Feed supplement or feed additive, feed premix or feed for animals of the order carnivora

Abstract

The present invention relates to a probiotic, feed supplement or feed additive, feed premix or feed for animals of the order Carnivora, said probiotic or animal feed comprising a strain of Weizmannia faecalis.

Description

[0001] Biochem Zusatzstoffe Handels- u. Produktionsgesellschaft mbH

[0002] Feed supplement or feed additive, feed premix or feed for animals of the order Carnivora

[0003] Field of the invention

[0004] The present application relates to a probiotic, feed supplement or feed additive, feed premix or feed for animals of the order Carnivora.

[0005] Incorporation by Reference

[0006] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there are any inconsistencies between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended.

[0007] Introduction

[0008] Animals of the order Carnivora typically eat feedstuff having a relatively high bacterial and viral load (raw meat, dead animals, etc). Even Carnivora held as pets (dogs, cats) eat such food at least occasionally, meaning that their digestive tract is exposed to high pathogenic burden. Furthermore, typical carnivore behaviour, like digging for feed in the ground, or sniffing at any conceivable objects, including dead animals or animal feces, exposes these animals to high pathogenic burden. On the other hand, in particular when held as pets, carnivores may suffer from deficiencies of the immune system, caused by monotonous lifestyle, obesity or overprocessed feed.

[0009] These deficiencies, combined with the increased exposure to pathogenic burden. May have an impact on the respective animal’s health, including longevity and overall fitness.

[0010] It is an object of the present invention to provide means and methods to support the health of animals of the order Carnivora.

[0011] It is another object of the present invention to provide means and methods to support the immune system of animals of the order Carnivora.

[0012] It is another object of the present invention to provide means and methods to support a balanced gut microbiota of animals of the order Carnivora.

[0013] It is another object of the present invention to provide means and methods to support the production of intestinal health-beneficial short chain fatty acids of animals of the order Carnivora.

[0014] It is another object of the present invention to provide means and methods to support feed digestion of animals of the order Carnivora.

[0015] It is another object of the present invention, to provide value added feed for animals of the order Carnivora.

[0016] The present invention addresses these issues.

[0017] Brief description of the Figures

[0018] Fig. 1. Impact of Weizmannia on fecal IgA level health-relevant taxa in control group and Weizmannia group. Fig. 2. Impact of Weizmannia on feed digestibility

[0019] Fig. 3: Impact of Weizmannia on short chain fatty acid profileFig. 4: Impact of Weizmannia on health-relevant intestinal bacteria

[0020] Detailed Description of the Invention

[0021] Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include singular and / or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

[0022] It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

[0023] Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.

[0024] According to a first aspect of the invention, a probiotic, feed supplement or feed additive, feed premix or feed for animals of the order Carnivora is provided, said probiotic or animal feed comprising a strain of Weizmannia faecalis. Members of Weizmannia, previously allocated to Bacillus, are lactic acid producing sporeforming bacteria. The type species, Bacillus coagulans, was first identified as a spoilage agent of milk products. Weizmannia can be used in the production lactic acid from cellulose and hemicellulose acid hydrolysate. Weizmannia also produces other commercially important chemicals like cyclodextrins and amylases.

[0025] Generally, probiotics are defined as “Live microorganisms, which when administered in adequate amounts confer a health benefit on the host” (\\ I IO and I \( ). 2< > I ). In animal feed, probiotics are typically used to improve weight gain and feed conversion. Due to their beneficial effect on health and stimulation of growth, probiotics are broadly used particularly for pigs and poultry. That type of formulas may contain one or more selected strains of microorganisms, and depending on the species and age of host animals they may be administered as a powder, suspension, capsules, pellet, gel or paste. They are used periodically or constantly, directly as an additive to feed and premixes. Administration of probiotic strains, both individual and combined, has been reported to have a significant effect on absorption and utilisation of feed, daily increase of body weight and total body weight of various animals, including turkeys, chicken, piglets, sheep, goats, cattle, and horses. An addition of probiotic microorganisms to feed results in improved quantity and quality of milk, meat and eggs.

[0026] As a result, probiotics are well established for being used in commercially relevant livestock, and their use results in improved commercial performance thereof. However, such commercial considerations play a minor role when it comes to animals of the order Carnivora (typically pets, like cats or small digs, or working animals, like shepherd dogs), where weight gain and other parameters of animal mass production are less important, while other factors of animal health (longevity, gut health, immune status, fitness and the like) have higher importance.

