Compositions containing guanidinoacetic acid for use in the treatment and / or prevention of coccidiosis-induced symptoms in poultry

Abstract

The present invention relates to a composition for use in the treatment and / or prevention of coccidiosis-induced symptoms in poultry, comprising guanidinoacetic acid, a salt of guanidinoacetic acid, or a mixture thereof.

Description

[Technical Field]

[0001] Necrotizing enterocolitis (NE) is one of the most common intestinal diseases in poultry and represents a high cost to the poultry industry worldwide (see Skinner et al., Avian Diseases 2010, 54(4):1237-40). With an annual loss of US$6 billion in global poultry production, this controllable disease is increasing. One reason is the voluntary or legally required reduction in antibiotic use in animal production. This trend is driven by the increasing emergence of antimicrobial resistance and consumer demand. Another reason is the decline in ionophores, which, in addition to their activity against coccidia, also show efficacy against Clostridium. When using live anticoccidia vaccines, the application of these ionophores is impossible, leading to an increase in Clostridium / necrotizing enterocolitis (Williams, 2005).

[0002] While this is a widespread problem in all poultry, necrotizing enterocolitis and coccidiosis are particularly serious health issues for broilers.

[0003] Necrotizing enterocolitis is a multifactorial disease. Its etiology is under investigation, but the main theories are as follows (see Fathima et al., Microorganisms 2022, 10(10):1958; Abd El-Hack et al., Poultry Science, 2022, 101(2):101590): Factors such as coccidiosis or heat stress weaken intestinal integrity. Coccidiosis is a disease specific to the commercial broiler industry and is caused by the development and proliferation of parasitic Eimeria species that can infect specific areas of the intestine and cause tissue damage to intestinal epithelial cells. Destruction of intestinal epithelial cells leads to malabsorption of nutrients and increased intestinal permeability (see Teng et al., Poultry Science 2020, 99(9):4203-4216) and can lead to a high risk of bacterial infection. Furthermore, active immunosuppression, utilized by parasites and remaining within the host, makes the host more susceptible to secondary infections, such as those caused by avian-specific Clostridium perfringens, which can lead to necrotizing enterocolitis.

[0004] Because coccidiosis is often part of disease development, anticoccidial agents are used to control Eimeria infection (see Mesa-Pineda et al., Frontiers in Veterinary Science 2021, 8:87653), and antibiotics are used to control pathogenic bacteria and avoid the development of intestinal disease. Similar to antibiotics, chemical anticoccidial or anticoccidial agents induce resistance in Eimeria species. Therefore, it is recommended to discontinue their use at least once a year throughout the broiler's growth cycle. Instead, live vaccines containing attenuated strains of Eimeria species are used. However, vaccination often carries a risk of mild coccidiosis. The ban on antimicrobial growth promoters and the rise of the "antibiotic-free" movement further exacerbate the impact of necrotizing enterocolitis. For this purpose, much recent research on necrotizing enterocolitis has focused on finding various ways to control the disease and understand its pathogenesis. Premixes or technological products that can improve gut health or directly affect infections but are not antibiotics can play a crucial role in controlling disease and its negative commercial effects.

[0005] Generally, necrotizing enterocolitis occurs in broiler chickens between 2 and 6 weeks of age. In its subclinical form, it is characterized by digestive disorders. The clinical form leads to serious problems and increased flock mortality in a very short period of time.

[0006] The clinical manifestations of necrotizing enterocolitis are characterized by acute, heavy diarrhea, followed by wet bedding after the first clinical signs appear, and a sudden increase in flock mortality of up to 1% per day (Ducatelle and Van Immerseel, 2010), sometimes totaling 50% mortality (Van der Sluis, 2013). The birds have disheveled feathers, are lethargic, and have anorexia.

[0007] Autopsy typically reveals a distended small intestine with a roughened mucosal surface, lesions, and a brownish (diphtheriae) pseudomembrane. Postmortem examination reveals a large amount of watery, brownish blood-stained fluid and a foul odor. The liver is dark, swollen, and distorted, and the gallbladder is distended (Hofacre et al., 2018).

[0008] In cases of hyperacute necrotizing enterocolitis, birds may die without showing any prior symptoms.

