Compositions comprising guanidinoacetic acid for treatment and / or prevention of coccidiosis-induced conditions in poultry

Abstract

The present invention relates to a composition for use in the treatment and / or prevention of coccidiosis-induced conditions in poultry, wherein the composition comprises guanidinoacetic acid, guanidinoacetate or a mixture of any of these.

Description

[0001] Necrotic enteritis (NE) is one of the most relevant intestinal diseases in poultry and causes high costs in the poultry industry worldwide (see Skinner et al., Avian Diseases 2010, 54(4): 1237-40). This is a situation that costs 6 billion dollars per year to global poultry production and the disease is on the rise. One reason is the voluntary or legally required reduction of antibiotics in animal production. This trend is driven by an increased incidence of antimicrobial resistance and consumer demand. Another reason is the reduction of ionophores, which in addition to their activity against coccidia, also show efficacy against clostridia. When an anticoccidial live vaccine is used, the application of these ionophores is not possible and clostridia / necrotic enteritis increase (Williams, 2005).

[0002] While this is a problem in all poultry, for broilers, in particular necrotic enteritis and coccidiosis, is a major health problem.

[0003] Necrotic enteritis is a multifactorial disease. While its etiology is under investigation, the main theories are as follows (see Fathima et al., Microorganisms 2022, 10(10): 1958; Abd El-Hak et al., Poultry Science 2022, 101(2): 101590): factors such as coccidiosis or heat stress weaken the intestinal integrity. Coccidiosis is an endemic disease in the commercial broiler industry and is caused by the development and reproduction of parasitic Eimeria species that can infect specific areas of the intestine and cause tissue damage in the intestinal epithelial cells. The destruction of the intestinal epithelial cells leads to impaired absorption of nutrients and increased intestinal permeability (see Teng et al., Poultry Science 2020, 99(9): 4203-4216), which can lead to a high risk of bacterial infection. In addition, the active immunosuppression exploited by the parasite to remain in the host makes the host more susceptible to secondary infections, such as infection by the bird-specific Clostridium perfringens, leading to necrotic enteritis. Eimeria Clostridium perfringens

[0004] ​​Since coccidiosis is usually part of the development of the disease, coccidiostats are used to control Eimeria infections (see Mesa-Pineda et al., Frontiers in Veterinary Science 2021, 8:87653) and antibiotics to control pathogenic bacteria, thus avoiding the development of enteric disease. Similar to antibiotics, chemical anticoccidials or coccidiostats induce the development of resistance in Eimeria species. Therefore, it is recommended to stop their use at least once a year throughout the growing cycle of broilers. In contrast, live vaccines containing attenuated strains of Eimeria species are used. However, vaccination usually carries the risk of mild coccidiosis. Due to the ban on antimicrobial growth promoters and the increase of the “never use antibiotics” movement, the impact of necrotic enteritis has further increased. For this reason, many recent studies on necrotic enteritis have focused on finding different ways to control the disease and understand its pathogenesis. Premixes or technical products that can improve intestinal health or have a direct effect on the infection but are not antibiotics can play an important role in controlling the disease and the negative commercial effects.

[0005] In general, necrotic enteritis occurs in broilers between 2 and 6 weeks of age. In the subclinical form, it is characterized by impaired digestion. The clinical form causes severe problems and increased group mortality in a very short time.

[0006] The clinical form of necrotic enteritis is characterized by acute dark diarrhea, leading to wet litter, and a sudden increase in group mortality, up to 1% per day (Ducatelle and Van Immerseel, 2010) after the first clinical signs appear, sometimes with a total mortality of up to 50% (Van der Sluis, 2013). Birds have ruffled feathers, lethargy and anorexia.

[0007] Autopsies usually show distended small intestine with rough mucosal surface, lesions and brown (diphtheritic) pseudomembrane. There is a large amount of watery, brownish, bloody fluid with a foul odor during the autopsy. The liver is dark in color, swollen, irregular in shape, and the gallbladder is distended (Hofacre et al., 2018).