[0027] Weizmannia faecalis DSM32016has been used as feed additive for fattening piglets as well as birds, including chicken (EFSA Journal 2020;18(6):6158). It has proven effective in weaned piglets and chickens for fattening at lxlO9CFU / kg complete feed. This conclusion was extended to suckling piglets, and to other birds for fattening and ornamental birds at the same use level. A specific Weizmannia faecalis strain is approved, inter alia, in Germany, as feed additive for piglets as well as birds, under reference no 4b 1900. Generally, piglets as well as birds live on a mixed diet comprising primarily plant-based feed ingredients. The inventors of the present invention have surprisingly shown that a probiotic, feed supplement or feed additive, feed premix or feed comprising a strain of Weizmannia faecalis stimulates the host immune machinery and contribute to a healthy gut environment including a more balanced microbiota in animals of the order Carnivora.

[0028] Not only live animals of the order Carnivora on a completely different diet than piglets and chickens, the former being mainly based on meat and other animal products. Furthermore, the effect shown in Carnivora - namely stimulation of the immune machinery - is completely different form the effect observed in piglets and chickens, where fattening and feed conversion rate (FCR) were improved.

[0029] Furthermore, the as part of their natural behaviour, carnivores typically take up large amounts of bacteria (e.g., by eating raw meat, mice, etc, or by sniffing in detritus and other animals’ feces) - unlike livestock animals, which typically receive sterilized feed. Therefore, a Carnivore’s digestive tract has to tackle higher bacterial burden than that of swine or birds.

[0030] Although Weizmannia faecalis has shown significant improvements in weight gain and feed conversion of livestock animals, this outcome cannot be automatically transferred to other animal species, e.g., belonging to the order Carnivora. The intestine and the associated microbiota and its composition is significantly impacted by host physiology as well as biotic and abiotic environmental factors such as dietary composition, social structure including population density, or medication such as the use of antibiotics (Moeller and Sanders 2020). These factors cause a selective pressure that shapes the microbial composition and activity, which in turn leads to animal specific host-microbe interaction including the effect on the immune system (Hauffe and Barelli 2019)). For example, in contrast to livestock animals which are living in a highly controlled environment including high population densities, household animals facing different environmental interactions and dietary challenges. These assumptions are reinforced by microbial analyses of different animal species and their intestinal microbial composition. For example, whereas the poultry gut is mainly colonized by Lactobacillaceae, Clostridiaceae, Lachnospiraceae, Ruminococcaceae, Enter obacteriaceae, and

[0031] Peptostreptococaceae ((Pourabedin and Zhao 2015)), the pig gut shows dominating taxa such as Prevotellaceae, Ruminococcacea, Lactobacillaceae, Veillonellaceae, Paraprevotellaceae, Streptococcaceae, and Cuccinivibrionaceae , ((Guevarra et al. 2019)). In contrast, the gut microbiota of dogs shows high abundances of Peptococcaceae, Lachnospiraceae, Clostridiaceae, Erysipelotrichaceae, Lactobacillaceae ((Mondo et al. 2020)). The differences in microbial composition and associated host-microbe interactions (including the immune system) between household and livestock animals can be also seen when focusing on the predominant bacterial pathogens. Whereas Clostridium perfringens and Enterococcus cecorum causing severe infections and high mortality rates in poultry ((Lee and Lillehoj 2021)), in swine especially enterotoxic Escherichia coli leading to severe diarrhea and hight mortality rates (Fairbrother et al. 2005). In household animals mainly Campylobacter causes intestinal disturbances and diarrhea ((Sandberg et al. 2002)) - a potential pathogen that does not lead to clear clinical signs in poultry ((Calenge and Beaumont 2012)).

[0032] The inventors have shown that dietary Weizmannia supplementation in dogs enhanced (a) fecal IgA level, (b) nutrition, (c) health-beneficial short chain fatty acids, and (c) fitness-relevant intestinal taxa. Therefore, IFezzzwazzzzza-based probiotics can be considered as feed additives supporting host immune defense against harmful bacteria and virus as well as maintaining a healthy and balanced gut environment in pets.

[0033] The inventors’ findings presented herein indicate that IFc / z / zzaz / z / za-based probiotics can stimulate the host immune machinery and contribute to a healthy gut environment in animals of the order Carnivora.