[0009] When birds suffer from a subclinical form of the disease, chronic damage to the intestinal mucosa and increased mucus production in the small intestine lead to impaired digestion and absorption of nutrients, reducing their growth capacity.

[0010] The deterioration of feed conversion and the resulting decline in performance become particularly pronounced around 35 days of age. Since feed accounts for approximately 65-70% of the input costs for producing broiler chickens, insufficient feed conversion increases production costs and significantly impacts profitability. In many cases, this asymptomatic disease remains untreated because there are no clear symptoms, permanently affecting production efficiency.

[0011] Therefore, there was still a need for a method to treat any type of coccidiosis-inducing symptoms in poultry. It was found that this problem could be solved by compositions containing guanidinoacetic acid, salts of guanidinoacetic acid, or mixtures thereof.

[0012] Therefore, one object of the present invention is a composition for use in the treatment and / or prevention of coccidiosis-induced symptoms in poultry, comprising guanidinoacetic acid, a salt of guanidinoacetic acid, or a mixture thereof.

[0013] The aforementioned composition is administered to poultry exhibiting coccidiosis-inducing symptoms.

[0014] In the context of this invention, the term poultry is used to refer to any type of domesticated bird raised for its usefulness. Examples of poultry include chickens (or broilers and laying hens), turkeys, geese, quail, and ducks raised for the production of meat or eggs. Preferably, in the context of this invention, the term poultry refers to chickens or broilers.

[0015] Guanidinoacetate supplementation leads to higher creatine availability in all tissue cells, not just muscle tissue, and therefore higher cellular energy availability in all cells. Furthermore, phosphorylated creatine acts as an energy buffer under cellular stress conditions, shifting energy production from glycolysis to a more efficient oxidative phosphorylation pathway. In addition, it leads to higher expression of creatine kinase. Therefore, guanidinoacetate supplementation improves energy digestibility by minimizing energy loss and energy availability for immune responses in immune cells, resulting in a more favorable metabolic profile in responses to Emelia infection or necrotizing enterocolitis.

[0016] Coccidiosis is caused by the development and proliferation of parasitic Eimeria species, which infect specific areas of the intestines and can cause tissue damage to intestinal epithelial cells. Furthermore, active immunosuppression utilized by the parasite and remaining within the host makes the host more susceptible to secondary infections, such as those caused by avian-specific Clostridium perfringens, which can lead to necrotizing enterocolitis. However, administration of guanidinoacetate supports poultry growth by improving the energy efficiency of poultry that may be impaired due to parasitic infection or secondary induced inflammation. In particular, guanidinoacetate administration improves limited mitochondrial energy, which is highly efficient for bird health. Therefore, guanidinoacetate administration improves growth, particularly in broilers, and resistance to multifactorial necrotizing enterocolitis challenges, including those caused by Eimeria species and Clostridium perfringens pathogens.

[0017] In one embodiment of the composition for use according to the present invention, coccidiosis is caused by Eimeria species and / or Clostridium perfringens.

[0018] In another embodiment of the composition for use according to the present invention, the coccidiosis-inducing symptoms are one or more of bacterial enteritis, necrotizing enterocolitis, diarrhea, and lesions.

[0019] Guanidinoacetic acid is soluble in water, but its solubility in water is considerably lower compared to some amino acids such as glycine or arginine. Nevertheless, the solubility of guanidinoacetic acid in water can be increased by converting the compound into an acid addition salt. Suitable acid addition salts of guanidinoacetic acid can be formed using hydrogen chloride, sulfuric acid, and phosphoric acid.

[0020] In further embodiments of the compositions for use according to the present invention, the salt of guanidinoacetic acid is an acid addition salt.

[0021] In preferred embodiments of the compositions for use according to the present invention, the salt of guanidinoacetic acid is hydrogen chloride guanidinoacetic acid, hydrogen sulfate guanidinoacetic acid, hydrogen phosphate guanidinoacetic acid, or a mixture thereof.

[0022] In principle, the compositions according to the present invention are not subject to any restrictions regarding the guanidinoacetic acid content. Rather, the guanidinoacetic acid content in the compositions according to the present invention is more or less determined by national or regional registration regulations concerning feed ingredients.

