Effect of berberine alkaloid in prevention and / or treatment of bowel diseases

Abstract

The present invention relates to berberine alkaloids, formulations thereof and their use in the prevention and / or treatment of infectious diseases in animals, in particular diseases caused by Clostridium perfringens and Eimeria. The invention also relates to improving feed conversion in food producing animals by administering a berberine composition.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of January 2, 2018, application number 201880032195.4, and invention name “Role of berberine alkaloids in the prevention and / or treatment of intestinal diseases”. Field of the Invention

[0002] The present invention relates to berberine alkaloids, their preparations and their use in preventing and / or treating infectious diseases in animals. In particular, the present invention relates to berberine alkaloids, their preparations and their use in preventing and / or treating infectious diseases including bacterial, viral, parasitic or fungal infections in food-producing animals. Background Art

[0003] Antibiotic use has been a major problem in global animal production for decades. An estimated 63,000 tonnes of antibiotics are used worldwide each year to feed cattle, chickens and pigs, roughly twice as much as doctors prescribe globally to fight infections in humans, and current trends suggest that worldwide consumption of antibiotics for animals will grow by two-thirds over the next 20 years.

[0004] Antibiotics have been supplemented into animal and poultry feeds not only to treat and control infections, but also as growth promoters at low doses and are thought to improve product quality, resulting in lower fat percentages and higher protein content in meat. According to the National Office of Animal Health (NOAH, 2001), they are used to “help growing animals digest their food more efficiently, get the most from it, and grow strong and healthy individuals,” thus providing an economic advantage to farmers. Therefore, it is important to increase and develop the armamentarium of agents that have the potential to act as antibiotics to combat infectious diseases and are cost-effective.

[0005] Antimicrobial resistance (AMR) is a natural process in which microorganisms evolve to be able to resist the effects of drugs, rendering them ineffective. Over time, this causes antibiotics to become less effective and, in extreme cases, ultimately useless. AMR is becoming an increasing problem because the rate of discovery of new antibiotics has greatly slowed, and therefore the number of new drugs is very limited. At the same time, the use of antibiotics has increased exponentially, increasing the development of resistance.

[0006] Recently, the use of antibiotics in food-producing animals has come under renewed scrutiny, with growing concerns that their overuse could contribute to the spread of antibiotic resistance genes by promoting the selection of antibiotic-resistant bacteria in animals. In addition, waste generated by animals may contain antibiotic residues, leading to their even wider spread in the environment. These are major issues with intensive livestock farming methods, the problems caused by their use being primarily a problem in developed countries rather than in developing countries.

[0007] Antimicrobial resistance (AMR) threatens effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi. AMR is a growing threat to global public health and requires action from all sectors of government and society. Widespread use and overuse of antibiotics in food-producing animals leads to the emergence of antibiotic-resistant bacteria that contaminate food and then to consumers who can in turn develop antibiotic-resistant infections. Figure 1 Mapping the spread of AMR from food-producing animals to humans.

[0008] The concern is that overuse of antibiotics in food-producing animals leads to the spread of resistant bacteria to humans, which in turn leads to “superbugs,” bacteria resistant to several classes of antibiotics. Superbugs are estimated to already cause more than 320,000 deaths per year in China and the United States, with the death toll expected to exceed 10 million by 2050, and cost the world more than $100 trillion.

[0009] The global burden of infections that are resistant to existing antimicrobial drugs is growing at an alarming rate. Methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae are the leading causes of hospital-acquired infections. Klebsiella pneumoniae is a common enterobacterium that has even become resistant to last-resort treatments by beta-lactam carbapenem antibiotics in some countries. In many parts of the world, treatment of urinary tract infections caused by Escherichia coli is now ineffective due to resistance to fluoroquinolone antibiotics.

[0010] The use of beta-lactam antibiotics and fluoroquinolones can lead to secondary infections and further complications, such as overgrowth of Clostridium difficile (CD). CD is a bacterium that can cause symptoms ranging from diarrhea to life-threatening inflammation of the colon. CD disease often affects older adults in long-term care facilities, often after treatment with antibiotic medications. However, studies have shown that CD infection rates are rising in populations not traditionally considered to be at high risk, such as young and healthy individuals with no history of antibiotic use or exposure to health care facilities. In the United States, about half a million people become ill each year due to the release of CD toxins, and in recent years, CD infections have become more frequent, severe, and difficult to treat with the rise of antimicrobial resistance. Ironically, the standard treatment for CD is other antibiotics: metronidazole for mild to moderate infections; vancomycin for more severe infections. However, up to 20% of people with CD will become ill again. After recurring two or more times, the rate of further recurrences increases to 65%.

[0011] Patients with infections caused by resistant bacteria are at risk of worse clinical outcomes and death, and consume more health care resources than patients infected with non-resistant strains of the same bacteria. Antimicrobial resistance is a complex issue that affects society as a whole and is driven by many interrelated factors. Single, isolated interventions have limited impact. Concerted action is needed to minimize the emergence and spread of antimicrobial resistance. It is important to develop new antimicrobial medicines as alternatives to address the global resistance problem facing human and animal health.

[0012] Major government regulators, including the European Union, FDA, and the Australian Department of Agriculture and Health, are now implementing strict new directives and legislation to control the use of antibiotics in food-producing animals to reduce antagonistic selection. Major companies in the food industry, such as McDonald's and Walmart, are launching their own initiatives to reduce the use of antibiotics in food.

[0013] Eliminating or banning the use of antibiotics in animals would and has led to many consequences. The National Institute of Animal Health estimates that without the use of growth-promoting antibiotics, the United States would need an additional 452 million chickens, 23 million cattle, and 12 million pigs to reach production levels achieved with current practices, resulting in a greater economic burden on agriculture.

[0014] More worryingly, the reduction or cessation of antibiotic use and changes in animal husbandry practices have caused certain animal diseases to become more common and prevalent. For example, necrotic enteritis in poultry. This has been reported in European countries (such as France and Scandinavia), where the ban on the use of antibiotic growth promoters was accompanied by a sharp increase in the incidence of necrotic enteritis, indicating that antibiotic growth promoters have a preventive role in controlling the disease. As more and more countries implement policies to reduce the use of antibiotics, the current cost of necrotic enteritis in the international poultry industry is estimated to be approximately US$ 2 billion per year and is expected to increase further. Other diseases cause significant losses to the poultry industry, such as coccidiosis and spotted liver disease, which have become the main causes of death in laying hens and reduce egg production.

[0015] The reduction or cessation of antibiotic use and changes in animal husbandry practices have also affected the swine industry, where diseases have become more prevalent and prevalent. Diarrhea associated with enterotoxigenic E. coli and outbreaks of swine dysentery associated with Brachyspira are responsible for high mortality and morbidity in pigs. Also detrimental to the swine industry is a group of conditions called ileitis that are associated with Lawsonia intracellulari and affect the small intestine. This group of conditions includes porcine intestinal adenosis, necrotic enteritis, regional ileitis, and proliferative hemorrhagic enteropathy.

[0016] Salmonellosis is one of the most common and widespread food poisonings and is caused by the bacterium Salmonella. It is estimated that tens of millions of human cases occur each year worldwide and the disease causes hundreds of thousands of deaths. Antimicrobial resistance of Salmonella serotypes has been a global problem. Surveillance data show that the overall antimicrobial resistance of Salmonella has increased significantly from 20% to 30% in the early 1990s to 70% in some countries in the early 21st century. Salmonella lives in the intestines of farmed animals, especially chickens and cattle. It can be found in water, food, or on surfaces contaminated with feces from infected animals or humans ( Figure 2 Describes various aspects of Salmonella infection and food poisoning).

[0017] Campylobacteriosis is a gastrointestinal disease caused by bacteria called Campylobacter (CB) and is a leading cause of foodborne illness. CB is primarily transmitted to humans through eating or drinking contaminated food (mainly poultry), water, or unpasteurized milk. CB can also be spread through contact with infected people or with cats, dogs, and farm animals that carry the bacteria. Figure 3 The epidemiology is shown.

[0018] Most people infected with CB develop diarrhea, cramping, abdominal pain, and fever that last one to two weeks. Symptoms usually appear two to five days after infection. The diarrhea may contain blood or mucus. In rare cases, CB can enter the bloodstream and cause more serious illness. Anyone can get campylobacteriosis, although very young children, the elderly, people with weakened immune systems, and people who work with livestock are at greater risk of infection. Treatment usually involves rehydration, but in severe or complicated cases, antibiotics such as erythromycin may be prescribed to reduce the duration of the illness.

[0019] More specifically, CB contamination of poultry carcasses / meat continues to occur. Methods to control CB contamination have focused on processing plants through washing and evisceration techniques. However, it is believed that if colonisation of the CB in the gut of birds could be controlled prior to slaughter, then contamination of processed birds could be reduced.

[0020] The mandatory reduction or cessation of antibiotic use has led to a "post-antibiotic era", which has led to the need to consider and develop alternatives to treat, control and protect food-producing animals (and humans) from disease. Currently, there is a need for medicines, including medicated feed, that can be used to alleviate the problems associated with the reduction or cessation of antibiotic use and the resulting disease outbreaks. To date, no cost-effective preventive or therapeutic agent has been found to replace antibiotics in animal feed. Summary of the invention

[0021] The present disclosure relates to methods for preventing and / or treating infectious diseases in animals, wherein the methods comprise administering a berberine alkaloid to the animal.

[0022] The present disclosure also relates to an animal feed comprising berberine alkaloids and animal foodstuff, wherein the amount of berberine alkaloids is about 0.001% w / w to 2% w / w of the animal foodstuff.

[0023] The present disclosure also relates to a dosing regimen comprising administering a berberine alkaloid or animal feed disclosed herein, wherein the berberine alkaloid or animal feed is administered for 1 to 6 weeks in an amount effective to prevent and / or treat infectious diseases in animals.

[0024] The present disclosure also relates to a method for reducing feed conversion ratio in a food producing animal, wherein the method comprises the step of administering to the food producing animal a berberine alkaloid disclosed herein or an animal feed.

[0025] The present disclosure also relates to methods for preventing or treating infectious diseases in animals comprising administering the animal feed disclosed herein.

[0026] The present disclosure also relates to methods of preventing or treating infectious enteric disease in animals comprising administering the animal feed disclosed herein.

[0027] The present disclosure also relates to a method for preventing or treating an infectious disease caused by Eimeria in an animal, comprising administering the animal feed disclosed herein.

[0028] The present disclosure also relates to a method for preventing or treating an infectious disease caused by a bacterium of the genus Clostridium in an animal, comprising administering the animal feed disclosed herein, wherein the bacterium is C. perfringen.

[0029] The present invention also relates to the use of berberine alkaloids in the preparation of a medicament for preventing and / or treating:

[0030] infectious diseases in animals;

[0031] infectious enteric diseases in animals;

[0032] Infections in animals caused by Eimeria spp.; or

[0033] An infectious disease caused by bacteria of the genus Clostridium, of which Clostridium perfringens is one.

[0034] The present disclosure also relates to the use of berberine alkaloids in the prevention and / or treatment of:

[0035] infectious diseases in animals;

[0036] infectious enteric diseases in animals;

[0037] Infections in animals caused by Eimeria spp.; or

[0038] An infectious disease caused by bacteria of the genus Clostridium, of which Clostridium perfringens is one.

[0039] The present disclosure also relates to berberine alkaloids for use in the prevention and / or treatment of:

[0040] infectious diseases in animals;

[0041] infectious enteric diseases in animals;

[0042] Infections in animals caused by Eimeria spp.; or

[0043] An infectious disease caused by bacteria of the genus Clostridium, of which Clostridium perfringens is one.

[0044] definition

[0045] As used herein, the term "acceptable excipient" refers to a solid or liquid filler, carrier, diluent or encapsulation material that can be safely used for administration. Depending on the specific route of administration, a variety of carriers well known in the art can be used. These carriers or excipients can be selected from sugars, starches, cellulose and derivatives thereof, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and pyrogen-free water. Excipients are discussed in, for example, Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams and Wilkins, 2005.

[0046] As used herein, the term "acceptable salt" refers to a salt that is toxicologically safe for systemic administration. Acceptable salts, including acceptable acidic / anionic or basic / cationic salts, are described in: PL Gould, International Journal of Pharmaceutics, November 1986, 33 (1-3), 201-217; SM Berge et al., Journal of Pharmaceutical Science, January 1977, 66 (1), 1; P. Heinrich Stahl, Camille G. Wermuth (eds.), Handbook of Pharmaceutical Salts: Properties, Selection and Use, Second Revised Edition, Wiley, 2011. Of course, acceptable salts of the acidic or basic compounds of the present invention can be prepared by conventional methods (e.g., reacting the free acid with the desired salt-forming base or reacting the free base with the desired salt-forming acid).

[0047] The acceptable salt of the acidic compound includes a salt with a cation, and can be selected from an alkali metal or alkaline earth metal salt, including sodium, lithium, potassium, calcium, magnesium, etc., and non-toxic ammonium, quaternary ammonium and amine cations, including but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine, etc., and salts formed with organic bases. Suitable organic bases include N-methyl-D-glucamine, arginine, benzathine penicillin, diethanolamine, ethanolamine, procaine and tromethamine.

[0048] Acceptable salts of basic compounds include salts with anions, and may be selected from organic or inorganic acids. Suitable anions include acetate, acyl sulfate, acyl sulfonate, adipate, ascorbate, benzoate, benzenesulfonate, bromide, camphorsulfonate, decanoate, hexanoate, octanoate, chloride, citrate, docusate, ethanedisulfonate, estolate, formate, fumarate, glucoheptanoate, gluconate, glucuronate, hippurate, hyclate. , hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, methanesulfonate, methyl bromide, methylsulfate, naphthenate, nitrate, octanoate, oleate, pamoate, phosphate, polygalacturonate, salicylate, stearate, succinate, sulfate, sulfonate, sulfosalicylate, tannate, tartrate, terephthalate, toluenesulfonate, triethiodide, etc.

[0049] Berberine is a positively charged quaternary ammonium cation. Acceptable salts of berberine include, but are not limited to, chloride, hemisulfate, and iodide salts.

[0050] As used herein, an "acceptable solvent" is a solvent that may not interfere with the biological activity of the solute for the purposes of the present disclosure. Examples of suitable solvents include, but are not limited to, water, ethanol and acetic acid, glycerol, liquid polyethylene glycols, and mixtures thereof. A particular solvent is water. The term "solvate" refers to a complex of variable stoichiometry formed by a solute (e.g., berberine alkaloid) and a solvent. In particular, the solvent used is an "acceptable solvent" as defined herein. When water is the solvent, the molecule is referred to as a hydrate.

[0051] As used herein, "IRP001" refers to berberine, which is a quaternary ammonium cation and a plant natural product with antimicrobial activity, as described herein. The terms "IRP001" and "berberine" are used interchangeably herein. As used herein, "IRP001 chloride" or "IRP001 Cl" means the chloride salt of berberine; and "IRP001 sulfate" is the hemisulfate salt of berberine. Therefore, it should be understood that the terms "IRP001 sulfate", "berberine sulfate", "IRP001 hemisulfate, and "berberine hemisulfate" are equivalent herein. The molecular structures of the berberine quaternary ammonium cation and the chloride and hemisulfate salts are as shown Figure 4 shown.

[0052] As used herein, the term "berberine alkaloids" refers to berberine and compounds having similar structures and characteristics to berberine and suitable for the compositions / methods / uses of the present invention. Such compounds include, but are not limited to, protoberberine: berberrubine, coreximine, tetrahydroafricanine, jatrorrhizine, 13-hydroxyberberine chloride, coralyne chloride, 7,8-dihydro-13-methylberberine, flavonoids (african alkaloids) and 13-benzylberberine.

[0053] Berberine alkaloids may exist in different isomers or different isomeric forms, such as various tautomers or tautomeric forms. It will be understood that the term "berberine alkaloids (classes)" encompasses different isomeric forms separated from each other as well as combinations.

[0054] Berberine alkaloids may also exist in various amorphous and crystalline forms (ie, polymorphs). It is also to be understood that the term "berberine alkaloid(s)" encompasses different amorphous and crystalline forms isolated from each other and in combination.

[0055] As used herein, the term "berberine alkaloid(s)" includes acceptable salts, solvates, solvates of the salts or prodrugs thereof.

[0056] As used herein, the term "food production animal" refers to an animal that is raised to produce food for consumption by another animal, such as a human. It should be understood that the term "food production animal" includes, for example, chickens or pigs.

[0057] It will be understood that the term "isomer" refers to structural or constitutional isomers, tautomers, regioisomers, geometric isomers or stereoisomers, including enantiomers or diastereomers. In addition, a racemate is understood to be an equimolar mixture comprising a pair of enantiomers.

[0058] It will be understood that the term "prodrug" refers to an inactive form of a compound that is converted to an active form in vivo. Suitable prodrugs include esters, phosphonates, and the like of the active form of the compound. Further discussion of prodrugs can be found in: Stella, VJ et al., "Prodrugs", Drug Delivery Systems, 1985, pp. 112-176, Drugs, 1985, 29, pp. 455-473 and "Design of Prodrugs", ed. H. Bundgard, Elsevier, 1985.

[0059] As used herein, a "safe" residue level of berberine is a level that causes no significant risk of disease, particularly cancer.

[0060] As used herein, the terms "treat", "treatment" and the like refer to the control, cure or improvement of a disease, disorder or condition, or the reduction in the rate of progression of a disease, disorder or condition, or the prevention or inhibition of symptoms or side effects, the reduction in the severity of the development of symptoms or side effects, and / or the reduction in the number or type of symptoms or side effects suffered by an animal subject, compared to not administering a pharmaceutical composition comprising a compound of the invention. The term "treatment" includes use in palliative setting.

[0061] As used herein, the terms "prevention," "prevention" and the like are intended to encompass treatment to delay or slow the development of a disease, disorder or condition, or its symptoms or side effects.

[0062] With respect to "prevention" and "treatment", as used herein, the term "effective amount" refers to an amount that achieves the desired effect when administered to an animal. For example, an effective amount of the composition disclosed herein is an amount that prevents or treats necrotic enteritis in chickens. The exact total effective amount of the antimicrobial depends on the purpose of the treatment and other factors, including the animal subject (e.g., chicken vs. pig), the route of administration, the body weight, and the severity of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Depicting the spread of AMR from food-producing animals to humans. https: / / www.cdc.gov / foodsafety / challenges / from-farm-to-table.html .

[0064] Figure 2 Describes various aspects of Salmonella infection and food poisoning. http: / / thelancet.com / journals / lancet / article / PIIS0140-6736(11)61752-2 / fulltext and https: / / www.epainassist.com / abdominal-pain / stomach / food-poisoning .

[0065] Figure 3 describes the epidemiology of Campylobacter. https: / / wwwnc.cdc.gov / eid / article / 10 / 6 / 04-0403-f1 .

[0066] Figure 4 The molecular structures of berberine quaternary ammonium cation, berberine chloride, and berberine hemisulfate are depicted.

[0067] Figures 5 to 12 Results of the pilot study of necrotic enteritis in chickens described in Example 1 are depicted.

[0068] Figure 5 is a graph of bird mortality before necropsy for each group.

[0069] Figure 6is a graph depicting the median small intestinal lesion score by treatment / challenge group.

[0070] Figure 7 Necrotic enterocolitis lesion scores are depicted.

[0071] Figure 8 Photographs of duodenum of birds from Group 9; NE challenged, IVP / berberine water 1.0 g / L

[0072] Fig. 9 Photographs of the duodenum of birds from Group 6; NE challenged, no berberine treatment

[0073] Fig.10 Photographs of duodenum of birds from Group 12; NE challenged with IVP / berberine diet 2.0 g / kg

[0074] Fig.11 Photographs of duodenum of birds from Group 4; no challenge IVP berberine water 1.0 g / L.

[0075] Fig.12 Photographs of the duodenum of birds from Group 6; NE challenged, no IVP / berberine.

[0076] Fig.13 Depicted are the molecular structures and names of representative compounds referred to in this disclosure.

[0077] Fig.14 The molecular structures and names of other representative compounds of the present invention are depicted:

[0078] Fig.15 Total individual water intake (Phase 1) for the necrotic enteritis pilot study described in Example 2 is depicted.

[0079] Fig.16 Total individual water intake for the necrotic enteritis pilot study described in Example 2 (Phase 2) is described.

[0080] Fig.17 Feed conversion rates from the necrotic enteritis pilot study described in Example 2 (Phase 1 and 2) are depicted.

[0081] Fig.18 Depicted is the housing arrangement with day-old chicks used for the studies described in Example 3.

[0082] Fig.19 Litter collected from the seeder barn on day 14 of the study described in Example 3 is described.

[0083] Fig. 20 Depicted is the 400 grams of litter distributed per barn on day 14 of the study described in Example 3.

[0084] Fig.21 Graph of average daily weight gain of birds in the study described in Example 3 by treatment group: average daily gain (ADG) (g / day) on the y-axis versus growth period (days) on the x-axis. Treatment Group 1 (Control): Treatment Group 2 (IVP 0.30 g / kg); Treatment Group 3 (IVP 0.10 g / kg); Treatment Group 4 (IVP 0.03 g / kg); Treatment Group 5 (Salino 60 ppm); Treatment Group 6 (Salino + Zn Bacitracin 50 ppm (Salino Zn Bac)).

[0085] Fig. 22 A comparison between the IVP used in a control finisher and in Example 3, Treatment Group 2 (IVP at a dose of 0.30 g / kg) is described.

[0086] Fig.23 Depicted are feces of birds from Treatment Group 2 of Example 3 on Day 42 (IVP dosed at 0.30 g / kg).