[0034] This finding has particular importance because, in animals of the order Carnivora, stress can be caused due to interruption of daily routines, vet visits, and new environments, which has the potential to lead to gastrointestinal disorders and disease or immunosuppression that increases the risk for pathogenic invasion (Fan et al. 2022).

[0035] As used herein the term “probiotic” relates to a composition comprising a live microorganism that is meant to be administered to an animal.

[0036] As used herein the terms “feed supplement” and feed additive” relate to products used in animal nutrition for purposes of improving the quality of feed.

[0037] As used herein, the term “feed premix” relates to a mixture of feed additives or mixtures of one or more feed additives with feed materials or water used as carriers. The premix is meant to be added to regular feed, and is not intended for direct feeding to animals.

[0038] According to one embodiment of the invention, the strain is Weizmannia faecalis DSM 32016 (WEIZMANNIA). DSM(Z) stands for “Deutsche Sammlung von Mikroorganismen und Zellkulturen” (German Collection of Microorganisms and Cell Cultures GmbH). Strains archived at the DSMZ receive an accession number “DSMXXXX”.

[0039] According to another aspect of the invention, a method for producing an animal feed premix or feed is provided, comprising adding a strain of Weizmannia faecalis composition to an animal feed premix or feed, wherein the animal feed premix or feed is for animals of the order Carnivora.

[0040] According to another aspect of the invention, a method for feeding an animal of the order Carnivora is provided, said method comprising administering a strain of Weizmannia faecalis or a probiotic, feed supplement or feed additive, feed premix or feed according to the invention to said animal.

[0041] According to another aspect of the invention, a method of modulation the gut microbiome of an animal of the order Carnivora is provided, said method comprising administering a strain of Weizmannia coagulans, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such strain, to said animal.

[0042] According to another aspect of the invention, a method of stimulating or supporting the immune system of an animal of the order Carnivora is provided, said method comprising administering a strain of Weizmannia coagulans, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such strain, to said animal.

[0043] According to another aspect of the invention, a method to support a balanced gut microbiota of animals of the order Carnivora is provided, said method comprising administering a strain of Weizmannia coagulans, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such strain, to said animal.

[0044] According to another aspect of the invention, a method to support the production of intestinal health-beneficial short chain fatty acids in animals of the order Carnivora is provided, said method comprising administering a strain of Weizmannia coagulans, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such strain, to said animal. According to another aspect of the invention, a method to support feed digestion of animals of the order Carnivora is provided, said method comprising administering a strain of Weizmannia coagulans, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such strain, to said animal.

[0045] According to another aspect of the invention, the use of a strain of Weizmannia coagulans, or of a probiotic, feed supplement or feed additive, feed premix or feed comprising such strain, is provided for (the manufacture of a medicament for) the treatment of an animal of the order Carnivora.

[0046] This language is deemed to encompass both the swiss type claim language accepted ins come countries (in this case, brackets are deemed absent) and EPC2000 language (in this case, brackets and content within the brackets is deemed absent).

[0047] According to embodiments of the invention, the animal of the order Carnivora is selected from the group consisting of Canidae and Felidae.

[0048] While the experiments shown herein have been made with dogs (male and female Beagles), there is sufficient plausibility, mainly based on their lifestyle, that the effects demonstrated can be extrapolated to other animals of the order Carnivora, in particular to cats.

[0049] Without being bound to theory, the difference in digestive tracts between herbivores (like birds) and omnivores (like swine) on the one hand side, and animals of the order Carnivora on the other hand side, may explain that in herbivores and omnivores, Weizmannia has been shown to improve fattening and feed conversion rate (FCR). This hypothesis is supported by the experimental results, according to which no weight gain was observed in test animal relative to control animals, while increased health parameters such as anenhanced IgA level, nutrition, short chain fatty acid production, and fitness-relevant intestinal bacteria was demonstrated.

[0050] Therefore, no reasonable expectation of success existed that effects shown in herbivores (like birds) and omnivores (like swine) could be translated to animals of the order Carnivora. At the same time, effects shown in dogs can be translated to other animals of the order Carnivora, due to similar functionality of the digestive tract. According to one embodiment of the invention, the animal of the order Carnivora belongs to the species Canis lupus. According to one embodiment of the invention, the animal of the order Carnivora is a dog.