[0023] In one embodiment, the composition for use according to the present invention comprises up to 1,500 ppm of guanidinoacetic acid, a salt of guanidinoacetic acid, or a mixture thereof. Preferably, the composition according to the present invention comprises up to 1,200 ppm of guanidinoacetic acid, a salt of guanidinoacetic acid, or a mixture thereof.

[0024] In addition to guanidinoacetic acid, the composition according to the invention may further comprise additional components, particularly components beneficial to the health of poultry. Efficient animal production is an act of balancing nutrition, health, and animal welfare, especially when reducing or eliminating the use of antibiotics. In this regard, reliable intestinal health is essential. Issues such as diseases, environmental factors, and the quality of feed ingredients can negatively affect the intestinal microbial balance, leading to growth retardation and performance decline, and ultimately economic losses. Not only microbial imbalance (dysbiosis), but also the overgrowth of Clostridium perfringens can also enable the growth of opportunistic bacteria such as Escherichia coli, resulting in various disorders. These disorders such as diarrhea, wet litter, necrotic enteritis, leaky gut (intestinal barrier dysfunction), intestinal inflammation (colitis), and poor immune status ultimately impair the feed efficiency, growth, and health of animals. Therefore, it is beneficial for the composition according to the invention to further comprise probiotics.

[0025] In another embodiment, the composition for use according to the invention further comprises probiotics.

[0026] Probiotics have been found to further improve the benefits of the composition according to the invention. In particular, guanidinoacetic acid and additional probiotics have been found to synergistically improve the growth of coccidiosis-infected poultry and the resistance to a multifactorial necrotic enteritis challenge including Eimeria species and Clostridium perfringens pathogens.

[0027] In a preferred embodiment of the composition for use according to the invention, the probiotics comprise spores of Bacillus species.

[0028] Probiotics containing Bacillus subtilis spores, such as Evonik's GutCare®, are thought to directly inhibit bacterial components, namely Clostridium perfringens, while simultaneously stabilizing the microbiome and reducing bacterial migration through leaky gut.

[0029] Probiotics containing Bacillus amyloliquefaciens spores, such as Evonik's EcoBiol®, improve animal health and production conditions, helping producers improve the profitability and quality of their products and address sustainability challenges. Specifically, feed supplemented with probiotics containing Bacillus amyloliquefaciens spores, such as Evonik's EcoBiol®, supports the balance of gut microbiota. These probiotics help reduce production costs through improved feed conversion and shorter time to slaughter.

[0030] The composition according to the present invention may further contain, either alone or in combination, one of the two types of probiotics described above.

[0031] In a more preferred embodiment of the composition for use according to the present invention, the probiotic comprises spores of Bacillus amyloliquefaciens and / or Bacillus subtilis.

[0032] For example, the probiotics include strains selected from B. subtilis (e.g., DSM 32315 or DSM 32540), B. amyloliquefaciens (e.g., CECT 5940), and / or mixtures of any of these.

[0033] In principle, the compositions according to the present invention can be administered to poultry at any, multiple, or all stages of their lifespan. The lifespan of a laying hen can be divided into the pre-laying stage and three production stages or three production periods, namely the initial production stage, the growth and juvenile production stage, and the nesting stage, which are sometimes called the pre-laying stage and stages I-III. The lifespan of birds captured and raised for meat production can be divided into three stages: the initiation stage, the rearing stage, and the finishing stage. For example, the total lifespan of a chicken can be 39 days, of which the days from d-0 to d-10 (d) are called the initiation stage, the days from d-10 to d-21 are called the rearing stage, and the days from d-21 to d-39 are called the finishing stage. Birds raised for egg production also have different growth and feeding periods. In principle, the compositions according to the present invention, administered to poultry exhibiting coccidiosis-inducing symptoms, such as poultry infected with coccidiosis, are not limited to any particular stage or period in the life of the poultry. Therefore, the compositions can be administered to poultry exhibiting coccidiosis-inducing symptoms, such as coccidiosis-infected poultry, at any conceivable point in the course of or between any of the stages, namely the initiation stage, the rearing stage, and / or the finishing stage. Nevertheless, it is preferable to administer the compositions to poultry exhibiting coccidiosis-inducing symptoms, such as coccidiosis-infected poultry, during or between the finishing stage.

[0034] In one embodiment, the composition for use according to the present invention is administered to poultry at any stage, multiple stages, or all stages of its life.