[0087] Fig.24 E. acervulina type lesions from Example 3 (from outside and inside the duodenum), score +1.

[0088] Fig.25 E. acervulina type lesions from Example 3, scores +2 and +3.

[0089] Fig.26 The E. acervulina type lesions of Example 3 had a score of +4.

[0090] Fig. 27 Ballooning of the intestine from Example 3.

[0091] Fig.28 The upper gut of Example 3 is congested (white arrows; left caption) and intestinal translucency (black arrows; right caption).

[0092] Fig.29 Aqueous intestinal contents from Example 3, including orange mucus.

[0093] Fig.30 Depicted is the correlation between corrected feed conversion ratio at 42 days and total intestinal coccidiosis lesion score at 21 days. The solid line shows the best fit line; the dashed lines show the 95% confidence intervals.

[0094] Specific embodiments of the present disclosure are described below. It will be appreciated that these embodiments are illustrative rather than restrictive. DETAILED DESCRIPTION OF THE INVENTION

[0096] The present disclosure relates to methods of preventing and / or treating infectious diseases in animals, wherein the methods comprise administering a berberine alkaloid or an acceptable salt thereof to the animal.

[0097] In the methods (and animal feeds; dosing regimens and uses) disclosed herein: the animal is preferably a human. The animal is preferably a non-human. Preferably, the non-human animal is a food-producing animal. The food-producing animal is preferably selected from a chicken or a pig. Preferably, the animal is an aquatic animal. The aquatic animal is preferably a finfish. Preferably, the aquatic animal is an aquatic shellfish. The aquatic shellfish is preferably selected from a crustacean or a mollusk. Preferably, the crustacean is selected from a crab, a crayfish, a lobster, a prawn and a shrimp. The mollusk is preferably selected from a clam, a mussel, an oyster, a scallop and a winkle. Preferably, the animal is a mammal. The mammal is preferably a human, a horse, a dog, a cat, a sheep, a cow, a pig or a primate. Preferably, the animal is a poultry. The poultry is preferably a chicken, a goose, a turkey or a duck.

[0098] Spotted liver disease

[0099] Preferably, the infectious disease is a liver disease or an intestinal disease. Preferably, the infectious disease is an intestinal disease. The liver disease is preferably spotted liver disease and the animal is a chicken. Preferably, the chicken is a laying hen. The spotted liver disease is preferably caused by bacteria of the genus Campylobacter. Preferably, the Campylobacter is antibiotic resistant.

[0100] Salmonellosis

[0101] Preferably, the infectious disease is related to food poisoning. Preferably, the food poisoning is salmonellosis. Preferably, salmonellosis is caused by an antibiotic resistant strain of Salmonella.

[0102] Campylobacteriosis

[0103] Preferably, the infectious disease is campylobacteriosis. Campylobacteriosis is preferably caused by an antibiotic-resistant strain of Campylobacter.

[0104] Infectious diseases caused by Escherichia coli: swine diarrhea / scour

[0105] Preferably, the infectious disease is caused by Escherichia coli.

[0106] Of all the diseases of sucking piglets, diarrhoea is the most common and probably the most important. In some outbreaks it is the cause of high morbidity and mortality. In a well run herd less than 3% of the litter should require treatment at any one time and piglet mortality from diarrhoea should be less than 0.5%. However, in severe outbreaks mortality levels may rise to 7% or more and in individual untreated piglets mortality levels may rise to as much as 100%. The main bacterial cause is Escherichia coli. Diarrhea in piglets can occur at any age during the suckling period but there are usually two peaks before 5 days of age and between 7 and 14 days of age.

[0107] Preferably, the infectious disease is diarrhea and the animal is a pig. Preferably, the infectious disease is diarrhea and the animal is a pig. Preferably, the infectious disease is dysentery and the animal is a pig.

[0108] Preferably, the infectious disease is caused by an antibiotic-resistant strain of E. coli.

[0109] Swine dysentery associated with Brachyspira

[0110] Swine dysentery (SD) is caused by a genus of cyclonic bacteria called Brachyspira, which includes Brachyspira hyodysenteriae, Brachyspira piloscoli, and Brachyspira hampsonii. The organism causes severe inflammation of the large intestine with bloody mucous diarrhoea. The high cost of the disease is associated with morbidity, mortality, growth inhibition and feed conversion efficiency as well as ongoing costs of in-feed medication.

[0111] Preferably, the infectious disease is caused by a bacterium of the genus Brachyspira. Preferably, the infectious disease is dysentery and the animal is a pig. Preferably, the infectious disease is caused by an antibiotic-resistant strain of Brachyspira.

[0112] The infectious disease is preferably caused by bacteria of the genus Lawsonia. Preferably, the infectious disease is caused by an antibiotic-resistant bacterial strain of the genus Lawsonia. The infectious disease is preferably caused by Lawsonia intracellularis.

[0113] Lawsonia intracellularis associated with porcine ileitis

[0114] Ileitis comprises a group of conditions that involve pathological changes in the small intestine associated with the bacterium Lawsonia intracellularis. The disease has four different forms. The first form is porcine intestinal adenosis (PIA), which is an abnormal proliferation of cells that line the intestine. PIA can develop into three other rare forms: necrotic enteritis (NE), in which the proliferating cells of the small intestine die and fall off, with a total thickening of the small intestine (hosepipe gut). Regional ileitis (RI), inflammation of the terminal part of the small intestine, and proliferative hemorrhagic enteropathy (PHE) or "bloody gut", in which the small intestine bleeds profusely. PHE is the most common form of ileitis in growing pigs. PHE is more common in pigs over 60 kg and in gilts.

[0115] Preferably, the infectious disease is represented by a group of conditions selected from the group consisting of porcine adenosis, necrotic enteritis, Crohn's disease and proliferative hemorrhagic enteropathy, and the animal is a pig.

[0116] Infectious diseases caused by Eimeria

[0117] Preferably, the infectious disease is caused by a parasite of the genus Eimeria. The parasite is preferably selected from E. maxima, E. acervuline and E. brunette. Preferably, the infectious disease is caused by an antibiotic-resistant parasite of the genus Eimeria. The antibiotic-resistant parasite is preferably selected from E. maxima, E. acervuline and E. brunette antibiotic-resistant bacterial strains. Preferably, the infectious disease is coccidiosis and the animal is a chicken.

[0118] Infectious diseases caused by Clostridium difficile

[0119] Preferably, the infectious disease is caused by a bacterium of the genus Clostridium. The bacterium is preferably selected from the group consisting of: Clostridium difficile and Clostridium perfringen.

[0120] Preferably, the bacterium is Clostridium difficile (C. difficile). Preferably, the infectious disease is diarrhea and the animal is a human. Preferably, the infectious disease is colitis and the animal is a human.

[0121] Clostridium perfringens and necrotic enteritis

[0122] Preferably, the infectious disease is caused by a bacterium of the genus Clostridium, wherein the bacterium is Clostridium perfringens. The infectious disease is caused by an antibiotic-resistant bacterium of the genus Clostridium, wherein the antibiotic-resistant bacterium is antibiotic-resistant Clostridium perfringens.

[0123] The infectious disease is preferably necrotic enteritis and the animal is a chicken. Preferably, the necrotic enteritis is caused by a Clostridium perfringens type A strain. The Clostridium perfringens type A strain is preferably Clostridium perfringens type A strain EHE-NE36. Preferably, the Clostridium perfringens type A strain is Clostridium perfringens type A strain EHE-NE18. Necrotic enteritis is preferably caused by a Clostridium perfringens type C strain.

[0124] Preferably, administration is via the chicken's feed or water. The feed is preferably in the form of crumbles or pellets.

[0125] Preferably, berberine alkaloids are administered in the feed of the chicken at a dose of 0.001 g / kg to 2.0 g / kg feed. Berberine alkaloids are preferably administered in the feed at a dose of 0.003 g / kg to 0.3 g / kg feed. Berberine alkaloids are preferably administered in the water of the chicken at a dose of 0.001 g / L to 1 g / L water.

[0126] Preferably, lesion scores are reduced and / or fecal oocyst counts are reduced. Preferably, lesion scores are reduced. Preferably, fecal oocyst counts are reduced. Preferably, morbidity is reduced. Preferably, mortality is reduced. Preferably, FCR is reduced. Preferably, there is an increase in average daily weight gain.

[0127] Feed safety and residue levels

[0128] For safety reasons, human and animal drugs and animal feed additives are strictly regulated. In Australia, the Therapeutic Goods Administration (TGA) is responsible for regulating therapeutic products for human use, while the Australian Pesticides and Veterinary Medicines Authority (APMVA) is responsible for the evaluation and registration of pesticides and veterinary drugs. In the United States, the Food and Drug Administration (FDA) is responsible for approving human and animal drugs and feed additives regulated by the Federal Food, Drug and Cosmetic Act (FD&C Act).

[0129] The FD&C Act requires that compounds used in food-producing animals be shown to be safe and that food produced by animals exposed to these compounds is safe for human consumption. Specifically, the regulations (21 CFR part 500, subpart E - Regulation of Carcinogenic Compounds for Use in Food-Producing Animals) prohibit the use in food-producing animals of any compound that has been found to induce cancer when ingested by humans or animals unless certain conditions are met (the so-called "DES Proviso"). Under the DES Proviso, the use of a suspected carcinogenic compound is not prohibited if it can be determined by prescribed inspection methods that "no residues" of the suspected carcinogenic compound are found in food produced by food-producing animals under conditions of use that are reasonably certain to be followed in practice.

[0130] Although the safety of berberine alkaloids is demonstrated by, for example, the widespread use of berberine alkaloids as human dietary supplements, berberine is suspected to be a carcinogen, although berberine itself has anticancer activity. (Ma, W.; Zhu, M.; Zhang, D.; Yang, L.; Yang, T.; Li, X.; and Zhang, Y. "Berberine inhibits the proliferation and migration of breast cancer ZR-75-30 cells by targeting Ephrin-B2" Phytomedicine 2017, 25: 45-51). Therefore, if the FDA decides to regulate berberine as a carcinogenic compound, US regulations prohibit the use of berberine in food-producing animals unless the "no residue" DES restrictive clause applies.

[0131] The term "residue-free" means that any residue remaining in the edible tissue is so low that it presents an insignificant risk of cancer to the consumer. More specifically, an insignificant risk of cancer is defined as a 1 in 1 million increased risk.

[0132] As used herein, a "safe" residue level of berberine is a level that poses no significant risk of disease, particularly cancer.

[0133] Preferably, the residual level of berberine alkaloids in the animal after the treatment period is low. Preferably, a safe residual level of berberine alkaloids is present in the animal after the treatment period.

[0134] Preferably, after the treatment period, there is a safe residual level of berberine alkaloids in the muscle tissue of the chicken. The residual level is at least less than about 13 ng berberine alkaloids per gram of muscle tissue.

[0135] Preferably, the residual level is about 10 ng berberine alkaloid per gram of muscle tissue. The residual content is preferably about 5 ng / g.

[0136] Preferably, berberine alkaloids have been administered in the feed of chickens at a rate of about 0.3 g / kg. The residual level of berberine alkaloids in the muscle tissue of chickens is preferably as follows:

[0137] About 6.1ng / g in the muscle tissue of chicken breast;

[0138] About 5.5 ng / g in the muscle tissue of the lower leg of chicken; and

[0139] The muscle tissue of the upper leg of chicken contains about 11.6 ng / g.

[0140] Preferably, berberine alkaloids are administered in the chicken's feed at a dosage of less than about 0.1 g / kg.

[0141] Preferably, berberine alkaloids are administered in the chicken feed at a dosage of about 0.03 g / kg. 35. The residual level of berberine alkaloids in chicken muscle tissue is preferably as follows:

[0142] Less than 2ng / g in chicken breast muscle tissue;

[0143] Less than 2 ng / g in chicken leg muscle tissue; and

[0144] The muscle tissue of chicken thigh is lower than 2ng / g.

[0145] Preferably, there are low residual levels of berberine alkaloids in the muscle tissue of the animal after the treatment period and the washout period. Preferably, there are safe residual levels of berberine alkaloids in the muscle tissue of the animal after the treatment period and the washout period.

[0146] Preferably, after the treatment period and the washout period, a safe residual level of berberine alkaloids is present in the muscle tissue of the chicken.

[0147] Preferably, the washout period is 1-2 weeks. The washout period is preferably selected from the following periods: between 1 day and 14 days; between 1 day and 7 days; between 1 day and 4 days; and between 1 day and 2 days. Preferably, the washout period is a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.

[0148] Preferably, after a washout period of 1 day, the residual levels of berberine alkaloids in the muscle tissue of chickens are as follows:

[0149] Chicken breast muscle tissue contains about 5.7 ng / g;

[0150] About 3.2 ng / g in chicken leg muscle tissue; and

[0151] About 6.0ng / g in chicken thigh muscle tissue.

[0152] Preferably, after a 2-day washout period, the residual levels of berberine alkaloids in the muscle tissue of chickens are as follows:

[0153] About 3.6ng / g in chicken breast muscle tissue;

[0154] About 3.1 ng / g in chicken leg muscle tissue; and

[0155] About 4.5ng / g in chicken thigh muscle tissue.

[0156] Preferably, the residual level of berberine alkaloids in the muscle tissue of chickens after a washout period of 4, 7 and 14 days is less than 2 ng / g.

[0157] Preferably, berberine alkaloids are administered in the chicken's feed at a dosage of about 0.3 g / kg.

[0158] The residual level is preferably at least less than 13 ng berberine alkaloids per gram of muscle tissue. The residual level is preferably about 10 ng berberine alkaloids per gram of muscle tissue. Preferably, the residual level is about 5 ng / g.

[0159] Preferably, berberine alkaloids are administered in the chicken's feed at a dosage of greater than about 0.1 g / kg.

[0160] Preferably, after the treatment period, low residual levels of berberine alkaloids are present in the liver and muscle tissue of the animal. Preferably, after the treatment period, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the animal.

[0161] Preferably, after the treatment period, there are safe residual levels of berberine alkaloids in the liver and muscle tissue of the chicken. The residual level of berberine alkaloids in the liver and muscle tissue of the chicken is preferably less than 2ng / g. Preferably, berberine alkaloids are administered in the feed of the chicken at a dose of about 0.03g / kg.

[0162] Preferably, after the treatment period and the washout period, there are low residual levels of berberine alkaloids in the liver and muscle tissue of the animal. Preferably, after the treatment period and the washout period, there are safe residual levels of berberine alkaloids in the liver and muscle tissue of the animal.

[0163] Preferably, after a treatment period and a washout period, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the chicken. The washout period is preferably a period between 1 week and 2 weeks. Preferably, the washout period is a period selected from the following: between 1 day and 14 days; between 1 day and 7 days; between 1 day and 4 days; and between 1 day and 2 days. The washout period is preferably a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.

[0164] Preferably, after a washout period of 1 day, the residual levels of berberine alkaloids in the muscle tissue of chickens are as follows:

[0165] Chicken breast muscle tissue contains about 5.7 ng / g;

[0166] About 3.2 ng / g in chicken leg muscle tissue; and

[0167] Chicken thigh muscle tissue contains about 6.0 ng / g.

[0168] The berberine alkaloid residue level in liver tissue of chicken was about 8.0 ng / g.

[0169] Preferably, after a 7-day washout period, the residual level of berberine alkaloids in the muscle tissues of the chicken breast, shank and thigh is less than 2 ng / g, and the residual level of berberine alkaloids in the liver tissue of the chicken is about 6.5 ng / g.

[0170] Preferably, after a 14-day washout period, the residual level of berberine alkaloids in the chicken's breast, leg and thigh muscle tissues is less than 2 ng / g, and the residual level of berberine alkaloids in the chicken's liver tissue is about 3.0 ng / g.

[0171] Preferably, berberine alkaloids are administered in the chicken's feed at a dosage of about 0.3 g / kg.

[0172] Preferably, after the treatment period, there are low residual levels of berberine alkaloids in the liver and muscle tissue of the animal. Preferably, after the treatment period, there are safe residual levels of berberine alkaloids in the liver and muscle tissue of the animal.

[0173] Preferably, after the treatment period, there are safe residual levels of berberine alkaloids in the liver and muscle tissue of the chicken. The residual level of berberine alkaloids in the liver tissue and the muscle tissue of the breast, calf and thigh of the chicken is preferably less than 2ng / g. Preferably, berberine alkaloids are administered in the feed of the chicken at a dose of about 0.03g / kg.

[0174] Preferably, after a treatment period and a washout period, a safe residual level of berberine alkaloids is present in the liver tissue of the chicken. The washout period is preferably a period selected from between 1 week and 2 weeks. Preferably, the washout period is a period selected from the following: between 1 day and 14 days; between 1 day and 7 days; between 1 day and 4 days; and between 1 day and 2 days. The washout period is preferably a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.

[0175] Preferably, after a 1-day washout period, the residual level of berberine alkaloids in the liver tissue of the chicken is about 8.0 ng / g. Preferably, after a 7-day washout period, the residual level of berberine alkaloids in the liver tissue of the chicken is about 6.5 ng / g. Preferably, after a 14-day washout period, the residual level of berberine alkaloids in the liver tissue of the chicken is about 3.0 ng / g. Berberine alkaloids are preferably administered in a dose of about 0.3 g / kg in the chicken feed.

[0176] Preferably, the treatment period is 35 days.

[0177] "Residue studies" are described elsewhere. Residue levels of berberine alkaloids can be determined experimentally. An example protocol for determining residue levels of berberine alkaloids in animal tissues using LC-MS / MS is as follows:

[0178] After euthanasia, breast, leg and thigh muscles as well as liver and kidney samples were removed from each bird. A known weight of tissue (approximately 1 g) was homogenized in 2 mL of water. The samples were centrifuged and a known volume of the supernatant was removed for analysis of berberine by LC-MS / MS to provide residual levels of berberine in muscle tissue (ng of berberine per g of muscle tissue).

[0179] Administration of berberine preparations

[0180] Preferably, the berberine alkaloid is berberine hemisulfate. Preferably, the berberine alkaloid is berberine chloride.

[0181] Preferably, the method further comprises an additive that masks the bitter taste of the berberine alkaloid or an acceptable salt thereof.

[0182] Berberine

[0183] Berberine is an isoquinoline alkaloid extracted from Rhizoma coptidis, Phellodendri chinensis cortex and other herbs. According to the Chinese Pharmacopoeia, the berberine contents of Rhizoma coptidis, Phellodendri chinensis, Phellodendron amurense and Berberidis radix are 5.5%, 3.0%, 0.6% and 0.6%, respectively. Rhizoma coptidis (Huanglian) belongs to the Ranunculaceae family and contains three main species of Coptis genus: Coptis chinensis (Weilian), Coptis deltoidea (Yalian) and Coptis teeta (Yunlian). Coptis chinensis is harvested in autumn and sliced ​​after removing the fibrous roots. Those Coptis chinensis with bright yellow parts and a very bitter taste are considered high quality. The bitter taste of berberine (and other berberine alkaloids disclosed herein) makes taste masking / palatability an important issue to consider when formulating berberine alkaloids for administration to animal subjects.

[0184] Berberine is a yellow powder. The chloride salt is slightly soluble in cold water, but readily soluble in boiling water. It is virtually insoluble in cold ethanol. The hemisulfate salt is soluble in about 30 parts of water and slightly soluble in ethanol. Berberine is a quaternary ammonium cation with the molecular formula C 20 H 18 NO4 + , molecular weight is 336.36. Figure 4 Molecular structures of berberine ammonium cation, berberine chloride salt, and berberine hemisulfate salt are depicted.

[0185] Berberine can be administered in any form acceptable for enteral administration. Suitable non-limiting forms for enteral administration include tablets, capsules, pastes, granules, chewable wafers, gels, oral solutions, injections, medicated water and medicated feed, and suppositories. However, for economically important food-producing animals, the preferred method of administration of berberine is through feed additives or medicated feeds in granular form. Berberine can also be administered through the drinking water of animal subjects by mixing water with a suitable solution or suspension of berberine.

[0186] The present disclosure also contemplates providing granules and liquid formulations that can be added to food and water, which make the formulations disclosed herein more palatable to, for example, food-producing animal subjects. For example, a palatable berberine alkaloid formulation may include berberine and an acceptable excipient suitable for forming a granular product. An acceptable excipient suitable for forming a granular product is, for example, corn starch or polyvinyl pyrrolidone (PVP). In an example, the liquid formulation is a liquid concentrate.

[0187] There are also many compounds with similar structures and properties to berberine, including protoberberine: berberrubine, bicuculline, tetrahydroafricanine, jatrorrhizine, 13-hydroxyberberine chloride, methoxyberberine chloride, 7,8-dihydro-13-methylberberine, flavonoids (africanine) and 13-benzylberberine. Protoberberine is suitable for use in the compositions / methods / uses of the present invention together with berberine and is referred to as "berberine alkaloids" in the specification.

[0188] Fructus Africana

[0189] Fibauera or fennel alkaloids are bitter alkaloids extracted from Fibauera recisa Pierre. According to the Chinese Pharmacopoeia, Fibauera recisa Pierre contains not less than 2.0% of fennel alkaloids. Other sources are the rhizomes of Coptis rhizoma, Coptis chinensis Franch, Coptis deltoidea and Coptisteeta Wall. Coptis rhizoma contains not less than 1.5% of fennel alkaloids.

[0190] Tetrandrine chloride is a yellow solid, soluble in hot water, slightly soluble in water, and slightly soluble in ethanol. Its melting point is 196-198°C. Its molecular formula is C 21 H 22 NO4Cl, molecular weight is 387.86. Fig.13 The molecular structure of the tetrandrine quaternium cation and the structure of the chloride salt are listed.