[0051] According to embodiments of the above discussed different aspects of the invention, Weizmannia faecalis is provided in the form of endospores. A spore is a dormant, tough, and non-reproductive structure produced by some bacteria associated to the family Bacillaceae. In a bacterium’s natural environment, spore formation is usually triggered by suboptimal environmental conditions such as a lack of nutrients or water. During sporulation, bacteria undergo an intricate sequence of cell differentiation resulting in a single, stress-resistant spore that allows a bacterium to survive unfavourable environmental conditions. The inner part of the spore contains of the bacterial DNA, ribosomes and large amounts of dipicolinic acid.

[0052] In vitro, starting from vegetative bacterial cells, spore production can be induced according to methods as described for example in Elisashvilj et al (2019).

[0053] Endospores can withstand harsh environments such as stomach acidity or heat and pressure during pelleting processes. When the environment becomes favorable, like in the animals’ intestine, the spores germinate into active probiotic cells.

[0054] According to embodiments of the invention, Weizmannia faecalis is present in the feed a final concentration of between > 1.0 x 107and < 1.0 xlO11CFU / kg)

[0055] In embodiments, Weizmannia faecalis is present on the feed in a final concentration of > 1 x 107, > 3 x 107, > 5 x 107, > 7 x 107, > 1 x 108, > 3 x 108, > 5 x 108, > 7 x 108, > 1 x 109, > 3 x 109, > 5 x 109or > 7 x 109CFU / kg.

[0056] In embodiments, Weizmannia faecalis is present on the feed in a final concentration of < 7 x 10n, < 5 x 10n, < 3 x 1011, < 1 x 10n, < 7 x 1010, < 5 x 1010, < 3 x 1010, < 1 x IO10, < 7 x 109, < 5 x 109, < 3 x 109or < 1 x 109CFU / kg. In embodiments, Weizmannia faecalis is present on the feed in a final concentration of 1 x 107CFU / kg, 5 x 107CFU / kg, 1 x 108CFU / kg, 5 x 108CFU / kg, 1 x 109CFU / kg, 5 x 109CFU / kg, 1 x 1010CFU / kg, 5 x 1010CFU / kg, 1 x 1011CFU / kg or 5 x 1011CFU / kg.

[0057] As used herein, the unit CFU / kg relates to colony -forming units per kg of complete feedstuff.

[0058] The following table discloses preferred upper and lower limit combinations:

[0059] Table 1: preferred combinations of upper and lower limit

[0060] The feedstuff may have a moisture content of between > 3 and < 30 % w / w.

[0061] In embodiments, the moisture content is > 3 % w / w, > 4 % w / w, > 5 % w / w, > 6 % w / w, > 7 % w / w, > 8 % w / w, > 9 % w / w, > 10 % w / w, > 11 % w / w, > 12 % w / w, > 13 % w / w, > 14 % w / w,

[0062] > 15 % w / w, > 16 % w / w, > 17 % w / w, > 18 % w / w, > 19 % w / w, > 20 % w / w, > 21 % w / w, >

[0063] 22 % w / w, > 23 % w / w, > 24 % w / w, > 25 % w / w, > 26 % w / w, > 27 % w / w, > 28 % w / w or >

[0064] 29 % w / w.

[0065] In embodiments, the moisture content is < 30 % w / w, < 29 % w / w, < 28 % w / w, < 27 % w / w,

[0066] < 26 % w / w, < 25 % w / w, < 24 % w / w, < 23 % w / w, < 22 % w / w, < 21 % w / w, < 20 % w / w, < 19 % w / w, < 18 % w / w, < 17 % w / w, < 16 % w / w, < 15 % w / w, < 14 % w / w, < 13 % w / w, < 12 % w / w, < 11 % w / w, < 10 % w / w, < 9 % w / w, < 8 % w / w, < 7 % w / w, < 6 % w / w, < 5 % w / w or < 4 % w / w.

[0067] In embodiments, the moisture content is 5 + / - 3 % w / w, 10 + / - 3 % w / w, 15 + / - 3 % w / w, 20 + / - 3 % w / w, 25 + / - 3 % w / w or 30 + / - 3 % w / w.

[0068] According to an embodiment of the invention, the feed comprises a final concentration of at least 20 % w / w of protein.