[0035] The daily weight gain of coccidiosis-infected birds treated with the composition according to the present invention was observed to be significantly higher than that of coccidiosis-infected birds treated with antibiotics via drinking water during the challenge period from day 16 to day 22, and throughout the entire challenge period from day 0 to day 42.

[0036] In another embodiment, the composition for use according to the present invention is administered to poultry that have started from the beginning of the initiation period until slaughter, or from the beginning of the rearing period until slaughter, or until any other life stage.

[0037] During the rearing period, coccidiosis-infected birds treated with the composition according to the present invention gained approximately 9 g / day more than negative controls, and an overall gain of approximately 2 g / day.

[0038] In a further embodiment, the composition for use according to the present invention is administered to poultry during the rearing stage.

[0039] In principle, the compositions according to the present invention are not subject to aggregation or specific states of the matrix.

[0040] In one embodiment, the composition for use according to the present invention is a liquid or solid composition.

[0041] When the composition according to the present invention is a solid composition, it is preferably an effervescent tablet. In that case, the composition contains a carbon dioxide generating compound and a gas-releasing compound. Preferably, the equivalent ratio of the gas-releasing compound to the carbon dioxide generating compound is 1:1 or more, and the molar ratio of the carbon dioxide generating compound to guanidinoacetic acid is 1:1 or more.

[0042] This composition is particularly suitable for water supply applications, preferably drinking water applications, because the carbon dioxide-producing compound reacts with the gas-releasing compound in the presence of water to release carbon dioxide. As a result, the dissolution process of guanidinoacetic acid is greatly accelerated.

[0043] For the purpose of providing or producing carbon dioxide, it is preferable that the carbon dioxide-producing compound is a salt of carbonate.

[0044] Therefore, in one embodiment of the composition according to the present invention, the carbon dioxide generating compound is an alkali carbonate, an alkaline earth carbonate, an ammonium carbonate, an alkali bicarbonate, an alkaline earth bicarbonate, an ammonium bicarbonate, or a mixture thereof.

[0045] In a preferred embodiment of the composition according to the invention, the carbon dioxide generating compound is sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, or a mixture of any of these.

[0046] In the case of the effervescent tablet, the gas-releasing compound reacts with the carbon dioxide generating compound in the presence of water to produce carbon dioxide. Thus, in principle, the underlying reaction is an acid-base reaction, and the hydroxonium ions (H3O + ) provided by the gas-releasing compound react with carbonate ions (CO3 2- ) and / or bicarbonate ions (HCO3 - ) under the formation of carbonic acid (H2CO3). However, the carbonic acid thus released is not thermodynamically stable and readily disintegrates into carbon dioxide and water, resulting in a foaming effect and an acceleration of the dissolution of guanidinoacetic acid.

[0047] To allow for the best possible release of carbon dioxide, the gas-releasing compound is preferably a stronger (or even much stronger) acid with respect to the pK a value than the carbon dioxide generating compound, particularly the salts of carbonic acid. This condition is typically met when the gas-releasing compound in the composition is an acid. In comparison, the carbonate in the salts of carbonic acid is a base. Carbonic acid has pKa values of pK a1 = 6.35 and pK a2 = 10.33. Thus, any gas-releasing compound suitable for use in the composition according to the invention should have a pKa value lower than that of carbonic acid.

[0048] In one embodiment of the composition according to the invention, the gas-releasing compound is an acid, such as an inorganic acid, an organic acid, or a mixture of any of these.

[0049] Preferably, the acid has one or more pK a values less than 6.35. This requirement is met by citric acid (pK a1 = 3.13, pKa2 = 4.76, pKa3 = 6.4), tartaric acid (pK a1=2.98, pK a2 =4.34) and malic acid (pK a1 =3.46, pK a2 It is filled with various organic acids such as (=5.10).

[0050] The use of solid organic acids makes it possible to provide the composition according to the present invention in solid form. The solid form of the composition according to the present invention is the most concentrated form of the composition and therefore requires a much smaller space than the liquid form, i.e., the solution of the composition. Therefore, the composition according to the present invention is preferably a solid composition.

[0051] Therefore, in preferred embodiments of the composition according to the present invention, the gas-releasing compound is a solid organic acid.