[0008] In each case of acute necrotic enteritis, the bird can die without showing any preliminary signs.

[0009] When birds have the subclinical form, chronic damage to the intestinal mucosa and an increase in the amount of mucus in the small intestine lead to impaired digestion and absorption of nutrients, resulting in poor growth performance.

[0010] Deteriorated feed conversion and resulting reduced performance become particularly evident at about 35 days of age. Since feed contributes about 65 to 70% of the input costs to produce broiler chickens, poor feed conversion increases production costs and significantly impacts profitability. Often, this subclinical disease remains untreated due to lack of overt symptoms and permanently affects production efficiency.

[0011] Therefore, there is still a need for a method for treating a coccidiosis-induced condition in poultry of any kind. This problem is solved by a composition comprising guanidino acetic acid, a guanidino acetate salt or a mixture of any of these.

[0012] Therefore, it is an object of the present application a composition for treating and / or preventing a coccidiosis-induced condition in poultry, wherein the composition comprises guanidino acetic acid, a guanidino acetate salt or a mixture of any of these.

[0013] The composition is administered to a poultry having a coccidiosis-induced condition.

[0014] The term poultry is used in the context of the present application to denote any kind of domesticated bird, which is captured due to its utility. Examples of poultry are domesticated poultry kept for the production of meat or eggs, including chickens (or broilers and layers), turkeys, geese, quails and ducks. Preferably, in the context of the present application, the term poultry refers to chickens or broilers.

[0015] Supplementation with guanidino acetic acid results in higher creatine availability in all tissue cells, not only in muscle tissue, thus resulting in higher cellular energy availability in all cells. Furthermore, phosphorylated creatine acts as an energy buffer under cellular stress conditions and shifts energy production from glycolysis to the more efficient oxidative phosphorylation pathway. In addition, it results in higher expression of creatine kinase. Thus, supplementation of guanidino acetic acid improves energy digestibility by minimizing energy loss, energy availability for immune responses in immune cells and results in a more favorable metabolic profile in response to Eimeria infection or necrotic enteritis.

[0016] Coccidiosis is caused by the development and reproduction of the parasitic Eimeria species, which can infect specific areas of the intestinal tract and cause tissue damage in the intestinal epithelial cells. In addition, the active immunosuppression exploited by the parasite to remain in the host makes the host more susceptible to secondary infections, such as for example a bird-specific Clostridium perfringens infection, leading to necrotic enteritis. However, the administration of guanidino acetic acid supports poultry growth by improving energy efficiency in poultry, which can be impaired due to parasitic infection or secondarily induced inflammation. In particular, the administration of guanidino acetic acid improves limited mitochondrial energy, which is efficient for the health of birds. Thus, the administration of guanidino acetic acid particularly improves broiler growth and resistance to the multi-factorial necrotic enteritis challenge involving Eimeria and Clostridium perfringens pathogens.

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

[0018] In another embodiment of the composition for use according to the present application, the coccidiosis-induced condition is one or more of bacterial enteritis, necrotic enteritis, diarrhea and lesions.

[0019] Guanidinoacetic acid is soluble in water, but has a rather low solubility in water compared to some amino acids such as glycine or arginine. However, 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 with hydrogen chloride, sulfuric acid and phosphoric acid.

[0020] In another embodiment of the composition for use according to the present application, the salt of guanidinoacetic acid is an acid addition salt.

[0021] In a preferred embodiment of the composition for use according to the present application, the salt of guanidinoacetic acid is guanidinoacetate hydrochloride, guanidinoacetate bisulfate, guanidinoacetate hydrogen phosphate or a mixture of any of these salts.

[0022] In principle, the composition according to the present application is not subject to any restrictions as to the content of guanidinoacetic acid. Rather, the content of guanidinoacetic acid in the composition according to the present application is more or less given by the national or regional registration regulations for feedstuffs.