[0069] Examples

[0070] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0071] Example 1

[0072] Taxonomic reclassification of the probiotic strain DSM 32016 to Weizmannia faecalis is attributed to substantial genetic differences, resulting in unique health-related potentials.

[0073] Bacterial species are classified taxonomically based on genetic differences, utilizing methods such as comparative genomics. This classification system aligns closely with the unique characteristics linked to certain modes of actions exhibited by each species. For instance, within the realm of probiotics, this genetic variance between different species dictates specific modes of action. Certain strains may possess genes encoding enzymes crucial for feed digestion, enabling them to ferment dietary fibers or proteins and produce short-chain fatty acids. Conversely, other strains may lack this specific capability. Therefore, understanding these genetic disparities not only helps in (a) taxonomic classification but also in (b) gaining insight into the potential modes of action of different probiotic species that contribute to intestinal health and overall well-being.

[0074] (a) Taxonomic Reclassification of DSM 32016: from Bacillus coasulans to Weizmannia faecalis

[0075] The bacterial species “Bacillus coagulans" has always been considered extraordinary due to its special combination of skills. Therefore, previous taxonomic assignments and related designations never appeared to be quite fitting to classify this multifaceted species. Throughout history, designations such as Lactobacillus sporogenes. Bacillus dexlrolaclicus. Bacillus lhermoacidif icons. Bacillus lhermoacidurans. Bacillus calidolaclis. Lactobacillus cereale. and eventually Bacillus coagulans were utilized, with the latter officially recognized as the species' name.

[0076] Even though the species was officially classified within the genus Bacillus, it was clear that Bacillus coagulans surpassed the typical attributes associated with Bacillus members - suggesting that the similarity with other Bacillus species is lower than expected. To reduce this genetical and physiological heterogeneity in the genus Bacillus a reclassification based on phylogenomic, and comparative genomic analyses was done in 2020. As result, all species not being closely related to Bacillus subtilis were reclassified into new genera, leading to a reassignment of Bacillus coagulans from the genera Bacillus to the new genera Weizmannia (= Weizmannia coagulans).

[0077] Further analysis through Average Nucleotide Identity (ANI) and core genome alignment revealed that the strain DSM 32016 shares a closer genetic relationship (98.550% identity) with a recently discovered bacterium, Weizmannia faecalis, isolated from the intestine of a healthy individual (Kieu et al., 2022), than with the type species Weizmannia coagulans (94.646% identity). Considering the genetic dissimilarity and the species identification threshold set at 95% (EFSA, 2021), it is evident that DSM 32016 belongs to the novel species W. faecalis rather than W. coagulans. Recently, species within the genus Weizmannia underwent another round of reclassification, now assigned to the genus Heyndrickxia. As a result, the currently scientifically recognized species name for DSM 32016 is Heyndrickxia faecalis. while Weizmannia faecalis is accepted as a synonym.

[0078] Table 1. ANI values (%) between DSM 32016 and type strains. Analysis conducted by BIOSAFE, Finland.

[0079] Type Strains Identity to DSM 32016 (%)

[0080] Weizmannia faecalis Marseille-P9853 98.6

[0081] Weizmannia coagulans DSM1 94.6

[0082] Weizmannia acidiproducens DSM 23148 76.1

[0083] Weizmannia ginsengihumi Gsoil 114 70.1

[0084] (b) Unique and health-related potentials of W. faecalis DSM 32016, distinguishing it from typical W. coasulans

[0085] Genetic analysis has revealed several unique genes in the genome of W. faecalis DSM 32016, distinctly setting it apart from typical W. coagulans strains. For instance, the genome of DSM 32016 harbors unique genes related to feed digestibility such as Arabinogalactanase, Alpha- xylosidase, Acetylxylan esterase, Endo-l,4-beta-xylanase, Bifunctional alpha- galactosidase / sucrose kinase, and Oligo-l,6-glucosidase whereas these genes are absent in typical W. coagulans strains like DSM1 (type strain), XZL4, or H-l . This example underscores that genetic disparities exceeding 5% translate into distinct genetic capabilities, potentially influencing health-promoting properties and species-specific host interactions.

[0086] Table 2. Digestive enzymes unique to DSM 32016 and their function related to feed digestibility.