[0052] Preferably, the gas-releasing compound is citric acid, tartaric acid, malic acid, or a mixture of any of these.

[0053] Therefore, in the context of the present invention, the term equivalent ratio represents the ratio of a gas-releasing compound to a carbon dioxide-producing compound required to yield one (or more) carbon dioxide molecules. For example, if the gas-releasing compound is citric acid having three carboxylic acid groups and the carbon dioxide-producing compound is sodium bicarbonate (NaHCO3), then 3 equivalents of citric acid and 3 equivalents of sodium bicarbonate react to produce 3 equivalents of carbon dioxide. Here, the equivalent ratio of the gas-releasing compound to the carbon dioxide-producing compound is 1:1, or 1. In another example, if the gas-releasing compound is citric acid having three carboxylic acid groups and the carbon dioxide-producing compound is sodium carbonate (Na2CO3), then 6 equivalents of citric acid react with 3 equivalents of sodium carbonate to produce 3 equivalents of carbon dioxide. Here, the equivalent ratio of the gas-releasing compound to the carbon dioxide-producing compound is 2:1, or 2.

[0054] Preferably, in the composition according to the present invention, the equivalent ratio of the gas-releasing compound to the carbon dioxide-producing compound is in the range of 1:1 to 3:1.

[0055] To ensure that carbon dioxide is generated or provided as completely as possible, the equivalent ratio of the released gas to the gas-producing compound is preferably at least 1.05:1.

[0056] When the feed according to the present invention is a solid matrix, it is preferable that the feed is a feed for poultry.

[0057] Another object of the present invention is a diet for use in the treatment and / or prevention of coccidiosis-inducing symptoms in poultry, wherein the diet comprises a composition according to the present invention.

[0058] Examples The in vivo study aimed to test the beneficial effects of GAA on necrotizing enterocolitis (NE). The general procedure and details of the floor-pen study to determine the efficacy of GAA in the prevention and adjuvant treatment of experimentally induced NE in broilers are described below.

[0059] The experimental design consisted of three treatments: the first was a basal diet (IUC) as a negative control; the second was a positive control using the same basal diet, but the birds received antibiotics via drinking water (20 mg / kg BW / day for 5 days) (ITC); and the last was the same basal diet with 0.12% (1.2 g / kg) supplemental guanidinoacetic acid (GAA), but without antibiotics in the drinking water. Feed treatment was applied throughout all rearing periods (initial period from day 0 to 16, growth period from day 16 to 23, and finishing period from day 23 to 42). Table 1 below summarizes the formulation and nutritional composition of the basal diet.

[0060] [Table 1]

[0061] The NE challenge was administered during the rearing period, and all birds in this period were challenged. The feed during the initial and finishing periods was a typical Northern European maize-soybean-wheat diet that was easily digestible and did not contain many anti-nutrient factors. The rearing diet contained a large amount of fish meal, wheat bran, and rye, resulting in a diet with a high crude protein content. These conditions and ingredients are known to interfere with intestinal health. In addition, on days 14 and 16, the broilers were administered orally in a 10-fold excess dose of live Paracox-8 Eimeria vaccine. On days 18-21, the broilers were also inoculated with Clostridium perfringens.

[0062] Each treatment was replicated in 12 pens, each with 18 male Ross 308 broilers in a completely randomized block design. Dietary formulations were based on Ross 308 broiler requirements for energy, amino acids, and macrominerals. The diet was provided as fragments during the initial stage and as pellets during the rearing and finishing stages. The chickens were free-feeding and had free access to water throughout the experiment. The birds were vaccinated against Newcastle disease at the hatchery. No other commercially available vaccines were administered during the study.

[0063] On days 21 and 22, four chickens per stall were euthanized for intestinal lesion scoring (adopted from Timbermont et al., Avian Pathology 2010, 39:117-221). The same chickens were also scored for typical coccidiosis lesions according to the method of Johnson and Reid (Experimental Parasitology 1970, 28(1):30-36), and assigned scores from 0 (no lesions) to 4 (severe lesions) for broiler-related species listed in this scoring system. The chickens in this study were scored for E. acervuline and E. maxima.

[0064] For statistical evaluation, Dunnett's test was used to compare antibiotic and GAA treatment with untreated challenged controls (UTC). The table below summarizes the mean and p-values ​​obtained for GAA treatment. Bold text highlights significant differences compared to IUC for antibiotic treatment as well.

