CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/364,587, filed May 12, 2022, which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] Coccidiosis and necrotic enteritis are infections of the intestines of domesticated animals that often occur simultaneously. Coccidiosis infection is caused by various pathogenic strains of protozoa including those of the genus Eimeria and necrotic enteritis is caused by pathogenic strains of Clostridia bacteria. The bacteria and protozoa are consumed inadvertently into the digestive tract of an animal as oocysts when foraging. Once inside the animal, the sporulated oocysts release sporocysts which proceed through the life cycle of the organism and release the initial infective stage sporozoites. The sporozoites invade the mucosal membranes of the jejunum, ileum, and caecum, where they reproduce un-sporulate oocytes. Their reproductive life cycle can cause mucosal tissue problems, such as imbalances in absorption of both water and electrolytes, and can lead to lesions throughout the intestines, resulting in diarrhea. During the infection, the new population of un-sporulated oocysts are released back into the environment via the animal’s feces.

[0003] Necrotic enteritis can be caused by Clostridia bacteria also consumed inadvertently by consumed by animals during foraging. Clostridia bacteria are a class of gram-positive, sporeforming, anaerobic bacteria that are present in the soil, vegetation, and as part of the normal flora in the gastrointestinal tract of animals. There are nearly 100 species in the genus of Clostridium that have been identified, yet only about two dozen are responsible for causing disease in humans and animals. Pathogenic species of Clostridium are known for producing several detrimental toxins and can be found throughout the gastrointestinal tract of animals resulting in several enteric diseases. Poultry that have prior mucosal damage in the intestine, such as that caused by a coccidiosis infection, can be particularly susceptible to further infection with the opportunistic pathogenic strain of Clostridia, Clostridium perfringens.

[0004] Coinfection with Eimeria and Clostridium is common in poultry populations, causing, among other symptoms, lesions, diarrhea, dehy dration, loss of appetite, weight loss, and death. Various antibiotic and anticoccidial treatments are available to effectively treat necrotic enteritis in poultry, but there is a need to shift away from antibiotic use to limit the generation of drug resistance in bacterial and protozoal populations. The emergence of drug-resistant strains of bacteria and protozoa and various regional regulatory constraints present a challenge to control coccidiosis and necrotic enteritis worldwide. Lack of disease management is an important concern globally due to production losses, increased mortality, increased veterinarian and medication costs, reduced welfare of birds, and food safety concerns. Thus, the need exists in the poultry industry for alternative management and dietary strategies to control coccidiosis and necrotic enteritis infections.

SUMMARY

[0005] In an aspect, the present disclosure provides a feed additive composition including a microbial fermentate product, where the microbial fermentate product is present in the feed additive composition in an amount effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa in an animal. The Clostridium bacteria can include Clostridium perfringens,' and the Eimeria protozoa can include one or more of Eimeria acervulina, Eimeria maxima, or Eimeria tenella. The inhibitory effect can include a direct or indirect inhibition of growth of Clostridium bacteria and a direct or indirect inhibition of the reproduction or shedding of Eimeria protozoa. The inhibitory effect against Clostridium bacteria and Eimeria protozoa can result in one or more beneficial effects for the animal, where the one or more beneficial effects include improved health measures or enhanced performance measures, or a combination thereof. In an aspect, the improved health measures can include reducing one or more of ceca lesion number, ceca lesion severity, liver lesion number, or liver lesion severity. In an aspect, the enhanced performance measures can include an increase in weight, a decrease in feed conversion ratio, an increase in egg production, a decrease in mortality, a decrease in morbidity, or any combination thereof.

[0006] In an aspect, the microbial fermentate product is a bacterial fermentate or a fungal fermentate. In an aspect, the microbial fermentate product is a postbiotic product.

[0007] In the aspects described herein, the microbial fermentate product can include a bacterial or a fungal fermentate of Acetobacter pasteurianus, Acetobacter peroxydans, Acetobacter okinawensis, Acidipropionibacterium acidipropionici, Arxula adeninivorans, Bacillus altitudinis, Bacillus atrophaeus, Bacillus aryabhattai, Bacillus cereus, Bacillus coagulans, Bacillus halotolerans, Bacillus megaterium, Bacillus pumilis, Bacillus safensis. Bacillus subtilis, Bacillus velezensis, Bacillus xiamenensis, Brevibacillus laterosporus, Candida carpophila, Candida fermentati,, Debaryomyces hansenii, Enterococcus mundtii, Kazachstania martiniae, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus pentosus, Lactobacillus pentosus plantarum, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc citreum, Mucor indicus, Paenibacillus taichungensis, Pediococcus acidilactici, Pediococcus pentosaceus, Phaffla rhodozyma, Pichia barkeri, Priceomyces metis sophilus, Rhodotorula graminis, Rhodosporidium toruloides, Saccharomyces cerevisiae, Schwanniomyces vanrijiae, and Weis sella confusa, or any combinations thereof.

[0008] In an aspect, the microbial fermentate product can include a bacterial fermentate of Bacillus megaterium, Lactobacillus lactis, Priceomyces melissophilus, Schwanniomyces vanrijiae, Saccharomyces cerevisiae, or Weissella confusa, or any combination thereof.

[0009] In an aspect, the feed additive compositions herein can include a solid or a liquid. In some aspects, the feed additive compositions herein can include a solid composition in the form of granules, flakes, pellets, powders, tablets, capsules, cubes, crumbles, pastes, gels, or any combination thereof. In some aspects, the feed additive composition can include a liquid spray, a liquid water additive, a liquid drench, or a liquid water dip for application.

[0010] In an aspect, the feed additive compositions herein contain an amount of microbial fermentate product effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa at an inclusion rate falling within a range from 0.01 kg/tonne to 15.0 kg/tonne. [0011] In an aspect, the feed additive compositions herein can be a component of a poultry feed product. The feed additive composition or poultry feed product can be formulated for a chicken or a turkey.

[0012] In an aspect, the microbial fermentate products of the feed additive compositions herein can include one or more molecular components including hpopeptides, polar lipids, isoprenoid-containing species, or combination thereof. In various aspects, the microbial fermentate product can include one or more of lipopeptide NO; gageostatin C; surfactin; neocopiamycin A; [Ile7] -surfactin; fengycin C; SNA-60-367-4; SNA-60-367-9; SNA-60-367-13; N-(3-oxodecanoyl)-L-homoserine lactone; serinolamide C; N-stearoylalanine; L-755807; aspochalasin J; (4E,6Z)-N-(5-ethyl-5-methyl-2-oxofuran-3-yl)-8-(3-heptyloxir an-2-yl)-3- hydroxyocta-4,6-dienamide; mooreamide A; bexarotene; 7-{[(2E,6Z)-3,7,l l-Trimethyl-2,6,10- dodecatrien-l-yl]oxy}-2H-chromen-2-one; diorcinol G, ammoresinol; cattienoid A; bisacremine G; neomarinone; methyl hydroxy-3, 4-dehydro-apo-8'-lycopenoate; kolanone; bacillaene; vicenistatin M; trienomycin B; prumycin; SF-1902-A3; or derivatives or isomers thereof. L0013J In an aspect, the feed additive compositions herein have a shelf life of up to approximately 30 months. In an aspect, the feed additive compositions herein are stable from 18 °C to 45 °C.

[0014] In an aspect, the present disclosure provides a method for feeding poultry the feed additive compositions described herein. In various aspects, the method can include feeding a chicken or a turkey the feed additive compositions described herein.

[0015] In an aspect, the present disclosure provides a method for inhibiting the growth of Clostridium bacteria and reproduction or shedding of Eimeria protozoa in poultry. The method can include administering a feed additive composition including a microbial fermentate product to the poultry. The microbial fermentate product can be present in the feed additive composition in an amount effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa. The Clostridium bacteria can include one or more of Clostridium baratii, Clostridium botulinum, Clostridium colinum, Clostridium difficile, Clostridium fallax, Clostridium perfringens, and Clostridium septicum. The Eimeria protozoa can include one or more of Eimeria acervulina, Eimeria maxima, or Eimeria tenella. The inhibitory' effect can include a direct or indirect inhibition of the growth of Clostridium bacteria and a direct or indirect inhibition of reproduction or shedding of Eimeria protozoa. In an aspect, the inhibitory' effect against Clostridium bacteria and reproduction or shedding of Eimeria protozoa can result in one or more beneficial effects for the poultry. The one or more beneficial effects can include improved health measures or enhanced performance measures, or a combination thereof. In an aspect, the improved health measures can include reducing one or more of ceca lesion number, ceca lesion severity, liver lesion number, or liver lesion severity. In an aspect, the enhanced performance measures can include an increase in weight, a decrease in feed conversion ratio, an increase in egg production, a decrease in mortality, a decrease in morbidity, or any combination thereof. In an aspect, producing the inhibitory effect in poultry fed the feed additive composition is observed relative to poultry fed a diet lacking the feed additive composition.

[0016] In an aspect, the method further can include administering the feed additive composition as a daily feed ration that is fed to the poultry on most days or on all days. The method further can include that the amount of microbial fermentate product effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa includes an inclusion rate falling within a range from 0.01 kg/tonne to 15.0 kg/tonne.

[0017] In an aspect, administering a feed additive composition including a microbial fermentate product includes a feed additive composition including a bacterial fermentate of Acetobacter pasteurianus, Acetobacter peroxydans, Acetobacter okinawensis, Acidipropionibacterium acidipropionici, Arxula adeninivorans, Bacillus altitudinis, Bacillus atrophaeus, Bacillus aryabhattai, Bacillus cereus, Bacillus coagulans, Bacillus halotolerans, Bacillus megaterium, Bacillus pumilis, Bacillus safensis, Bacillus subtilis, Bacillus velezensis, Bacillus xiamenensis, Brevibacillus laterosporus , Candida carpophila, Candida fermentati, Debaryomyces hansenii, Enterococcus mundtii, Kazachstania martiniae, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus pentosus, Lactobacillus pentosus plantarum, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc citreum, Mucor indicus, Paenibacillus taichungensis, Pediococcus acidilactici, Pediococcus pentosaceus, Phaffia rhodozyma, Pichia barkeri, Priceomyces melissophilus, Rhodotorula graminis, Rhodosporidium toruloides, Saccharomyces cerevisiae, Schwanniomyces vanrijiae, and Weissella confusa, or combinations thereof.

[0018] In an aspect, a method for reducing or preventing a coccidiosis and necrotic enteritis coinfection in poultry, including administering a feed additive composition including a microbial fermentate product to the poultry, where the microbial fermentate product is present in the feed additive composition in an amount effective to produce an inhibitory effect against Clostridium perfringens and Eimeria protozoa. The Eimeria protozoa include one or more of Eimeria acervulina, Eimeria maxima, or Eimeria tenella. The inhibitory effect can result in one or more beneficial effects for the poultry. The inhibitory effect can include a direct or indirect inhibition of the growth of Clostridium bacteria and a direct or indirect inhibition of reproduction or shedding of Eimeria protozoa. The one or more beneficial effects can include improved health measures or enhanced performance measures, or a combination thereof. In an aspect, the improved health measures can include reducing one or more of ceca lesion number, ceca lesion severity, liver lesion number, or liver lesion severity. In an aspect, the enhanced performance measures can include an increase in weight, a decrease in feed conversion ratio, an increase in egg production, a decrease in mortality, a decrease in morbidity, or any combination thereof.

[0019] In an aspect, the method further can include challenging the poultry with Clostridium perfringens at a concentration of from 1.0 x 10° to 1.0 x 10 ¹⁰ CFU and Eimeria protozoa from 1.0 * 10° to 1.0 * 10 ¹⁰ sporulated oocysts or sporozoites in a daily ration of poultry feed per poultry. In an aspect, the method further can include where the poultry have acquired an infection with Clostridium perfringens and Eimeria protozoa in their habitat in an amount effective to cause disease in the poultry. L0020J In an aspect, the present disclosure provides a method for enhancing performance measures in poultry. The method can include administering a feed additive composition including a microbial fermentate product to poultry, where the microbial fermentate product is present in the feed additive composition in an amount effective for enhancing performance measures in the poultry. In an aspect, the method includes enhancing performance measures in poultry that are coinfected with Clostridium perfringens and one or more of Eimeria acervulina, Eimeria maxima, or Eimeria tenella. In an aspect, the enhanced performance measures can include an increase in weight, a decrease in feed conversion ratio, an increase in egg production, a decrease in mortality, a decrease in morbidity, or any combination thereof.

[0021] In an aspect, the increase in weight can include an average weight increase of from 0.5% to 30% in a population of poultry fed the feed additive composition as compared to an average weight in a population of poultry fed a diet lacking the feed additive composition. In an aspect, the decrease in feed conversion ratio can include an average decrease of from 0.5% to 10% in the feed conversion ratio of a population of poultry fed the feed additive composition as compared to an average feed conversion ratio in a population of poultry fed a diet lacking the feed additive composition. In an aspect, the increase in egg production can include an average increase of from 0.5% to 25% in the egg production of a population of poultry fed the feed additive composition as compared to a population of poultry fed a diet lacking the feed additive composition. In an aspect, the decrease in mortality can include an average decrease of from 0.5% to 40% in the mortality' of a population of poultry fed the feed additive composition as compared to a population of poultry fed a diet lacking the feed additive composition. In an aspect, the decrease in morbidity can include an average decrease of from 0.5% to 40% in the morbidity of a population of poultry fed the feed additive composition as compared to a population of poultry fed a diet lacking the feed additive composition.