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

[0024] In addition to guanidinoacetic acid, the composition according to the present application can comprise further components, in particular components that are beneficial to the health of poultry. Effective animal production is a balancing act of nutrition, health and animal welfare, especially when antibiotic use is reduced or eliminated. In this context, it is absolutely essential to ensure intestinal health. Challenges such as disease, environmental factors and feed ingredient quality can have a negative impact on the intestinal microbial balance, leading to slower growth and reduced performance, which in turn results in economic losses. Microbiota imbalance (dysbiosis) and overgrowth of Clostridium perfringens enable the growth of opportunistic bacteria such as E. coli, which can lead to different pathologies. These pathologies, such as diarrhea, wet litter, necrotic enteritis, intestinal leakage (impaired intestinal barrier function), intestinal inflammation (colitis) and adverse immune status ultimately impair the feed efficiency, growth and health of the animals. Therefore, it is beneficial that the composition according to the present application further comprises a probiotic.

[0025] In another embodiment, the composition used according to the present application further comprises a probiotic.

[0026] It was found that probiotics further improved the benefits of the composition according to the present application. In particular, it was found that guanidino acetic acid and additional probiotics synergistically improved the growth and resistance of poultry infected with coccidiosis against multi-factorial necrotic enteritis challenges involving Eimeria spp. and Clostridium perfringens pathogens. Eimeria

[0027] In a preferred embodiment of the composition used according to the present application, the probiotic comprises Bacillus spores.

[0028] A probiotic comprising Bacillus subtilis spores, such as Evonik’s GutCare ® , was found to directly inhibit the bacterial component, i.e. Clostridium perfringens, while stabilizing the microbiome and reducing leaky gut induced bacterial translocation.

[0029] A probiotic comprising Bacillus amyloliquefaciens Bacillus amyloliquefaciens spores, such as Evonik’s EcoBiol ® , improves the health and production conditions of animals and helps producers to improve their product quality, increase the profitability of their business and address sustainability challenges. In detail, feed supplemented with a probiotic comprising Bacillus amyloliquefaciens spores, such as Evonik’s EcoBiol ® , supports the microbial balance in the gut. The probiotic helps to reduce production costs by improving feed conversion rate and reducing slaughter time.

[0030] The composition according to the present application can further comprise one of the two types of probiotics described above, alone or in combination.

[0031] In another preferred embodiment of the composition used according to the present application, the probiotic comprises spores of Bacillus amyloliquefaciens and / or Bacillus subtilis.

[0032] For example, the probiotic comprises a strain selected from Bacillus subtilis (e.g. DSM 32315 or DSM 32540), Bacillus amyloliquefaciens (e.g. CECT 5940) and / or a mixture of any of these.

[0033] ​In principle, the composition according to the present application can be administered to poultry at any, multiple or all stages of rearing the poultry, i.e. at any, multiple or all stages of its life. The life of a laying hen can be divided into a pre-laying period and three production stages or periods, i.e. a pre-laying period, a growing young bird production stage and a laying period, sometimes also referred to as a pre-laying period and a period I to III. The life cycle of a caged broiler can be divided into three stages: a starter stage, a grower stage and / or a finisher stage. For example, the entire life cycle of a chicken can be up to 39 days, wherein day 0 to day 10 is referred to as the starter stage, day 10 to day 21 is referred to as the grower stage and day 21 to day 39 is referred to as the finisher stage. The growing and feeding period of a laying bird also varies. In principle, the administration of the composition of the present application to poultry having a coccidiosis-induced condition, e.g. coccidiosis-infected poultry, is not limited to any particular stage or period in the life cycle of the poultry. Thus, the composition can be administered to poultry having a coccidiosis-induced condition, e.g. coccidiosis-infected poultry, at any possible time point at any, multiple or all stages, i.e. at any, multiple or all stages of the starter stage, the grower stage and / or the finisher stage. Nonetheless, it is preferred that the composition is administered to poultry having a coccidiosis-induced condition, e.g. coccidiosis-infected poultry, during or at the finisher stage.