[0087] Digestive Enzyme Function

[0088] Acetylxylan esterase Deacetylation of xylan and xylooligosaccharides

[0089] Alpha-xylosidase Hydrolysis of terminal, non-reducing end of xylan (exo acting)

[0090] Arabinogalactanase Hydrolysis of arabinogalactan and arabinogalactan-proteins Bifunctional alpha-galactosidase / sucrose Hydrolysis of alpha-galactosides derived kinase from sucrose (e.g., raffinose)

[0091] Endo-l,4-beta-xylanase Hydrolysis xylan (endo acting)

[0092] Conclusion:

[0093] The comprehensive findings underscore distinct genetic disparities between DSM 32016 and the type strains of W. coagu Ians, warranting its categorization as a novel species, Weizmannia faecalis. As such, W. faecalis DSM 32016 represents the initial exploration of its kind within animals of the order Carnivora (and Suidae), targeting the support of intestinal health, feed conversion, and immune defense (as well as growth performance).

[0094] Example 2

[0095] Materials and Methods

[0096] Experimental design

[0097] The study was based on 10 adult male and female Beagles from the institute of animal nutrition. The experiment was conducted in a cross-over design with two 35-day feeding periods separated by a 7-day washout period. For both feeding periods dogs were assigned to two treatments, each group was based on 5 dogs (Table 2).

[0098] Table 2. Experimental design and probiotic dosage

[0099] CONTROL WEIZMANNIA

[0100] Dogs (n) 10 10

[0101] Probiotic none 1.0 x 109CFU / kg food

[0102] Feeding Period 1 Dog 1-5 Dog 6-10

[0103] 7-day washout

[0104] Feeding Period 2 Dog 6-10 Dog 1-5

[0105] Experimental diets and procedure

[0106] The diets of both groups were calculated to be iso-nutritive and to meet the nutrient requirements for adult dogs (Table 3). In both experimental feeding phases two basal dry feed diets were provided either lacking (CONTROL) or containing Weizmannia faecalis DSM 32016 (WEIZMANNIA). Table 3. Experimental diet composition

[0107] Ingredient CONTROL WEIZMANNIA

[0108] Rice flour (%) 40 40

[0109] Greave flour (%) 25 25

[0110] Poultry meal - low ash (%) 20 20

[0111] Rapeseed oil (%) 7.8 7.8

[0112] Fiber blend (%) 5.0 5.0

[0113] Premix minerals and vitamins (%) 1.5 1.5

[0114] Titanium dioxide (%) 0.2 0.2

[0115] Calcium carbonate (%) 0.005 0.005

[0116] W.faecalis DSM 32016 (CFU / kg) - 1.0 x 109

[0117] The probiotic was produced and provided by Biochem Zusatzstoffe Handels- und Produktionsges. mbH (Lohne, Germany). Strains were obtained from DSM as vegetative bacterial cells, and spore production was induced according to methods as described for example in Elisashvili et al (2019).

[0118] All final diets were registered and produced in the institute of Animal Nutrition at the Free University of Berlin (Germany). The diets were manufactured and stored in a cool, dry place until needed for feeding. The doghouse was temperature (20°C)-, humidity (50-60%)-, and lighting (12 h per day)-controlled.

[0119] Fecal sampling and analytical methods

[0120] Individual body weight was monitored at day 1, 7, 14, 21, 28, and 35 of each feeding period. Feed intake and individual fecal quality was documented daily. For fecal IgA quantification and microbial analysis, feces were collected individually from day 31 to day 35 of each feeding period and immediately frozen. Fecal IgA quantification was based on an enzyme-linked immunosorbent assay (ELISA). Microbial analysis was conducted by LABOKLIN (Labor fur klinische Diagnostik GmbH, Bad Kissingen, Germany), based on qPCR, and focused on the abundance of health-relevant taxa.

[0121] Apparent fecal digestibility of nutrients and minerals was determined using titanium dioxide (TiO2) as an indigestible marker. The concentration of titanium dioxide (TiO2) in the feed and feces was measured photometrically (Ultrospec 2100 pro photometer, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA).

[0122] All experimental feeds were ground to pass through a 0.25-mm sieve before analysis. Nutrients (CP, crude fat, dry matter, crude fiber, crude ash) and organic matter (OM) were analyzed in the feed and feces according to the VDLUFA method (dry matter: VDLUFA III 3.1; crude protein: VDLUFA III 4.1. 1 modified according to the macro-N determination (vario Max CN); crude fiber: VDLUFA III 6.1.4; ash: VDLUFA III 8.1; crude fat: VDLUFA III 5.1.1).