BRIEF DESCRIPTION OF THE FIGURES

[0022] Not applicable.

DETAILED DESCRIPTION

[0023] Reference will now be made in detail to various aspects of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter. 100241 As described above, coinfection with Clostridium bacteria and Eimeria protozoa can cause a dual disease state leading to both coccidiosis and necrotic enteritis in poultry, which can have devastating effects on the health of these animals. Coinfection with Clostridium and Eimeria can lead to an increase in morbidity and mortality and can have devastating economic impacts for the poultry industry. Necrotic enteritis and coccidiosis infections combined can be attributed to a loss of nine billion dollars worldwide. As the poultry industry shifts away from the use of antibiotics and anticoccidials to control such infections, there exists a need to develop alternative interventions to control disease outbreaks. In accordance with various aspects herein, feed additive compositions and methods for feeding poultry are included to prevent or reduce coinfection with Clostridium bacteria and Eimeria protozoa in poultry, as well as to improve various health measures and to enhance performance measures in the animals.

[0025] The present disclosure provides feed additive compositions that can include a microbial fermentate product in an amount effective to produce an inhibitory effect against coinfection with Clostridium bactena and Eimeria protozoa in an animal. The coinfection with Clostridium bacteria and Eimeria protozoa can include an infection in the animal that can be a clinical infection or a subclinical infection. The coinfection with Clostridium bacteria and Eimeria protozoa can be naturally acquired in an animal’s habitat in an amount effective to cause disease in the animal, or the coinfection with Clostridium bacteria and Eimeria protozoa can be acquired by challenging an animal in a controlled setting. The present disclosure provides the feed additive compositions in an amount effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa by reducing or preventing a coinfection with Clostridium bacteria and Eimeria protozoa through Clostridium growth inhibition and inhibition of Eimeria reproduction or shedding. The effective amount of feed additive composition in the diet can directly or indirectly inhibit the growth of Clostridium bacteria and the reproduction or shedding of Eimeria protozoa and can confer one or more beneficial effects to the animal(s).

[0026] As used herein, a “beneficial effect” can refer to one or more improved health measures or one or more enhanced performance measures in an animal or group of animals. Various improved health measures can include a reduction in one or more of ceca lesion number, ceca lesion severity, liver lesion number, or liver lesion severity. Various enhanced performance measures can include an increase in weight, a decrease in feed conversion ratio, an increase in egg production, a decrease in mortality, a decrease in morbidity, or any combination thereof. In various aspects, the beneficial effects observed herein can be a direct or indirect result of the inhibition of the growth of Clostridium bacteria and can be a direct or indirect result of the reproduction or shedding of Eimeria protozoa. Both improved health measures and enhanced performance measures are described in more detail elsewhere herein.

[0027] As used herein, “microbial fermentate product” can refer to a composition containing one or more of non-viable microorganism(s), spores produced by fermentation of said microorganism(s) prior to inactivation, and metabolites produced by fermentation of said microorganism(s) prior to inactivation. For example, a microorganism can be subject to a fermentation process to form a fermentate which, after inactivation, includes non-viable microorganisms, spores, cell fractions, and metabolites (e.g., fatty acids, polysaccharides, phenols, peptides, proteins, vitamins, amino acids, and the like) obtained from the fermentation process. Various spore-forming microorganisms suitable for generating the microbial fermentate products herein can generate spores during fermentation. Spores generated during fermentation can remain a component of the microbial fermentate product, or they can be removed from the microbial fermentate product. Various non-spore forming microorganisms suitable for generating the microbial fermentate products herein do not generate spores, and any fermentates produced by non-spore forming microorganisms can be referred to further herein as a “postbiotic product.” The microbial fermentate product herein can be a bacterial fermentate or a fungal fermentate, where the fungal fermentate further can include a yeast fermentate.

[0028] Examples of microorganisms suitable for generating the microbial fermentate products herein include, but are not limited to, Acetobacter pasteurianus, Acetobacter peroxydems, Acetobacter okinawensis, Acidipropionibacterium acidipropionici, Arxula adeninivorans, Bacillus altitudinis, Bacillus atrophaeus, Bacillus aryabhattai, Bacillus cercus, Bacillus coagulans, Bacillus halotolerans, Bacillus megaterium, Bacillus pumilis, Bacillus safensis, Bacillus subtilis, Bacillus velezensis, Bacillus xiamenensis, Brevibacillus laterosporus, Candida carpophila, Candida fermentati, Debaryomyces hansenii, Enterococcus mundtii, Kazachstania martiniae, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus pentosus, Lactobacillus pentosus plantarum, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc citreum, Mucor indicus, Paenibacillus taichungensis, Pediococcus acidilactici, Pediococcus pentosaceus, Phaffia rhodozyma, Pichia barkeri, Priceomyces melissophilus, Rhodotorula graminis, Rhodosporidium toruloides, Saccharomyces cerevisiae, Schwanniomyces vanrijiae, and Weissella confusa, alone or in any combination thereof. In some aspects, the suitable bacteria can include Bacillus velezensis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Pediococcus pentosaceus, Priceomyces melissophilus, and Schwanniomyces vanrijiae, alone or in any combination thereof. The microbial fermentate products are suitable for use in the methods described elsewhere herein

Feed Additive Compositions

[0029] The feed additive compositions herein can include a microbial fermentate product or they can include a microbial fermentate product plus other components as described below. The feed additive compositions can include a microbial fermentate product in the feed additive composition in an amount effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa in poultry. The inhibitory effect against Clostridium bacteria and Eimeria protozoa can result in one or more beneficial effects for the animals feed the feed additive compositions, as will be discussed in more detail below.

[0030] The feed additive compositions can be fed directly to poultry or they can be fed to poultry as a component of a poultry feed. The feed additive compositions can be in any suitable form, including a solid or a liquid. For example, the feed additive composition can be a solid composition in the form of granules, flakes, pellets, powders, tablets, capsules, cubes, crumbles, pastes, gels, and the like. In various aspects, the feed additive composition can be a dry solid. In some aspects, the feed additive composition can be a liquid spray, a liquid water additive, or a liquid water dip bath for application to a feed product. In various aspects, the food additive compositions herein can include a liquid drench. It will be appreciated that the liquid drench can include one or more of a suspension, a solution, or an emulsion. In some aspects, the feed additive compositions herein can include a mixture of a solid and liquid component. The feed additive composition can be suitable for administration in the diet of various species of poultry as described in more detail below.

[0031] The feed additive compositions herein can have both shelf-life stability and temperature stability. The feed additive compositions herein further can be stable during processing and storage. The feed additive compositions can have a shelf life of approximately 24 months. In various aspects, the feed additive compositions can have a shelf life of up to approximately 30 months. In various aspects, the feed additive compositions can be stable at any storage temperature from 18 °C to 45 °C. In various aspects, the feed additive compositions herein further can be stable at various additional storage temperatures ranging from -80 °C to 18 °C. In various aspects, the feed additive compositions herein further can be stable at processing temperatures that meet or exceed 90 °C during processing, such as during pelleting, extrusion, and the tike. [0032] The amount of feed additive composition administered in the diet of an animal can vary depending on the stage of animal growth and the nutritional requirements of the animal. The amount of feed additive composition can be determined based on the concentration of microbial fermentate product required to provide an amount effective to produce an inhibitory effect against Clostridium bacteria. In various aspects, the inhibitory effect can be a direct or indirect inhibition of the growth of Clostridium bacteria. Tn various aspects, the microbial fermentate products herein can reduce or prevent an infection in an animal against various species of Clostridium bacteria, including, but not to be limited to Clostridium baratii, Clostridium botulinum, Clostridium colinum, Clostridium difficile, Clostridium fallax, Clostridium perfringens, and Clostridium septicum.

[0033] In an aspect, the amount of feed additive composition used herein can be determined based on the concentration of microbial fermentate product required to provide an amount effective to inhibit the growth of Clostridium perfringens. In various aspects, the microbial fermentate products herein can be used in an amount effective to inhibit the growth of Clostridium perfringens types A, B, C, D, E, and/or G. In various aspects, the microbial fermentate products herein can be used in an amount effective to inhibit the growth of Clostridium perfringens type A/G.

[0034] The amount of feed additive composition further can be determined based on the concentration of microbial fermentate product also required to provide an amount effective to produce an inhibitory effect against Eimeria protozoa. In various aspects, the inhibitory effect can be a direct or indirect inhibition of the reproduction or shedding, or both, of Eimeria protozoa. In various aspects, the microbial fermentate products herein can reduce or prevent an infection in an animal against various species of Eimeria protozoa, including, but not to be limited to Eimeria acervulina, Eimeria brunetti, Eimeria hagani, Eimeria maxima, Eimeria mitis, Eimeria mivati, Eimeria necatrix, Eimeria praecox, or Eimeria tenella.

[0035] In an aspect, the amount of feed additive composition used herein can be determined based on the concentration of microbial fermentate product required to provide an amount effective to inhibit the reproduction or shedding, or both, of Eimeria protozoa. In various aspects, the microbial fermentate products herein can be used in an amount effective to inhibit reproduction or shedding, or both, of one or more Eimeria protozoa species.

[0036] In various aspects, the feed additive compositions herein can include a microbial fermentate product that is sorbed by a carrier and fed to an animal. The microbial fermentate products can be absorbed by a carrier material, adsorbed by a carrier material, or both. Suitable earners for use herein can include, but are not limited to, soy hulls, wheat hulls, or combinations thereof. In some aspects, the carrier can include an inert insoluble fiber carrier such as a cellulose powder, hemicellulose, lignin, or combinations thereof. In some aspects, the carrier can include a mineral such as a calcium carbonate. The microbial fermentate products herein can be sorbed by the carriers, including absorbed or adsorbed, in any ratio suitable to transport the effective amount to the ceca of poultry. In some aspects, the feed additive compositions herein can be added as a component of a poultry feed and then fed to an animal.

[0037] The microbial fennentate product herein can be added to the diet of poultry in an amount effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa. In various aspects, the microbial fermentate product can be present at an amount effective at a concentration of from 0.01 kg/tonne, 0.05 kg/tonne, 0.10 kg/tonne, 0.15 kg/tonne, 0.20 kg/tonne, 0.25 kg/tonne, 0.30 kg/tonne, 0.35 kg/tonne, 0.40 kg/tonne, 0.45 kg/tonne, 0.50 kg/tonne, 0.55 kg/tonne, 0.60 kg/tonne, 0.65 kg/tonne, 0.70 kg/tonne, 0.75 kg/tonne, 0.80 kg/tonne, 0.85 kg/tonne, 0.90 kg/tonne, 0.95 kg/tonne, 1.0 kg/tonne, 1.25 kg/tonne, 1.5 kg/tonne, 1.75 kg/tonne, 2.0 kg/tonne, 2.25 kg/tonne, 2.5 kg/tonne, 3.0 kg/tonne, 3.5 kg/tonne, 4.0 kg/tonne, 4.5 kg/tonne, 5.0 kg/tonne, 5.5 kg/tonne, 6.0 kg/tonne, 6.5 kg/tonne, 7.0 kg/tonne, 7.5 kg/tonne, 8.0 kg/tonne, 8.5 kg/tonne, 9.0 kg/tonne, 9.5 kg/tonne, 10.0 kg/tonne, 10.5 kg/tonne, 11.0 kg/tonne, 11.5 kg/tonne, 12.0 kg/tonne, 12.5 kg/tonne, 13.0 kg/tonne, 13.5 kg/tonne, 14.0 kg/tonne, 14.5 kg/tonne, or 15.0 kg/tonne, or can be any amount falling within a range of any of the forgoing. In some aspects, the microbial fermentate product can be added to the diet of an animal at an inclusion rate that can include more than 15.0 kg/tonne microbial fermentate if such an inclusion rate does not preclude the animal from being supplied the nutritional requirements necessary to promote and maintain the normal growth and health of the animal. In some aspects, it will be understood that for various control treatment groups no microbial fermentate product is included in the diet of untreated poultry for the sake of comparison. Various beneficial effects and the measurement thereof are discussed elsewhere herein. As used herein, the terms “inclusion rate,” and “amount effective,” are used interchangeably unless otherwise noted.