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

[0035] It was observed that the daily weight gain of coccidiosis-infected birds receiving the composition of the present application was significantly higher than the daily weight gain of coccidiosis-infected birds receiving antibiotics via drinking water, both during the challenge period (from day 16 to day 22) and overall (from day 0 to day 42).

[0036] In another embodiment, the composition for use according to the present application is administered to poultry from the start of the starter period until slaughter or from the start of the grower period until slaughter or any other stage of life.

[0037] During the grower period, coccidiosis-infected birds receiving the composition according to the present application gained about 9 g / day more than the negative control and overall they gained about 2 g / day weight gain more.

[0038] In another embodiment, the composition for use according to the present application is administered to poultry in the grower period.

[0039] In principle, the composition according to the present application is not subjected to a particular state of aggregation or matrix.

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

[0041] When the composition according to the application is a solid composition, it is preferably an effervescent tablet. In this case, the composition comprises 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 the guanidino acetic acid is 1 : 1 or more.

[0042] The composition is particularly suitable for water supply applications, preferably drinking water applications, as the carbon dioxide generating compound and the gas releasing compound react in the presence of water to release carbon dioxide. Thus, the dissolution process of the guanidino acetic acid is greatly accelerated.

[0043] To provide or generate carbon dioxide, preferably the carbon dioxide generating compound is a salt of carbonic acid.

[0044] Thus, in one embodiment of the composition according to the application, the carbon dioxide generating compound is an alkali metal carbonate, an alkaline earth metal carbonate, an ammonium carbonate, an alkali metal bicarbonate, an alkaline earth metal bicarbonate, an ammonium bicarbonate or a mixture of any of these.

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

[0046] In the case of an effervescent tablet, the gas releasing compound and the carbon dioxide generating compound react in the presence of water to generate carbon dioxide. In principle, the underlying reaction is thus an acid-base reaction, the hydronium ion (H3O + ) provided by the gas releasing compound reacts with the carbonate (CO3 2- ) and / or the bicarbonate ion (HCO3 - ), forming carbonic acid (H2CO3). However, the carbonic acid thus released is not thermodynamically stable and readily disintegrates into carbon dioxide and water, which leads to a fizzing effect and thus accelerated dissolution of the guanidino acetic acid.

[0047] To allow the best possible release of carbon dioxide, it is preferred that, in terms of pK a values, the gas releasing compound is a stronger (or even much stronger) acid than the carbon dioxide generating compound, in particular a salt of carbonic acid. This condition is generally met when the gas releasing compound in the composition is an acid. In contrast, the carbonate in the carbonates is a base. Carbonic acid has pka values pK a1 = 6.35 and pK a2 = 10.33. Thus, the pKa value of any gas releasing compound suitable for the composition of the application should be lower than the pKa values of carbonic acid.

[0048] In an embodiment of the composition according to the application, 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 values lower than 6.35. a This requirement is met by a variety of organic acids, such as citric acid (pK a1 = 3.13, pKa2= 4.76 and pKa3= 6.4), tartaric acid (pK a1 = 2.98 and pK a2 = 4.34) and malic acid (pK a1 = 3.46 and pK a2 = 5.10).

[0050] The use of solid organic acids allows to provide the composition according to the application in solid form. The solid form of the composition according to the application is the most concentrated form of said composition and thus requires much less space than the liquid form, i.e. a solution of said composition. Therefore, it is preferred that the composition according to the application is a solid composition.