[0191] The total effective amount or dosage of the antimicrobial compound in the prepared feed may be 0.001 g / kg to 2 g / kg. Exemplary amounts of the total amount of antimicrobial compound in the prepared feed are: 0.001 g / kg (0.0001%), 0.003 g / kg (0.0003%), 0.01 g / kg (0.001%), 0.03 g / kg (0.003%), 0.1 g / kg (0.01%), 0.3 g / kg (0.03%), 1.0 g / kg (0.1%) and 2 g / kg (0.2%).

[0192] The present disclosure also relates to an animal feed comprising a berberine alkaloid and an animal food, wherein the amount of the berberine alkaloid is about 0.001% to 1% w / w of the animal food.

[0193] The amount of berberine alkaloids in the food may be in the range of 0.001 g / kg to 2 g / kg, i.e. 0.001% to 0.2% w / w. Exemplary contents of berberine alkaloids in food are: 0.001 g / kg (0.0001%), 0.003 g / kg (0.0003%), 0.01 g / kg (0.001%), 0.03 g / kg (0.003%), 0.1 g / kg (0.01%), 0.3 g / kg (0.03%), 1.0 g / kg (0.1%) and 2.0 g / kg (0.2%).

[0194] The feed is preferably in crumble; pellet form; or aqueous form.

[0195] The present disclosure also relates to a dosing regimen comprising administering to an animal a berberine alkaloid or an animal feed disclosed herein, wherein the berberine alkaloid or the composition or the animal feed is administered for 1 to 6 weeks in an amount effective for preventing and / or treating an infectious disease in the animal.

[0196] Preferably, the berberine alkaloid or the animal feed is fed for 1, 2, 3, 4, 5 or 6 weeks. Preferably, the berberine alkaloid or the animal feed is fed for 1 to 6 weeks; 2 to 5 weeks; or between 3 and 4 weeks.

[0197] Preferably, berberine alkaloids are administered at a concentration of about 0.6 g / L water or about 1.2 g / kg feed. The amount of berberine alkaloids in feed may range from 0.001 g / kg to 2 g / kg, i.e., 0.0001% to 0.2% w / w. Exemplary amounts of berberine alkaloids or acceptable salts in food are: 0.001 g / kg (0.0001%), 0.003 g / kg (0.0003%), 0.01 g / kg (0.0001%), 0.03 g / kg (0.0003%), 0.1 g / kg (0.01%), 0.3 g / kg (0.03%), 1.0 g / kg (0.1%) and 2 g / kg (0.2%).

[0198] The present disclosure also relates to a method for reducing feed conversion ratio in a food producing animal, wherein the method comprises the step of administering a berberine alkaloid to the food producing animal.

[0199] Preferably, the food producing animal is disease free. Preferably, the food producing animal is diseased. Preferably, the food producing animal is selected from chicken or pig. Preferably, the food producing animal is a chicken.

[0200] The present disclosure also relates to a method for preventing or treating an infectious disease in an animal, comprising administering the animal feed disclosed herein.

[0201] The present disclosure also relates to a method for preventing or treating infectious enteric disease in an animal, comprising administering the animal feed disclosed herein.

[0202] The present disclosure also relates to a method for preventing or treating an infectious disease caused by Eimeria in an animal, comprising administering the animal feed disclosed herein.

[0203] Preferably, the infectious disease is caused by antibiotic-resistant parasites of the genus Eimeria. Preferably, the infectious disease is coccidiosis and the animal is a chicken.

[0204] Preferably, the infectious disease is necrotic enteritis and the animal is chicken.

[0205] The present disclosure also relates to the use of berberine alkaloids in the preparation of a medicament for the prevention and / or treatment of:

[0206] infectious diseases in animals;

[0207] infectious enteric diseases in animals;

[0208] Infections in animals caused by Eimeria spp.; or

[0209] An infectious disease caused by bacteria of the genus Clostridium, of which Clostridium perfringens is one.

[0210] The present disclosure also relates to the use of berberine alkaloids in the prevention and / or treatment of:

[0211] infectious diseases in animals;

[0212] infectious enteric diseases in animals;

[0213] Infections in animals caused by Eimeria spp.; or

[0214] An infectious disease caused by bacteria of the genus Clostridium, of which Clostridium perfringens is one.

[0215] The present disclosure also relates to berberine alkaloids for use in the prevention and / or treatment of:

[0216] infectious diseases in animals;

[0217] infectious enteric diseases in animals;

[0218] Infections in animals caused by Eimeria spp.; or

[0219] An infectious disease caused by bacteria of the genus Clostridium, of which Clostridium perfringens is one.

[0220] The development of formulations, doses, and regimens for preventing or treating infectious diseases in animals is described in the following studies.

[0221] Formulation and palatability studies

[0222] A study was conducted to determine feed palatability and bird productivity following administration of four IRP001 formulations to broiler chickens.

[0223] Study Design - Upon receipt, two hundred and forty (240) day old commercial broiler chicks were evenly distributed into individual floor pens and acclimatized for 7 days. On day 7, the birds were weighed and assigned sequentially to sixteen (16) groups of 15 birds each. Feed intake, water intake, weight gain and mortality were used as outcome parameters.

[0224] Investigational Veterinary Products (IVP)

[0225] Table 1 IVPs used for formulation and palatability studies

[0226] name Components Dose level (g / kg)* Masked IRP001 Chloride 30% IRP001 2.7, 5.3 and 10.7 Masked IRP001 Sulfate 10% IRP001 8.0, 16.0 and 32.0 Unmasked IRP001 Chloride 100% IRP001 0.4, 0.8 and 1.6 Unmasked IRP001 Sulfate 100% IRP001 0.4, 0.8 and 1.6

[0227] *Dosage based on fixed concentration of IRP001 in feed

[0228] Study animals were dosed according to the treatment schedule detailed below in Table 2. The medicated feed was provided ad libitum to the birds in the relevant treatments, as was drinking water which was also provided ad libitum as the only source of feed for the birds.

[0229] Table 2 Treatment options

[0230]

[0231] Table 3 Timeline of events

[0232]

[0233]

[0234] Based on the above, the food and water intake and body weight of the animals (and organs after euthanasia) can be recorded. The average weight gain over the treatment period, the average daily weight gain, and the feed conversion ratio (FCR) can be calculated. The performance of the animals can be evaluated by these parameters. Similarly, the food and water intake parameters can indicate the palatability of the drug, while the weight gain and feed conversion ratio (FCR) parameters can provide the antibiotic effect of the IVP, that is, the degree to which the IVP promotes growth.

[0235] Feed conversion efficiency research

[0236] A study to determine the feed conversion efficiency and tissue residues of IRP001 when administered via feed to commercial broiler chicks. Residues with a washout period of 1 week were also studied.

[0237] Investigational Veterinary Products (IVP)

[0238] Table 4 IVPs for feed conversion efficiency studies

[0239] name Components Dose level (g / kg) IRP001 Chloride 100% IRP001 Chloride 1.0, 0.1, 0.03 and 0.01

[0240] Table 5 Treatment options

[0241]

[0242]

[0243] Table 6 Timeline of events

[0244]

[0245] After a 1-week washout period, the residual level of IRP0001 was determined by the following experiment:

[0246] After euthanasia, muscle samples of breast, shank and thigh as well as liver and kidney were removed from each bird. A known weight of tissue (approximately 1 g) was homogenized in 2 mL of water. The samples were centrifuged and a known volume of the supernatant was removed for analysis of IRP001 by LC-MS / MS to provide residual levels of berberine in muscle tissue (ng berberine per g muscle tissue).

[0247] Study on the efficacy of IRP001 on industrial standard Zinc Bacitracin

[0248] The efficacy of IRP001 in the prevention or treatment of necrotic enteritis was determined by administration of IRP001, including studies of dose response, feed conversion, tissue retention and safety. Using a validated experimental model, IRP001 was administered via feed to broiler chicks artificially challenged with pathogenic strains of Eimeria spp and Clostridium perfringens. The current industry standard treatment, bacitracin zinc, was used for efficacy and FCR comparison.

[0249] Study Design (Necrotic Enteritis Challenge) - Commercial broiler chickens housed in isolators were orally challenged at 9 days of age with 5,000 sporulated oocysts of attenuated vaccine strains each of E. maxima and E. acervuline and 2,500 sporulated oocysts of E. brucei in 1 mL of 1% (w / v) sterile saline.

[0250] Six days after oocyst challenge (day 15), a known pathogenic strain of Clostridium perfringens (type A strain NE18) was administered, it ( 8.0 log10 cfu / chicken). Two birds from each of all 42 groups were sacrificed on day 17 to define the lesion score.

[0251] Feed intake, body weight gain, mortality and NE lesion scores at necropsy were used as outcome parameters.

[0252] Investigational Veterinary Products (IVP) - IRP001

[0253] Table 7 IVP and dose levels for efficacy study of IRP001 against industrial standard bacitracin zinc

[0254] name composition Dose level (g / kg) IRP001 100% IRP001 1.0、0.3、0.1、0.03 Bacitracin Zinc Industry Standards Industry Standards

[0255] Table 8 Challenge and treatment schedule

[0256]

[0257]

[0258] Table 9 Timeline of events

[0259]

[0260]

[0261] The residual level of IRP0001 can be determined by the following experiment:

[0262] Muscle samples of breast, shank and thigh as well as liver and kidney were removed from each bird after euthanasia, and a known weight of tissue (approximately 1 g) was homogenized in 2 mL of water. The samples were centrifuged and a known volume of supernatant was removed for analysis of IRP001 by LC-MS / MS to provide residual levels of berberine in muscle tissue (ng of berberine per g of muscle tissue).

[0263] Dose rate studies

[0264] The study objectives are to evaluate the efficacy of three dose rates of IRP001 in the feed against a moderate mixed coccidiosis challenge (Eimeria species) in commercial broiler chickens and to assess any occurrence of necrotic enteritis or nonspecific enteritis. Safety data and tissue residue data will be obtained.

[0265] Study Design (Eimeria Challenge) - Commercial broiler chicks housed in barns were challenged at 14 days of age (day 14) with wild-type Eimeria oocysts; approximately 12,000 E. tenella, 40,000 E. acervuline and as many E. maxima oocysts per bird.

[0266] Seven days after oocyst challenge (day 21), groups of four birds were randomly selected from each experimental barn and humanely euthanized.

[0267] Overall intestinal health (enteritis) and lesion scores were assessed on day 21 and at necropsy, feed intake, body weight gain and mortality were used as outcome parameters, and feed conversion ratio was calculated for each time period.

[0268] Investigational Veterinary Products (IVP) - IRP001

[0269] Table 10 IVP and controls for dose rate studies

[0270] name composition Dose level (g / kg) IRP001 100% IRP001 1.0, 0.3, and 0.1 Salinomycin Industry Standards 60ppm Salinomycin + Bacitracin Zinc Industry Standards 60ppm+50ppm

[0271] Table 11 Treatment and challenge regimen

[0272]

[0273]

[0274]

[0275]

[0276] Table 12 Timeline of events

[0277]

[0278]

[0279]

[0280] The residual level of IRP0001 can be determined by the following experiment:

[0281] Muscle samples of breast, shank and thigh as well as liver and kidney were removed from each bird after euthanasia, and a known weight of tissue (approximately 1 g) was homogenized in 2 mL of water. The samples were centrifuged and a known volume of supernatant was removed for analysis of IRP001 by LC-MS / MS to provide residual levels of berberine in muscle tissue (ng of berberine per g of muscle tissue).

[0282] Residue research

[0283] This study and protocol was designed to determine the residual depletion profile of naturally occurring IVPs administered at maximum label dose rates by quantifying marker tissue residues in broiler chicks treated via feed throughout the production cycle.

[0284] background

[0285] Antimicrobials are widely used in livestock farming to control and prevent potentially deadly disease outbreaks in intensive animal husbandry. Some believe this is responsible for the development of resistant microorganisms, and government regulators are now implementing directives to control the use of these antimicrobials.

[0286] The inventors have identified several naturally occurring compounds that could be used as natural antibiotics to replace antibiotics currently used in food producing animals such as poultry and pigs.

[0287] Candidate formulations are tested to meet regulatory standards, such as those required by the Australian Pesticides and Veterinary Medicines Authority (APVMA) and the US Food and Drug Administration (FDA). In this regard, determining the residual consumption profile of animal health treatments is an integral part of the product development process. This allows government regulators to set an appropriate with-holding period (WHP) to protect human health and agricultural trade.

[0288] IRP001 has been selected as a candidate IVP because it has been determined to be safe and non-toxic. Poultry has been selected as the target animal species due to the extensive reliance of the chicken industry on antimicrobials to prevent or treat a variety of diseases caused by enteropathogens. These clinically important enteropathogens may potentially respond to IRP001.

[0289] Compliance

[0290] This tissue residue depletion study should be conducted according to an agreed-upon protocol using SOPs and good scientific practice.

[0291] Study Design

[0292] a. Experimental Unit: The experimental unit and observation unit are both animals, and the statistical unit is the treatment group.

[0293] b. Animal model: Feed intake, daily water intake, body weight change, mortality and marker residues in tissues were used as outcome parameters.

[0294] c. Inclusion Criteria: Animals will be selected for the study if they meet the criteria outlined in Section 10 below.

[0295] d. Exclusion and Removal Criteria: Animals that are deemed by the investigator to be debilitated, sick, injured, or otherwise unsuitable for inclusion in the study upon receipt will be excluded.

[0296] After selection, animals that may be considered unsuitable for continued study will be removed only with the written consent of the Sponsor or Investigator. The reason for any removal will be fully documented and justified in the original data and study report. Animals removed from the study will receive appropriate veterinary care.

[0297] e. Allocation: Broiler chicks: Upon receipt, one hundred and eighty (180) broiler chicks meeting the inclusion criteria will be randomly allocated to eighteen (18) separate treatment groups of ten (10 birds per group) after removal from the shipping containers. The method of allocation and randomization will be described in the primary data and study report.

[0298] f. Blinding: Not applicable.

[0299] Investigational Veterinary Products (IVP)

[0300] Record all drug product details including batch number, expiry date, receipt and use.

[0301] a. Veterinary product under investigation: IRP001 Cl as 100% IRP001 Cl.

[0302] b. Source: IVP will be provided by the Sponsor.

[0303] c. Storage: IVP should be stored at ambient temperature within the designated temperature zone. Record the storage location and conditions of the IVP.

[0304] d. Security: The sponsor provides an SDS or its equivalent (if any).

[0305] e. Analysis: A Certificate of Analysis (if available) was provided to the IVP.

[0306] f. Medication Disposal: Record the disposition of all remaining IVP.

[0307] treat

[0308] a. Dose calculation: The dose is based on a fixed concentration of IRP001 Cl in the feed (0.03 or 0.1 g / kg IRP001 Cl).

[0309] b. Dose preparation: Powdered IRP001 Cl is incorporated into commercial feed raw material ingredients and then mixed thoroughly in, for example, a "concrete mixer" type equipment to provide the outlined final concentrations in the feed.

[0310] c. Dosing Methods: Study animals were dosed according to the treatment regimen detailed in Table 1 below. During the relevant treatments, the birds were provided with medicated feed ad libitum as their sole source of feed.

[0311] Table 13 Treatment regimen - feed conversion rate

[0312]

[0313]

[0314] *Euthanasia

[0315] **Note: Medicated feed was withdrawn from Groups 6 and 12 on Day 28 to allow for a 14-day washout period for these groups.

[0316] Timeline of events

[0317] Table 14 Timeline of events

[0318]

[0319]

[0320]

[0321] Test system

[0322] The detailed information of the animals was recorded in the raw data, namely: species, broiler chickens; number, 180; source, commercial (batch of 90); age, one day old.

[0323] Animal Management

[0324] a. Animal Welfare: Research animals are managed similarly with due consideration for their welfare. Research animals are observed and the “Animal Care Record” is completed in accordance with the requirements of the Animal Ethics Committee (AEC).

[0325] b. Health management: If necessary, any routine preventive treatment should be carried out as soon as possible and recorded (product name, batch number, expiration date, dosage, route of administration and date).

[0326] Study animals were observed twice daily at appropriate locations according to standard operating procedures (SOPs) beginning on day 0. Any health issues requiring further investigation were recorded.

[0327] All health problems and adverse events must be reported to the investigator within one business day. Any adverse event that the investigator believes is product related, causes death, is life-threatening, involves a large number of animals, or involves adverse events in humans must be documented and reported to the sponsor and AEC within one business day.

[0328] Routine veterinary care and procedures may be performed and described in the original data. Concomitant medications may be used with prior approval by the Investigator and Sponsor (if relevant) for standard regulatory practices and humane reasons. No treatment similar to IVP was administered. All concomitant medications were recorded to provide identification of the material used (product name, batch number and expiration date), animal ID, reason for use, route of administration, dose and date administered, and included in the original data (trial log) and study report.

[0329] If injury or illness results in euthanasia or death of a research animal, this should be documented and a necropsy performed by a veterinarian when possible. A “necropsy report” including the probable cause of death should be included in the raw data.

[0330] All health problems, adverse events, and animal mortality were included in the study reports, including their relationship to treatment.

[0331] c. House Environment: Treatment groups were housed in dedicated chicken floor houses in two separate and discrete controlled environment rooms within an approved animal facility. One room housed all untreated birds from Groups 13 to 18, inclusive, and the second room housed all treated birds from Groups 1 to 12, inclusive. The floor space of each house was approximately 1.5 m 2 The chickens were raised on litter in accordance with normal commercial practice.

[0332] There were 18 floor pens with 10 birds per pens up to day 49. At the end of the study, the maximum bird weight in each pens was well below the 40kg / m2 maximum recommended for broiler chicks in the Australian Standard of Practice. 2 .

[0333] NOTE - Birds from Groups 13 to 18 inclusive (untreated control animals) were maintained in similar but physically separate isolators from drug treated birds from Groups 1 to 12 to ensure no cross contamination during the study.

[0334] d. Experimental diet: A formulated commercial starter ration followed by a grower ration was administered throughout the study. Copies of the feed bag labels or equivalent showing the feed ingredients were included with the original data.

[0335] e. Feed and water intake: Weigh and record the feed added each day, and calculate the daily feed intake (by treatment group). Measure and record the daily water volume, and calculate the daily water intake (by treatment group).

[0336] f. Animal Disposition: Study animals were humanely euthanized as approved by the AEC and recorded at the intervals listed in the timeline of events (Table 14).

[0337] Study Procedure

[0338] a. Trial Log: Record all planned and unplanned events during the study.

[0339] Effect evaluation

[0340] a. Body weight: Weigh the birds on day 0 (group weight) and on days 7, 14, 21, 28 and 35 - record the weight of each animal. Before and after weighing, check the weighing scale with a calibrated test weight and record. Body weights at the end of the study will be compared between groups to determine the effect of treatment, if any.

[0341] b. Examination: A separate clinical examination is performed on euthanasia at the time of gross pathology and tissue collection. The clinical examination is recorded. Digital still images may be recorded as appropriate.

[0342] c. Observations: The general health of the birds was checked twice daily, usually before 8 am and after 4 pm. Thus, the typical interval between observations was 9 hours during the day and 15 hours overnight. Birds showing abnormal clinical signs were recorded, carefully observed, and euthanized if the investigator considered them to be significantly suffering from abnormal clinical signs (e.g., marked weakness with a low probability of recovery).

[0343] d. Necropsy examination: Between days 35 and 49, all birds were euthanized and necropsied according to the schedule - Table 14.

[0344] e. Gross Pathology: All birds from all Groups 1 to 18 were necropsied and examined for gross visual pathological changes which were described and scored (per individual bird) as appropriate.

[0345] f. Tissue Residue Analysis: Duplicate representative samples of liver, kidney, breast muscle (1), leg muscle (2) [upper and lower thigh] and whole skin with intact fat were collected from the six (6) heaviest birds in each group (Groups 1 to 18 (inclusive)) according to the schedule, Table 1, and stored frozen (<10 degrees Celsius) for subsequent marker residue analysis. Birds from Groups 13 to 18 were sacrificed on day 35 as untreated control birds and tissues were collected to meet tissue assay requirements.

[0346] Samples will be labeled with a self-adhesive label listing the study number, animal ID, time point, date, sample type, and replicate.

[0347] For residue analysis, samples were thawed and a known weight of tissue (approximately 1 g) was homogenized in 2 ml of water. Samples were centrifuged and a known volume of supernatant was removed for analysis by LC-MS / MS.

[0348] Table 15 Analysis matrix

[0349]

[0350]

[0351] If analysis is required for assay validation and verification.

[0352] g. Storage, Transfer, and Disposal of Samples: Record the storage, transfer, and disposal of samples. Transport replicate 1 tissue samples frozen on wet ice to the analytical laboratory at the times outlined in Section 10. Transfer samples with the accompanying temperature data logger and chilled water bottles in accordance with the Standard Operating Procedure (SOP). Store replicate 2 tissue samples frozen for a period of 6 months following the last sample collection time point. Beyond this point, they may be discarded at the discretion of the study site unless specifically requested otherwise by the Sponsor representative.

[0353] Statistical analysis

[0354] Methods are documented in the study report.

[0355] Data Recording

[0356] The protocol specifications will supersede the facility SOPs. Study forms may be added or modified as needed during the study without requiring a protocol amendment or deviation.

[0357] a. Protocol Approval: Before the study begins, the protocol is approved and signed by all relevant personnel (see page 1).

[0358] b. Modification / Deviation: A modification is a change or modification to the protocol prior to performing the changed or modified task. The modification must state the reason for the change and have written documented authorization by the sponsor. The modification must be signed by the investigator and the sponsor.