Poultry Feed

[0038] The feed additive compositions herein can be administered directly to any suitable poultry species or can be administered to poultry as a component of a poultry feed. The poultry feed suitable for use herein can be a complete poultry feed to which the feed additive composition is added. A complete poultry feed can include a nutritionally adequate feed for poultry that is compounded to be fed as the sole ration and can maintain life and/or promote growth and production without any additional substances being consumed except water. Complete poultry feeds can be compounded mixtures containing any combination of nutrients, as described below, plus various energy sources such as grains, fat, and protein. In addition, various major vitamins and minerals can be added to the complete poultry feed. A complete poultry feed can include ingredients such as, but not to be limited to, soybean, corn, wheat, soybean meal, meat and bone meal, fats and oils (e.g., soya oil), limestone, monocalcium phosphate (CaH ₄ P ₂ O ₈ ), sodium bicarbonate (NaHCO3), sodium chloride (NaCl), ammino acids (e.g., L-lysine HCl, DL- methionine, and L-threonine), vitamins (e.g., vitamin A (retinyl-acetate), vitamin D3 (cholecalciferol), vitamin E (DL-α-tocopherol), vitamin K3 (menadione), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B6 (pyridoxine-HCL), vitamin B12 (cyanocobalamin), vitamin b3 (niacin), D-pantothenic acid, vitamin B9 (folic acid), vitamin B9 (biotin), salts, organic acids, choline chloride (C ₅ H ₁₄ ClNO), potassium iodide (KI), ferrous(II) sulfate monohydrate oxide (FeSO ₄ .H ₂ O), cupric sulfate (CuSO4.5H ₂ O), manganese (II) oxide (MnO), zinc sulfate (ZnSO ₄ .H ₂ O), sodium selenite (Na2SeO3), enzymes, phytase, and various combinations thereof. [0039] Methods of preparing poultry feed are described, for example, in Feeding Poultry: The Classic Guide to Poultry Nutrition for Chickens, Turkeys, Ducks, Geese, Gamebirds, and Pigeons, G.F. Heauser, Norton Creek Press, 2003 and Commercial Poultry Nutrition, 3 ^rd Edition, Leeson et al., University Books, 2005. [0040] The total protein in the poultry feed can be from about 10 wt.% to about 40 wt.%, from about 12 wt.% to about 35 wt.%, from about 15 wt.% to about 30 wt.%, or from about 16 wt.% to about 26 wt.%. Total protein in the poultry feed can be from 5 wt.%, 6 wt.%, 7 wt.%, 8 wt.%, 9 wt.%, 10 wt.%, 11 wt.%, 12 wt.%, 13 wt.%, 14 wt.%, 15 wt.%, 16 wt.%, 17 wt.%, 18 wt.%, 19 wt.%, 20 wt.%, 21 wt.%, 22 wt.%, 23 wt.%, 24 wt.%, 25 wt.%, 26 wt.%, 27 wt.%, 28 wt.%, 29 wt.%, 30 wt.%, 31 wt.%, 32 wt.%, 33 wt.%, 34 wt.%, 35 wt.%, 36 wt.%, 37 wt.%, 38 wt.%, 39 wt.%, or 40 wt.% of the poultry feed, or can be any amount falling within a range of any of the forgoing. The total protein in the poultry feed can be variable depending on the formulation and intended use of the feed. [0041] Total fat (e.g., oil, fat, and/or lipids) in the poultry feed can be from about 0.5 wt.% to about 10 wt.%, from about 1.0 wt.% to about 8 wt.%, from about 1.5 wt.% to about 7 wt.%, or from about 3 wt.% to about 6 wt.%. Total fat in the poultry feed can be from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, or 10.0% by percent weight of the poultry feed, or can be any amount falling within a range of any of the forgoing. The total fat in the poultry feed can be variable depending on the formulation and intended use of the feed. For example, a poultry feed formulated for a chicken can include from about 1.5 wt.% to about 10.0 wt.% fat. A poultry feed formulated for a turkey can include from about 0.5 wt.% to about 10.0 wt.% fat.

[0042] The feed additive compositions herein can be any suitable feed product designed for mixing with another composition, such as a base feed, to form the poultry feed. The feed additive composition further can include a premix, a concentrate, a base mix, a supplement, a top dress, or a combination thereof.

[0043] A base feed can be a commercially available feed or other animal feed. A base feed suitable for poultry can refer to a ration that contains any of the various cereal grains, their byproducts, and other sources of primary nutrition (e.g., fat, starch, and protein) such as barley, blood meal, bone meal, com (e.g., whole or meal), poultry meal, hominy, soybeans (e.g., whole or meal), tallow, wheat (e.g., whole, bran, or middlings), or a combination thereof.

[0044] A premix can be a composition that can include vitamins, minerals, appropriate medications, carriers, and combinations thereof, and are typically less than 1 wt.% of the diet but can be higher. The carrier can increase bulk to improve distribution in compounding to prepare a more complete feed material. Such premixes can be used to formulate concentrates and complete feeds.

[0045] A concentrate can be a composition that can include high-protein feed components and can also include vitamins, minerals, appropriate medications, and combinations thereof. A concentrate is typically 5 wt.% to 40 wt.% of the diet but can be higher or lower. A concentrate can include additives. Concentrates can be used to make complete feeds by adding available grams or other energy sources. An additive can include an ingredient or a chemical preparation or combination of ingredients which is added to the base feed to fulfill a specific nutritional requirement. It can be used in micro quantities and may have no nutritional value but is added to the feed to improve its quality and efficacy . Feed additives can include, but are not limited to, acidifiers, antioxidants, aromatics, deodorizing agents, flavor enhancers, mold inhibitors, pellet binders, preservatives, sweeteners, toxin binders, and the like.

[0046] A base mix can be similar to a supplement but can contain just a portion of the animal's (e.g., the poultry’s) protein requirements, so it can be used with high protein ingredients and grain (e.g., ground grain and protein source, such as soybean meal) to form the poultry feed. A base mix can include a mixture of one or more macro-mineral sources and one or more micro- ingredient sources such as vitamin premixes, trace mineral premixes, essential ammo acids, and feed additives, that when mixed with sources of protein and energy form a complete feed.

[0047] A supplement can include a feed ingredient, or a chemical preparation or combination of feed ingredients, intended to supply any deficiencies in an animal (e.g., poultry) feed and/or improve the nutritive balance or performance of the animal or poultry feed.

[0048] A top dress can include a supplement that can be added at specific time intervals to the bird’s ration to provide a specific supplement or supplements over a time period that makes it inconvenient or difficult to include in a complete feed.

[0049] The feed additive compositions herein can be added to a premix, a concentrate, a supplement, a top dress, or a base mix that is added to a poultry feed and can be formulated such that the feed additive composition is any suitable proportion of the diet, such as 30 wt.% or less of the poultry feed, or 10 wt.% or less of the poultry feed. In various aspects, the feed additive compositions herein can make up any suitable proportion of the diet, such as from 0. 1 wt.% to 30 wt.%, 1 wt.% to 30 wt.%, 1 wt.% to 15 wt.%, 1 wt.% to 5 wt.%, 15 wt.% to 30 wt.%. In vanous aspects, the feed additive compositions herein can make up any suitable proportion of the diet, such as from about 1 wt.%, 2 wt.%, 3 wt.%, 4 wt.%, 5 wt.%, 6 wt.%, 8 wt.%, 10 wt.%, 12 wt.%, 14 wt.%, 16 wt.%, 18 wt.%, 20 wt.%, 22 wt.%, 24 wt.%, 26 wt.%, 28 wt.%, or 30 wt.% of the poultry feed, or can be any amount falling within a range of any of the forgoing. It will be appreciated that in some aspects, the feed additive composition can be present at greater than 30 wt.% of the poultry' feed.

[0050] The feed additive compositions described herein can form any suitable proportion of a poultry feed as a portion of a premix, base mix, concentrate, supplement, top dress, or a combination thereof. The feed additive composition can be about 0.01 wt.% to about 99.9 wt.%, about 0.1 wt.% to about 95 wt.%, or about 0.5 wt.% to about 90 wt.% of the poultry feed, such that the final poultry feed includes 0.01 kg/tonne to 15.0 kg/tonne of the feed additive composition. It will be appreciated that the amount of microbial fermentate product present in the feed additive compositions and effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa can include a concentration falling within the range from 0.01 kg/tonne to 15.0 kg/tonne. Furthermore, the amount of microbial fermentate product effective to produce a beneficial effect, including improved health measures and enhanced performance measures, in animals coinfected with Clostridium bacteria and Eimeria protozoa can include a concentration falling within a range from 0.01 kg/tonne to 15.0 kg/tonne. 10051 1 For example, the microbial fermentate product can be added to a poultry feed in an amount effective to produce one or more beneficial effects in an animal coinfected with Clostridium bacteria and Eimerict protozoa. In various aspects, the microbial fermentate product can be added to a poultry feed at concentrations including from 0.01 kg/tonne, 0.05 kg/tonne, 0.10 kg/tonne, 0. 15 kg/tonne, 0.20 kg/tonne, 0.25 kg/tonne, 0.30 kg/tonne, 0.35 kg/tonne, 0.40 kg/tonne, 0.45 kg/tonne, 0.50 kg/tonne, 0.55 kg/tonne, 0.60 kg/tonne, 0.65 kg/tonne, 0.70 kg/tonne, 0.75 kg/tonne, 0.80 kg/tonne, 0.85 kg/tonne, 0.90 kg/tonne, 0.95 kg/tonne, 1.0 kg/tonne, 1.25 kg/tonne,

1.5 kg/tonne, 1.75 kg/tonne, 2.0 kg/tonne, 2.25 kg/tonne, 2.5 kg/tonne, 3.0 kg/tonne, 3.5 kg/tonne, 4.0 kg/tonne, 4.5 kg/tonne, 5.0 kg/tonne, 5.5 kg/tonne, 6.0 kg/tonne, 6.5 kg/tonne, 7.0 kg/tonne,

7.5 kg/tonne, 8.0 kg/tonne, 8.5 kg/tonne, 9.0 kg/tonne, 9.5 kg/tonne, 10.0 kg/tonne, 10.5 kg/tonne, 11.0 kg/tonne, 11.5 kg/tonne, 12.0 kg/tonne, 12.5 kg/tonne, 13.0 kg/tonne, 13.5 kg/tonne, 14.0 kg/tonne, 14.5 kg/tonne, or 15.0 kg/tonne, or can be any amount falling within a range of any of the forgoing. It will be understood that for various control treatment groups, no microbial fermentate product is included in the diet of untreated poultry for the sake of comparison.

[0052] The inclusion rate of the microbial fermentate products suitable for use herein can be expressed as kg/tonne, Ib/ton, or wt.%. It will be appreciated that a kg/tonne value can be converted to a wt.% by dividing the kg/tonne value by 1000 and multiplying the resulting value by 100. It will be appreciated that a “tonne” is also referred to in the art as a “metric ton,” where the value of a tonne is equivalent to approximately 1000 kg (i.e., 2204.62 pounds). Unless otherwise noted, as used herein the value for “ton” present in the units Ib/ton is equivalent to approximately 2000 pounds.

Molecular Components of Microbial Fermentate Products

[0053] The microbial fermentate products obtained during of the fermentation of the bacterial strains identified herein can include one or more molecular components, including, but not to be limited to, lipopeptides, polar lipids, isoprenoid-containing compounds, or combination thereof.

[0054] Molecular components of the microbial fermentate products can be characterized using various methods, including high performance liquid chromatography (HPLC). In some aspects, the HPLC process can include detection using ultraviolet (UV) detection methods. In other aspects, the molecular components of the microbial fermentate products can be characterized using ultra high-performance liquid chromatography coupled to high-resolution accurate-mass spectrometry (UHPLC-HRAM). Lipopeptides

[0055] Exemplary lipopeptides present in some of the microbial fermentate products herein can include those falling into the categories of short lipopeptides and long lipopeptides. The short lipopeptides can be characterized as having amino acid sequences of approximately seven amino acid residues covalently linked to lipids having hydrocarbon chains with anywhere from 10-16 carbons in length (i.e., C10-C16). In some aspects, the short lipopeptides can include from five to nine amino acids. The long lipopeptides can be characterized as having amino acid sequences of approximately 10 amino acid residues covalently linked to lipids having hydrocarbon chains with anywhere from 17-20 carbons in length (i.e., C17-C20). In some aspects, the long lipopeptides can include from nine to 14 amino acids. In various aspects, the amino acids in the short lipopeptides or long lipopeptides can include other alkylated amino acids.

[0056] Exemplary lipopeptides present in some of the microbial fermentate products herein can include, but are not to be limited to, one or more of lipopeptide NO (C50H87N7O13); gageostatin C (C51H89N7O13); surfactin and surfactin variants (C52H91N7O13); neocopiamycin A (C53H93N3O17); [Ile7] -surfactin (C53H93N7O13); fengycin C (C72H110N12O20); SNA-60-367-4 (C73H112N12O20); SNA-60-367-9 (C74H114N12O20); SNA-60-367-13 (C75H116N12O20); or derivatives or isomers thereof. By way of non-limiting examples, the structures of various lipopeptides found in some of the microbial fermentates herein are presented in Table 1.

Table 1. Chemical Structures of Various Lipopeptide Compounds

Polar Lipids

[0057] Exemplary polar lipids present in some of the microbial fermentate products herein can include those that contain long-chain fatty acids with a polar amine-containing or an amide- containing head group. Exemplary polar lipids present in some of the microbial fermentate products herein can include, but are not to be limited to, one or more of N-(3-oxodecanoyl)-L- homoserine lactone (C ₁₄ H ₂₃ NO ₄ ); serinolamide C (C ₂₁ H ₄₁ NO ₃ ); N-stearoylalanine (C ₂₁ H ₄₁ NO ₃ ); L-755807 (C ₂₄ H ₃₅ NO ₄ ); aspochalasin J (C ₂₄ H ₃₅ NO ₄ ); (4E,6Z)-N-(5-ethyl-5-methyl-2-oxofuran- 3-yl)-8-(3-heptyloxiran-2-yl)-3-hydroxyocta-4,6-dienamide (C ₂₄ H ₃₇ NO ₅ ); mooreamide A (C ₂₄ H ₃₉ NO ₃ ); or derivatives or isomers thereof. By way of non-limiting examples, the structures of various polar lipids found in some of the microbial fermentate products herein are presented in Table 2.