[0051] In a preferred embodiment of the composition according to the application, the gas releasing compound is thus 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] Thus, in the context of the present application, the term equivalent ratio denotes the ratio of the gas releasing compound to the carbon dioxide generating compound which is required to give one (or more) molecule of carbon dioxide. For example, in case the gas releasing compound is citric acid having 3 carboxylic acid groups and the carbon dioxide generating compound is sodium bicarbonate (NaHC03), 3 equivalents of citric acid react with 3 equivalents of sodium bicarbonate to generate 3 equivalents of carbon dioxide. Here, the equivalent ratio of the gas releasing compound to the carbon dioxide generating compound is 1 : 1, or simply 1. In another example, in case the gas releasing compound is citric acid having 3 carboxylic acid groups and the carbon dioxide generating compound is sodium carbonate (Na2C03), 6 equivalents of citric acid react with 3 equivalents of sodium carbonate to generate 3 equivalents of carbon dioxide. Here, the equivalent ratio of the gas releasing compound to the carbon dioxide generating compound is 2: 1, or simply 2.

[0054] Preferably, in the composition according to the application, the equivalent ratio of the gas releasing compound to the carbon dioxide generating 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, it is preferred that the equivalent ratio of the released gas to the gas generating compound is at least 1.05: 1.

[0056] When the diet according to the present application is a solid matrix, it is preferred that the diet is a poultry diet.

[0057] It is another object of the present application a diet for use in the treatment and / or prevention of a condition induced by coccidiosis in poultry, wherein said diet comprises a composition according to the present application.

[0058] Example: The in vivo study aimed at testing the beneficial effect of GAA on necrotic enteritis (NE). The general procedure and details for the 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 3 treatment groups: the first one consisted of a basal diet as negative control (IUC), the second one was a positive control with the same basal diet but birds received antibiotics through the drinking water (20 mg / kg BW / day for 5 days) (ITC), and the last one was the same basal diet with 0.12% (1.2 g / kg) of supplemental guanidinoacetic acid (GAA) but no antibiotics in the drinking water. Feed treatments were applied in all feeding phases (start phase from day 0 to day 16, growing phase from day 16 to day 23 and fattening phase from day 23 to day 42). The following Table 1 summarizes the formulation and nutritional composition of the basal diet.

[0060] Table 1 : Formulation and nutritional composition of the basal diet for the start, growing and fattening phases.

[0061] The NE challenge was performed during the growing phase and all birds of this phase were challenged. The feed for the start and fattening phases was a typical corn-soy-wheat diet for Northern Europe, which is easily digestible and does not contain many anti-nutritional factors. The start diet included a high proportion of fish meal, wheat bran and rye, resulting in a diet with a high crude protein content. These conditions and raw ingredients are known to be detrimental to gut health. In addition, at day 14 and 16, the broilers received a 10-fold excess of Paracox-8 live Eimeria vaccine by oral administration. The broilers also received a Clostridium perfringens inoculation at each of the days from day 18 to 21.

[0062] Each treatment was replicated in 12 pens, with 18 male Ross 308 broilers each in a completely randomized block design. The feed formulation of the diets was based on the requirements of Ross 308 broilers for energy, amino acids and macro minerals. Diets were provided as crumbles for the starter phase and as pellets for the grower and finisher phase. Throughout the experiment, the chickens were fed ad libitum and had free access to water. The birds were vaccinated against Newcastle disease in the hatchery. No other commercial vaccines were provided during the study.

[0063] On days 21 and 22, four chickens per pen were euthanized for intestinal lesion scoring (adapted from Timbermont et al., Avian Pathology 2010, 39: 117-221). The same chickens were scored for typical lesions of coccidiosis according to the method of Johnson and Reid (Johnson and Reid, Experimental Parasitology 1970, 28(1): 30-36), with scores ranging from 0 (no lesion) to 4 (severe lesion) for the species of chicken relevant 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 treatments with the untreated challenge control (UTC). The table below summarizes the resulting means and p-values for GAA treatments. Capital letters highlight significant differences with the IUC, as well as significant differences of antibiotic treatments with the IUC.