[0123] An Agilent 6890 N gas chromatograph equipped with an HP-88 capillary column (60 m x 0.25 mm i.d. with 0.20 pm film thickness, Agilent) and a flame ionization detector was used to determine the fatty acid profile in the dried food residues (DFR).

[0124] Safety, quality, and ethical statements

[0125] The study was conducted in accordance with the German Animal Welfare Act and approved by the State Office for Health and Social Affairs. Furthermore, the study was following the EFSA administrative / technical guidance and Commission Regulation.

[0126] The dogs used in the study were handled in accordance with the European Union Directive 2010 / 63 / EU on the protection of animals used for experimental and other purposes and Commission Recommendation 2007 / 526 / EC on the accommodation and care of animals used for experimental and other scientific purposes. All dogs are observed twice daily for abnormalities, abnormal behavior, and clinical signs of disease. The study investigator was obliged to immediately inform the study sponsor and external monitor of any adverse events.

[0127] Results

[0128] Overall, all dogs were healthy throughout the trial and maintained body weight with only slight differences. The fecal consistency can be representative for the conditions of the gut including a healthy digestion. Dog faeces should be solid and not too watery. In this study, the fecal water content was 2.3% lower of IFc / z / j / a / z / z / a-supplemented animals compared to the control group animals (Fig. l), suggesting a better fecal consistency of animals fed with Weizmannia. Furthermore, immunological analysis indicated that Weizmannia supplementation increases the average concentration of intestinal IgA by 108%, indicating an enhanced immune activity and defense against potentially harmful bacteria and viruses (Fig. 1). The total feed digestibility was improved by 1.1%, whereby especially the fiber digestion was enhanced by Weizmannia (+10.5%). This fermentative utilization of fiber is associated with an increased amount of health-promoting short chain fatty acids. Thus, Weizmannia supplementation enhances the concentration of propionate (+11.4%), butyrate (+1.4%), valerate (+3.6%), and lactate (+177%).

[0129] Furthermore, Weizmannia supplementation enhanced health-beneficial bacteria such as members of Fusobacteria by 2.2% and Clostridium hiranonis by 5.1% (Fig. 1, Fig. 4). Faecalibacterium prausnitzii and members of Blautia where slightly enhanced by 0.3% and 0.9%, respectively, due to probiotic supplementation. In contrast, Escherichia coli was found to be reduced by -6.6% in feces of dogs receiving dietary probiotics (Fig. 1, Fig. 4).

[0130] Discussion

[0131] The present study shows that Weizmannia-based probiotics can enhance intestinal IgA levels, support digestion, as well as stimulate fitness-relevant short chain fatty acid production and associated bacteria in healthy adult dogs. Probiotic-increased IgA levels are an indication of a generally stimulated immune machinery involving the activity and proliferation of immune cells and their bioactive agents such as cytokines (Tezuka and Ohteki 2019). IgA belongs to the first line of defense against infections (Janeway 2001). Furthermore, IgA also inhibits the adhesion of harmful bacteria and viruses to epithelial cells and neutralizes bacterial toxins and viruses (Janeway 2001). Thus, JFezzmawma-enhanced fecal IgA levels suggest an improved immune defense against potential harmful bacteria and viruses in associated animals.

[0132] Microbial analysis indicated an enhanced abundance of Fusobacleriiim. Blautia, and F. prausnitzii. Based on their microbial activities, these taxa are associated with overall fitness. For example, Fusobacterium, Blautia, and F. prausnitzii are using carbohydrates and simple sugars to produce a variety of short chain fatty acids such as butyrate, acetate, and lactate (Liu et al. 2021). Microbial-produced short chain fatty acids have various health beneficial- properties. Butyrate, for example, is well known (a) as energy source for colonocytes, (b) to promote the mucin production and tight junctions, which strengthens the intestinal barrier, (c) for its immune-stimulative and anti-inflammatory properties, and (d) to reduce oxidative stress (Liu et al. 2018). In contrast, acetate can (a) be a co-substrate for butyrate production, (b) increase colonic blood flow and oxygen update (c) act anti-inflammatory, and (d) be an energy source for muscles and neurons (Yehualashet and Yikna 2021). Lactate is known to have a comprehensive impact on various immune cells and processes (Manoharan et al. 2021). In this way lactate can enhance immune function and reduces inflammation, barrier function, increase immune response to viral challenges, or increases resistance against inflammations caused due to endotoxins (Garrote, Abraham and Rumbo 2015). Furthermore, lactate is known to improve the intestinal barrier function, lowers pro-inflammatory cytokine levels, and is an important energy source in brain and heart (Garrote, Abraham and Rumbo 2015; Riske et al. 2017). Additionally, fecal propionate and valerate was increased due to Weizmannia supplementation. Propionate is an important energy source in the liver and plays a crucial role in reducing lipopolysaccharide-induced intestinal permeability (Hosseini et al. 2011; Den Besten et al. 2013), whereas valerate is an amino acid precursor and known as an regulator of intestinal homeostasis (Gao et al. 2022).