Table 2. Chemical Structures of Various Polar Lipids Isoprenoid-Containing Compounds

[0058] Exemplary isoprenoid-containing compounds present in some of the microbial fermentate products herein can include, but are not to be limited to, one or more of bexarotene (C ₂₄ H ₂₈ O ₂ ); 7-{[(2E,6Z)-3,7,ri-Trimethyl-2,6,10-dodecatrien-l-yl]oxy}-2H -chromen-2-one (C ₂₄ H ₃₀ O ₃ ); diorcinol G (C ₂₄ H ₃₀ O ₃ ), ammoresinol (C ₂₄ H ₃₀ O ₄ ); cattienoid A (C ₂₄ H ₃₂ O ₄ ); bisacremine G (C ₂₄ H ₃₂ O ₅ ); neomarinone (C ₂₆ H ₃₂ O ₅ ); methyl hydroxy-3, 4-dehydro-apo-8’- lycopenoate (C ₃₁ H ₄₂ O ₃ ); kolanone (C ₃₂ H ₄₂ O ₄ ); bacillaene (C ₃₄ H ₅₀ N ₂ O ₆ ); or derivatives or isomers thereof. By way of non-limiting examples, the structures of various isoprenoid-containing compounds found in some of the microbial fermentate products herein are presented in Table 3.

Table 3. Chemical Structures of Various Isoprenoid-Containing Compounds

Other Compounds

[0059] Other additional compounds present in some of the microbial fermentate products herein can include but are not to be limited to, vicenistatin M (C ₃₀ H ₄₇ NO ₅ ); trienomycin B (C ₃₄ H ₄₈ N ₂ O ₇ ); prumycin (C ₈ H ₁₇ N ₃ O ₄ ); SF-1902-A3 (C ₃₁ H ₅₅ N ₅ O ₈ ); or derivatives or isomers thereof. By way of non-limiting examples, the structures of various additional compounds found in some of the microbial fermentate products herein are presented in Table 4.

Table 4. Chemical Structures of Various Additional Compounds

Poultry Animals

[0060] The feed additive compositions herein can be formulated for use in any suitable species of poultry and at any suitable life stage. The term “poultry” as used herein, refers to domestic fowls, including chickens, turkeys, geese, ducks, ostriches, quails, and pheasants raised to produce meat or eggs. Poultry can include birds characterized as “broilers,” defined as birds reared for meat production, and can also include birds characterized as “layers,” defined as birds reared for laying eggs. Poultry further can include birds characterized as “breeders,” defined as birds that have reached the age of sexual maturity and can lay eggs. In some aspects, the poultry described herein can be selected from the group including a chicken, a turkey, a duck, and a goose. In some aspects, the poultry is a chicken. As used herein, the terms “chicken,” “broiler chicken,” “broiler,” “layer,” and “bird,” are used interchangeably unless otherwise noted.

[0061] Chickens (Gallus gallus domesticus) suitable for use in herein can include, but are not limited to, breeds and strains such as Hy-Line, Lohmann, H&N, Hendrix, Ross, Cobb, Hybro, Heritage, Hubbard, ISA, Shaver, Arbor Acres, Indian River, Peterson, and Dekalb.

[0062] Turkeys (Meleagris gallopavo domesticus) suitable for use herein can include, but are not limited to, breeds and strains such as Broad Breasted White, Nicholas, British United Turkeys, Hybrid Turkeys, Broad Breasted Bronze, and Standard Bronze.

Methods for Feeding Poultry

[0063] The present disclosure provides methods for feeding poultry the feed additive compositions described herein. The methods for feeding poultry can include feeding an animal or group of animals a microbial fermentate product in the feed additive composition in an amount effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa to provide a beneficial effect to the poultry. The beneficial effect can be determined as one or more improved health measures or enhanced performance measures, or a combination thereof, in an animal or group of animals coinfected with Clostridium bacteria and Eimeria protozoa. In various aspects, a beneficial effect resulting from an enhancement in performance measures can be a direct result of a beneficial effect due to improved health measures in animals coinfected with Clostridium bacteria and Eimeria protozoa. In other aspects, a beneficial effect resulting from an enhancement in performance measures can be an indirect result of a beneficial effect due to improved health measures in animals coinfected with Clostridium bacteria and Eimeria protozoa. In yet other aspects, a beneficial effect resulting from an enhancement in performance measures can be a direct or an indirect result, or both, of a beneficial effect due to improved health measures in animals coinfected with Clostridium bacteria. It will be appreciated that the beneficial effects described herein can be observed in poultry having a clinical or subclinical infection with Clostridium bacteria and Eimeria protozoa. It will further be appreciated that in various instances, animals that are not coinfected with Clostridium bacteria and Eimeria protozoa can also exhibit a beneficial effect when fed the feed additive compositions herein, including one or more improved health measures or enhanced performance measures, or a combination thereof. Beneficial Effects

[0064] Producing a beneficial effect in an animal can be a result of the direct or indirect inhibition of the growth of Clostridium bacteria and the reproduction or shedding, or both, of and Eimeria protozoa in response to the administration of the feed additive compositions herein. In an aspect, the beneficial effects against Clostridium bacteria and Eimeria protozoa can be measured in the animal(s) as one or more improved health measures. Tn various aspects, the improved health measures can include a reduction in the number of necrotic lesions in the animal and a reduction in the severity of the necrotic lesions in the animal. In various aspects, the improved health measures can include a reduction in ceca lesion number, a reduction in ceca lesion severity, a reduction in liver lesion number, and/or a reduction in liver lesion severity, or a combination thereof. The reduction in the number of necrotic lesions in poultry coinfected with Clostridium bacteria and Eimeria protozoa can be compared relative to a corresponding method using poultry feed that does not include the feed additive compositions.

[0065] In vanous aspects, the number of ceca lesions and/or liver lesions can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 75%, or a range falling within any of the forgoing, as compared to poultry coinfected with Clostridium bacteria and Eimeria protozoa that have not been fed the feed additive compositions. A reduction in lesion severity (i.e., a reduction in ceca lesion severity or number and/or a reduction in liver lesion severity or number) in poultry coinfected with Clostridium bacteria and Eimeria protozoa can be determined by scoring the number and severity of lesions present in the liver and ceca of infected birds. Lesion severity for both ceca lesions and liver lesions can be seen as an increase as the lesion score increases from 0 to 4. An exemplary lesion scoring schema is described in Table 5, which can be applied in full or in part depending on which portion of the animal (e.g., ceca, liver, or both) is being analyzed during a course of treatment.

Table 5. Lesion Scoring Schema for Ceca and Liver Lesions

[0066] In some aspects, ceca lesion severity can be measured as the average ceca lesion score or as the percent of lesions with a high ceca lesion score. For example, more ceca lesions scored as a 0, 1, or 2 as opposed to a 3 or 4, can be an indication of less severe ceca lesions in the poultry. A reduction in ceca lesion severity upon feeding poultry a poultry feed containing the feed additive compositions described herein can be a reduction in average ceca lesion score of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to poultry fed diet lacking the feed additive compositions. A reduction in ceca lesion severity upon feeding poultry' the feed additive compositions described herein can be a reduction in the percent of ceca lesions scored as a 3 or 4, such that less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, or less than 10% of ceca lesions are scored a 3 or a 4, or such that the percent of ceca legions scored a 3 or 4 is reduced relative to poultry fed a diet lacking a feed additive composition.

[0067] In some aspects, liver lesion severity can be measured as the average liver lesion score or as the percent of lesions with a high liver lesion score. For example, more liver lesions scored as a 0, 1, or 2 as opposed to a 3 or 4, is an indication of less severe liver lesions in the poultry. A reduction in liver lesion severity upon feeding the poultry a poultry feed containing the feed additive compositions described herein can be a reduction in average liver lesion score of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to poultry fed a poultry feed lacking the feed additive compositions. A reduction in liver lesion severity upon feeding the poultry the feed additive compositions containing poultry feed described herein can be a reduction in percent of liver lesions scored as a 3 or 4, such that less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, or less than 10% of liver lesions are scored a 3 or a4, or such that the percent of liver legions scored a 3 or 4 is reduced relative to poultry fed a diet without the feed additive compositions.

[0068] The beneficial effects against Clostridium bacteria and Eimeria protozoa can also be measured in the animal(s) as one or more enhanced performance measures. Poultry fed the feed additive compositions by the methods herein can exhibit enhanced performance measures, including an increase in weight, a decrease in feed conversion ratio, an increase in egg production, a decrease in mortality, a decrease in morbidity, or any combination of these traits when coinfected with Clostridium bacteria and Eimeria protozoa The enhanced performance measures in poultry fed the feed additive compositions herein can include an increase in weight, a decrease in feed conversion ratio, an increase in egg production, a decrease in mortality, a decrease in morbidity, or combinations thereof, relative to poultry fed a feed lacking the feed additive compositions. Birds fed the feed additive compositions by the methods herein further can exhibit prevention of infection with Clostridium bacteria and Eimeria protozoa or improved treatment efficacy and/or reduction of severity of infection when coinfected with Clostridium bacteria and Eimeria protozoa. The enhanced performance measures described herein can be observed in poultry coinfected with Clostridium bacteria and Eimeria protozoa through a challenge in a controlled setting or in poultry coinfected with Clostridium bacteria and Eimeria protozoa via an environmentally acquired infection in their habitat in an amount effective to cause disease in the poultry. It will be appreciated that the enhanced performance measures described herein can be observed in birds fed the feed additive compositions herein even when the birds are not comfected with Clostridium bacteria and Eimeria protozoa in an amount effective to cause disease in the poultry.

[0069] The methods described herein can increase the weight of poultry fed the feed additive compositions. The weight can be increased by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, or at least 30%, or any amount falling within a range of the forgoing, relative to poultry fed an equivalent poultry feed lacking the feed additive compositions. For example, the increase in weight can include an average weigh increase of from at least 0.5% to 30%, or from at least 0.5% to 10%, or from at least 10% to 30% in a population of poultry fed the feed additive composition as compared to an average weight in a population of poultry fed a diet lacking the feed additive composition.

[0070] Weight of individual birds can be measured using standard weighing techniques at any predetermined time interval over the course of the lifespan of the bird. By way of example, birds can be weighed at birth and then weighed each day following birth until the animal expires. In some aspects, the birds can be weighed twice a day, every other day, weekly, or monthly during the lifespan of the bird. Weight can be compared across treatment groups and to controls not fed the feed additive compositions over one or more time periods during the lifespan of a bird(s). Comparison of weight gain at various time intervals, across treatment groups, and to controls (e.g., both positive and negative controls) can be made to determine the effect of a given feed additive composition on weight gain.

[0071] The methods described herein can decrease the feed conversion ratio of poultry fed the feed additive compositions. The feed conversion ratio (FCR) is the measure of an animal(s) efficiency at converting the total amount of feed that the animal(s) consumes over a given period of time against the total weight output (including body mass and/or eggs produced) that the animal(s) gains from consuming the feed during the given period of time. FCR is a unitless value that correlates to the amount of total weight output as measured by body mass and/or eggs produced by the animal(s) fed the feed additive compositions. FCR can be determined using the following formula:

[0072] Various factors can affect FCR, including, but not to be limited to the age of an animal or population of animals, feed quality, genetic makeup of the animal(s), type of animal, water consumption, environmental conditions, and animal management practices. FCR ratios for poultry treated with the microbial fermentates herein can include, but are not to be limited to, FCR ratios of from 0.5 to 10, or from 0.5 to 5. In various aspects, the FCR for poultry feed the feed additive compositions herein can be from 1.300 to 1.800. In various aspects the FCR can be reported as points from 0.5 to 10. It will be appreciated that a lower feed conversion ratio can correlate to a greater efficiency by the animal(s) at converting the total amount of feed consumed over a known period of time into total weight output (e.g., as measured by body mass and/or egg production).

[0073] The feed conversion ratio can be decreased by at least 0.5%, at least 1.0%, at least 1.5% at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10%, or any amount falling within a range of the forgoing, relative to the feed conversion ratio in poultry fed an equivalent poultry feed lacking the feed additive compositions. For example, the decrease in feed conversion ratio can include an average feed conversion ratio decrease of from 0.5% to 10% in a population of poultry fed the feed additive composition as compared to an average feed conversion ratio in a population of poultry fed a diet lacking the feed additive composition.

[0074] The methods described herein can increase egg production of the poultry. Egg production can be increased by at least 0.5%, at least 1.0%, at least 1.5% at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, or at least 25%, or any amount falling within a range of the forgoing, relative to poultry fed an equivalent feed lacking the feed additive compositions. For example, the increase in egg production can include an average increase of from at least 0.5% to 25%, or from at least 0.5% to 10%, or from at least 10% to 25% in the egg production of a population of poultry fed the feed additive composition as compared to a population of poultry fed a diet lacking the feed additive composition.

[0075] Egg production can be calculated over the course of the lifespan of the birds being fed the feed additive compositions herein. Egg production can be determined by monitoring and recording the number of eggs laid by each individual bird or of the number of eggs laid by the entire flock of birds. In some aspects, the number of eggs laid by each individual bird, or the entire flock of birds, can be recorded once a day, every other day, weekly, or monthly during the lifespan of the birds. Comparison of egg production at various intervals, across treatment groups, and to controls (e.g., both positive and negative controls) can be made to determine the effect of a given feed additive composition on egg productivity. L0076J The methods described herein can decrease mortality of the poultry. Mortality can be decreased by at least 0.5%, at least 1.0%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, or at least 40%, or any amount falling within a range of the forgoing, relative to poultry fed an equivalent poultry feed lacking the feed additive compositions. For example, the decrease in mortality can include an average decrease of from at least 0.5% to 40%, or from at least 0.5% to 10%, or from at least 10% to 40% in the mortality of a population of poultry fed the feed additive composition as compared to a population of poultry fed a diet lacking the feed additive composition.