[0065] Table 1: Summary of growth performance parameters and corresponding p-values for all treatments.

[0066] Table 1 summarizes the growth performance parameters of broilers for all treatments and all feeding periods, including p-values. In general, infected antibiotic-treated birds (ITC) performed better than infected untreated birds (UTC). The large differences in body weight (BW), daily weight gain (DWG), and feed conversion ratio (FCR) indicate that antibiotics are an effective treatment for NE in broilers. During the challenge period from d 16 to d 22, the daily weight gain (DWG) of GAA-supplemented birds was significantly higher than the DWG of IUC birds. During the growth phase, GAA-supplemented birds gained about 9 g / day more than the negative control, and overall, they gained about 2 g / day more. GAA-treated birds had no significant effect on daily feed intake (DFI) at any period compared to untreated birds, except for a small effect in the pre-challenge period. Due to the higher DWG and similar DFI, the FCR of GAA-treated birds was also significantly lower than the FCR of untreated birds during the challenge period and overall. Finally, the percentage of NE-related mortality was seen to greatly decrease with GAA supplementation and was comparable to the antibiotic-treated control overall.

[0067] Table 2: Summary of intestinal health scores and corresponding p-values for all treatments.

[0068] Table 2 summarizes the effect of treatment on intestinal health scores. The effect of antibiotic treatment on improving NE scores, Eimeria maxima lesions, and total lesion score (TMLS) was clear and strong compared to untreated controls at d 21. GAA-supplemented birds also had lower amounts of E. maxima and coccidiosis lesions at d 21 and lower NE scores and lower percentage of birds affected by NE at d 22, similar to antibiotic treatment.

[0069] From these results, it can be concluded that GAA supplementation helps birds to use energy from feed more effectively to combat NE infection, potentially in a more efficient way to support the energy- demanding immune response. In particular, this results in a lower mortality of NE, which also increases overall profitability under real-world conditions. In addition, weight gain and feed conversion ratio are improved, while feed intake is not affected. In summary, GAA supplementation supports broiler health during NE challenge and can be a cost-effective and animal welfare-supporting solution, especially in commercial “never use antibiotics” farms.

Claims

1. A composition for use in the treatment and / or prevention of a coccidiosis-induced condition in poultry, wherein the composition comprises guanidino acetic acid, a salt of guanidino acetic acid or a mixture of any of these.

2. The composition for use according to claim 1, wherein the coccidiosis is caused by Eimeria ( Eimeria spp ) and / or Clostridium perfringens ( Clostridium perfringens ).

3. The composition for use according to claim 1 or 2, wherein the coccidiosis-induced condition is one or more of bacterial enteritis, necrotic enteritis, diarrhoea and lesions.

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

5. The composition for use according to any one of claims 1 to 4, wherein the salt of guanidino acetic acid is guanidino acetic acid hydrochloride, guanidino acetic acid bisulfate, guanidino acetic acid bisphosphate or a mixture of any of these.

6. The composition for use according to any one of claims 1 to 5, wherein the composition comprises up to 1,500 ppm of guanidino acetic acid, a salt of guanidino acetic acid or a mixture of any of these.

7. The composition for use according to any one of claims 1 to 6, further comprising a probiotic.

8. The composition for use according to claim 7, wherein the probiotic comprises Bacillus (… Bacillus spp ) spores.

9. The composition for use according to claim 7 or 8, wherein the probiotic bacteria comprise spores of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) and / or Bacillus subtilis ( Bacillus subtilis ).

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

11. The composition for use according to any one of claims 1 to 10, wherein the composition is administered to poultry starting from the starter phase until slaughter or from the grower phase until slaughter or any other life phase.

12. The composition for use according to any one of claims 1 to 11, wherein the composition is administered to the poultry in the grower phase.

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

14. A diet for use in the treatment and / or prevention of a coccidiosis-induced condition in poultry, wherein the diet comprises a composition according to any one of claims 1 to 13.

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