of research and development. Shikimic acid is a monomer chemical that is derived from the Chinese herb anise, which is a widely used medication. It is undeniable that the state has fostered the production of this chemical.

To know the prevention additives used in poultry feed, we interview the Poultry Disease Research Specialist, Prof. Ye Weicheng.

We spoke with Prof. Ye, a specialist in the study of poultry diseases, to better grasp the situation as well as the practical need for disease prevention in poultry farming. This interview led us to adopt this as the objective of our next stage of the assignment. Poultry usually has the shorter lifespan, thus it is higher desired for effeciency and quickly control. Details are as follows:

a. To prevent drug resistance, all antibiotics used for humans are strictly prohibited for the prevention and treatment of poultry diseases.
b. It is advised to take into account the product's price and the product's antiviral effects when adding Chinese medicine sources to the meal.
c. Vaccination is one of the most used methods for poultry disease prevention, different species employ it in various ways.
d. The usage of the product necessitates a high level of effectiveness due to the short (three months to a year) breeding period for chickens.

4 Safety consideration and addition way of shikimic acid

According to his suggestion and further checking the regulations for GMOs feed, we finally decided the premixed compound feed as our final product form.

a. He advocated for the need to add feed additives during the production of feed additives rather than during the farming of pigs or poultry. Farmers cannot produce shikimic acid on their own and that it must be added to the production process as a disease-preventive drug.
b. Shikimic acid is a naturally occurring compound that can be used as a feed additive, and according to Chinese law, genetically modified (GM) products can be added to feed.
c. He agreed with that the microbial alternative fermentation to produce shikimic acid is a better way compared with plant extraction. But it is necessary to focus on biosafety and market access regulations.

5 Suggestions of the project's experimental design

• Dr. Feng
• Research field: bioinformatics

According to a study of the literature, due to its rapid growth rate and well-defined metabolic pathway, E. coli makes an excellent host for the synthesis of shikimic acid. Therefore, to manufacture shikimic acid, we would like to use an E. coli strain. We consulted Dr. Feng to gain his assistance in determining the final metabolic network system to better define the experimental design ideas and which genes need to be modified. In addition, he also mentioned that the introduction of glk and glf genes can also increase yields. Please see the details in our model page.

a. In accordance with the metabolic pathways and model predictions, (1) Increase PEP production: (i) blocking the phosphate transport pathway (PTS system) by knocking out the ptsG gene, preventing PEP from being converted to pyruvic acid. (ii) blocking PEP branching pathways, such as the lactic, acetic, and ethanol synthesis pathways (knocking out the poxB gene). (2) Increase E4P content: tktA and talB gene overexpression. (3) Constructing and enhancing the pathway for the synthesis of shikimic acid:(i) To increase the metabolic flow of shikimic acid production from PEP and E4P, aroG, aroB, aroD, and aroE genes are overexpressed. (ii) aroK and aroL genes are knocked down to prevent the shikimic acid catabolic pathway.
b. Additionally, he suggested that it has been demonstrated that ptsG knockdown results in a partial loss of the pts system's functionality in E. coli and that the glf and glk genes can be added to recombine E. coli and increase production. So, we looked through the literature to find information on glf-glk.

6 Access to the market and production of genetically modified goods must be governed

• National regulations and announcements

We learned about the operational procedures for adding genetically modified products to feeds and the proportion of ingredients to be added to premixed feeds from the relevant regulations. According to our project, the product is finalized as premixed compound feed, which needs to pass the safety evaluation by the relevant provisions of the "Regulations on the Safety Management of Agricultural Genetically Modified Organisms", and obtain the certificate of safety of agricultural genetically modified organisms, and then be evaluated by the relevant provisions of the "Measures for the Administration of New Feeds and New Feed Additives", and finally obtain the license for the production of feeds and feed additives, which will permit the production of the product.

a. the Announcement of the Ministry of Agriculture of the People's Republic of China No. 2045"[9], Article 4 suggests that: The Ministry of Agriculture of the People's Republic of China has announced that the production of feed additives derived from genetically modified organisms must comply with the Regulations on the Safety Management of Agricultural Genetically Modified Organisms and undergo safety evaluation under the "Measures for the Administration of New Types of Feed and New Types of Feed Additives".
b. Article 23 of the revised document "Measures for the Administration of the Production License of Feed and Feed Additives" of the Ministry of Agriculture and Rural Affairs Decree No. 1 [12] dated January 7, 2022: The Ministry of Agriculture and Rural Affairs' Decree No. 1 defines complementary pre-mixed feed as a combination of minerals, trace elements, vitamins, amino acids, and other feed additives, carriers, and diluents in a certain proportion. The amount added to compounded feed, concentrate supplements, or drinking water for animals should not exceed 0.1% and 10%.

7 Proposed implementations of our product in future

For pigs and poultry, 300-400g of the star anise-derived shikimic acid can be added to 1 ton of feed for three to five days, according to information given by Jinan Qimautang Bio-technology Co., Ltd.
Before we can suggest additional decisive programs, such as the validation of effectiveness, addition amount, and addition period, we still need to carefully validate and gather more experimental data. This goes for both the HP study findings and any future ones. Thus, this has become the main goal of the next phase of our project.

Summary

Based on the findings of the aforementioned research, we have a comprehensive grasp of the present poultry/pig industry. According to the experts, we have gradually narrowed our goal to animal farming, such as pigs. We gained clarity on shikimic acid's potential as a preventative medication and its safety, enabling us to confidently create our goods. Additionally, we looked for solutions to the problem of how the product should be made and concluded that the finished item would be a premixed compound feed. Additionally, we carefully examined the national legislation and defined the product's manufacturing procedure, which calls for several steps before it can be approved for production. We intend to keep researching the impact of the product and additional problems to eventually commercialize our idea.