[0359] When a deviation is identified, the Investigator should document, sign, and date the deviation from the protocol or applicable SOP. The impact on the overall outcome or integrity of the study will be evaluated. The Sponsor must be informed of the deviation as soon as practicable.

[0360] All protocol modifications and deviations are recorded accordingly and numbered sequentially based on the date they occurred or were identified.

[0361] c. Document Notes: Document notes are recorded to clarify events or circumstances that might not otherwise be apparent from the raw data. Document notes must be communicated to the sponsor as soon as practicable.

[0362] d. Changes in Research Investigators: Changes in study investigators or other responsible research personnel should be recorded accordingly.

[0363] e. Raw Data: All initial raw data pages are paginated, identified with the study number, signed, and dated by the person making the observations and recording the information.

[0364] f. Correspondence Log: The investigator maintains copies of all correspondence related to the study. Any telephone conversations that result in changes to the study documentation, design, conduct, or report of the study are recorded.

[0365] g. Permits: The research detailed in this protocol should be covered by a governmental agency permit (e.g., APVMA Small Trial Permit).

[0366] Research Report

[0367] The study report is prepared by the investigator or designee. It includes a list of data for each variable measured. The study investigator's compliance statement is included in the study report. The original signed study report with raw data and statistical reports is submitted to the sponsor and filed.

[0368] Salmonella and Campylobacter research

[0369] The present disclosure also contemplates preventing or treating infectious diseases caused by Salmonella or Campylobacter.The following describes studies investigating the effectiveness of berberine alkaloids or berberine alkaloid compositions in preventing or treating diseases caused by Salmonella or Campylobacter infection. These studies were modeled on published protocols: Alali, WQ et al. “Effect of essential oil compound on shedding and colonization of Salmonella entericserovar heidelberg in broilers”, Poultry Science, 2013, 92:836-841; Berghaus, R. et al. “Enumeration of Salmonella and Campylobacter in environmental farm samples and processing plant carcass rinses from commercial broiler chicken flocks”, Appl. Environ. Microbiol. 2013, 1-37; Cochran, WG, and GM Cox, Experimental Design. 2nd ed. John Wiley & Sons, New York, NY. pp. 582-583, 1992 (Cochran and Cox, 1992).

[0370] Salmonella Research

[0371] The objective of this study was to evaluate the effectiveness of IVP as a means of controlling Salmonella heidelberg in broiler chickens.

[0372] Experimental design

[0373] In this twelve (12) barn study, six hundred (600) chicks were allocated to three (3) treatment groups, each with four (4) replicate blocks and divided into fifty (50) birds per barn.

[0374] Treatment groups were assigned to the housing using a randomized complete block design (Cochran and Cox, 1992). The treatment groups were as follows:

[0375] 1. No treatment - Salmonella Heidelberg challenge control

[0376] 2. Treatment 1 - Salmonella Heidelberg Challenge

[0377] 3. Treatment 2 - Salmonella Heidelberg Challenge

[0378] The study started with placement of the birds (day of hatching; DOT 0), at which time the birds were assigned to the experimental barns. Only birds that appeared healthy were assigned to the study, and the final number and disposition of all unassigned birds were recorded. No birds were replaced during the study. The weight of the birds in the barns (kg) was recorded at the beginning (DOT 0), DOT 35 and end (DOT 42) of the study.

[0379] Materials and methods

[0380] Birds. Six hundred (600) day-old Ross x Ross straight-run broiler chicks were obtained. Birds received routine vaccinations (HVTSB1) and breeder flock information was recorded. All birds were vaccinated with a commercial coccidiosis vaccine at the recommended dose.

[0381] Housing and Environmental Control. At the start of the study, fifty (50) broiler chicks were assigned to twelve (12) floor pens measuring 5 x 10 (1.00 ft 2 / bird stocking density). The facility was fan-cooled. A thermostatically controlled gas heater was the primary heat source. Auxiliary heat lamps (one [1] lamp per barn) provided heat when needed. Birds were housed at ambient humidity and provided a lighting program based on the recommendations of the lead stockman. When placed, each barn contained approximately four (4) inches of fresh pine shavings. Bedding was not changed during the study. Each barn contained one (1) tube feeder and one (1) bell drinker, resulting in a ratio of fifty (50) birds per feeder and drinker.

[0382] Method of administration and drinking water: unlimited.

[0383] Diets. Rations were administered as follows: DOT 0 to DOT 14 for starter, DOT 14 to DOT 35 for finisher, and DOT 35 to DOT 42 for finisher. Diets were administered as crumbles (starter diet) or pellets (finisher and finisher diets). Diet formulations for this study consisted of non-medicated commercial broiler starter, finisher, and finisher diets blended with appropriate feeds calculated to meet or exceed NRC standards, and no antibiotics were added to any feed unless specifically specified as part of the treatment regimen. Experimental treatment diets were prepared from a basal starter diet, and the amounts of all basal feeds and test articles used to prepare treatment batches were recorded. To ensure uniform distribution of all test articles, treatment feeds were blended and pelleted in a California pellet mill at 80°C (recording pelleting temperature). After blending was completed, the feeds were distributed to the barns for the designated treatment groups. Test articles were stored in the SPRG climate-controlled storage area. All diet, formulation, and other feed information was recorded.

[0384] Feed Change. Birds received a treatment-appropriate feed from DOT 0 to DOT 42. The ration was changed from brooding to finishing at DOT 14 and from finishing to finishing at DOT 35. At that time, all previous feed was removed from each barn, weighed individually and replaced with finishing feed. At DOT 42, all unconsumed finishing feed was removed from the barns, weighed individually and discarded.

[0385] Salmonella inoculation. On DOT 0, twenty-five (25) chicks (50% sowers) per barn were tagged, color coded (for identification), and orally dosed (by gavage) for 10 7 CFU of Salmonella Heidelberg resistant to nalidixic acid.

[0386] Salmonella sampling. Bootsocks swab samples were collected from all barns DOT 14 and DOT 42 to determine environmental contamination of Salmonella. Gloves were changed between each swab to reduce potential cross-contamination of samples. Pre-moistened Bootsock swabs (Solar Biologicals, Inc., catalog number BT SW-001) were taken out of sterile bags, placed on feet covered with clean new plastic boots, walked around and inside the barn, took out the Bootsock, and then placed it in a sterile bag marked with the barn number. After repeating the process for each barn, the samples were appropriately stored and then submitted for Salmonella analysis.

[0387] Cecal Salmonella Culture. Cecal sampling was completed at DOT 42. Ten (10) horizontally exposed (untagged) birds were removed from each barn at DOT 42, euthanized (by cervical dislocation), and the cecum of each bird was aseptically removed. After the cecal sample was removed, it was placed in one (1) sterile plastic sample bag (Fisher Scientific), labeled, stored on ice, and submitted for Salmonella analysis.

[0388] Isolation and Identification of Salmonella. All samples submitted for isolation and identification of Salmonella (bootsock swabs and / or cecum) were stored on ice in sterile Whirl Pack bags prior to analysis. Upon arrival, tetrothionate broth was added to the Bootsock swab samples, the cecum was weighed, sterile saline was added, and the samples were stomachered. One (1) mL aliquot was removed for MPN analysis, 10X tetrathionate broth (Difco) solution was added, and the samples were incubated overnight at 41.5°C. A circle of the sample was tapped onto a xylose lysine tergitol-4 agar (XLT-4, Difco) plate, which was incubated overnight at 37°C. Using Poly-O Salmonella-specific antiserum (MiraVista, Indianapolis, IN), up to 3 (three) black colonies were selected and confirmed as Salmonella positive. (Berghaus et al., 2013; Alali et al., 2013)

[0389] Salmonella Enumeration Procedure (MPN Method). For all ten (10) horizontal exposure (unlabeled) and five (5) direct challenge (labeled) samples, one (1) ml homogenized (stomachered) peptone broth sample was transferred to three (3) adjacent wells in the first row of a 96-well two (2) ml deep block. A 0.1 ml aliquot of the sample was transferred to 0.9 ml of tetrathiosulfate broth in the second row, and the above procedure was repeated for the remaining rows (to produce five (5) ten-fold dilutions), and the blocks were incubated (at 42°C for 24 hours) (Table 16). One (1) μl of each well was transferred to XLT-4 agar (containing nalidixic acid) using a pin tool replicator, the plates were incubated (at 37°C for 24 hours), the final dilution of each sample was recorded, and entered into the MPN calculator (to determine the sample MPN). Suspected Salmonella isolates were confirmed using Poly-O Salmonella-specific antiserum (MiraVista, Indianapolis, IN) (Berghaus et al., 2013; Alali et al., 2013).

[0390] Table 16 Salmonella count

[0391]

[0392] Disease and Coccidiosis Control. All birds were vaccinated with a USDA approved coccidiosis vaccine at one (1) day of age via a spray cabinet. No concomitant medication was used during the study. To prevent cross contamination, plastic disposable boots were worn when entering the barns and were changed between each barn.

[0393] Identification of birds. The barn is a unit of measurement. The security of the barn will prevent birds from migrating.

[0394] Monitoring. Monitor all birds for general flock condition, temperature, lighting, water, feed, litter conditions, and unexpected house conditions / events. Record findings twice daily during normal working hours (record one (1) observation on the last study day). Record one (1) observation on Saturdays, Sundays, and observed holidays.

[0395] Mortality. Check the barn for mortality daily. Cull birds only to reduce suffering. Record the date and removal weight (kg) of any bird culled (or found dead). Perform a necropsy on all culled (or dead) birds and record the following information: sex and possible cause of death.

[0396] Bird and Feed Disposal. All birds, mortalities and surplus feed (including mixer flushes) are disposed of by appropriate ethical methods.

[0397] Source Data Control and Processing. Data is recorded in indelible ink with legible entries and each source data sheet is signed (or initialized) and dated by a separate record entry. All source data errors (and / or changes) are initialized, dated and a brief explanatory statement or error code is written directly on the sheet.

[0398] Data management. Performed data management and statistical analysis of weight gain, feed consumption, feed conversion and Salmonella results.

[0399] Events Calendar

[0400] Table 17 Salmonella Research Event Calendar

[0401]

[0402]

[0403] Campylobacter Research

[0404] This study was conducted to determine the efficacy of an investigational veterinary product (IVP) in reducing shedding (horizontal transmission) and colonization of Campylobacter jejuni in the cecum of broiler chickens.

[0405] Experimental design

[0406] One hundred and twenty (120) day-old (non-SPF) commercial broiler chickens were received. Five (5) birds were euthanized by cervical dislocation and their ceca were cultured for Campylobacter jejuni. The remaining one hundred and five (105) selected birds were randomly divided into three (3) groups in an isolation room divided into three parts, with 35 birds in each group. The experimental variables are as follows. All birds were fed a broiler starter crumble diet and treated as specified below.

[0407] Number of rooms - 1 divided into 3 poultry spaces

[0408] Total number of chicks - 120

[0409] Number of chicks euthanized immediately - 05

[0410] Number of birds that can be subdivided into treatment groups - 105 Treatment groups - 3

[0411] Repeat Block - N / A

[0412] Poultry per room subdivision - 35

[0413] Treatment Group

[0414] 1. No treatment - Campylobacter jejuni challenge

[0415] 2. Treatment 1-Campylobacter jejuni challenge

[0416] 3. Treatment of 2-Campylobacter jejuni Challenge

[0417] Materials and methods

[0418] Birds. One hundred and ten (110) day-old Ross 708 male broiler chicks were obtained. Birds were sexed, received routine vaccinations (HVTSB1), and breeder flock information was recorded. Birds received one (1) dose of a commercially approved coccidial vaccine at one (1) day of age according to the manufacturer's recommendations.

[0419] Housing and Environmental Control. At the start of the study, one hundred and five (105) day-old Ross 708 male broiler chicks were assigned to one (1) isolation room. The room was subdivided into three (3) equal bird spaces. Thirty-five (35) chicks were placed in each room. The dimensions of each room were 13.4' x 15.7' (approximately 2.0 feet 2The environment of the isolation room was controlled by an independent HEPA filtration system and a heat pump unit with one (1) heat lamp that provided supplemental heat during brooding. Birds were housed at ambient humidity. When placed, each barn contained approximately four (4) inches of kiln-dried bags of fresh pine sawdust. The litter was not changed during the course of this study. Each room contained one (1) tube feeder and one (1) bell drinker (35 birds / feeder to drinker ratio). Light was provided to the birds for twenty-four (24) hours per day.

[0420] Method of administration and watering: Unrestricted.

[0421] Diet. Birds were fed a broiler starter diet throughout the study. A non-medicated commercial broiler starter diet was mixed with appropriate feeds calculated to meet or exceed NRC guidelines, and no antibiotics were added to any feed unless specifically specified as part of the treatment regimen. Diets were prepared from a basal starter diet. Once mixed, the feeds were distributed to the barns for the designated treatment groups. Test articles were stored in a climate-controlled area. Documentation was maintained for all diets, preparations, and feeds.

[0422] Feed change. Birds receive a starter feed from DOT 0 to DOT 35.

[0423] Administration of Campylobacter jejuni: At DOT 14, 35 birds were orally gavaged with 0.1 ml of Campylobacter jejuni JB strain broth containing approximately 10 6 CFU / ml (chicken dose is about 10 5 CFU / ml).

[0424] Campylobacter colonization assessment: At DOT 0, five (5) birds were cultured for the prevalence of Campylobacter jejuni; at DOT 35, thirty-three (33) birds were euthanized by cervical dislocation per treatment. The cecum of each bird was aseptically removed and placed in a sterile plastic sampling bag (Fisher Scientific) for Campylobacter isolation analysis. All samples were stored on ice prior to Campylobacter analysis.

[0425] Campylobacter Enumeration Procedure: Campylobacter Enumeration Procedure (Direct Enumeration). For each sample, one (1) ml homogenized Bolton broth sample was transferred to three (3) adjacent wells in the first row of a 96-well two (2) ml deep module. An aliquot of 0.1 ml was transferred to 0.9 ml of Bolton broth in the second row, and the procedure was repeated for the remaining rows (generating twelve (12) ten-fold dilutions), and then 0.1 ml from each well was spread onto Campy Cefex agar (Table 18). The plates were incubated (42°C for 24 hours) in the presence of Campylobacter gas and the final dilution of each sample was recorded. Suspected Campylobacter isolates were confirmed by Gram staining.

[0426] Table 18 Campylobacter counts

[0427]

[0428] Disease control. No concomitant medication will be used during the study. To prevent cross contamination, disposable plastic boots will be worn when entering the room and changed between each room.

[0429] Identification of birds. The barn is a unit of measurement. The security of the barn will prevent birds from migrating.

[0430] Monitoring. Monitor all birds for overall flock condition, temperature, lighting, water, feed, litter conditions, and unexpected house conditions / events. Record findings twice daily during normal working hours (record one (1) observation on Day 35). Record one (1) observation on Saturdays, Sundays, and observed holidays.

[0431] Mortality. Check the room daily for mortality. Cull birds only to reduce suffering. Record the date and removal weight (kg) of any bird culled (or found dead). Perform a necropsy on all culled (or dead) birds and record the following information: sex and possible cause of death.

[0432] Bird and Feed Disposal. All birds, mortalities and surplus feed (including mixer flushes) are disposed of by appropriate ethical methods.

[0433] Source Data Control and Processing. Data is recorded in indelible ink with legible entries and each source data sheet is signed (or initialized) and dated by a separate record entry. All source data errors (and / or changes) are initialized, dated and a brief explanatory statement or error code is written directly on the sheet.

[0434] Data management. Performed data management and statistical analysis of weight gain, feed consumption, feed conversion, and Campylobacter outcomes.

[0435] Events Calendar

[0436] Table 19 Campylobacter Event Calendar

[0437]

[0438] Example

[0439] Necrotic enteritis

[0440] Necrotic enteritis is an intestinal infection found in food-producing animals such as poultry. First described by Parish in 1961, it is caused in poultry by the bacterium Clostridium perfringens and can present as an acute clinical disease or a subclinical illness. Although C. perfringens is considered the causative agent of necrotic enteritis, other contributing factors are often required to predispose the animal to disease. It is well recognized that necrotic enteritis is a multifactorial disease process with many risk factors including infection with Eimeria, removal of antibiotic growth promoters, environmental and management conditions, physiological stress and immunosuppression, and the nature and form of the diet.

[0441] Necrotic enteritis is a potentially fatal disease that can cause up to 1% flock mortality per day for several days during the last few weeks of rearing, rising to a cumulative total mortality of 30-50%. In subclinical forms, damage to the intestinal mucosa leads to reduced digestion and absorption, reduced weight gain and increased feed conversion, resulting in reduced commercial performance. It is this manifestation of the disease that is reported to cause the greatest economic losses in the poultry production industry. In addition, C. perfringens in poultry poses a risk of transmission to humans through the food chain, where C. perfringens is one of the bacterial pathogens frequently isolated in human food-borne disease outbreaks.

[0442] Necrotic enteritis was previously controlled by well-known antimicrobial drugs such as virginiamycin, bacitracin, etc. More and more countries have banned the use of antibiotics in food-producing animals, resulting in the emergence of necrotic enteritis, which poses a serious threat to animal and public health.

[0443] Clostridium perfringens is a Gram-positive anaerobic bacterium found in soil, dust, feces, feed, poultry litter and intestinal contents. It is extremely prolific and is capable of producing a variety of toxins and enzymes. Based on the production of four toxins (α, β, ε and ι), Clostridium perfringens strains are classified into five toxin types (A, B, C, D and E). It has been proposed that necrotic enteritis is caused by type A and, to a lesser extent, by type C, where type A strains produce chromosomally encoded alpha toxins, while type C strains produce alpha toxins as well as beta toxins.

[0444] Alpha toxin is a phospholipase C sphingomyelinase that hydrolyzes phospholipids and promotes disorganization of membranes, inducing the synthesis of mediators such as leukotrienes, thromboxanes, platelet aggregation factors, and prostacyclins. These mediators lead to vasoconstriction, platelet aggregation, and myocardial dysfunction, resulting in acute death. Beta toxin induces hemorrhagic necrosis of the intestinal mucosa, although the exact mechanism is unclear. The pathology of necrotizing enterocolitis is being reevaluated while other virulence factors are being sought. Recently, evidence has emerged that alpha toxin may not have a major role in the proposed pathogenesis of necrotizing enterocolitis, with studies reporting that the use of non-wild-type alpha toxin results in an impaired ability to cause the disease. Evidence suggests that molecules from the supernatant of Clostridium perfringens cultures recapitulate disease-like pathology when injected into the intestine. Recent evidence also suggests that NetB toxin from Clostridium perfringens may play a key role in the pathogenesis of necrotizing enterocolitis.

[0445] C. perfringens is naturally present in the intestine at low levels, but disturbances in the normal intestinal microflora can lead to rapid proliferation of the bacterium, resulting in the development of necrotic enteritis. Chickens are most commonly infected between 2 and 6 weeks of age, but necrotic enteritis may occur in birds between 7 and 16 weeks of age, or even up to 6 months of age.

[0446] The clinical feature of the disease is a sudden increase in mortality in the flock, usually without warning, although wet litter is sometimes an early indicator. Clinical signs may include weakness, dehydration, lethargy, ruffled feathers, diarrhea and reduced feed consumption, although the duration of clinical illness before death is short, so the reduction in weight gain is not obvious. Visible lesions can be seen in the small intestine. The duodenum, jejunum and ileum become thin-walled, friable, distended and filled with gas. In addition, the mucosal surface is covered with gray-brown to yellow-green amphipathic membranes or pseudomembranes. Lesions may also be found in other organs and atrophy of red blood cells and mucous bursae. The subclinical form of necrotic enteritis is quite difficult to identify, and sick birds that respond to antibiotic analog treatment are usually considered to have the disease. Although wet litter does not always originate from Clostridium, wet litter often prompts poultry farms to immediately take antibiotic treatment. In addition, mild necrosis of the intestinal mucosa has been reported in subclinical necrotic enteritis. Example 1 describes the use of berberine sulfate (IRP001 sulfate) in the prevention or treatment of necrotic enteritis.

[0447] Example 1

[0448] A pilot study to determine the dose response, efficacy, and safety of IRP001 sulfate when administered prophylactically (orally, in feed) and therapeutically (orally, in drinking water) to specific pathogen-free chickens artificially challenged with Clostridium perfringens using a validated experimental model.

[0449] Materials and methods

[0450] Study Design (Necrotic Enteritis Challenge) - Commercial broiler chickens raised in isolators were orally challenged at 9 days of age with 5,000 sporulated oocysts of attenuated vaccine strains each of E. maxima and E. acervuline and 2500 sporulated oocysts of E. brucei in 1 mL of 1% (w / v) sterile saline.

[0451] Five and six days after oocyst challenge (days 14 and 15), a known pathogenic strain of C. perfringens (type A strain EHE-NE36, CSIRO Livestock Industries, Geelong, Australia) was administered. 8.0 log10 cfu / chicken). All NE cohort birds were sacrificed and necropsied on day 16. NE lesion scores and mortality at necropsy were used as outcome parameters and are shown below in Tables 22 and 23. Feed and water intake and body weight gain were also measured.