[0065] [Table 2]

[0066] Table 1 summarizes broiler growth performance parameters for all treatments and rearing periods, including p-values. In general, infected birds treated with antibiotics (ITC) performed better than infected, untreated birds (UTC). Significant differences in body weight (BW), daily weight gain (DWG), and feed conversion ratio (FCR) indicate that antibiotics are an efficient and effective treatment for NE in broilers. Daily weight gain (DWG) in birds supplemented with GAA was significantly higher than that of IUC birds during the challenge period from day 16 to day 22. During the rearing period, GAA-supplemented birds gained approximately 9 g / day more than negative controls, and approximately 2 g / day more overall. Except for a small effect in the pre-challenge period, there was no significant effect on daily feed intake (DFI) between GAA-treated birds and untreated birds at any period (p>0.05). Because DWG was higher and DFI was similar, the FCR of GAA-treated birds was significantly lower than that of untreated birds during the challenge period and overall. Finally, overall, the rate of NE-related mortality was found to be significantly reduced with GAA supplementation, comparable to that of antibiotic-treated controls.

[0067] [Table 3]

[0068] Table 2 summarizes the effects of treatment on the intestinal health score. The effect of antibiotic treatment was clear, being potent in improving NE score, E. maxima lesions, and overall coccidiosis lesions (TMLS) compared to untreated controls at day 21. Birds supplemented with GAA also had less E. maxima and coccidiosis lesions at day 21, lower NE scores at day 22, and a lower percentage of birds affected by NE, similar to antibiotic treatment.

[0069] These results suggest that GAA supplementation may help broilers more effectively utilize energy from their feed to combat NE infection and more effectively support the energy-demanding immune response. In particular, this leads to a reduction in mortality from NE, which also increases overall profitability in real-world situations. Furthermore, weight gain and feed conversion ratios improve, while feed intake remains unaffected. In conclusion, GAA supplementation supports the health of broilers during NE challenges and is likely a cost-effective and animal welfare-supporting solution, especially in commercial "antibiotic-free" farms.

Claims

1. A composition for use in the treatment and / or prevention of coccidiosis-inducing symptoms in poultry, the composition comprising guanidinoacetic acid, a salt of guanidinoacetic acid, or a mixture thereof.

2. The composition for use according to claim 1, wherein the coccidiosis is caused by a species of the genus Eimeria and / or Clostridium perfringens.

3. The composition for use according to claim 1 or 2, wherein the coccidiosis-inducing symptoms are one or more of bacterial enteritis, necrotizing enterocolitis, diarrhea, and lesions.

4. The composition for use according to any one of claims 1 to 3, wherein the salt of guanidinoacetic acid is an acid addition salt.

5. The composition for use according to any one of claims 1 to 4, wherein the salt of guanidinoacetic acid is guanidinoacetic acid hydrogen chloride, guanidinoacetic acid hydrogen sulfate, guanidinoacetic acid hydrogen phosphate, or a mixture thereof.

6. The composition for use according to any one of claims 1 to 5, comprising up to 1,500 ppm of guanidinoacetic acid, a salt of guanidinoacetic acid, or a mixture thereof.

7. A composition for use according to any one of claims 1 to 6, further comprising probiotics.

8. The composition for use according to claim 7, wherein the probiotic comprises spores of a species of the genus Bacillus.

9. The composition for use according to claim 7 or 8, wherein the probiotic comprises spores of Bacillus amyloricefaciens and / or Bacillus subtilis.

10. The composition for use according to any one of claims 1 to 9, which is administered to poultry at any stage, multiple stages, or all stages of its life.

11. The composition for use according to any one of claims 1 to 10, which is administered to poultry that has started from the beginning of the initiation period until slaughter, or from the beginning of the rearing period until slaughter, or until any other life stage.

12. The composition is a composition for use according to any one of claims 1 to 11, administered to poultry during the rearing period.

13. The composition for use according to any one of claims 1 to 12, wherein the composition is a liquid or solid composition.

14. A diet comprising the composition described in any one of claims 1 to 13, for use in the treatment and / or prevention of coccidiosis-inducing symptoms in poultry.

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