[0077] Mortality can be monitored over the course of a lifespan of the birds being fed the feed additive compositions herein. Mortality' can be recorded as the death of one bird or a percentage of a population of birds. Comparison of mortality at various intervals, across treatment groups, and to controls (e.g., both positive and negative controls) can be made to determine the effect of a given feed additive composition on mortality over the course of a treatment.

[0078] The methods described herein can decrease morbidity in the poultry. For example, morbidity can be decreased by at least 0.5%, at least 1.0%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, or at least 40%, or any amount falling within a range of the forgoing, relative to poultry fed an equivalent poultry feed lacking the feed additive compositions. For example, the decrease in morbidity can include an average decrease of from at least 0.5% to 40%, or from at least 0.5% to 10%, or from at least 10% to 40% in the morbidity of a population of poultry fed the feed additive composition as compared to a population of poultry fed a diet lacking the feed additive composition.

[0079] Morbidity can be monitored over the course of a lifespan of the birds being fed the feed additive compositions herein. Morbidity can include the incidence of one or more type of illness identified by various symptoms of necrotic enteritis or other illness, including but not to be limited to, inactivity, listlessness, diarrhea, bloody stools, lesions, cloudy eyes, pus in the eyes, tissue mass, scaly legs, loss of appetite, respiratory distress, paralysis, and the like. Morbidity' can be recorded as symptomatic illness in one bird or a percentage of a population of birds exhibiting symptomatic illness. Comparison of morbidity at various intervals, across treatment groups, and to controls (e.g., both positive and negative controls) can be made to determine the effect of a given feed additive composition on morbidity.

[0080] Feeding poultry with the feed additive compositions can begin upon hatching and can extend throughout an animal’s lifecycle. Feeding poultry the feed additive compositions can also be performed over a narrower time span or life stage. It will be appreciated that the feed additive compositions can be administered as a daily feed ration that is fed to the poultry on most days or on all days. In various aspects, the feed additive composition can be administered to the poultry one, two, three, four, five, six, or seven days of the week, or a range falling within any of the forgoing, for a predetermined time span, or for the life of the animal.

[00811 The feed additive compositions herein can be fed to the animal(s) during any of its discrete life stages, throughout the lifespan of the animal, or only when the animal has an active infection. By way of example, in some aspects the feed additive compositions can be fed at any inclusion rate described herein to an animal throughout the duration of an active infection and until the infection is cleared, where the animal can then be switched back to a diet that lacks the feed additive compositions. In some aspects, the feed additive compositions herein can be fed prophylactically to the animals to prevent disease. In some aspects, the feed additive compositions herein can be fed prophylactically to the animals at a first inclusion rate, and if the animals become infected with a pathogen the animal could be switched to a diet including a feed additive composition at a second inclusion rate, where the second inclusion rate is higher than the first inclusion rate. Once an animal clears an infection consuming a diet at a second inclusion rate, the animal can be returned to a diet at a first inclusion rate or a diet having no feed additive composition included therein. The first inclusion rate and second inclusion rate can include any inclusion rate or a range of inclusion rates as described elsewhere herein.

[0082] The methods herein can include feeding poultry a feed additive composition directly and/or feeding poultry a poultry feed containing the feed additive composition. The feed additive compositions fed to the poultry can include one or more microbial fermentate products, or it can include one or more microbial fermentate products plus other components as described elsewhere herein. The methods of feeding the poultry herein can inhibit the growth of Clostridium bacteria and the reproduction and shedding, or both, of Eimeria protozoa in the poultry. In an aspect, the amount of feed additive composition can be determined based on the concentration of microbial fermentate product required to provide an amount effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa, such as directly or indirectly inhibiting the growth of Clostridium perfringens and the reproduction or shedding, or both, of Eimeria protozoa. The methods using the feed additive compositions herein can provide various advantages to poultry as compared to corresponding methods using poultry feed that does not include the feed additive compositions. The methods using the feed additive compositions herein can produce a beneficial effect in an animal fed the feed additive composition as observed relative to poultry fed a diet lacking the feed additive composition. |0083J The methods herein can include any suitable methods of admimstenng the feed additive compositions to poultry. In some aspects, the methods herein are directed to administering the feed additive compositions chickens or turkeys. The methods can include mixing the feed additive compositions with a base feed, a premix, a concentrate, a base mix, a supplement, a top dress, or a combination thereof as described elsewhere herein, and providing the mixture to poultry for ingestion. Tn some aspects, the methods can include administering the feed additive compositions directly to poultry for ingestion. The methods can include administering a feed additive composition at a concentration in an amount effective to produce an inhibitory effect against Clostridium bacteria in a range from 0.01 kg/tonne, 0.05 kg/tonne, 0.10 kg/tonne, 0.15 kg/tonne, 0.20 kg/tonne, 0.25 kg/tonne, 0.30 kg/tonne, 0.35 kg/tonne, 0.40 kg/tonne, 0.45 kg/tonne, 0.50 kg/tonne, 0.55 kg/tonne, 0.60 kg/tonne, 0.65 kg/tonne, 0.70 kg/tonne, 0.75 kg/tonne, 0.80 kg/tonne, 0.85 kg/tonne, 0.90 kg/tonne, 0.95 kg/tonne, 1.0 kg/tonne, 1.25 kg/tonne, 1.5 kg/tonne,

I.75 kg/tonne, 2.0 kg/tonne, 2.25 kg/tonne, 2.5 kg/tonne, 3.0 kg/tonne, 3.5 kg/tonne, 4.0 kg/tonne, 4.5 kg/tonne, 5.0 kg/tonne, 5.5 kg/tonne, 6.0 kg/tonne, 6.5 kg/tonne, 7.0 kg/tonne, 7.5 kg/tonne, 8.0 kg/tonne, 8.5 kg/tonne, 9.0 kg/tonne, 9.5 kg/tonne, 10.0 kg/tonne, 10.5 kg/tonne, 11.0 kg/tonne,

I I.5 kg/tonne, 12.0 kg/tonne, 12.5 kg/tonne, 13.0 kg/tonne, 13.5 kg/tonne, 14.0 kg/tonne, 14.5 kg/tonne, or 15.0 kg/tonne, or any amount falling within a range of any of the forgoing. It will be understood that in some aspects, such as for various control treatment groups, no feed additive composition is included in the diet of poultry. In some aspects, the methods can include use of a feed additive compositions in the diet of an animal at an inclusion rate that can include more than 15.0 kg/tonne feed additive composition as long as the inclusion rate does not preclude the animal from being supplied the nutritional requirements necessary to promote and maintain the normal growth and health of the animal.

[0084] The methods herein can include inhibiting the growth of Clostridium bacteria and inhibit the reproduction and shedding of Eimeria protozoa in poultry. The methods can include feeding poultry by administering the feed additive composition containing a microbial fermentate product to the poultry. The microbial fermentate product can be present in the feed additive compositions administered to the poultry in an amount effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa, where the inhibitory effect results in one or more beneficial effects for the poultry. The feed additive compositions can be administered to poultry in an amount effective to directly or indirectly inhibit the growth of Clostridium bacteria. The Clostridium bacteria include one or more of Clostridium baratii, Clostridium botulinum, Clostridium colinum, Clostridium difficile, Clostridium fallax, Clostridium perfringens, and Clostridium septicum. The feed additive compositions further can be administered to poultry in an amount effective to directly or indirectly inhibit the reproduction or shedding of Eimeria protozoa, including, but not to be limited to Eimeria acervulina, Eimeria brunetti, Eimeria hagani, Eimeria maxima, Eimeria mitis, Eimeria mivati, Eimeria necatrix, Eimeria praecox, or Eimeria tenella.

[0085] The methods described herein can reduce or prevent a Clostridium and Eimeria coinfection in poultry. Administering the feed additive composition containing a microbial fermentate product to the poultry as described herein can reduce or prevent a Clostridium and Eimeria coinfection in the poultry. The microbial fermentate product can be present in the feed additive compositions administered to the poultry in an amount effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa, where the inhibitory effect results in one or more beneficial effects for the poultry. When poultry fed the feed additive compositions described herein are challenged with or infected via an environmentally acquired infection in their habitat with Clostridium bacteria and Eimeria protozoa the incidence of an infection in the poultry can be reduced relative to incidence of an infection in poultry fed a poultry feed lacking the feed additive compositions. For example, incidence of Clostridium and Eimeria coinfection can be reduced at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, or any amount falling within a range of the forgoing, as compared to the incidence of infection in poultry fed a feed lacking the feed additive compositions. In various aspects, the incidence of Clostridium and Eimeria coinfection can be reduced to more than 70%.

[0086] The methods described herein can treat or reduce the severity of a Clostridium and Eimeria coinfection. Administering the feed additive composition containing a microbial fermentate product to the poultry as described herein can reduce the severity of a Clostridium and Eimeria coinfection in the poultry. The microbial fermentate product can be present in the feed additive compositions administered to the poultry in an amount effective to produce an inhibitory effect against Clostridium bacteria and Eimeria protozoa, where the inhibitory effect results in one or more beneficial effects for the poultry. The severity of a Clostridium and Eimeria coinfection can be measured as the mortality rate, or culling rate, of birds coinfected with Clostridium bacteria and Eimeria protozoa. For example, a reduction in the mortality rate can indicate a reduction in the severity of a Clostridium and Eimeria coinfection. The methods described herein can treat a Clostridium and Eimeria coinfection in poultry by reducing one or more symptoms of the infection, reducing mortality rate, reducing the morbidity rate, or increasing the number of birds that clear the infection. [0087] The methods described herein include a method for enhancing performance measures in poultry. The method can include administering a feed additive composition including a microbial fermentate product to poultry, where the microbial fermentate product is present in the feed additive composition in an amount effective for enhancing performance measures in the poultry. In an aspect, the method includes enhancing performance measures in poultry that are coinfected with Clostridium perfringens and one or more of Eimeria acervulina, Eimeria maxima, or Eimeria tenella. In an aspect, the enhanced performance measures include an increase in weight, a decrease in feed conversion ratio, an increase in egg production, a decrease in mortality, a decrease in morbidity, or any combination thereof. The enhanced performance measures are described in more detail elsewhere herein.

[0088] The methods herein can include challenging a population of poultry' with Clostridium perfringens and one or more of Eimeria acervulina, Eimeria maxima, or Eimeria tenella. The challenge can include administering the Clostridium perfringens in an amount of from 1.0 x 10° to 1.0 x 10 ¹⁰ colony forming units (CFU) in a dietary ration of an animat or from 1.0 x 10 ³ to 1.0 x 10 ⁹ , or from 1.0 x 10 ⁸ to 60.0 x 10 ⁸ CFU in 1-3 ml volumes mixed into from 15 to

30 grams of poultry feed per bird. For example, the methods can include challenging a population of poultry with Clostridium bacteria at an amount of from at least 1.0 x 10° CFU, at least 1.0 x 10 ¹ CFU, at least 1.0 x 10 ² CFU, at least 1.0 x 10 ³ CFU, at least 1.0 x 10 ⁴ CFU, at least 1.0 x 10 ⁵

CFU, at least 1.0 x 10 ⁶ CFU, at least 1.0 x 10 ⁷ CFU, at least 1.0 x 10 ⁸ CFU, at least 1.5 x 10 ^s

CFU, at least 2.0 x 10 ⁸ CFU, at least 2.5 x 10 ⁸ CFU, at least 3.0 x 10 ⁸ CFU, at least 3.5 x 10 ⁸

CFU, at least 4.0 x 10 ⁸ CFU, at least 4.5 x 10 ⁸ CFU, at least 5.0 x 10 ⁸ CFU, at least 5.5 x 10 ⁸

CFU, at least 6.0 x 10 ⁸ CFU, at least 6.5 x 10 ⁸ CFU, at least 7.0 x 10 ⁸ CFU, at least 7.5 x 10 ⁸

CFU, at least 8.0 x 10 ⁸ CFU, at least 8.5 x 10 ⁸ CFU, at least 9.0 x 10 ⁸ CFU, at least 9.5 x 10 ^s

CFU, at least 10 x 10 ⁸ CFU, at least 20 x 10 ⁸ CFU, at least 30 x 10 ⁸ CFU, at least 40 x 10 ⁸ CFU, at least 50 x 10 ⁸ CFU, at least 60 x 10 ⁸ CFU, at least 70 x 10 ⁸ CFU, at least 80 x 10 ⁸ CFU, at least 90 x 10 ⁸ CFU, at least 1.0 x 10 ⁹ CFU, or at least 1.0 x 10 ¹⁰ CFU, or an amount falling within a range of any of the forgoing, in a daily ration of poultry feed per bird. In various aspects, the poultry feed used to deliver the dose of Clostridium perfringens does not contain a feed additive composition.

[0089] In some aspects, the dose of Clostridium perfringens can be administered during a challenge of a population of poultry in a way that it is not included in a daily ration. In some aspects, the dose of Clostridium perfringens can be administered directly to each bird in an enclosure, such as via oral gavage. In some aspects, the dose of Clostridium perfringens can be administered directly to a predetermined number of birds per enclosure, where those birds are then returned to the enclosure with any remaining non-infected birds, and where the directly infected birds act as seeder birds that then go on to infect the remainder of birds in the enclosure through direct contact or through indirect contact such as through excrement in the environment. It will be appreciated that challenging a population of poultry can be performed using any strain of Clostridium bacteria, or mixtures thereof, as described elsewhere herein.