[0452] Investigational Veterinary Product (IVP) - IRP001 Berberine Hemisulfate (IRP001 Sulfate)

[0453] Table 20 Challenge and treatment regimen

[0454]

[0455]

[0456] Table 21 Timeline of events

[0457]

[0458]

[0459] result

[0460] Table 22 Summary data of median lesion scores

[0461]

[0462] Table 23 Mortality of broiler chickens before autopsy

[0463]

[0464] The introduction of IRP001 sulfate at 1.0 g / L in water or 2.0 g / kg in feed resulted in a significant reduction in mortality in NE-challenged broiler chickens relative to no treatment and treatment groups at 0.1 g / L in water or 0.2 g / kg in feed (see Figure 5The mortality rates of the no treatment, 0.1 g / L water and 0.2 g / kg in feed groups were not significantly different from those of NE-challenged broilers.

[0465] The introduction of IRP001 sulfate at 1.0 g / L in water or 2.0 g / kg in feed resulted in a significant reduction in small intestinal lesion scores relative to the no treatment group in broiler chickens challenged with NE (see Figures 6 to 12 In contrast, introduction of IRP001 at 0.1 g / L in water or 0.2 g / kg in feed did not result in a decrease in median lesion score relative to no treatment in NE-challenged broiler chickens.

[0466] Example 2

[0467] A follow-up study to determine feed palatability, feed and water consumption, and bird productivity following incorporation of a single formulation of IRP001 hemisulfate (unmasked) given to broiler chicks in the feed or in the water. The study explored the optimal treatment regimen in terms of treatment start date.

[0468] Materials and Methods

[0469] Study Design

[0470] Phase 1: Upon receipt, on Day 0, two hundred and seventy (270) day-old commercial broiler chicks were sequentially distributed to receive the chicks in sixteen (16) individual floor pens, with 16 or 17 birds per pens.

[0471] Phase 2: Upon receipt, on day 22, ninety (90) day old commercial broiler chicks were sequentially distributed to receive the chicks in four (4) separate floor pens, 22 or 23 birds per pens.

[0472] Feed intake, water intake, weight gain and mortality were used as outcome parameters.

[0473] Investigational Veterinary Products (IVP)

[0474] Table 24 IVP of Example 2

[0475]

[0476] Table 25 Stage 1 - Day 0 Age Chicks

[0477]

[0478]

[0479] Table 26 Stage 2 - Day 22 received chicks

[0480] Group preparation IVP concentration in feed (g / kg) IVP concentration in water (g / L) Treatment days Execution Day 17 none - - - 42 18 none - - - 42 19 IVP 2 - 25-42 42 20 IVP 2 - 25-42 42

[0481] Table 27 Event timeline

[0482]

[0483]

[0484] result

[0485] Individual daily feed intake and individual daily water intake data (by house and then by treatment group) for Phase 1 and Phase 2 (and for the entire trial for birds continuing both phases in groups / houses 2, 15 and 16) were calculated using the total feed and water figures provided to each house each day divided by the number of birds in each house. If errors occurred in weighing, administering / watering or recording (or other unexplained losses of feed and water), the means were corrected using the same house means for 1-2 days on either side of the apparent error. Similarly, group mean body weights for Phase 1 were calculated using total body weight / total number of birds on Day 0 and individual body weights on Days 7, 14, 21, 28, 35 and 42. The total (individual) feed consumed / treatment was calculated and feed conversion ratio / treatment was calculated using the following expression: total (individual) feed / total (individual) weight gain.

[0486] Individual daily feed intake and individual daily water intake were statistically compared for treatments within each phase and between Barns 2 and 15 / 16 in both phases using the following linear model and Tibco SPOTFIRE S+8.2 (2010):

[0487] (parameter) treatment+day+animal house+treatment:day.

[0488] "Day" was included in the model to allow for variation over time, "Housing" was included as each treatment consisted of 2 housing units, and the interaction term "Treatment: Day" was included to allow for a treatment x time effect. Model suitability was confirmed by inspection of residual plots; in all cases, the statistical model was appropriate.

[0489] Phase 1:

[0490] Table 28 Summary data of phase 1

[0491]

[0492]

[0493] Feed intake: "Treatment" was significant, "Day" was highly significant, and "Housing" was not significant. However, in individual pairwise comparisons of treatments, no significant differences were observed (p<0.05).

[0494] Water intake: "Treatment" was significant and "Day" was highly significant. The number of pairwise comparisons of treatments was significant, but the results were not conclusive. There did appear to be a moderate trend for the group receiving the longer duration of the trial treatment (unmasked in the drinking water) to drink less water than the group treated for a shorter duration and the untreated control group (see Fig.13 ).

[0495] Body weight "treatment" was significant, "day" was highly significant, and "housing" was not significant. However, in individual pairwise comparisons of treatments, no significant differences were observed (p<0.05).

[0496] During Phase 1, unmasked treatments delivered via drinking water over different periods did not appear to affect feed intake or body weight, although treated birds tended to drink less water.

[0497] Phase 2:

[0498] Table 29 Summary data of Phase 2

[0499]

[0500] Feed intake: "Treatment" and "Housing" were not significant, while "Day" was highly significant. However, for pairwise comparisons of the two treatments, no significant differences were observed (p<0.05).

[0501] Water intake: "Treatment" was highly significant, "Day" was highly significant. For pairwise comparisons of the two treatments, a significant difference was observed, with treated birds (those receiving treatment in the feed) drinking more water (see Fig.14 ).

[0502] Body weight: "Treatment" and "Housing" were not significant (although "Day" was significant as expected), and for pairwise comparisons of the two treatments no significant differences were observed (p<0.05).

[0503] Treatments in Phase 2 (in the feed) did not appear to affect feed intake or body weight, while treated birds tended to drink more water (in contrast to Phase 1, where birds tended to drink less water when the unmasked treatment was applied in the drinking water).

[0504] Phase 1+2 (untreated group - barn 2 vs 15 / 16, treated in water days 6-42):

[0505] Feed Intake: Although "Treatment" was not significant in the overall model ("Day" was highly significant), there was a significant difference (p<0.05) for the pairwise comparison of the two treatments, with the untreated birds consuming -0.14 kg more feed throughout the trial.

[0506] Water intake: "Treatment" was highly significant, "Day" was highly significant. For pairwise comparisons of the two treatments, a significant difference was observed (p<0.05), with untreated birds drinking more water than untreated birds throughout the experiment.

[0507] Body Weight: Although "Treatment" was significant in the model (and "Day" was highly significant as expected), no significant differences were observed for pairwise comparisons of the two treatments (p<0.05).

[0508] When the combination of significantly higher feed intake and similar (not significantly different) body weights is considered through feed conversion, treatment over 6-42 days appears to have a moderate advantage over no treatment (see Fig.15 Treated birds consumed 3.84 kg of feed and gained 2.35 kg (FCR of 1.6 kg of feed: 1 kg gain), while untreated birds consumed 4.17 kg of feed and gained 2.14 kg (FCR of 1.95 kg of feed: 1 kg gain).

[0509] Example 3

[0510] To evaluate the efficacy of three dose rates of in-feed IVP berberine chloride against a mixed moderate coccidiosis challenge in commercial broiler chicks and assess any occurrence of necrotic enteritis or non-specific enteritis. This study provided a scoping project for the most likely effective dose rate of IVP in broiler chicks and assessed the likely success in controlling coccidiosis and subsequent necrotic enteritis compared to industry standards.

[0511] Materials and Methods

[0512] Field samples of coccidian oocysts (species of Eimeria from broiler and laying hen sources) were obtained and transported to the laboratory where they were filtered, sporulated, disinfected and stored. The species of Eimeria present were then identified by PCR and the oocysts were counted. These were propagated from chicks used for the first experiment to produce a number sufficient to meet the challenge inoculation of the sower birds. Thirty one-day-old broiler chicks were obtained from a commercial hatchery and placed in the hatchery cages of the chicken house in the experimental facility, with 10 chicks per cage. They were given a non-medicated ration and on the 7th day, sporulated oocysts were administered by gavage. On the 13th day, the birds were euthanized and their intestines removed. The upper and lower small intestines and cecum were separated, placed in 2% potassium dichromate, left at 4°C for 3-5 days, and then scraped to remove the mucosa. It was passed through a coarse sieve into a fresh potassium dichromate solution. Oocysts were sporulated, examined and counted under a microscope, and identified to species by PCR.

[0513] One thousand one hundred and eighty (1180) one-day-old Ross 308 chicks were obtained from a commercial hatchery where they were vaccinated against infectious bronchitis and Newcastle disease. The chicks were transported to the experimental facility and randomly assigned to 30 floor pens with 36 chicks per pens ( Fig.18 ). The stalls are reduced to half their normal size by means of dividers, providing 3.5m 2 of floor space, aiming to provide approximately 30kg / m 2 Two additional full-size barns were housed with 50 birds each (these served as seeders for the coccidiosis challenge).

[0514] Feeds were formulated based on appropriate balanced basal rations (starter, grower and finisher). Products were added to each basal ration as follows (Table 30).

[0515] Table 30 Treatment groups / feed

[0516]

[0517]

[0518] The feed was distributed to the barns on a randomized complete block basis. Each barn was provided with feed as follows: 0.7 kg feed per bird during the starter period (approximately day 0-14), 1.2 kg feed per bird during the finisher period (approximately day 15-28) and then during the finisher period until termination (day 42). The sower barns received ration number 1 (without medication).

[0519] On day 6, the birds in the sower barn were provided with oocyst inoculations (approximately 0.5 mL per bird) by individual gavage using a step-through pipette. Three samples of sporulated oocysts from different chicken farms were used and given to approximately one-third of the birds in each sower barn. The litter in the sower barn was slightly tilted on days 12, 13, and 14. On day 14, the top 2-3 cm of the litter in the sower barn was collected, thoroughly mixed together, and weighed ( Fig.19 The total weight of the litter was divided by 30 and this amount of litter was then distributed to each test barn (each barn received 400 gm of mixed seeded litter, Fig. 20 ).

[0520] Four subsamples of the mixed litter will be collected and oocyst counts performed by suspending 7 gm of litter in 75 mL of saturated sucrose and counting the total number of visible oocysts in a Whitlock Universal counting chamber at 100X magnification.

[0521] Birds were weighed on a barn basis on days 0, 14, 21, 28 and 42. Feed consumption was measured on days 14, 21, 28 and 42. Feed conversion ratio (FCR) was calculated for each time period and corrected for bird losses and removals.

[0522] Record any dead or culled birds and weigh and examine them at necropsy. Pay particular attention to lesions in the intestine consistent with coccidiosis or enteritis. Record sex.

[0523] On day 21, four birds were randomly selected from each experimental animal house, humanely euthanized, and their intestines and caeca were scored for coccidiosis lesions in four intestinal segments (upper, middle, lower intestine and caeca), and lesions typical of Eimeria species were recorded. Overall intestinal quality was also visually assessed at this point (looking for enteritis).

[0524] On day 21, four individual fecal samples were collected from each barn, combined, and assessed for oocyst counts.

[0525] On day 42, all surviving birds were euthanized and their carcasses disposed of by collecting contaminated waste (not slaughtered for human consumption).

[0526] result

[0527] Table 31 shows the characteristics of Eimeria species in the inoculum given to seeders birds as determined by PCR by Birling Avian laboratories. This PCR is only qualitative, but the relative abundance of each species can be estimated (if the abundance is greater, it is shown as an increase in the number of "+" symbols).

[0528] Table 31 Eimeria species detected in the challenge inoculum of sower birds

[0529]

[0530] +, ++ indicate relative amounts present; ND = not detected.

[0531] Table 32 summarizes the oocyst counts per gram of mixed litter samples (samples counted in quadruplicate) taken from the sower's barn 7 days after inoculation. The size of the oocysts could be clearly assessed, but the species could not be accurately determined. Sporulation can be determined using this technique.

[0532] Table 32 Oocyst counts on mixed litter samples from sower birds used as challenge for each barn

[0533]

[0534] 1 large oocyst, typical of Eimeria maxima or Eimeria brucei

[0535] 2 Medium oocysts, typical of E. tenella, E. necroticus, or E. precocious

[0536] 3 small oocysts, typically E. acervulina or E. mitis

[0537] Based on the oocyst counts shown in Table 32, on Day 14, each barn received approximately 6.3 million oocysts in the distributed seeded litter.

[0538] Several oocysts of typical sizes for Eimeria were observed when counting challenge seeded litter (percentages estimated in Table 32). However, only the small oocysts appeared to sporulate, while the other sized oocysts showed little evidence of sporulation at the time the litter was spread.

[0539] Table 33 shows the average body weight at each weighing time, and Table 34 shows the average weight gain during each period. Fig.21 The mean daily weight gain by treatment is shown.

[0540] Table 33 Average live weight of each age group (gm)

[0541]

[0542] A, B, C-mean values ​​with different superscripts are significantly different (P<0.05), ANOVA, separated by Duncan's multiple range test.

[0543] Table 34 Weight gain by period

[0544] Mean weight gain (gm) in each period (days)

[0545]

[0546]

[0547] A, B, C-mean values ​​with different superscripts are significantly different (P<0.05), ANOVA, separated by Duncan's multiple range test.

[0548] Body weights at 14 days showed clear differences with treatment, with birds receiving 0.3g / kg IRP weighing significantly less than the negative control, and both IRP dose rates were low. Both salinomycin-containing feeds were at intermediate levels at 14 days. This trend became apparent at day 7, but was not significant for this age group, which was also reflected in the weight gain during these periods. By day 21, the average weight of birds in the 0.3g / kg IVP treatment group was significantly lower than any other treatment. The IVP at 0.03g / kg had the highest average weight on day 21 and was significantly higher than the salinomycin + bacitracin group and the 0.3g / kg IRP group. Birds receiving IVP 0.3g / kg remained significantly lighter than all other groups until the end of the experiment, although there was no difference in the rate of gain after day 21 between the two groups. The coccidiosis challenge experienced by the negative control did not significantly reduce growth rate compared to the treatment groups.

[0549] Starting on day 14, the IVP 0.3 g / kg group weighed significantly less than the control and lower IVP dose groups. This group (Feed #2) consumed less feed than any other group in the trial and more than the other two groups treated with IVP (Table 35). The color of the feed for this group was bright yellow ( Fig. 22 ), and by 42 days, undigested feed particles were visible in the feces of these birds ( Fig.23 ). Slower growth rate with IVP 0.3 g / kg was seen in Fig.21 . The poultry always appeared healthy.

[0550] Coccidiosis challenge did not reduce the growth rate of the negative control group during the challenge week (days 15-21).

[0551] Throughout the experiment, the growth rates of the lower IVP dose groups and the salinomycin and salinomycin + bacitracin groups were statistically similar to the control group.

[0552] Table 35 shows the feed intake per bird and Table 36 shows the feed conversion ratio (FCR = feed:weight gain ratio) corrected for bird losses.

[0553] The feed intake of birds receiving IVP 0.3 g / kg during days 0-14 and 0-21 and the feed intake of both salinomycin-containing diets during days 0-21 had significantly lower feed intake than the control group. The feed intake of IVP 0.1 g / kg and 0.03 g / kg was similar to the control group. No significant feed intake differences were observed thereafter.

[0554] Table 35 Feed intake per bird

[0555]

[0556] The FCR (corrected for bird losses and removals) showed significant changes only after the entire experimental period (days 0-42). The FCR of both diets containing salinomycin (#5 and #6) was significantly better than the control, while the FCR of the diet also containing bacitracin (#6) was significantly better than the IVP 0.1 g / kg group (#3). The minor differences in sex ratios determined between the groups had no significant effect on the performance of the birds (not shown).

[0557] Table 36 FCR adjusted for mortality

[0558]

[0559] A, B, C-mean values ​​with different superscripts are significantly different (P<0.05), ANOVA, separated by Duncan's multiple range test.

[0560] Table 37 shows the results of coccidiosis lesion scores at day 21 (7 days after exposure to contaminated litter).

[0561] Table 37 Coccidiosis lesion scores

[0562]

[0563] A, B, C-mean values ​​with different superscripts are significantly different (P<0.05), ANOVA, separated by Duncan's multiple range test.

[0564] The lesions of coccidiosis were mainly typical of E. acervulina. PCR of the challenge litter showed the presence of E. maxima, E. tenella and E. mitis. This was consistent with the pre-challenge oocyst data, as only the smaller oocysts (E. acervulina and E. mitis) appeared to have good levels of sporulation when the oocysts were counted before challenge.

[0565] Negative controls and the lowest IVP levels showed the highest lesion scores in the duodenum, jejunum, and total intestine. The location of the lesions and their appearance were typical of E. acervulina (see Figure 24 to Figure 26 ). In the duodenum, only salinomycin plus bacitracin significantly reduced lesions compared to the control group and the two lower dose rates of IVP. Jejunal lesions were generally lower, but there were some significant differences. Overall, the highest levels of feeds containing IRP and salinomycin (#5 and #6) significantly reduced total intestinal lesion scores.

[0566] Pooled fecal samples from each barn were evaluated for oocyst content. Table 38 shows the results. The results showed some consistency, but one barn (0.1 g IVP / kg group) had extremely high oocyst counts in fecal samples (checked twice). This individual barn also showed very high coccidiosis lesion scores, which skewed the results for that treatment group. The observed raw oocyst counts were not uniform (significant by Levene's test), so the counts were converted to base 10 logarithms to overcome this problem for ANOVA analysis. The transformed log 10 The results are also shown in Table 38. Fecal oocyst counts were numerically reduced for the IVP treated feed groups (#2, #3 and #4), and only the feeds containing salinomycin (#5 and #6) significantly reduced fecal oocyst counts compared to the negative control group.

[0567] Table 38 Fecal oocyst counts

[0568]

[0569] A, B-means with different superscripts are significantly different (P<0.05), ANOVA, separated by Duncan's multiple range test.

[0570] Table 39 shows the intestinal pathology score, which is based on Tierlynck et al., Avian Pathology, 2011, 40: 139-144 (Tierlynck et al., 2011). This is a scoring system designed to quantify the level of dysbiosis present in a group of birds, attributing certain obvious visible abnormalities to the score. Higher total scores (maximum 10) reflect higher levels of dysbiosis, although coccidiosis may be affected if present. For our purposes, the intestinal score reflects only the overall intestinal pathology. Figure 27 to Figure 29 Some examples of observed intestinal abnormalities are shown.

[0571] In this experiment, the mean intestinal integrity score was lower on day 21 than on day 28. On day 21, the intestinal areas with increased intestinal scores were coelom ballooning, congestion, translucency, and abnormal content in the upper intestine and the presence of undigested feed particles in the rectum. On day 28, the areas with higher scores were coelom ballooning, congestion, translucency, and tension in the upper intestine.

[0572] There were no significant differences in total gut health scores among all treatments at 21 or 28 days, but the higher two levels of IVP (0.3 and 0.1 g / kg) and both salinomycin-containing diets reduced upper intestinal translucency scores and the presence of undigested feed particles in the rectum compared to the control group and the lowest level of IVP (0.03 g / kg) at 21 days. At 28 days, the 0.3 g / kg IVP produced a total gut health score that approached significance (P=0.06) compared to the control group.

[0573]

[0574] Table 40 shows significant correlations (Pearson correlation coefficients) with observed values.

[0575]

[0576] There was a strong and significant positive correlation (r = 0.70) between the corrected feed conversion ratio on day 42 and the total coccidiosis lesion score on day 21 (i.e., higher lesion scores were associated with higher FCR, and these lesions accounted for 83% of the variation in FCR). Fig.30 The results are presented graphically in the Figure 2. Coccidiosis lesion scores were moderately positively correlated with fecal oocyst counts on Day 21 (one accounted for 62% of the variation in the other). Intestinal luminal distension on Day 21 was moderately correlated with fecal oocyst counts on Day 21 and FCR on Day 42. The presence of higher levels of undigested feed particles in the rectum on Day 21 was associated with higher coccidiosis lesion scores and worse (higher) FCR on Day 42. Total intestinal integrity scores on Day 28 were also moderately positively correlated with coccidiosis lesion scores on Day 21 and FCR on Day 42.

[0577] Discussion and Conclusion

[0578] Although the coccidia challenge applied contained several species of Eimeria, only the E. acervulina type showed good sporulation upon challenge. It is generally believed that the conditions for sporulation are the same for all species, so this observation is unusual and the reason for this is unknown. This observation is certainly accurate because only E. acervulina type lesions were observed during the 21st day inspection. E. acervulina is a low-level pathogenic species that is unlikely to cause mortality and has a minor effect on growth rate. However, it may cause diarrhea and affect feed conversion efficiency.

[0579] The challenge applied produced moderate coccidiosis lesions in the negative control group, which were significantly reduced by the feed containing salinomycin and the feed containing 0.30 g / kg IVP; but not by the lower dose rates. At day 21, only the feed containing salinomycin significantly reduced the level of oocysts in the feces, while all groups receiving IVP levels were numerically lower than the control. Therefore, IVP appears to have some effect on Eimeria acervulina.

[0580] Chicks receiving the diet containing 0.30 g / kg IVP had significantly lower early growth rates than all other groups (up to 21 days) and although their growth rates subsequently improved, they remained the lightest birds in the experiment. This was associated with lower feed intake per bird by day 21. The feed with the higher IVP level was bright yellow in colour and birds consuming it showed moist, yellowish excreta. Whether this lower feed intake was due to palatability cannot be determined with certainty and requires further evaluation, the predominance of loose excreta may indicate an adverse effect of some nature at this introduction rate.