[0133] Furthermore, this study indicates the ability of Weizmannia to stimulate the abundance of C. hiranonis. C. hiranonis plays an important role in secondary breakdown of host bile acids such as deoxycholic- and lithocholic acid (Guzior and Quinn 2021). This specific microbial activity is a key mechanism in the enterohepatic host metabolism and, thus fundamental in keeping intestinal balance (Guard et al. 2019). Several studies have indicated that a reduced abundances of C. hiranonis is associated to gut dysbioses and potential intestinal disorders (Pilla et al. 2020).

[0134] E. coli is a commensal mostly harmless inhabitant of a healthy gut in human as well as animals (Ramos et al. 2020). Only specific E. coli strains such as EHEC can cause a cytotoxicity to intestinal epithelial cells and increase gut permeability (Lukyanenko et al. 2011). The abundance of E. coli in this study lays in the normal range observed in healthy dogs (LABOKLIN: 107-l 09CFU / g feces). As commensal intestinal bacterium, E. coli is relevant in contributing to a comprehensive immune stimulation and, thus, modulation of the intestinal immune system (Belkaid and Hand 2014). This sensitive equilibrium between commensal and pathogenic E. coli is strongly dependent on the overall capacity of the gut microbiota to suppress harmful E. coli. Several studies have shown the inhibitory properties of probiotics against pathogenic E. coli strains (Cordonnier et al. 2017), indicating the supportive effect of probiotics to keep intestinal balance. The Weizmannia- wea reduction of E. coli in this study might be an implication of this health-beneficial probiotic mode of action.

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Claims

What is claimed is:

1. A probiotic, feed supplement or feed additive, feed premix or feed for animals of the order Carnivora, said probiotic or animal feed comprising a strain of Weizmannia faecalis.

2. The probiotic, feed supplement or feed additive, feed premix or feed according to claim 1, wherein the strain is Weizmannia faecalis DSM 32016 (WEIZMANNIA).

3. A method of producing an animal feed premix or feed, comprising adding a strain of Weizmannia faecalis to an animal feed premix or feed, wherein the animal feed premix or feed is for animals of the order Carnivora.

4. A method of feeding an animal of the order Carnivora, said method comprising administering a strain of Weizmannia faecalis or a probiotic, feed supplement or feed additive, feed premix or feed comprising such strain, to said animal.

5. A method of modulation the gut microbiome of an animal of the order Carnivora, said method comprising administering a strain of Weizmannia faecalis, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such strain, to said animal.

6. A method of stimulating or supporting the immune system of an animal of the order Carnivora, said method comprising administering a strain of Weizmannia faecalis, or a probiotic, feed supplement or feed additive, feed premix or feed comprising such strain, to said animal.

7. Use of a strain of Weizmannia faecalis, or of a probiotic, feed supplement or feed additive, feed premix or feed comprising such strain, for (the manufacture of a medicament for) the treatment of an animal of the order Carnivora.

8. The probiotic, feed supplement or feed additive, feed premix or feed according to any one of the aforementioned claims wherein the animal is selected from the group consisting of Canidae and Felidae.

9. The probiotic, feed supplement or feed additive, feed premix or feed according to any one of the aforementioned claims, wherein Weizmannia faecalis is provided in the form of endospores.

10. The feed or method according to any one of claims, in which Weizmannia faecalis is present in the feed in a final concentration of between > 1.0 x 107and < 1.0 xlO11CFU / kg.

11. The feed or method according to any one of the aforementioned claims, wherein the feed comprises a final concentration of at least 20 % w / w of protein.

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