[0090] The challenge further can include challenging a population of poultry with one or more of Eimeria acervulina, Eimeria maxima, or Eimeria tenella at an amount of from of from 1.0 x 10° to 1.0 x 10 ¹⁰ , or from 1.0 x 10 ³ to 1.0 x 10 ⁹ sporulated oocysts or sporozoites in a dietary ration of an animal, or from 1.0 x 10 ⁸ to 60.0 x 10 ⁸ sporulated oocysts or sporozoites in 1-3 ml volumes mixed into from 15 to 30 grams of poultry feed per bird. For example, the methods can include challenging a population of poultry with one or more of Eimeria acervulina, Eimeria maxima, or Eimeria tenella at an amount of from 1.0 x 10°, at least 1.0 x 10 ¹ , at least 1.0 x 10 ² , 1.0 x 10 ³ CFU, at least 1.0 x 10 ³ , at least 1.0 x 10 ⁴ , at least 1.0 x 10 ⁵ , at least 1.0 x 10 ⁶ , at least 1.0 x 10 ⁷ , at least 1.0 x 10 ⁸ , at least 1.5 x 10 ⁸ , at least 2.0 x 10 ⁸ , at least 2.5 x 10 ⁸ , at least 3.0 x 10 ⁸ , at least 3.5 x 10 ⁸ , at least 4.0 x 10 ⁸ , at least 4.5 x 10 ⁸ , at least 5.0 x 10 ⁸ , at least 5.5 x 10 ⁸ , at least 6.0 x 10 ⁸ , at least 6.5 x 10 ⁸ , at least 7.0 x 10 ⁸ , at least 7.5 x 10 ⁸ , at least 8.0 x 10 ⁸ , at least 8.5 x 10 ⁸ , at least 9.0 x 10 ⁸ , at least 9.5 x 10 ⁸ , at least 10 x 10 ⁸ , at least 20 x 10 ⁸ , at least 30 x 10 ⁸ , at least 40 x 10 ⁸ , at least 50 x 10 ⁸ , at least 60 x 10 ⁸ , at least 70 x 10 ⁸ , at least 80 x 10 ⁸ , at least 90 x 10 ⁸ , at least 1.0 x 10 ⁹ , or at least 1.0 x 10 ¹⁰ sporulated oocysts or sporozoites, or an amount falling within a range of any of the forgoing, in a daily ration of poultry feed per bird. In various aspects, the poultry feed used to deliver the dose of Eimeria protozoa does not contain a feed additive composition.

[0091] In some aspects, the dose of Eimeria protozoa can be administered during a challenge of a population of poultry in a way that it is not included in a daily ration. In some aspects, the dose of Eimeria protozoa can be administered directly to each bird in an enclosure, such as via oral gavage. In some aspects, the dose of Eimeria protozoa can be administered directly to a predetermined number of birds per enclosure, where those birds are then returned to the enclosure with any remaining non-infected birds, and where the directly infected birds act as seeder birds that then go on to infect the remainder of birds in the enclosure through direct contact or through indirect contact such as through excrement in the environment. It will be appreciated that challenging a population of poultry can be performed using any strain of Eimeria protozoa, or mixtures thereof, as described elsewhere herein. 100921 Challenging a population of poultry with Clostridium perfringens can include administering an amount of from 1.0 x 10° to 1.0 x 10 ¹⁰ colony forming units (CFU) in a dietary ration of an animal, of from 1.0 x 10 ³ CFU to 1.0 x 10 ⁹ CFU, or from 1.0 x 10 ⁸ CFU to 60.0 x 10 ⁸ CFU of Clostridium perfringens, and an amount of from 1.0 x 10° to 1.0 x 10 ¹⁰ sporulated oocysts or sporozoites, of from 1.0 x 10 ³ to 1.0 x 10 ⁹ sporulated oocysts or sporozoites, or from 1 0 x 10 ⁸ to 60.0 x 10 ⁸ sporulated oocysts or sporozoites of Eimeria protozoa in 1 -3 ml volumes mixed into from 15 to 30 grams in a daily ration of poultry feed per bird. The birds can be allowed to consume the mixture for anywhere from 0.5 to 4 hours post administration and then returned to the diet containing the feed additive compositions. In some aspects, the birds can have their feed trays emptied for 0 to 8 hours prior to administration of the mixture containing Clostridium perfringens and Eimeria protozoa. It will be appreciated that a challenge with Clostridium bacteria and Eimeria protozoa, can include a challenge in a controlled setting where the bacteria are administered directly to the poultry. While in some aspects herein poultry can acquire an infection during a challenge in a controlled setting, in other aspects the poultry treated with the feed additive compositions herein can acquire an infection with Clostridium bacteria and Eimeria protozoa in their habitat. It will be appreciated that naturally acquired coinfections of Clostridium bacteria and Eimeria protozoa can include those acquired in the animals’ habitat where the bacteria are ingested in an amount effective to cause disease in the animal(s). The beneficial effects described using the feed additive compositions herein can be observed in poultry coinfected with Clostridium bacteria and Eimeria protozoa through a challenge in a controlled setting and in poultry coinfected with Clostridium bacteria and Eimeria protozoa via an environmentally acquired infection in their habitat in an amount effective to cause disease in the poultry. The infections herein with Clostridium bacteria and Eimeria protozoa can include those that are subchnical or clinical.

EXAMPLES

[0093] Various aspects of the present disclosure can be better understood by reference to the following Examples, which are offered by way of illustration. The present disclosure is not to be limited to the Examples given herein.

Example 1; Microbial Strains

[0094] Bacterial and fungal strains for generating the microbial fermentate products herein include one or more of: Acetobacter okinawensis, Acetobacter pasteurianus, Acetobacter peroxydans, Acetobacter okinawensis, Acidipropionibacterium acidipropionici, Arxula adeninivorans, Bacillus altitudinis, Bacillus atrophaeus, Bacillus aryabhattai, Bacillus cercus, Bacillus coagulans, Bacillus halotolerans, Bacillus megaterium, Bacillus pumilis, Bacillus safensis, Bacillus subtilis, Bacillus velezensis, Bacillus xiamenensis, Brevibacillus laterosporus, Candida carpophila, Candida fermentati, Debaryomyces hansenii, Enterococcus mundtii, Kazachstania martiniae, Lactobacillus brevis, Lactobacillus paracasei, Lactobacillus pentosus, Lactobacillus pentosus plantarum, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc citreum, Mucor indicus, Paenibacillus taichungensis, Pediococcus acidilactici, Pediococcus pentosaceus, Phaffia rhodozyma, Pichia barkeri, Priceomyces melissophilus, Rhodotorula graminis, Rhodosporidium toruloides, Saccharomyces cerevisiae, Schwanniomyces vanrijiae, and Weissella confusa. All microbial strains listed were sourced from Cargill, Inc. (Wayzata, MN) unless otherwise indicated.

Example 2: Preparation of Microbial Fermentates for /n Vitro Analysis

[0095] For in vitro analysis, microbial derived fermentates of the various microbial strains described in Example 1 were prepared in 250 ml baffled shake flasks as follows. Bacterial cultures were grown in TSB (Tryptic Soy Broth) containing the following composition: Bacto™ Tryptone (Pancreatic Digest of Casein, Thermo Fisher Scientific, Waltham, MA, USA) 17.0 g/L, Bacto™ Soytone (Peptic Digest of Soybean Meal, Thermo Fisher Scientific, Waltham, MA USA) 3.0 g/L, glucose 2.5 g/L, sodium chloride 5.0 g/L, and dipotassium hydrogen phosphate 2.5 g/L. Overnight seed cultures were generated using 13 ml round bottom tubes containing 3 ml of TSB medium. Seed culture medium was inoculated with fresh biomass from a single colony sampled from prepared agar plates using an inoculation loop. Seed cultures were incubated for 18-24 hours in a shaker at 250 rpm, 30 °C, and 75% humidity. Any fungal-denved fermentates using the various microbial strains described in Example 1 were prepared in YPD Medium (Yeast Extract Peptone Dextrose, Thermo Fisher Scientific, Waltham, MA USA) unless otherwise indicated.

[0096] Working cultures for obtaining fermentates were generated using 250 ml baffled shake flasks containing 50 ml of TSB medium that were inoculated at initial ODeoo (i.e., optical density at 600 nm) of 0.1 using an inoculum from the overnight seed cultures. Working cultures were incubated on an agitation shaker at 250 rpm, 30 °C, and 75% humidity for 24 hours. After 24 hours, the agitation was turned off in the shaker and the cultures were incubated for an additional 24 hours. After 48 hours post inoculation, the 250 ml flasks were removed from the shaker and heated to 60 °C and held at that set point for one hour to heat inactivate the microbial culture biomass. Fermentates were packaged and stored at -80 °C until further chemical and biological characterization. Upon use in the in vitro analyses herein, the fermentates were thawed and heated to 75 °C for 15 min for an additional heat inactivation step. The contents were then split between 4 tubes, vortexed, and used immediately or stored at -20 °C for additional future use and testing.

Example 3: Growth Inhibition of Clostridium yerfrinsens by Microbial Fermentates

[0097] The assay described in this example includes a single dose screening assay that evaluates the effects of various bacterial fermentates against Clostridium perfringens using the bacterial strains described in Example 1 and prepared according to Example 2. Two multi-drug resistant strains of Clostridium perfringens, referred to herein as multi-drug resistant strain 1 and multi-drug resistant strain 2 were assayed.

[0098] Clostridium perfringens bacterial cultures were prepared in 250 ml baffled shake flasks containing 50 ml of sterile Clostridial Differential Broth (CDB) (Thermo Fisher Scientific, Waltham, MA USA). The flasks were inoculated using a single colony that was picked from a culture plate, and grown overnight for 18-24 hours in an anaerobic chamber supplemented with 85% N, 10% CO ₂ and 5% H at 250 rpm and 39 °C. Following incubation, the culture was diluted to an OD ₆₀₀ of 0.1 in sterile CDB medium using a clean cuvette in an optical spectrophotometer. [0099] Each well of the first row of a 96-well round bottom microtiter plate was prepared with 160 pl of sterile CDB medium. For each fermentate sample assayed, a 20 pl aliquot at a 1:10 ratio, or 0.1 dilution (i.e., 10 percent volume (10% v/v)), was added to three replicate wells and incubated at 39 °C for 24 hours. Approximately 5 x 10 ⁴ CFU of Clostridium perfringens in 10 pl of sterile CDB medium was added to each well. Control samples were included in the assays as a non-inoculated dilution senes containing no fermentate. A “negative control plate” without bacterial culture was also included to monitor for sterility.

[0100] After incubation of Clostridium perfringens with the bacterial fermentates at 39 °C for 24 hours, direct plating of each Clostridium perfringens sample was performed by serially diluting each sample and streaking the cultures onto selective and differential CDB plating medium. The bacterial plates were incubated at 39 °C in an anaerobic chamber for 18-24 hours. Following incubation, colonies on each plate were counted, converted to log value, and findings were recorded.

[0101] Results from using bacterial fermentates from Pediococcus pentosacous, Bacillus altitudinis, Bacillus laterosporus, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus paracasei, Bacillus velezensis to determine their effects on the grow th of Clostridium perfringens are presented in Table 6. Samples were run in triplicate (n=3), and results are reported as average CFU/ml of Clostridium perfringens at a fermentate dilution of 10% v/v.

Table 6. Effects of Various Bacterial Fermentates on the Growth of Clostridium perfringens

[0102] The bacterial fermentates tested in this example exhibited a minimum of a 1-log reduction in colony forming units (CFU) against Clostridium perfringens growth when compared to an untreated control. In many cases, the bacterial fermentates exhibited a minimum of a 2-log reduction in colony forming units (CFU) against Clostridium perfringens growth when compared to an untreated control. Results reported herein were conducted using multi-drug resistant strain

1 of Clostridium perfringens. Similar results were reported (not shown) for multi-drug resistant strain 2 of Clostridium perfringens.

Example 4: Analysis of the Minimum Inhibitory Concentration of Microbial Fermentates on Growth Inhibition of Clostridium perfringens

[0103] The assay described in this example includes a dose titration assay that evaluates the minimum inhibitory concentrations of bacterial fermentates required to inhibit Clostridium perfringens growth using fermentates from bacterial strains as described in Example 1 and prepared according to Example 2. Both multi -drug resistant strain 1 and multi -drug resistant strain

2 of Clostridium perfringens were assayed.

[0104] Clostridium perfringens bacterial cultures were initiated using one or more colonies that were picked from a culture plate and placed into in 250 ml baffled shake flasks containing 50 ml of sterile Clostridial Differential Broth (CDB) (Thermo Fisher Scientific, Waltham, MA USA) and grown overnight in an anaerobic chamber supplemented with 85% N, 10% CO2 and 5% H at 39 °C. The culture was diluted to an ODeoo of 0.1 in sterile CDB medium using a clean cuvette and an optical spectrophotometer.

[0105] Each well of a 96-well round bottom microtiter plate was prepared for the bacterial fermentates. The bacterial fermentates were added to individual wells of the first column of the 96-well plate in the following volumes (percent volume (% v/v) for final dilutions in parenthesis): 80 pl (40% v/v dilution), 40 pl (20% v/v dilution), 20 pl (10% v/v dilution), 10 pl (5 % v/v dilution), 5 pl (2.5 % v/v dilution) and 2.5pl (1.25 % v/v dilution). Respective amounts of sterile CDB medium to bring the volume up to 100 pl were added to each well to achieve the recited dilutions. The bacterial fermentates added to column 1 were mixed thoroughly five to six times with the CDB medium. Following mixing, 100 pl of the mixture in column 1 was transferred to 100 pl of sterile CDB medium in column 2 and mixed thoroughly as before. Following mixing, 100 pl of the mixture in column 2 was transferred to 100 pl of sterile CDB medium in column 3 and mixed thoroughly as before. The dilution procedure was repeated through column 6. Upon reaching column 6, the mixture was mixed thoroughly and 100 pl of the mixture was discarded before proceeding.