[0581] Corrected feed conversion ratio was slightly reduced by all treatments throughout the trial (days 0-42), but only the diet containing salinomycin significantly reduced the aforementioned feed conversion ratio compared to the negative control. FCR was strongly correlated with the coccidiosis score at day 21, and moderately correlated with the number of fecal oocysts and certain intestinal integrity scores (body cavity distension, undigested feed particles in the rectum at day 21 and total intestinal score at day 28). 83% of the variation in FCR at day 42 could be statistically explained by the variation in the coccidiosis score at day 21. Intestinal body cavity distension is a frequently described symptom associated with coccidiosis. Since the intestinal score at day 28 also had a moderate correlation with the coccidiosis score at day 21, we can assume that the effects of coccidiosis infection continue to exist in the intestine after the lesions have resolved (no coccidiosis was observed at day 28). The intestinal scoring system is designed to quantify the presence and level of a condition called dysbacteriosis in broiler chicks, which is believed to be caused by coccidiosis. The gut integrity scores were higher (i.e. more severe) on day 28 compared to day 21, suggesting some level of dysbacteriosis. The treated feeds reduced the gut scores compared to the negative control, which was statistically significant (P=0.06). In this regard, IVP provided similar improvements to salinomycin and salinomycin plus bacitracin zinc. This would be reasonable evidence that IVP may have some protective effect against dysbacteriosis.

[0582] Campylobacteriosis

[0583] Campylobacteriosis is a gastrointestinal illness caused by bacteria called Campylobacter (CB). In Australia, CB is one of the most common causes of bacterial gastroenteritis and is frequently associated with eating contaminated poultry. Infection can occur at any time of the year but is more common during the warmer months. In 2011, Campylobacter was the fourth leading cause of foodborne illness in the United States.

[0584] Most people infected with CB experience diarrhea, cramping, abdominal pain, and fever that lasts one to two weeks. Symptoms usually appear 2 to 5 days after infection. The diarrhea may contain blood or mucus. In rare cases, CB can enter the bloodstream and cause more serious illness.

[0585] CB is primarily transmitted to humans through eating or drinking contaminated food (primarily poultry), water, or unpasteurized milk. CB can also be spread through contact with infected people or with cats, dogs, and livestock that carry the bacteria ( Figure 3 epidemiology is shown).

[0586] Anyone can get campylobacteriosis, although very young children, the elderly, people with weakened immune systems, and people who work with livestock are at greater risk.

[0587] Most people recover from campylobacteriosis by resting and staying hydrated. Recovery usually takes one week but may take up to two weeks. Treatment usually involves rehydration solutions obtained from your pharmacist to help resolve dehydration caused by diarrhea. In severe or complicated cases, antibiotics such as erythromycin may be prescribed to reduce the duration of the illness.

[0588] CB contamination of poultry carcasses / meat continues to occur. Methods to control CB contamination have focused on the processing plant through washing and evisceration techniques. However, it is believed that if CB colonization of the bird's intestine could be controlled prior to slaughter, contamination of processed birds would be reduced.

[0589] Example 4 discloses the antimicrobial activity of certain natural compounds against Campylobacter.

[0590] Example 4

[0591] Laboratory work

[0592] The identified natural compounds can potentially be used for the prevention and treatment of diseases caused by Campylobacter The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were tested in vitro.

[0593] 1. Berberine Chloride

[0594] 2. Berberine Sulfate

[0595] 3. Arecoline

[0596] 4. Pulsatilla

[0597] 5. Matrine

[0598] 6. Oxymatrine

[0599] 7. Andrographolide

[0600] 8. Tetrandrine

[0601] 9. Baicalin

[0602] Materials and methods

[0603] Clinical and Laboratory Standards Institute (CLSI) guidelines were used for this project. Ten representative strains were selected. The test concentrations of each compound were: 1000, 500, 250, 125, 62.5 μg / ml. The positive control used was tetracycline.

[0604] Table 41 MIC and MBC of tested Campylobacter strains

[0605]

[0606]

[0607] result

[0608] Table 42 Campylobacter in vitro results

[0609] test result* MIC Both compounds displayed a MIC of 62.5 μg / ml. MBC The same two compounds displayed an MBC of 62.5 μg / ml.

[0610] *Tetracycline results were consistent with the reference standard for MIC and MBC.

[0611] Pig Diseases

[0612] E. coli-diarrhea (diarrhea)

[0613] Of all the diseases of sucking piglets, diarrhoea is the most common and probably the most important. In some outbreaks it is responsible for high morbidity and mortality. In a well run herd less than 3% of the litter should require treatment at any one time and piglet mortality from diarrhoea should be less than 0.5%. However, in severe outbreaks mortality levels may rise to 7% or more and in individual untreated litters mortality levels may rise to as much as 100%. The main bacterial cause is Escherichia coli. Diarrhea in piglets can occur at any age during the suckling period but there are usually two peaks before 5 days of age and between 7 and 14 days of age.

[0614] With acute disease, the only sign may be finding a perfectly fine pig dead. Necropsy reveals acute enteritis so severe that suddenly there may be no outward signs of diarrhea. Clinically affected piglets huddle together and shiver or lie in a corner. The skin around the rectum and tail is wet. Looking around the barn there may be signs of diarrhea of ​​a watery to salad cream consistency. In many cases there is a distinctive odor. As diarrhea progresses, piglets become dehydrated, their eyes become sunken, and their skin becomes thick and leathery. The diarrhea often adheres to the skin of other piglets, giving them an orange to white color. Prior to death, piglets may be found paddling on their sides and foaming at the mouth.

[0615] In subacute disease, symptoms are similar but the impact on piglets is less severe, prolonged longer and mortality is often lower. This diarrhoea is usually seen between 7 and 14 days of age and presents as watery to thin salad cream consistency diarrhoea that is usually white to yellow in colour.

[0616] It is estimated that piglet diarrhoea costs the Australian pig industry more than A$7 million per year. The incidence and type of diarrhoea, sanitation costs and recovery rates determine the extent of individual pig losses. Antidiarrheal drugs, such as bentonite or kaolin, are used to protect the intestinal lining. Electrolytes are often added to drinking water. Antibiotics are used to reduce the number of intestinal bacterial populations, although overuse of these drugs should be avoided because of the development of resistance. Current antibiotic drugs are listed in Table 43 below.

[0617] Table 43 Antibiotics used to treat diarrhea in piglets

[0618]

[0619] *Prohibited in some countries

[0620] Example 5 discloses the antimicrobial activity of certain natural compounds against porcine diseases.

[0621] Example 5

[0622] Laboratory work

[0623] Natural compounds have been identified as potential for the prevention and treatment of infectious enteric diseases in pigs, including Escherichia coli-induced diarrhea. Minimum inhibitory concentration (MIC) in vitro testing. The compounds tested are.

[0624] 1. Berberine Chloride

[0625] 2. Berberine Sulfate

[0626] 3. Arecoline

[0627] 4. Pulsatilla

[0628] 5. Matrine

[0629] 6. Oxymatrine

[0630] 7. Andrographolide

[0631] 8. Tetrandrine

[0632] 9. Baicalin

[0633] Materials and methods

[0634] This project used the Clinical and Laboratory Standards Institute (CLSI) guidelines to assess MICs following a method adapted from: Wiegland et al. "Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances" Nature Protocols 2008; 3(2): 163-175. Representative strains were selected. The test concentrations of each compound were: 1000, 500, 250, 125, 62.5 μg / ml. The positive control used was tetracycline.

[0635] Table 44 Swine disease strains tested for MIC

[0636]

[0637]

[0638] result

[0639] The MICs of berberine and tetrandrine against all five strains of E. coli causing diarrhea were 125 μg / ml. The results for tetracycline were consistent with the standard results obtained for MIC.

[0640] Clostridium difficile

[0641] Clostridium difficile (CD) is a bacteria that can cause conditions ranging from diarrhea to life-threatening inflammation of the colon. Illness caused by CD most often affects the elderly or those in long-term care facilities and often occurs after antibiotic drug therapy. However, studies show that rates of CD infection are increasing among populations not traditionally considered to be at high risk, such as young and healthy individuals with no history of antibiotic use or exposure to health care facilities. Approximately 500,000 people in the United States develop CD disease each year, and in recent years, CD infections have become more frequent, severe, and difficult to treat with the rise of antimicrobial resistance.

[0642] Some people carry C. difficile in their intestines but never get sick, although they can still spread the infection. Signs and symptoms usually appear within five to ten days of starting antibiotics but may appear as early as the first day or up to two months later. The most common symptoms of mild to moderate CD infection are diarrhea and mild abdominal cramping. In severe cases, people tend to become dehydrated and may need to be hospitalized. The colon becomes inflamed (colitis), and sometimes small patches of raw tissue may form, which may bleed or produce pus.

[0643] The antibiotics most often used to treat CD infections include fluoroquinolones, cephalosporins, penicillin, and clindamycin. Ironically, the standard treatment for CD is another antibiotic. For mild to moderate infections, oral metronidazole is often prescribed, although it is not FDA-approved. For more severe cases, oral vancomycin is prescribed. Fidamicin is another approved option for treating CD, but it is more expensive. Up to 20% of people with CD get sick again. After two or more relapses, further relapse rates increase to 65%. Treatment for CD relapses usually involves vancomycin. Fecal microbiota transplants, or fecal transplants, may be considered, but they are not FDA-approved.

[0644] Therefore, the present disclosure relates to a method of preventing or treating an infectious disease caused by Clostridium bacteria in humans, the method comprising administering berberine alkaloids.

[0645] The present disclosure also contemplates that the berberine alkaloids or animal feed disclosed herein can inhibit spore formation. Overgrowth of spores after antibiotic treatment is considered a problem in humans. Therefore, the present disclosure relates to preventing overgrowth of Clostridium difficile spores after antibiotic treatment by administering the berberine alkaloids or animal feed disclosed herein.

[0646] Example 6 discloses the antimicrobial activity of certain natural compounds against Clostridium.

[0647] Example 6

[0648] Laboratory work

[0649] The natural compounds identified could potentially be used for prevention and treatment of C. difficile. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were tested in vitro. C. perfringens was also tested. The natural compounds tested were:

[0650] 1. Berberine Chloride

[0651] 2. Berberine Sulfate

[0652] 3. Arecoline

[0653] 4. Pulsatilla

[0654] 5. Matrine

[0655] 6. Oxymatrine

[0656] 7. Andrographolide

[0657] 8. Tetrandrine

[0658] 9. Baicalin

[0659] Materials and Methods

[0660] This project adopted the Clinical and Laboratory Standards Institute (CLSI) guidelines. The guidelines were adopted to follow the method of evaluating MIC adapted from: Wiegland et al. "Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances" Nature Protocols 2008; 3 (2): 163-175, and the method of evaluating MBC adapted from: Chen "Novel therapeutic approaches targeting Clostridium difficile", in: Biology Dissertations, Boston (Mass.): Northeastern University, 2014. Representative strains were selected. The test concentrations of each compound were: 1000, 500, 250, 125, 62.5 μg / ml. The positive control used was vancomycin.

[0661] result

[0662] Table 45 Clostridium in vitro results

[0663]

[0664] * Results for vancomycin were consistent with the MIC and MBC criteria.

[0665] Experimental data

[0666] Minimum inhibitory concentration (MIC) determinations were performed on necrotic enteritis strains of Clostridium perfringens and clinical isolates of Clostridium difficile using berberine sulfate as a test agent and vancomycin as an established control. Berberine sulfate was obtained as a natural extract from Sichuan BioFarm Inc with a purity of 98.0%. The MIC of berberine against Clostridium perfringens was 125 μg / ml, but partial inhibition of growth was still seen at a concentration of 62.5 μg / ml, indicating that the true MIC is between these two values.

[0667] The minimum bactericidal concentration (MBC) of berberine against C. perfringens was equal to the MIC (125 ug / ml), at which concentration 100% of viable cells were observed to be killed. The MIC of berberine against C. difficile was found to be 500-1000 ug / ml (variation between replicates). The MBC of berberine against C. difficile was 1000 ug / ml. The MIC and MBC values ​​of berberine against C. difficile were equal to or less than 2-fold dilutions of the values ​​previously studied. Vancomycin MICs were within the expected range for both C. perfringens and C. difficile.

[0668] Example 7

[0669] This study aimed to determine the tissue residues of the naturally occurring plant compound IRP001 chloride (berberine chloride) when administered orally via feed to commercial broiler chicks.

[0670] Summarize

[0671] Broiler chickens received 0.3g / kg or 0.03g / kg IRP001 chloride mixed into feed, or received regular feed without additives (i.e., control group). Treatment was started immediately after the birds were penned into the barn (10 per group), and continued for 35 days. The birds were euthanized on the 35th day to collect tissues, or fed with regular feed for up to 7 days after the 35th day to check the residues after the cleanout period. The other two groups received IRP001 chloride added to feed for 28 days, and IRP001 chloride was added to their feed (i.e., 0.3g IRP001 chloride in 1kg feed or 0.03g IRP001 chloride in 1kg feed) at 0.3g / kg or 0.03g / kg and then before euthanasia and tissue collection, a 14-day cleanout period was applied with regular food.

[0672] IRP001 chloride was extracted from 1 g samples of three muscle tissues collected from each bird (in each case from the breast, thigh and calf). The residual mass of IRP001 chloride was determined using LC-MS / MS. The method allows the detection of IRP001 with a lower limit of 2 ng IRP001 / g tissue. The assay was fully validated during each assay and was shown to have a quantitative accuracy better than ±20% at 5 ng / g tissue. Levels below 2 ng IRP001 / g were found to be within the baseline noise of the assay and below the lower limit of detection (LLOD), i.e., IRP001 could not be detected.

[0673] In one embodiment, the method was optimized so that IRP001 chloride could be detected with certainty at 2 ng / g tissue. The assay was fully validated during each assay run and demonstrated a quantitative precision of better than +20% at 4 ng / g or 5 ng / g tissue. Levels of 1 ng / g tissue or less were found to be within the baseline noise of the assay and below the lower limit of quantitation (LLOQ).

[0674] After 35 days of administration at high IRP001 chloride concentrations, berberine residues were detectable and quantifiable. The mean residue levels (n=3) at high feed additive concentrations without clearance after 35 days of administration were 6.1 ng, 5.5 ng and 11.6 ng per gram of mammary gland, calf and thigh tissues, respectively. There was a clear clearance effect at high feed additive concentrations in all three muscle tissues, reaching levels of approximately 1 ng / g after 4 days of clearance, which is below the LLOQ. In all cases (regardless of clearance), at low concentrations of feed additive, the mean residue levels were below 1 ng / g, which is below the LLOQ.

[0675] Even when measured after feeding 0.3 g IRP001 chloride / kg feed for 35 days without a washout period, all residue levels determined in this study were below the nominal safe residue level of 13 ng / g.

[0676] After high feed additive concentrations, residual levels in the liver were above 13 ng / g in the absence of clearance, but below 13 ng / g after one day of clearance. Given the limited consumption of liver on average in chicks, the levels of IRP001 in the liver are not worrisome.

[0677] Overall, the data suggest that the risk of cancer in chicken meat from chickens fed IRP001 chloride is less than 1 in 1 million at feed additive levels equal to or below 0.3 g berberine / kg feed.

[0678] Berberine levels in chicken muscle (i.e., chicken meat) were below LLOD after administration of 0.03 IRP001 / kg feed or 4 days after washout after administration of 0.3 g IRP001 / kg feed.

[0679] introduce

[0680] Berberine alkaloids, including berberine, are safe. Berberine has been used by humans as a dietary supplement for many years and is available in capsule form from several manufacturers. It is marketed for use once or twice daily, with doses of up to 400 mg berberine chloride per capsule. Furthermore, in experiments leading to the present invention, no adverse reactions or unexpected events were observed in broiler chickens treated with berberine at a dose of 1 g berberine in 1 kg of commercial feed over 42 days (see Example 8).

[0681] As described elsewhere, in the United States, the Food and Drug Administration (FDA) is responsible for approving human and animal drugs and feed additives regulated by the Federal Food, Drug, and Cosmetic Act (FD&C Act).

[0682] The FD&C Act requires that compounds intended for use in food-producing animals be demonstrated to be safe, and that foods produced by animals exposed to these compounds be demonstrated to be safe for human consumption. Specifically, the regulations (21 CFR part 500, subpart E - Regulation of Carcinogenic Compounds for Use in Food-Producing Animals) prohibit the use in food-producing animals of any compound found to induce cancer when ingested by humans or animals unless certain conditions are met (the so-called "diethylstilbestrol (DES) proviso"). Under the DES proviso, the use of a suspected carcinogenic compound is not prohibited if it can be determined by prescribed inspection methods that "residues" of the compound will not be found in food produced by food-producing animals under conditions of use that are reasonably certain to be followed in practice.

[0683] Therefore, if the FDA decides to regulate berberine as a carcinogenic compound, U.S. regulations would prohibit the use of berberine in food-producing animals unless the “no residue” DES restriction applies.

[0684] The term "no residue" means that any residue remaining in edible tissue is at such a low level that it presents an insignificant risk to the consumer. More specifically, insignificant risk is defined as a 1 part per million increase in risk.

[0685] Despite berberine’s documented safety, toxicology studies commissioned by the U.S. government (National Center for Toxicology Research) found potential carcinogenicity in high-dose chronic rodent studies.

[0686] Therefore, to obtain GRAS status, it is necessary to estimate the maximum acceptable residue level of berberine in chicken (taking into account the typical lifetime consumption of chicken). To ensure that the risk of cancer from eating chicken is less than 1 in 1 million, the maximum acceptable residue level is estimated to be 13 ng of berberine per gram of chicken (i.e., breast or leg muscle tissue).

[0687] To investigate whether the disclosed feed additive is safe and suitable for GRAS status at the indicated dosages, appropriate residue trials were conducted. Invetus Pty Ltd was contracted to conduct the trials and collect tissues, and Monash University was contracted to analyse berberine in tissue samples.

[0688] Residual study design

[0689] The protocol for this study using broiler chickens is attached to the Examples as Appendix B. Two concentrations of IRP0001 chloride were studied: 0.3 g / kg feed and 0.03 g / kg feed, representing high and low concentrations of the feed additive, respectively.

[0690] One hundred and eighty birds were divided into 18 pens, each containing ten birds. To represent the typical rearing of broiler chicks, the experimental birds received a diet containing the additive at either a high or low concentration for 35 days. After 35 days, one group of each additive concentration was euthanized for tissue collection (the six largest birds in each pens).

[0691] In order to study whether IRP001 chloride is significantly eliminated (metabolized and excreted) after the feed containing IRP001 chloride is replaced with regular feed, other groups receive IRP001 chloride for 35 days, and then receive regular feed 1, 2, 4 or 7 days before euthanasia and tissue collection. The other two groups receive IRP001 chloride for 28 days, and then receive regular feed for 14 days (i.e., 14 days of removal). The parallel control group is treated with exactly the same treatment method, except that the control birds receive regular feed throughout the study. In all cases, samples are taken from three areas of muscle tissue (breast, upper and lower thighs). Collect samples, freeze and transport for analysis. Table 46 summarizes the study design, which shows the IRP001 concentration and feeding regimen used in each of the 18 groups of birds in the residual study.

[0692] Table 46 Summary of feeding regimen for each group of broiler chickens

[0693]

[0694] NB-control group received regular feed without additives

[0695] Performance of LC-MS / MS analysis

[0696] Details of the assay methods used for tissue extraction and LC-MS / MS are outlined in Appendix A. The berberine assay was initially calibrated in simple solutions and subsequently validated following tissue extraction.

[0697] The berberine peak in tissue samples can be detected at concentrations as low as 2 ng / g of tissue. Due to interference from tissue matrix effects and analyte carryover at 1 ng / g of tissue, it is difficult to quantify IRP001 at this concentration or lower. At 5 ng / g (or 4 ng / g in some cases), the assay can be validated as accurate at ±20% of the true analyte concentration. In the results section, IRP001 levels above 5 ng / g are cited as absolute values, IRP001 levels between 2 and 5 ng / g are considered below the lower limit of quantitation (LLOQ), and outputs with indicated values below 2 ng / g are considered within baseline noise, below the lower limit of detection (LLOD), and thus undetectable.

[0698] In one embodiment, the berberine peak from tissue samples can be detected at concentrations as low as 1 ng / g of tissue. Due to interference from tissue matrix effects and analyte carryover at 1 ng / g of tissue, it is difficult to quantify IRP001 at this concentration or lower. At 5 ng / g (or 4 ng / g in some cases), the assay can be validated as accurate at ±20% of the true analyte concentration. In fact, concentrations less than 2 ng / g can be considered below the lower limit of quantitation (LLOQ). The lower limit of peak detection is 1 - 2 ng / g.

[0699] Results

[0700] Tissue samples from 3 birds per feed additive group were received by the Monash analysis team and analyzed by LC-MS / MS. Individual samples from each control group were assayed.

[0701] Table 47 shows the mean concentration and standard deviation of berberine assayed in each muscle tissue excised from 3 birds per group. One representative from each control group was assayed and these values were found to be effectively zero and were represented as "<LLOD" (below the LLOD), i.e., undetectable, in the results table.

[0702] Generally speaking, breast tissue samples, thigh and shank muscle samples were comparable and although the concentrations determined were low, the data still showed a clear and logical trend. At the low feed additive concentration of 0.03 g / kg of feed, the mean residual levels of berberine were undetectable (i.e., below the LLOD and LLOQ) in all cases, with or without clearance.

[0703] At the higher IRP001 concentration of 0.3 g / kg feed, the mean berberine residues after 35 days were within the quantifiable range. For breast it was 6.1 ± 1.6, for shank it was 5.5 ± 3.0 ng / g, and for thigh tissue it was 11.6 ± 6.6 ng / g. In these two tissues, a gradual clearance was evident. Berberine residues decreased after 1 and 2 days, and the berberine levels were below the LLOD after 4 days.

[0704] Table 47 Residues of IRP001 chloride in muscle tissues

[0705]

[0706]

[0707] NB

[0708] <LLOD = below the limit of detection (i.e., not detectable)

[0709] * Asterisk indicates estimated value <LLOQ (below the limit of quantitation validated)

[0710] Table 48 shows the mean concentration and standard deviation of berberine determined in liver tissues excised from 3 birds per group. One representative from each control group was assayed and these values were found to be virtually zero and are represented as <LLOD, i.e., not detectable, in the results table.

[0711] Table 48 Residues of IRP001 chloride in muscle tissues

[0712]

[0713]

[0714] NB

[0715] <LLOD = below the limit of detection (i.e., not detectable)

[0716] * Asterisk indicates estimated value <LLOQ (below the limit of quantitation validated)

[0717] Conclusion

[0718] All residue levels in muscle tissues (chicken meat) determined in this study were below the nominal safety residue level of 13 ng / g, even when assayed after 35 days of feeding with 0.3 g berberine / kg feed without a withdrawal period. In all cases, the residue levels at the lower IRP001 concentration of 0.03 g / kg feed were determined to be less than 2 ng / g tissue and can be considered not detectable.

[0719] After feeding birds with 0.3g IRP001 / kg feed, the residue level in the liver was above the limit of quantification, which decreased after a 7-day washout period and fell below the limit of quantification after a 14-day washout period. After feeding birds with 0.03g IRP001 / kg feed, the residue level in the liver was below the detection limit before and after washout.

[0720] Appendix A

[0721] Analytical methods

[0722] Berberine was determined by LC-MS / MS using tetrahydropalmitine as an internal standard.

[0723] Preparation of tissue samples

[0724] 1. Cut out approximately 1 g of tissue and weigh into an M tube. Store the tissue in a -20 °C freezer until ready for homogenization.

[0725] 2. For each gram of tissue, add 2 volumes of MilliQ water to the tube.

[0726] 3. Connect the M tube to the GentleMACS homogenizer and run the program method RNA_01_01 (60 seconds) 3 times to ensure complete homogenization of the tissue.

[0727] 4. Dispense the tissue homogenate into Eppendorf tubes in 200 μL aliquots.

[0728] 5. Add 10 μL of internal standard solution to each 200 μL aliquot of tissue homogenate, followed by 600 μL of 100% methanol. Vortex the samples 3 x 10 seconds at maximum setting, then centrifuge at 10,000 rpm for 3 minutes.

[0729] 6. Transfer 100 μL of supernatant to LC vials for analysis.

[0730] Method validation

[0731] 1. The selectivity, linearity, LLOQ, accuracy, precision, recovery, stability and matrix effect of the method were verified.

[0732] 2. Selectivity was evaluated by preparing samples spiked with individual analytes at concentrations up to 500 ng / g, with 5 replicates per sample. Peak signals were compared with calibration standards (spiked with analytes) to ensure there was no interference.

[0733] 3. To evaluate the LLOQ, 5 ng / g and 10 ng / g standards were prepared in 6 replicates. The LLOQ was determined at the lowest concentration of the calibration curve with a precision and accuracy of ≤20%.

[0734] 4. To demonstrate accuracy and precision, four concentration levels were prepared, namely 20, 50, 100 and 500 ng / g (each repeated 5 times). Accuracy was expressed as the deviation (%) from the nominal concentration, and precision was expressed as the relative standard deviation (RSD) of the replicates.

[0735] 5. To assess recovery, prepare matrix recovery samples by extracting blank tissue and then spiking with analyte solution to obtain various concentration levels up to 500 ng / g (5 replicates each). Recovery is defined by the ratio of the mean peak area of ​​the extracted samples to the mean peak area of ​​the matrix recovery samples.

[0736] 6. To evaluate benchtop stability, four concentration levels of 20, 50, 100, and 500 ng / g were prepared, each with five replicates, and allowed to stand at room temperature for 30 minutes prior to extraction. The average peak area was compared to that of the freshly prepared standards.

[0737] 7. To evaluate the matrix effect (ME), 4 concentration levels of 20, 50, 100 and 500 ng / g were prepared in neat solution, and each concentration level was repeated 5 times. ME was defined as the ratio of the average peak area of ​​the recovered samples to the average peak area of ​​the neat standard samples.

[0738] Table 49 LCMS determination conditions

[0739]

[0740]

[0741] Appendix B

[0742] Compliance

[0743] Tissue residue depletion studies were performed according to an agreed protocol utilizing SOPs and good scientific practices.

[0744] Study Design

[0745] a. Experimental Unit: The experimental unit and observation unit are both individual animals. The statistical unit is the treatment group.

[0746] b. Animal model: Feed intake, daily water intake, body weight change, mortality and marker residues in tissues were used as outcome parameters.

[0747] c. Inclusion Criteria: Animals were selected if they met the criteria outlined below.

[0748] d. Exclusion and Removal Criteria: Animals that are deemed by the investigator to be weak, sick, injured, or otherwise unsuitable for inclusion in the study upon receipt will be excluded.

[0749] After selection, animals that may be considered unsuitable for continued study will be removed only with the written consent of the Sponsor or Investigator. The reason for any removal will be fully documented and justified in the original data and study report. Animals removed from the study will receive appropriate veterinary care.

[0750] e. Allocation: Broiler chicks: Upon receipt, one hundred and eighty (180) broiler chicks meeting the inclusion criteria will be randomly allocated to eighteen (18) separate treatment groups of ten (10 birds per group) after removal from the shipping containers. The method of allocation and randomization will be described in the primary data and study report.

[0751] f. Blinding: Not applicable.

[0752] Investigational Veterinary Products (IVP)

[0753] Record all drug product details including batch number, expiry date, receipt and use.

[0754] a. Veterinary product under investigation: IRP001 Cl as 100% IRP001 Cl.

[0755] b. Source: IVP will be provided by the Sponsor.

[0756] c. Storage: IVP should be stored at ambient temperature within the designated temperature zone. Record the storage location and conditions of the IVP.

[0757] d. Security: The sponsor provides an SDS or its equivalent (if any).

[0758] e. Analysis: A Certificate of Analysis (if available) was provided to the IVP.

[0759] f. Medication Disposal: Record the disposition of all remaining IVP.

[0760] treat

[0761] a. Dose calculation: The dose is based on a fixed concentration of IRP001 Cl in the feed (0.03 or 0.1 g / kg IRP001 Cl).

[0762] b. Dose preparation: Powdered IRP001 Cl is incorporated into commercial feed raw material ingredients and then mixed thoroughly in, for example, a "concrete mixer" type equipment to provide the outlined final concentrations in the feed.

[0763] c. Dosing Methods: Study animals were dosed according to the treatment regimen detailed in Table 49 below. During the relevant treatments, the birds were provided with medicated feed ad libitum as their sole source of feed.

[0764] Table 50 Treatment regimen - feed conversion rate

[0765]

[0766] *Euthanasia

[0767] **Note: Medicated feed was withdrawn from Groups 6 and 12 on Day 28 to allow for a 14-day washout period for these groups.

[0768] Timeline of events

[0769] Table 51 Event timeline

[0770]

[0771]

[0772] Test system

[0773] The detailed information of the animals was recorded in the raw data, namely: species, broiler chickens; number, 180; source, commercial (batch of 90); age, one day old.

[0774] Animal Management

[0775] a. Animal Welfare: Research animals are managed similarly with due consideration for their welfare. Research animals are observed and the “Animal Care Record” is completed in accordance with the requirements of the Animal Ethics Committee (AEC).

[0776] b. Health management: If necessary, any routine preventive treatment should be carried out as soon as possible and recorded (product name, batch number, expiration date, dosage, route of administration and date).

[0777] Study animals were observed twice daily at appropriate locations according to standard operating procedures (SOPs) beginning on day 0. Any health issues requiring further investigation were recorded.

[0778] All health problems and adverse events must be reported to the investigator within one business day. Any adverse event that the investigator believes is product related, causes death, is life-threatening, involves a large number of animals, or involves adverse events in humans must be documented and reported to the sponsor and AEC within one business day.

[0779] Routine veterinary care and procedures may be performed and described in the original data. Concomitant medications may be used with prior approval by the Investigator and Sponsor (if relevant) for standard regulatory practices and humane reasons. No treatment similar to IVP was administered. All concomitant medications were recorded to provide identification of the material used (product name, batch number and expiration date), animal ID, reason for use, route of administration, dose and date administered, and included in the original data (trial log) and study report.

[0780] If injury or illness results in euthanasia or death of a research animal, this should be documented and a necropsy performed by a veterinarian when possible. A “necropsy report” including the probable cause of death should be included in the raw data.

[0781] All health problems, adverse events, and animal mortality were included in the study reports, including their relationship to treatment.

[0782] c. House Environment: Treatment groups were housed in dedicated chicken floor houses in two separate and discrete controlled environment rooms within an approved animal facility. One room housed all untreated birds from Groups 13 to 18, inclusive, and the second room housed all treated birds from Groups 1 to 12, inclusive. The floor space of each house was approximately 1.5 m 2 The chickens were raised on litter in accordance with normal commercial practice.

[0783] There were 18 floor pens with 10 birds per pens up to day 49. At the end of the study, the maximum bird weight in each pens was well below the 40kg / m2 maximum recommended for broiler chicks in the Australian Standard of Practice. 2 .

[0784] NOTE - Birds from Groups 13 to 18 inclusive (untreated control animals) were maintained in similar but physically separate isolators from drug treated birds from Groups 1 to 12 to ensure no cross contamination during the study.

[0785] d. Experimental diet: A formulated commercial starter ration followed by a grower ration was administered throughout the study. Copies of the feed bag labels or equivalent showing the feed ingredients were included with the original data.

[0786] e. Feed and water intake: Weigh and record the feed added each day, and calculate the daily feed intake (by treatment group). Measure and record the daily water volume, and calculate the daily water intake (by treatment group).

[0787] f. Animal Disposition: Study animals were humanely euthanized as approved by the AEC and recorded at the intervals listed in the timeline of events (Table 50).

[0788] Study Procedure

[0789] a. Trial Log: Record all planned and unplanned events during the study.

[0790] Effect evaluation

[0791] a. Body weight: Weigh the birds on day 0 (group weight) and on days 7, 14, 21, 28 and 35 - record the weight of each animal. Before and after weighing, check the weighing scale with a calibrated test weight and record. Body weights at the end of the study will be compared between groups to determine the effect of treatment, if any.

[0792] b. Examination: A separate clinical examination is performed on euthanasia at the time of gross pathology and tissue collection. The clinical examination is recorded. Digital still images may be recorded as appropriate.

[0793] c. Observations: The general health of the birds was checked twice daily, usually before 8 am and after 4 pm. Thus, the typical interval between observations was 9 hours during the day and 15 hours overnight. Birds showing abnormal clinical signs were recorded, carefully observed, and euthanized if the investigator considered them to be significantly suffering from abnormal clinical signs (e.g., marked weakness with a low probability of recovery).

[0794] d. Necropsy examination: Between days 35 and 49, all birds were euthanized and necropsied according to the schedule - Table 14.

[0795] e. Gross Pathology: All birds from all Groups 1 to 18 were necropsied and examined for gross visual pathological changes which were described and scored (per individual bird) as appropriate.

[0796] f. Tissue Residue Analysis: Duplicate representative samples of liver, kidney, breast muscle (1), leg muscle (2) [upper and lower thigh] and whole skin with intact fat were collected from the six (6) heaviest birds in each group (Groups 1 to 18 inclusive) and stored frozen (<10 degrees Celsius) for subsequent marker residue analysis as per the schedule, Table 50. Birds from Groups 13 to 18 were sacrificed on day 35 as untreated control birds and tissues were collected to meet tissue assay requirements.

[0797] Samples will be labeled with adhesive labels listing the study number, animal ID, time point, date, sample type, and replicate.

[0798] For residue analysis, samples were thawed and a known weight of tissue (approximately 1 g) was homogenized in 2 ml of water. Samples were centrifuged and a known volume of supernatant was removed for analysis by LC-MS / MS.

[0799] Table 52 Analysis Matrix

[0800]

[0801] If analysis is required for assay validation and verification.

[0802] g. Storage, Transfer, and Disposal of Samples: Record the storage, transfer, and disposal of samples. Transport replicate 1 tissue samples frozen on wet ice to the analytical laboratory at the times outlined in Section 10. Transfer samples with the accompanying temperature data logger and chilled water bottles in accordance with the Standard Operating Procedure (SOP). Store replicate 2 tissue samples frozen for a period of 6 months following the last sample collection time point. Beyond this point, they may be discarded at the discretion of the study site unless specifically requested otherwise by the Sponsor representative.

[0803] Statistical analysis

[0804] Methods are documented in the study report.

[0805] Data Recording

[0806] The protocol specifications will supersede the facility SOPs. Study forms may be added or modified as needed during the study without requiring a protocol amendment or deviation.

[0807] a. Protocol Approval: Before the study begins, the protocol is approved and signed by all relevant personnel (see page 1).

[0808] b. Modification / Deviation: A modification is a change or modification to the protocol prior to performing the changed or modified task. The modification must state the reason for the change and have written documented authorization by the sponsor. The modification must be signed by the investigator and the sponsor.

[0809] When a deviation is identified, the Investigator should document, sign, and date the deviation from the protocol or applicable SOP. The impact on the overall outcome or integrity of the study will be evaluated. The Sponsor must be informed of the deviation as soon as practicable.

[0810] All protocol modifications and deviations are recorded accordingly and numbered sequentially based on the date they occurred or were identified.

[0811] c. Document Notes: Document notes are recorded to clarify events or circumstances that might not otherwise be apparent from the raw data. Document notes must be communicated to the sponsor as soon as practicable.

[0812] d. Changes in Research Investigators: Changes in study investigators or other responsible research personnel should be recorded accordingly.

[0813] e. Raw Data: All initial raw data pages are paginated, identified with the study number, signed, and dated by the person making the observations and recording the information.

[0814] f. Correspondence Log: The investigator maintains copies of all correspondence related to the study. Any telephone conversations that result in changes to the study documentation, design, conduct, or report of the study are recorded.

[0815] g. Permits: The research detailed in this protocol should be covered by a governmental agency permit (e.g., APVMA Small Trial Permit).

[0816] Research Report

[0817] The study report is prepared by the investigator or designee. It includes a list of data for each variable measured. The study investigator's compliance statement is included in the study report. The original signed study report with raw data and statistical reports is submitted to the sponsor and filed.

[0818] Example 8

[0819] This study evaluated the safety of IRP001 chloride in broiler chickens by histological examination.

[0820] Summary and Conclusion

[0821] The histological results are shown in Table 53.

[0822] Table 53 Histology

[0823]

[0824] From the above, the cumulative pathology and enteritis scores were equal to or lower than the control treatment 1 group. In summary, all gastrointestinal tract (GIT) histological lesions identified were within normal limits for broiler chicks in a production environment. All liver histological lesions identified were within normal limits for broiler chicks in a production environment.

[0825] Experimental Description and Protocol

[0826] Purpose

[0827] The objective of this study was to examine the general safety of IRP001 chloride in broiler chickens raised to market weight by histological examination.

[0828] Experimental design

[0829] The experiment consisted of the following treatments (1 barn per treatment, Table 54).

[0830] Table 54 Treatment

[0831] TRT Poultry Type Start Day Adding medication to feed G / Kg 1 broiler 0 none 0.0 2 broiler 0 IRP001 0.05 3 broiler 0 IRP001 0.5 4 broiler 0 IRP001 1.0

[0832] Description and management of ground-level housing

[0833] The poultry are kept in an area of ​​4 x 10 = 40 ft2 The barn is built with clean wood shavings as a bedding layer, which is about 4 inches thick. The barn has 5-foot high sides and the bottom 1 1 / 2 feet is solid wood to prevent birds from migrating.

[0834] The temperature of the building was monitored. The environmental conditions (temperature) during the trial were appropriate (optimal) for the age of the animals. Lighting was provided by fluorescent lamps placed above the barns. Each barn was provided with diet ad libitum in a tube feeder. From D0 to D7, feed was also supplied on trays placed on the bedding in each barn. Each barn was provided with water ad libitum from a Plasson drinker.

[0835] Standard floor housing practices were used throughout the experiment. Animals and housing facilities were inspected twice daily to observe and record general health, consistent feed and water supply and temperature, removal of all dead birds, and identification of incidents. Birds found dead during the study were recorded in the daily mortality log and were not replaced. Housing number, date of death, sex, weight, and diagnosis were recorded.

[0836] birds

[0837] Cobb male chicks were obtained on the day of hatching and ten male broiler chicks were placed in each barn. All test animals and any other avian quantifiables were recorded on the animal handling sheet. The birds were sexed at the hatchery. The breeder flock history and vaccination records of the hatchery were recorded. The weights of the birds in the barns were recorded at D0 and 42.

[0838] feed

[0839] All feeds were prepared as crumbles / pellets and fed.

[0840] Recorded quantities of all base feeds and items used to prepare treatment batches. Mixed and bagged each batch of feed separately. Identified study number, mixing date, feed type, and correct treatment number for each bag. Maintained complete records of feed mixes and test item inventories.

[0841] Feed samples

[0842] Collect treatment feed samples (each 150g) and mixed: one sample is taken from the beginning, middle and end of each batch of treatment diet. Samples will be retained until shipped or discarded within two months after the report is submitted.

[0843] Administration schedule

[0844] All weights are per barn. Feed treatment brooder feed from D0 to 21. On D21, weigh unconsumed brooder feed per barn and discard. Send out finisher feed and apply until D35. On D35, weigh unconsumed finisher feed per barn and discard. Feed finisher feed until D42. On D42, weigh unconsumed finisher feed per barn and discard.

[0845] diet

[0846] Dietary details are shown in Tables 55 and 56.

[0847] Table 55 Nutrition

[0848]

[0849]

[0850] The main ingredients used are corn, soy meal and animal by-products.

[0851] Table 56 Ingredients

[0852]

[0853] 1 Vitamin blend providing the following (per kg diet): Thiamine mononitrate, 2.4 mg; Niacin, 44 mg; Riboflavin, 4.4 mg; D-calcium pantothenate, 12 mg; Vitamin B 12 (cobalamin), 12.0μg; pyridoxine HCl, 4.7mg; D-biotin, 0.11mg; folic acid 5.5mg; sodium bisulfite menadione complex, 3.34mg; choline chloride, 220mg; cholecalciferol 27.5ug; trans-retinyl acetate, 1,892ug; all-racemic alpha-tocopheryl acetate, 11mg; ethoxyquin 125mg

[0854] 2 The trace mineral mixture provided the following (per kg diet): manganese (MnSO4·H2O), 60 mg; iron (FeSO4·7H2O), 30 mg; zinc (ZnO), 50 mg; copper (CuSO4·5H2O), 5 mg; iodine (ethylenediamine dihydroiodide), 0.15 mg; selenium (NaSe03), 0.3 mg.

[0855] The basal diets did not contain any probiotic / prebiotic feed additives, NSPases, anticoccidials or antibiotic growth promoters. All diets contained phytase.

[0856] Histological samples

[0857] On the day the study ended (D42), five birds per barn were humanely euthanized and upper, middle, and lower intestinal sections as well as liver lobes were collected and stored in neutral buffered formalin. These samples were shipped for analysis.

[0858] program

[0859] 1. Standard ground animal housekeeping practices were used throughout the experiment. The temperature of the building was monitored. Environmental conditions (temperature) during the experiment were appropriate (optimal) for the age of the animals. Fluorescent lamps placed above the animal house provided lighting. The lighting scheme was 24 hours on from D0 to D14, and then 18 hours on until D42.

[0860] 2. Each barn was provided with diet ad libitum in a tube feeder. From day 1 to day 7, feed was also supplied on trays placed on the litter in each barn.

[0861] 3. Method of application and drinking water: unlimited.

[0862] 4. Environmental control. There is environmental humidity.

[0863] 5. Disease control. No concomitant drug therapy was used during the study.

[0864] 6. Poultry identification. The barn is a unit of measurement. The safety of the barn prevents poultry from migrating.

[0865] 7. During the study, twice daily observations of general flock conditions were recorded. Observations included feed and water availability, temperature control, and any abnormalities. Birds were carefully observed for any abnormal reactions.

[0866] Data Entry and Analysis

[0867] Source data are entered using indelible ink. Entries are legible, signed or initialized, and dated by the person making the observation. Each source data sheet is signed by the person responsible for the data. Any errors or changes to the source data are initialized and dated, and a correction code or statement is added as to the reason for the change.

[0868] Poultry and feed handling

[0869] All birds and feed were buried as per SOP. Disposal records are included in the source data.

[0870] Location of source data

[0871] The original source data sheet and final report are sent to the sponsor. Exact copies of the documents and final report are retained.

[0872] Example 9

[0873] This study measured the anticoccidial efficacy / susceptibility of IRP001 against a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella.

[0874] Experimental design

[0875] The experiment consisted of 72 cages, starting with 8 male chicks. Treatments were replicated in 6 blocks, each randomly assigned within a block of 12 cages. The randomization procedure for housing assignments for treatments and blocks was provided by Southern Poultry Research, Inc. (SPR, Athens, GA 30607), which conducted the study for the sponsor.

[0876] The treatment groups are listed in Table 57.

[0877] Table 57 Treatment Group

[0878] Trt describe 1 No Healing / No Attack 2 No healing / attacks 3 IRP001-0.03g / kg 4 IRP001-0.1g / kg

[0879] manage

[0880] 1. Perform a thorough inspection of the facility to ensure all cages have accessible water and feed in each cage. Maintain building temperatures appropriate for the age of the birds.

[0881] 2. Provide uniform and continuous lighting through fluorescent lamps hung vertically along the wall.

[0882] 3. Provide feed and water without restriction.