[0106] Approximately 5 * 10 ⁴ CFU of Clostridium perfringens in 10 pl of sterile CDB medium was added to each well. Control samples were included in the assays as a non-inoculated dilution series containing no-fermentate and inoculated dilution series containing no fermentate. A “negative control plate” without bacterial culture was also included to monitor for sterility.

[0107] After incubation of Clostridium perfringens with the bacterial fermentates at 39 °C for 18-24 hours, direct plating of each Clostridium perfringens sample was performed using serial dilutions of each sample into selective CDB medium. The bacterial plates were read at 600 nm to determine average Clostridium perfringens quantity in CFU/ml. After determining the ODeoo, results were converted to log value, and findings were recorded.

[0108] Results from using bacterial fermentates from Pediococcus pentosacous, Bacillus altitudinis, Bacillus laterosporus, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus paracasei, Bacillus velezensis to determine their effects on the grow th of Clostridium perfringens are presented in Table 7. Samples were run in triplicate (n=3), and results are reported as average CFU/ml of Clostridium perfringens at fermentate dilutions of 40 % v/v, 20 % v/v, 10 % v/v, 5 % v/v, 2.5 % v/v, and 1.25 % v/v. Table 7. Effects of Various Bacterial Fermentates on the Growth of Clostridium perfringens in a Dose Titration Assay

[0109] The bacterial fermentates tested in this example exhibited a minimum of a l-log reduction in colony forming units (CFU) against Clostridium perfringens growth when compared to an untreated control. In many cases, the bacterial fermentates exhibited a minimum of a 2-log reduction in colony forming units (CFU) against Clostridium perfringens growth when compared to an untreated control. Results reported herein were conducted using multi-drug resistant strain 1 of Clostridium perfringens. Similar results were reported (not shown) for multi-drug resistant strain 2 of Clostridium perfringens. Example 5: Preparation of 2L Microbial Fermentates for In Vivo Analysis

[0110] For in vivo analysis, bacterial derived fermentates of the various bacterial strains described in Example 1 were prepared in a 2 L stirred bioreactor as follows. Bacterial cultures were grown in TSB (Tryptic Soy Broth) containing the following composition: Bacto™ Tryptone (Pancreatic Digest of Casein, Thermo Fisher Scientific, Waltham, MA USA) 17.0 g/L, Bacto™ Soytone (Peptic Digest of Soybean Meal, Thermo Fisher Scientific, Waltham, MA USA) 3.0 g/L, glucose 2.5 g/L, sodium chloride 5.0 g/L, and dipotassium hydrogen phosphate 2.5 g/L. Overnight seed cultures were generated using 500 ml baffled shake flasks with 100 ml of TSB medium. Seed culture medium was inoculated with fresh biomass of a single colony from prepared agar plates using an inoculation loop. Seed cultures were incubated for 18-24 hours in a shaker at 250 rpm, 30 °C and 75% humidity.

[0111] Working cultures for obtaining fermentates were generated using 2 L stirred bioreactors containing 2 L of TSB medium that were inoculated at initial ODeoo of 0.1 using an inoculum from the overnight seed cultures. Working cultures were incubated in the stirred bioreactor at 700 revolutions per minute (rpm) while aerated, with a gas flow rate of 0.25 VVM (vessel volume per minute; i.e., 0.5 SLMP (standard liters per minute)), at 30 °C for 24 hours. A 50% solution of anti-foaming agent (Ivanhoe 1163B, Ivanhoe Industries, Inc.; Zion, Illinois, USA) was added to the bioreactor at a 0.1% flow rate during the incubation to prevent excess foaming in the bioreactor vessel. After 24 hours, the agitation and aeration were turned off in the bioreactor and the cultures were incubated for an additional 24 hours. After 48 hours post inoculation, 2 L vessels were heated to 60 °C and held at that set point for one hour to heat inactivate the microbial culture biomass. Fermentates were packaged and stored at -80 °C until further chemical and biological characterization.

[0112] The 2 L volumes of bacterial fermentates were dried and prepared for storage until use in animal studies. Before drying, each bacterial fermentate was mixed with a soy hull (or other suitable) carrier in a 1: 1 ratio on a dry matter basis. The soy hulls were sized at approximately 1.0 mm in diameter. The bacterial fermentates, were then individually mixed thoroughly and dried in an oven at 60 °C Drying time varied between 24 hours to up to 5 days depending on volume of bacterial fermentate to be dried. After incubation, the dried fermentate mixture was ground using a grinder equipped with a 0.5 mm screen until uniform consistency was achieved. In at least some batches, a tracer compound, such as a metal oxide, was added at to the dried bacterial fermentate compositions of approximately 5 g per inclusion rate of product. Fully dried bacterial fermentates were stored in polymeric air-tight packaging, labeled, and stored in a -20 °C freezer until use. Example 6; Analysis of Microbial Fermentates on Clostridium Perfrinsens Reduction Using an Infection Model in Broiler Chickens

[0113] Assays described in this example evaluated the effects of bacterial fermentates from Bacillus velezensis, Pediococcus pentosaceus, and Lactobacillus plantarum on Clostridium Perfringens infection reduction in broiler chickens.

[0114] Male broiler chickens (Cobb 500, Cobb-Vantress Inc.) were separated into six treatment groups, each treatment group having 12 pens and each pen starting with 8 chickens on day zero (DO). The groups included an unchallenged negative control group (CON); a challenged, positive control group (POS); an antibiotic control group challenged and treated with the antibacterial BMD® (Bacitracin-Methylene-Disalicylate; Zoetis, USA); a challenged group treated with Bacillus velezensis fermentate; a challenged group treated with Lactobacillus pediococcus fermentate; and a challenged group treated vcdh Lactobacillus plantarum fermentate. The birds were vaccinated for Mareks upon hatching. On study day zero (DO), the birds were vaccinated for Newcastle disease and infectious bronchitis by spray application using a spray cabinet. Each treatment group is outlined in Table 8.

Table 8. Treatment Groups - Experimental Design

[0115] Birds were fed a diet of poultry feed with the various treatments at the inclusion rates as outlined in Table 8 until the end of the study. Vanous performance metrics were monitored and recorded during the study. Body weight in grams was recorded on study days 0, 14, 21, and 28. Body weight gain in grams was compared over the periods including days 0-14, 0-21, 0-28, 14-21, and 14-28. Mortality-adjusted feed conversion rate (FCR; recorded as points) was determined over days 0-14, 0-21, 0-28, 14-21, and 14-28. Livability (in %) was recorded from days 0-14 and 0-28. Necrotic enteritis ceca lesion scores were recorded using the scoring schema as outlined in Table 9.

Table 9. Lesion Scoring

[0116] The various performance metrics that were monitored and recorded during the study and are presented and summarized below in reference to Tables 10-12. | () 1 171 Weight Results indicate that the negative control birds were significantly heavier than challenged birds at Day 21 and Day 28, with no differences observed in weight gain across the challenged groups. The negative control birds showed significantly higher weight gain than challenged birds at all time intervals except Day 0-14. No differences were observed across the challenged groups, except that birds that received the Lactobacillus plantarum fermentate had significantly lower weight gain than other challenged treatments at Day 14-28. Weight results are outlined in Table 10.

Table 10. Body Weight for All Treatment Groups

[0118] Feed Conversion Rate (FCR)'. Results indicate that negative control birds had significantly improved FCR compared to challenged groups at all time intervals containing challenge periods after Day 14. No significant differences were observed in FCR at any time interval across challenged groups, except that birds that received the Lactobacillus plantarum fermentate had significantly higher FCR from Day 0-28 and Day 14-28. The FCR was adjusted for mortality within each treatment group. FCR results are outlined in Table 11. Table 11. FCR for All Treatment Groups

[0119] Livability (%): No significant differences were observed at any time intervals across treatment groups.

[0120] Lesion Score: Results indicate that the positive control challenge birds had significantly higher lesion scores than the negative control birds (i.e., 1. 19 ± 0.07 vs. 0.00). Birds that received the Bacillus velezensis fermentate and the Lactobacillus pediococcus fermentate had significantly reduced lesion scores compared to the positive control challenged birds (i.e., 0.75 ± 0 09 and 0.78 ± 0.10 vs. 1.19 ± 0.07, respectively). The birds that received BMD® antibiotic treatment had numerically but not statistically significant lower lesion scores than the positive control challenged birds (i.e., 1.08 ± 0.078 vs. 1.19 ± 0.07). Birds that received the Bacillus velezensis fermentate and the Pediococcus pentosaceus fermentate had lesion scores that were significantly lower than BMD® treated birds. Lesion scores are outlined in Table 12.

Table 12. Lesion Scores for Each Treatment Group

[0121] The Bacillus velezensis fermentate and the Pediococcus pentosaceus fermentate treatments significantly reduced lesion scores in the necrotic enteritis challenge model as compared to the non-treated positive control treatment.

Example 7: Preparation of Eimeria Oocysts

[0122] Eimeria oocysts were prepared for analysis to determine the effects of fermentates on various types of Eimeria protozoa.

[0123] Unsporulated Eimeria tenella and maxima oocysts were obtained following two passages in broiler chickens. An oocyst and fecal slurry was stored in an air-tight container in 2% (w/v) potassium di chromate at 4 °C until needed for use. Aliquots of 10 milliliters (ml) were removed from storage and placed into a T-25 tissue culture flask in 2.5% (w/v) potassium dichromate solution and incubated at room temperature in a shaking incubator for 72-120 hours. A small aliquot was observed under a microscope at 40/ magnification to determine sporulation percentage of at least 85% sporulation to excise sporozoites. Once 85-90% sporulation was achieved, viability was checked using ethidium bromide dye. Oocysts were then cleaned as described below.

[0124] An aliquot of the oocyst-fecal suspension was removed, cleaned, and concentrated by following a bicarbonate-ether technique. Briefly, the oocyst-fecal matter suspension was harvested and added to a 50 ml centrifuge tube, spun for 5 minutes and the supernatant was discarded. The sediment was resuspended in 35 ml of 1% sodium bicarbonate. A 3-5 ml layer of ether was added and the contents were mixed by vigorous shaking. The mixture was centnfuged for 5 minutes at 1600 x g and the debris plug and supernatant were discarded. The remaining sediment was washed by being resuspend in water, centrifuged for 1600 x g for 5 minutes and the supernatant discarded. The remaining sediment was resuspended in 2.5% (w/v) potassium di chromate solution and 1% antibiotics. Washed oocysts were stored at 4 °C until ready for use

Example 8; In Vitro Analysis of Microbial Fermentates on Eimeria Viability

[0125] A trypan blue staining assay was performed to analyze Eimeria viability following incubation with microbial fermentates. The trypan blue staining assay allows for a direct identification and enumeration of Eimeria protozoa at various life cycle phases. Viability of Eimeria oocysts can be detennined by trypan blue exclusion from the interior of the oocyst. A viable oocyst has a clear cytoplasm in the presence of trypan blue, whereas a nonviable oocyst has a blue cytoplasm in the presence of trypan blue stain. Trypan blue exclusion staining can be utilized to stain Eimeria protozoa at various life stages, including but not to be limited to oocysts, sporocysts, and sporozoites.

[0126] Cleaned samples of Eimeria tenella and Eimeria maxima oocysts were centrifuged at 1600 x g for 10 minutes to remove the potassium dichromate and antibiotic storage solution. The pelleted oocysts were washed three times with phosphate buffered saline (PBS), centrifuging at 1600 x g for 10 mins each time. The washed oocysts were resuspended at a concentration of about 1 x 10 ⁶ oocyst/ml in PBS, and each well of a 96-well round bottom plate was inoculated with 10 pl, or 1 x 10 ⁴ oocyst per well in PBS. Fermentates were thawed from individual 100 pl stocks and filter sterilized with a 0.2-micron polyethylene terephthalate (PET) filter. Each well was brought to a total volume of 200 pl by adding 170 pl of sterile PBS and 20 pl of each sterilized fermentate in duplicate. A total of 343 fermentates were assayed. Control wells were included as follows: PBS only, fermentate alone, and untreated oocysts with and without trypan blue exclusion dye.

[0127] The inoculated oocysts were incubated at 39 °C, with 5% CO2 for 24 hours. After 24 hours the plate was removed from the incubator and 2 pl of 0.4% (w/v) trypan blue was added to each well while aspirating gently 5-8 times. The plate was placed back into the incubator at 39 °C, with 5% CO2 for 5 minutes. Cell viability was read on Cytation 5 (Agilent Technologies, CA, USA) plate reader at 590 nm within 10 minutes of trypan blue addition.

[0128] Fermentates that resulted in death of Eimeria oocysts were recorded according to the presence of compromised membranes as observed by trypan blue assay. The effects of the 343 fermentates against sporulated Eimeria oocysts was examined by staining with trypan blue. Of the 343 fermentates, 29 fermentates showed significant difference as compared to controls for Eimeria maxima, and 26 fermentates showed significant difference as compared to controls for Eimeria tenella. Effective fermentates were further screened using the motility assay described in Example 5 below.