[0883] 4. Check cages twice daily. Record observations including feed availability, water, temperature and any abnormalities.

[0884] 5. After removing dead birds from cages, record the cage number, date, bird weight, sex and possible cause of death in the daily mortality record.

[0885] Experimental rations

[0886] An unmedicated commercial brooding ration was formulated to be mixed with a commonly used feed in northern Georgia. This ration (in paste form) was administered ad libitum from the date of chick arrival until the end of the study. The experimental diets were prepared from a uniform basal diet. The quantities of all basal diets and test articles used to prepare the treatment batches were recorded. The treatment diets were mixed to ensure uniform distribution of the test article. The mixer was flushed between medicated treatment diets. The feed was transferred to Building 2 and distributed to cages of the same treatment.

[0887] Weigh feeds issued and kept at DOT 14 and 20.

[0888] Feed samples

[0889] The beginning, middle and end of each treatment diet were collected and mixed to form a composite sample. For each treatment, one sample was taken from the composite and held until the study was completed.

[0890] animal

[0891] Day-of-hatch chicks (Cobb 500) were obtained for the study. Strain, source, and vaccination records were recorded. Upon arrival, chicks were assigned to treatment house cages. Chicks (DOT 0) were divided into groups of 8, weighed, and placed in designated cages. The total number of birds participating in the test was 576. The quantifiable information for all birds was recorded in the source data.

[0892] Birds were weighed at DOT 0, 14 and 20 according to cage.

[0893] Oocyst inoculation

[0894] The coccidia oocyst inoculation procedure is described in the SPR SOP. At DOT 14 of the study, all T1 birds received 1 ml of distilled water via an oral pipette (oral). All other birds received a coccidia inoculum diluted to a volume of 1 ml (oral). The inoculum was a mixture of Eimeria acervulina (100,000 oocysts / bird), Eimeria maxima (50,000 oocysts / bird) and Eimeria tenella (75,000 oocysts / bird).

[0895] Oocysts per gram of fecal material

[0896] At DOT 19, all fecal collection pans were cleaned. At DOT 20, fecal samples were collected from all treatment cages. Feces collected from each cage were mixed thoroughly and prepared for fecal flotation. Each sample was examined for the number of oocysts per gram of fecal material.

[0897] Lesion scoring

[0898] All birds in each cage were scored for lesions at DOT 20. The coccidiosis lesion scoring method of Johnson and Reid (1970) was used to score the infected area of ​​the intestine (Johnson J, Reid WM. "Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chickens" Exp Parasitol. 1970 Aug; 28(1): 30-6).

[0899] Collecting data

[0900] 1. Data collection follows the following schedule:

[0901] DOT 0 issues feed, weighs it by cage and distributes it to the birds.

[0902] Birds were vaccinated against coccidia (except T1) at DOT 14.

[0903] DOT 19 Cleaning fecal tray

[0904] Birds were weighed per cage at DOT 20. Remaining feed was weighed. Fecal material was collected per cage. All birds were scored for coccidiosis lesions.

[0905] 2. Autopsy Record the death weight to determine the possible cause of death.

[0906] Clinical observations were recorded twice daily.

[0907] Handling of test animals and feed

[0908] All birds and remaining feed were buried in SPR pits as per SPR SOP. Disposal records were included in the source data.

[0909] Data analysis

[0910] The mean values ​​of group weight gain, feed consumption, feed conversion ratio, opgs, coccidiosis score and mortality were calculated. The raw data were analyzed according to the SPR standard operating procedure for data analysis. The raw data were analyzed using the STATIX program LSD test. When the ANOVA F value was significant (p≤0.05), a P value of 0.05 was used to separate the means.

[0911] Record

[0912] The final report and original source data sheets are sent to the sponsor. An exact copy is retained by Southern Poultry Research, Inc.

[0913] result

[0914] The results of feed intake, corrected feed conversion ratio (Adj. FCR), weight gain (Wt. Gain), mortality (% Cocci Mort.); lesion scores; and fecal oocyst counts (E. acer, E. maxima, and E. tenella) are shown in Table 58.

[0915] Table 58 Results of Example 9

[0916]

[0917]

[0918] Those skilled in the art will appreciate that, without departing from the scope of the invention as broadly described, various changes and / or modifications may be made to the invention as shown in the specific embodiments. Therefore, the present embodiments should be considered in all aspects as illustrative and not restrictive.

[0919] This application includes the following specific implementation schemes:

[0920] 1. A method for preventing and / or treating an infectious disease in an animal, wherein the method comprises administering a berberine alkaloid to the animal.

[0921] 2. The method according to embodiment 1, wherein the animal is a human.

[0922] 3. The method according to specific embodiment 1, wherein the animal is a non-human animal.

[0923] 4. The method of embodiment 1, wherein the non-human animal is a food-producing animal.

[0924] 5. The method according to embodiment 4, wherein the food producing animal is selected from chickens or pigs.

[0925] 6. The method according to any one of embodiments 1 to 5, wherein the infectious disease is an intestinal disease.

[0926] 7. The method according to any one of embodiments 1 to 6, wherein the infectious disease is caused by a parasite of the genus Eimeria.

[0927] 8. The method according to embodiment 7, wherein the parasite is selected from the group consisting of E. maxima, E. acervuline and E. brunette.

[0928] 9. The method according to any one of embodiments 1 to 6, wherein the infectious disease is caused by antibiotic-resistant parasites of the genus Eimeria.

[0929] 10. The method according to embodiment 9, wherein the antibiotic resistant bacterial parasite is selected from the group consisting of Eimeria maxima, Eimeria acervulina and Eimeria brunneri antibiotic resistant parasites.

[0930] 11. The method according to any one of embodiments 7 to 10, wherein the infectious disease is coccidiosis and the animal is a chicken.

[0931] 12. The method according to any one of embodiments 1 to 6, wherein the infectious disease is caused by a bacterium of the genus Clostridium, wherein the bacterium is C. perfringens.

[0932] 13. The method according to any one of embodiments 1 to 6, wherein the infectious disease is caused by an antibiotic-resistant bacterium of the genus Clostridium, wherein the antibiotic-resistant bacterium is antibiotic-resistant Clostridium perfringens.

[0933] 14. The method of embodiment 12 or embodiment 13, wherein the infectious disease is necrotic enteritis and the animal is a chicken.

[0934] 15. The method according to embodiment 14, wherein the necrotic enteritis is caused by a type A strain of Clostridium perfringens.

[0935] 16. The method according to embodiment 15, wherein the Clostridium perfringens type A strain is Clostridium perfringens type A strain EHE-NE36.

[0936] 17. The method according to embodiment 15, wherein the Clostridium perfringens type A strain is Clostridium perfringens type A strain EHE-NE18.

[0937] 19. The method according to embodiment 14, wherein the necrotic enteritis is caused by a type C strain of Clostridium perfringens.

[0938] 20. The method according to any one of embodiments 14 to 19, wherein the administration is performed via the feed or water of the chicken.

[0939] 21. The method according to embodiment 20, wherein the feed is in the form of crumbles or pellets.

[0940] 22. The method according to embodiment 20 or embodiment 21, wherein the berberine alkaloid is administered in the chicken's feed at a dose of 0.001 g / kg to 2 g / kg feed.

[0941] 23. The method according to embodiment 20, wherein the berberine alkaloid is administered in the feed of the chicken at a dosage of 0.001 g / L to 0.1 g / L water.

[0942] 24. The method of any one of Embodiment 11 or Embodiments 14 to 23, wherein the lesion score is reduced and / or the fecal oocyst count is decreased.

[0943] 25. The method according to any one of embodiments 14 to 24, wherein the morbidity is reduced.

[0944] 26. The method according to any one of Embodiment 11 or Embodiments 14 to 25, wherein the mortality rate is reduced.

[0945] 27. The method of any one of embodiments 14 to 26, wherein after the treatment period, a safe residual level of berberine alkaloid is present in the muscle tissue of the chicken.

[0946] 28. The method of embodiment 27, wherein the residual level is at least less than about 13 ng berberine alkaloid per gram of muscle tissue.

[0947] 29. The method according to embodiment 27, wherein the residual level is about 10 ng of berberine alkaloid per gram of muscle tissue.

[0948] 30. The method of embodiment 27, wherein the residual level is about 5 ng berberine alkaloid per gram of muscle tissue.

[0949] 31. The method of embodiment 27 or embodiment 28, wherein the berberine alkaloid is administered in the chicken's feed at a dose of about 0.3 g / kg.

[0950] 32. The method according to embodiment 31, wherein the residual level of berberine alkaloids in the muscle tissue of the chicken is as follows:

[0951] About 6.1ng / g in chicken breast muscle tissue;

[0952] About 5.5 ng / g in chicken leg muscle tissue; and

[0953] There are about 11.6ng / g in the muscle tissue of chicken thigh.

[0954] 33. The method according to any one of embodiments 27 to 30, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of about less than 0.1 g / kg.

[0955] 34. The method according to any one of embodiments 27 to 30, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of about 0.03 g / kg.

[0956] 35. The method according to embodiment 34, wherein the residual level of berberine alkaloids in the muscle tissue of the chicken is as follows:

[0957] Less than 2ng / g in chicken breast muscle tissue;

[0958] Less than 2 ng / g in chicken leg muscle tissue; and

[0959] The muscle tissue of chicken thigh is lower than 2ng / g.

[0960] 36. The method of any one of embodiments 14 to 26, wherein after the treatment period and washout period, there is a safe residual level of berberine alkaloid in the muscle tissue of the chicken.

[0961] 37. The method of embodiment 36, wherein the washout period is a period between 1 week and 2 weeks.

[0962] 38. The method of embodiment 36, wherein the clearance period is selected from the following periods: between 1 day and 14 days; between 1 day and 7 days; between 1 day and 4 days; and between 1 day and 2 days.

[0963] 39. The method of embodiment 36, wherein the clearance period is a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.

[0964] 40. The method of embodiment 39, wherein after a washout period of 1 day, the residual level of berberine alkaloids in the muscle tissue of the chicken is as follows:

[0965] Chicken breast muscle tissue contains about 5.7 ng / g;

[0966] About 3.2 ng / g in chicken leg muscle tissue; and

[0967] About 6.0ng / g in chicken thigh muscle tissue.

[0968] 41. The method of embodiment 39, wherein after a 2-day washout period, the residual level of berberine alkaloids in the muscle tissue of the chicken is as follows:

[0969] About 3.6ng / g in chicken breast muscle tissue;

[0970] About 3.1 ng / g in chicken leg muscle tissue; and

[0971] About 4.5ng / g in chicken thigh muscle tissue.

[0972] 42. The method of embodiment 39, wherein the residual level of berberine alkaloids in the muscle tissue of the chicken after a washout period of 4, 7 and 14 days is less than 2 ng / g.

[0973] 43. The method according to any one of embodiments 36 to 42, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of about 0.3 g / kg.

[0974] 44. The method of embodiment 36 or embodiment 37, wherein said residual level is at least less than 13 ng of said berberine alkaloid per g of muscle tissue.

[0975] 45. The method of embodiment 36 or 37, wherein the residual level is about 5 ng / g.

[0976] 46. ​​The method according to any one of embodiments 36, 37, 44 or 45, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of greater than about 0.1 g / kg.

[0977] 47. The method of any one of embodiments 14 to 26, wherein after the treatment period, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the chicken.

[0978] 48. The method according to embodiment 47, wherein the residual level of berberine alkaloids in the liver and muscle tissue of the chicken is less than 2 ng / g.

[0979] 49. The method according to embodiment 48, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of about 0.03 g / kg.

[0980] 50. The method of any one of embodiments 14 to 26, wherein after the treatment period and washout period, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the chicken.

[0981] 51. The method of embodiment 50, wherein the washout period is between 1 week and 2 weeks.

[0982] 52. The method of embodiment 50, wherein the clearance period is a period selected from the group consisting of: between 1 day and 14 days; between 1 day and 7 days; between 1 day and 4 days; and between 1 day and 2 days.

[0983] 53. The method of embodiment 50, wherein the clearance period is a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.

[0984] 54. The method of embodiment 53, wherein after a washout period of 1 day, the residual level of berberine alkaloids in the muscle tissue of the chicken is as follows:

[0985] Chicken breast muscle tissue contains about 5.7 ng / g;

[0986] About 3.2 ng / g in chicken leg muscle tissue; and

[0987] Chicken thigh muscle tissue contains about 6.0 ng / g.

[0988] The berberine alkaloid residue level in liver tissue of chicken was about 8.0 ng / g.

[0989] 55. The method of embodiment 53, wherein after a 7-day washout period, the residual level of berberine alkaloids in the breast, calf and thigh muscle tissues of the chicken is less than 2 ng / g, and the residual level of berberine alkaloids in the liver tissue of the chicken is about 6.5 ng / g.

[0990] 56. The method of embodiment 53, wherein after a 14-day washout period, the residual level of berberine alkaloids in the breast, calf and thigh muscle tissues of the chicken is less than 2 ng / g, and the residual level of berberine alkaloids in the liver tissue of the chicken is about 3.0 ng / g.

[0991] 57. The method according to any one of embodiments 50 to 56, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of about 0.3 g / kg.

[0992] 58. The method of any one of embodiments 14 to 26, wherein after the treatment period, safe residual levels of berberine alkaloids are present in the liver and muscle tissue of the chicken.

[0993] 59. The method according to embodiment 58, wherein the residual level of berberine alkaloids in the liver tissue and the muscle tissues of the breast, calf and thigh of the chicken is less than 2 ng / g.

[0994] 60. The method of Embodiment 58 or Embodiment 59, wherein the berberine alkaloid is administered in the chicken's feed at a dose of about 0.03 g / kg.

[0995] 61. The method of any one of embodiments 14 to 26, wherein after the treatment period and the washout period, there is a safe residual level of berberine alkaloids in the liver tissue of the chicken.

[0996] 62. The method of embodiment 61, wherein the clearance period is a period selected from between 1 week and 2 weeks.

[0997] 63. The method of embodiment 61, wherein the clearance period is a period selected from the group consisting of: between 1 day and 14 days; between 1 day and 7 days; between 1 day and 4 days; and between 1 day and 2 days.

[0998] 64. The method of embodiment 61, wherein the clearance period is a period selected from 1 day, 2 days, 4 days, 7 days and 14 days.

[0999] 65. The method of embodiment 64, wherein the residual level of berberine alkaloids in the liver tissue of the chicken after a washout period of 1 day is about 8.0 ng / g.

[1000] 66. The method of embodiment 64, wherein the residual level of berberine alkaloids in the liver tissue of the chicken after a 7-day washout period is about 6.5 ng / g.

[1001] 67. The method of embodiment 64, wherein the residual level of berberine alkaloids in the liver tissue of the chicken after a 14-day washout period is about 3.0 ng / g.

[1002] 68. The method according to any one of embodiments 61 to 67, wherein the berberine alkaloid is administered in the feed of the chicken at a dose of about 0.3 g / kg.

[1003] 69. The method according to any one of embodiments 27 to 68, wherein the treatment period is 35 days.

[1004] 70. The method according to any one of embodiments 1 to 69, wherein the berberine alkaloid is berberine hemisulfate.

[1005] 71. The method according to any one of embodiments 1 to 69, wherein the berberine alkaloid is berberine chloride.

[1006] 72. The method according to any of the preceding embodiments, further comprising an additive that masks the bitter taste of the berberine alkaloids.

[1007] 73. An animal feed comprising berberine alkaloid and animal food, wherein the berberine alkaloid is present in an amount of 0.001% w / w to 2% w / w of the animal food.

[1008] 74. The animal feed of embodiment 73, wherein the feed is in the form of a crumble; a pellet; or an aqueous form.

[1009] 75. A dosing regimen comprising administering a berberine alkaloid or an animal feed according to Embodiment 73 or Embodiment 74, wherein the berberine alkaloid or the animal feed is administered for 1 to 6 weeks in an amount effective to prevent or treat an infectious disease in an animal.

[1010] 76. A method for reducing feed conversion ratio in a food producing animal, wherein said method comprises the step of administering to said food producing animal a berberine alkaloid or an animal feed according to Embodiment 73 or Embodiment 74.

[1011] 77. The method of embodiment 76, wherein the food producing animal is disease free.

[1012] 78. The method of embodiment 77, wherein the food producing animal is diseased.

[1013] 79. The method of any one of embodiments 76 to 78, wherein the food producing animal is selected from a chicken or a pig.

[1014] 80. The method of embodiment 79, wherein the food producing animal is a chicken.

[1015] 81. A method for preventing and / or treating infectious diseases in animals, comprising administering an animal feed according to Embodiment 73 or Embodiment 74.

[1016] 82. A method for preventing and / or treating infectious enteric diseases in animals, comprising administering an animal feed according to embodiment 73 or embodiment 74.

[1017] 83. A method for preventing and / or treating an infectious disease caused by Eimeria in an animal, comprising administering an animal feed according to Embodiment 73 or Embodiment 74.

[1018] 84. The method of embodiment 82, wherein the infectious disease is caused by antibiotic-resistant parasites of the genus Eimeria.

[1019] 85. The method of Embodiment 82 or Embodiment 83, wherein the infectious disease is coccidiosis and the animal is a chicken.

[1020] 86. A method for preventing and / or treating an infectious disease in an animal caused by a bacterium of the genus Clostridium, comprising administering an animal feed according to Embodiment 73 or Embodiment 74, wherein the bacterium is Clostridium perfringens.

[1021] 87. The method of embodiment 86, wherein the infectious disease is caused by antibiotic-resistant Clostridium perfringens.

[1022] 88. The method of embodiment 86 or embodiment 87, wherein the infectious disease is necrotic enteritis and the animal is a chicken.

[1023] 89. Use of berberine alkaloids in the preparation of a medicament for preventing and / or treating:

[1024] infectious diseases in animals;

[1025] infectious enteric diseases in animals;

[1026] Infections in animals caused by Eimeria spp.; or

[1027] An infectious disease caused by bacteria of the genus Clostridium, of which Clostridium perfringens is one.

[1028] 90. Use of berberine alkaloids in the prevention and / or treatment of:

[1029] infectious diseases in animals;

[1030] infectious enteric diseases in animals;

[1031] Infections in animals caused by Eimeria spp.; or

[1032] An infectious disease caused by bacteria of the genus Clostridium, of which Clostridium perfringens is one.

[1033] 91. Berberine alkaloids for use in the prevention and / or treatment of:

[1034] infectious diseases in animals;

[1035] infectious enteric diseases in animals;

[1036] Infections in animals caused by Eimeria spp.; or

[1037] An infectious disease caused by bacteria of the genus Clostridium, of which Clostridium perfringens is one.

Claims

1. Use of a granular product in the preparation of an animal feed for reducing feed conversion efficiency in a food producing animal infected or potentially infected with an antibiotic resistant strain of a disease causing microorganism selected from the group consisting of Escherichia coli, Eimeria, Clostridium and Salmonella, said reducing the feed conversion efficiency of the food producing animal comprising administering to the food producing animal said animal feed, said animal feed consisting essentially of: (a) an antimicrobial active ingredient selected from berberine, berberine chloride, berberine hemisulfate, berberine sulfate, berberrubine, bicuculline, tetrahydroafricanine, jatrorrhizine, 13-hydroxyberberine chloride, methoxyberberine chloride, 7,8-dihydro-13-methylberberine, berberine (african pine), 13-benzylberberine and mixtures thereof and (b) corn starch or polyvinylpyrrolidone, wherein the antimicrobial active ingredient has a concentration of 0.0001% to 2% w / w in the animal feed.

2. The use according to claim 1, wherein the animal feed is administered for a treatment period such that the residual level of the antimicrobial active ingredient found in the muscle tissue of the food producing animal is below 13 ng / g muscle tissue.

3. The use according to claim 2, wherein the residual level is less than 10 ng / g muscle tissue.

4. The use according to claim 2, wherein the residual level is less than 2 ng / g muscle tissue after a washout period of 4, 7 and 14 days.

5. The use according to claim 1, wherein the antimicrobial active ingredient is administered to food-producing animals at a dose of 0.03 g / kg to 0.3 g / kg.

6. The use according to claim 1, wherein the antimicrobial active ingredient is administered to food-producing animals at a dose of 0.1 g / kg to 0.3 g / kg.

7. The use according to claim 1, wherein the antimicrobial active ingredient is administered to food-producing animals at a dose of 0.3 g / kg.

8. The use of claim 1, wherein the animal feed is administered for a treatment period of 1 to 6 weeks and in an amount effective to prevent or treat an infectious disease in the food producing animal.

9. The use according to claim 8, wherein the treatment period is 35 days.

10. The use according to claim 1, wherein the active ingredient is berberine hemisulfate.

11. The use according to claim 1, wherein the active ingredient is berberine chloride.

12. The use according to claim 1, wherein the antimicrobial active ingredient has a concentration of 0.001% to 2% w / w in the animal feed.

13. The use according to claim 1, wherein the food producing animal is disease free.

14. The use according to claim 1, wherein the food producing animal is diseased.

15. The use according to claim 1, wherein the food producing animal is a chicken or a pig.

16. Use according to claim 15, wherein the food producing animal is a chicken.

17. The use according to claim 1, wherein the disease-causing microorganism is Eimeria.

18. The use according to claim 17, wherein the animal is a chicken.

19. The use according to claim 1, wherein the disease-causing microorganism is Clostridium perfringens.

20. The use according to claim 19, wherein the animal is a chicken.

21. The use according to claim 1, wherein the antimicrobial active ingredient has a concentration of 0.0001% to 0.2% w / w in the animal feed.

22. Use according to claim 21, wherein the antimicrobial active ingredient has a concentration of 0.001% to 0.2% w / w in the animal feed.

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