Example 9; In Vitro Analysis of Microbial Fermentates on Eimeria Sporozoite Motility

[0129] Motility analysis of Eimeria sporozoites was assayed using a sporozoite motility screening assay as follows

[0130] Sporulation of Eimeria. Suspended oocysts and fecal slurry at or around 1.5 x 10 ⁵ oocysts per milliliter (mL) were stored in an air-tight container in 2.5% (w/v) potassium dichromate at 4 °C until needed for use. Aliquots of 50-200 mL (oocyst and 2.5% (w/v) potassium dichromate) were removed from storage and placed directly into Erlenmeyer flask and incubated at room temperature (23 °C) with a forced aeration pump, 110 Air Dual (Marine Metal Products, Clearwater Florida, USA) for 72-120 hours. Every 24 hours a small aliquot was observed using an inverted microscope (Olympus, Corp., Waltham, MA, USA) at 20* magnification to determine complete or partial sporulation. Complete sporulation consists of four sporocyst, each containing two sporozoites. Once sporulation reached at least 85% sporulation, oocysts were stored at 4 °C prior to cleaning.

[0131] Cleaning of Eimeria oocysts: The potassium di chromate storage media was removed. The oocysts were washed and centrifuged at 1000 times the relative centrifugal force (RCF) for 10 minutes in deionized water five separate times. The oocysts were sterilized in a 10% (v/v) sodium hypochlorite sterilization step in an ice bath for 5-10 minutes, followed by centrifugation of the solution at 1000 x RCF for 10 minutes. The supernatant was discarded.

[0132] Isolation of the oocysts from fecal slurry: The fecal slurry was resuspended in a saturated sodium chloride solution (20-35 mL). The solution was then overlayed with 2-5 mL distilled water. The solution was centrifuged for 5 minutes at 1000 x RCF. The cleaned and separated oocysts were localized to the interface between the NaCl and FEO. The oocysts were collected from the interface with a syringe and 16-gauge needle and placed into a clean conical vial. This step was repeated, starting with the overlay solution, to collect additional oocysts from the fecal slurry . After the second collection, 25 mL of Hank’s Balanced Salt Solution (HBSS) (Thermo Fischer, Co) was added to the oocysts, and the oocysts were then washed with centrifugation three times at 1000 x RCF. The oocysts were ready for use and were stored at 4 °C in distilled H2O.

[0133] Isolation and purification of sporozoites: The oocyst suspension was counted using a hemocytometer, and the number required for the assay were removed (approximately 2 x 10 ⁶ oocysts). The oocysts were placed into a 2 mL round bottom centrifuge tube and were centrifuged for 30 seconds at 10,000 x RCF to pellet the oocysts. The pelleted oocysts was suspended in 1 mL HBSS and 0.25 mL - 0.5 mL of 0.5 mm diameter (Jencons, No 8. Ballottini) glass beads were added. The tube was placed into a bead beater for 10 seconds. An aliquot (25 pl) of the solution was removed and the contents were checked to see how many of the oocysts were lysed, and the tube was placed back into the bead beater as needed, without exceeding three cycles and stopping once free sporozoites were seen. The sporozoites were transferred from the glass beads by repeated washes of HBSS medium into a clean 15 mL conical tube (3-4 washes).

[0134] For excystation of the sporozoites, 0.25-0.5% (w/v) Trypsin, 1% taurocholic acid, and 0.3% bile salts were added to the sporozoites, which were then incubated at 39-41 °C for 60- 90 minutes, depending on species. The sporozoites were checked every 30 minutes to determine when most of the sporozoites were free. The sporozoites and debris were washed in Dulbecco’s Modified Eagles Medium (DMEM) (Dulbecco’s, Fischer Scientific) and centrifuged as before to remove excess trypsin. The washing and centrifugation were performed two times, to form pelleted sporozoites. Once recovered, the sporozoites were utilized in assays in the same day.

[0135] Following incubation, the 96-well plates were analyzed using an 1X81 Olympus (Olympus, Corp., Waltham, MA, USA) inverted microscope and Well Plate Navigator software (Olympus, Corp., Waltham, MA, USA). Each well was observed under 40 x magnification and live video acquisition was performed. Each sample was scanned to find from 20-60 motile Eimeria in a single field of view (FOV). The magnification was reduced to 20< refocused, and live video was recorded for 30 seconds to 1 minute. Video was acquired for each well at 20* magnification in triplicate and saved to hard drive memory for sporozoite motility analysis using EthoVision XT software (Noldus Information Tech., Leesburg, VA, USA).

[0136] Results indicate that the fermentates showed inhibitory effects against the Eimeria tenella sporozoites after incubation using the tracking software to track and analyze the movement and activity of the sporozoite as compared to the control.

Example 10: Preparation of 20 L Microbial Fermentates for In Vivo Analysis

[0137] For in vivo analysis, bacterial derived fermentates of the various bacterial strains described in Example 1 were prepared in a 20 L stirred bioreactor as follows. Bacterial cultures were grown in TSB (Tryptic Soy Broth) containing the following composition: Bacto™ Tryptone (Pancreatic Digest of Casein, Thermo Fisher Scientific, Waltham, MA USA) 17.0 g/L, Bacto™ Soytone (Peptic Digest of Soybean Meal, Thermo Fisher Scientific, Waltham, MA USA) 3.0 g/L, glucose 2.5 g/L, sodium chloride 5.0 g/L, and dipotassium hydrogen phosphate 2.5 g/L. Overnight seed cultures were generated using 500 ml baffled shake flasks with 100 ml of TSB medium. Seed culture medium was inoculated with fresh biomass of a single bacterial colony from prepared agar plates using an inoculation loop. Seed cultures were incubated for 18-24 hours in a shaker at 250 rpm, 30 °C and 75% humidity.

[0138] Fungal derived fermentates of the various microbial strains described in Example 1 were prepared in a 20 L stirred bioreactor as follows. In specific, yeast cultures were grown in YPD Medium (Yeast Extract Peptone Dextrose, Thermo Fisher Scientific, Waltham, MA USA). Overnight seed cultures were generated using 500 ml baffled shake flasks with 100 ml of YPD medium. Seed culture medium was inoculated with fresh biomass of a single yeast colony from prepared agar plates using an inoculation loop. Seed cultures were incubated for 18-24 hours in a shaker at 250 rpm, 30 °C and 75% humidity.

[0139] Working bacterial cultures for obtaining bacterial fermentates were generated using 20 L stirred bioreactors containing 18 L of TSB medium with 0.6% airflow that were inoculated at initial ODeoo of 0. 1 using an inoculum from the overnight seed cultures. Working bacterial cultures were incubated in the stirred bioreactor at 500 revolutions per minute (rpm), with a gas flow rate of 0.25 VVM (vessel volume per minute; i.e., 4.5 SLMP (standard liters per minute)), at 30 °C for 24 hours. A 50% solution of anti-foaming agent (Ivanhoe XFO-324K, Ivanhoe Industries, Inc.; Zion, Illinois, USA) was added to the bioreactor at a 0.1% flow rate during the incubation to prevent excess foaming in the bioreactor vessel. Cultures were allowed to mature for an additional 24 hours at 30 °C with no agitation and no airflow.

[0140] Working yeast cultures for obtaining yeast fermentates were generated using 20 L stirred bioreactors containing 18 L of YPD medium that were inoculated at initial ODeoo of 0.1 using an inoculum from the overnight seed cultures. Working yeast cultures were incubated in the stirred bioreactor at 500 revolutions per minute (rpm), with a gas flow rate of 0.25 VVM (vessel volume per minute; i.e., 4.5 SLMP (standard liters per minute)), at 30 °C for 24 hours. A 100% solution of XFO-324K anti-foaming agent (Ivanhoe Industries, Inc.; Zion, Illinois, USA) was added to the bioreactor including a feedback probe having low to medium sensitivity with a flow pump at 10 % flow rate for 5 second periods during the incubation to prevent excess foaming in the bioreactor vessel.

[0141] For both the fermentation of bacteria and yeast, after 24 hours, the agitation was turned off in the bioreactor and the cultures were incubated for an additional 24 hours. After 48 hours post inoculation, the 20 L vessels were heated at 60 °C with agitation at 200 rpm and no air flow for one hour to heat inactivate the microbial culture biomass. Fermentates were packaged, chilled to 10 °C, and stored at -80 °C until further chemical and biological characterization.

[0142] The 18 L volumes of bacterial or yeast fermentates were dried and prepared for storage until use in animal studies. Before drying, each fermentate was mixed with a soy hull (or other suitable) carrier in a 1 : 1 ratio on a dry matter basis. The soy hulls were sized at approximately 1 0 mm in diameter. The fermentates, were then individually mixed thoroughly and dried in an oven at 60 °C. Drying time varied between 24 hours to up to 5 days depending on volume of each fermentate to be dried. After incubation, the dried fermentate mixture was ground using a grinder equipped with a 0.5 mm screen until uniform consistency was achieved. In at least some batches, a tracer compound, such as a metal oxide, was added at to the dried fermentate compositions of approximately 5 g per inclusion rate of product. Fully dried fermentates were stored in polymeric air-tight packaging, labeled, and stored in a -20 °C freezer until use.

Example 11: In vivo Analysis of Microbial Fermentates on Eimeria Reduction Using an Infection Model in Broiler Chickens

[0143] Experiments described in this example evaluate the effects of bacterial fermentates on Eimeria acervulina, Eimeria maxima, and Eimeria tenella infection reduction in broiler chickens.

[0144] Broiler chickens (Cobb 500, Cobb-Vantress Inc.) will be separated into by treatment groups, each treatment group starting with 18 chickens on day zero (DO). The treatment groups will be replicated into 12 cages with a total of 216 birds per treatment group. On day 7, the birds will be counted and adjusted to 16 birds per pen, where any extra birds will be removed, weighed, and recorded. The birds will be vaccinated for Mareks upon hatching. On study day zero (DO), the birds will be vaccinated for Newcastle disease, infectious bronchitis, and coccidiosis by spray application using a spray cabinet. Each Treatment Group is outlined in Table 13.

Table 13. Treatment Groups, Experimental Design

[0145] The birds are provided water and feed ad libitum throughout the study unless otherwise noted. The birds will be observed twice daily for clinical symptoms, and weights of each chicken were recorded on days 0, 14, 28, and 35. Feed will be included in the diet for each treatment group from day 0 of the study. The feed will be weighed in on day 0 and non-consumed feed weighed on days 14, 28, and 35. Feed intake will be calculated from days 0-14, 14-28, 0-28, 0-35, and 28-35. An unmedicated commercial type chicken starter compounded with feedstuffs commonly used in the United States is formulated for daily use as a basal starter feed. Experimental treatment feeds are prepared from the basal starter feed. Quantities of all basal feed and test articles used to prepare treatment batches will be documented. Treatment diets are mixed to assure a uniform distribution of respective fermentate.

[0146] Average bird weight per pen on days 14, 28, and 35 are to be calculated. The average feed conversion ratio (FCR) will be calculated on approximate study days 14, 28, and 35 (i.e , days 0-14, 14-28, 0-28, 0-35, and 28-35) using the total feed consumption forthe pen divided by the total weight of the surviving birds, and as described elsewhere herein. Adjusted feed conversion ratios will be calculated using the total feed consumption in the pen divided by the total weight of surviving birds plus the weight of birds that died or were removed from a respective pen.

[0147] Birds will be challenged with one or more of three species of Eimeria, including Eimeria acervulina, Eimeria maxima, and Eimeria tenella. The Eimeria inoculum mixtures will be administered in 1 ml volume by oral gavage on Day 14 to the birds in the infected treatments outlined above. The challenge will include the following amounts of oocysts per treatment group: approximately 4,000 Eimeria maxima oocysts per bird, approximately 10,000 acervulina Eimeria oocysts per bird, and approximately 2,000 Eimeria tenella oocysts per bird in 1 ml volumes via oral gavage.

[0148] On approximately days 21 and 28, excreta will be collected from three birds per pen by squeezing the bird’s hind ends. Samples from all three birds will be collected individually and assayed for oocyst counts using the McMasters method or equivalent counting method. Oocysts per gram (OPG) of excreta will be recorded. On days 21 and 28 the three birds assayed for oocyst count will be assayed for ceca lesion presence using lesion scoring methods as discussed elsewhere herein. Morbidity and mortality will also be documented.

[0149] On Days 21 and 28, three birds from each group will be used to collect excreta samples and will be scored for lesions and assayed for the presences of Eimeria acervulina, Eimeria maxima, and Eimeria tenella using a scoring schema as outlined herein. The intestinal mucosa will be scraped onto a glass slide and the contends will be observed under a microscope and scored for the presence of oocysts according to the scoring metrics outlined in Table 14.

Table 14. Intestinal Mucosa Oocyst Scoring Schema

[0150] Mean data for group weight gain, feed consumption, feed conversion, lesion scores, oocysts excreted per gram (OPG), and mortality is to be calculated and analyzed by standard statistical procedures. Birds exhibiting abnormal behavior will be documented and removed from the study as needed.

[0151] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference is to be considered supplementary to that of this document: for irreconcilable inconsistencies, the usage in this document controls.

[0152] Values expressed in a range format are to be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” is to be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0153] Unless expressly stated, ppm (parts per million), percentage, and ratios are on a by weight basis. Percentage on a by weight basis (% w/w) is also referred to as weight percent (wt.%) or percent by weight (% wt.) herein.