WO2023172440A1

WO2023172440A1 - METHODS, COMPOSITIONS, AND FOOD ADDITIVES COMBINING A F4/F18 VACCINE AND β-MANNANASE AND/OR A PROBIOTIC FOR THE TREATMENT AND PREVENTION OF ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC) IN PIGS

Info

Publication number: WO2023172440A1
Application number: PCT/US2023/014443
Authority: WO
Inventors: Pedro Sanchez URIBE
Original assignee: Elanco Us Inc.
Priority date: 2022-03-07
Filing date: 2023-03-03
Publication date: 2023-09-14
Also published as: AR128708A1

Abstract

Methods, compositions, and food additives combining a vaccine and β-mannanase and/or a probiotic for the treatment and prevention of enterotoxigenic Escherichia coli (ETEC) in pigs, optionally a F4/F18 vaccine, are provided.

Description

METHODS, COMPOSITIONS, AND FOOD ADDITIVES COMBINING A F4/F18 VACCINE AND p-MANNANASE AND/OR A PROBIOTIC FOR THE TREATMENT AND PREVENTION OF ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC) IN PIGS

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This International Patent Application claims priority to U.S. Provisional Patent Application No. 63/317,422, filed on March 7, 2022, the disclosure of which is incorporated herein in its entirety.

BACKGROUND

Field

[0002] The present disclosure relates to the use of a F4/F18 Escherichia coli vaccine (F4/F18) in combination with P-mannanase or a probiotic for the treatment and/or prevention of diarrhea in pigs.

Description of Related Art

[0003] The use of vaccines against Escherichia coli (E coli) directly delivered to piglets is one of the useful interesting tools in preventing intestinal disorders during nursery and. Accordingly, there is a need in the agriculture industry for improved vaccine compositions to treat and/or prevent diarrhea in piglets caused by enterotoxigenic Escherichia coli (ETEC).

SUMMARY

[0004] In an embodiment, a method for the treatment and/or prevention of E. coli diarrhea in pig can comprise administering a F4/F18 vaccine and an effective amount of composition comprising P-mannanase to a mammal in need thereof.

[0005] In an embodiment, a method for the treatment and/or prevention of E. coli diarrhea in pig can comprise administering a F4/F18 vaccine and an effective amount of a composition comprising a probiotic to a mammal in need thereof.

[0006] In an embodiment, a method for the treatment and/or prevention of E. coli diarrhea in pig can comprise administering a F4/F18 vaccine and an effective amount of composition comprising P-mannanase and a priobiotic to a mammal in need thereof.

[0007] In an embodiment, a method for the improvement in intestinal health, decreased inflammation in the gut, and/or an improved microbiome in the intestine in a pig can comprise administering a F4/F18 vaccine and an effective amount of composition comprising P- mannanase to a pig in need thereof.

[0008] In an embodiment, the method can comprise administering a composition comprising the vaccine, P-mannanase, probiotic, prebiotic, or a combination thereof. The method can comprise administering a composition comprising the vaccine and P-mannanase. [0009] In an embodiment, a method for the treatment and/or prevention of E. coli diarrhea in pig can comprise administering a vaccine and an effective amount of composition comprising P- mannanase to a mammal in need thereof. The vaccine can be a F18-E. coli vaccine, F4- . coli vaccine, or a F4/F18 combination vaccine. The vaccine can be an or a avirulent live vaccine. [0010] In an embodiment, the F4/F18 vaccine can comprise a combination of live non- pathogenic E. coli O8:K87 (F4ac) and live non-pathogenic E. coli 0141 :K94 (F18ac). The F4/F18 vaccine can comprise between about l.OxlO⁸ and 9xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli O8:K87 (F4ac). The F4/F18 vaccine can comprise between about 1.3xl0⁸ and 9xl0⁸ CFU (colony forming Units) of live non-pathogenic E. coli O8:K87 (F4ac). The F4/F18 vaccine can comprise between about 2.0xl0⁸ and 4xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli 0141 :K94 (F18ac). The F4/F18 vaccine can comprise between about 2.8xl0⁸ and 3xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli 0141 :K94 (F18ac). In some aspects, the live non-pathogenic E. coli may not be attenuated. The F4/F18 vaccine can be administered orally. The F4/F18 vaccine can be administered orally via drinking water.

[0011] In an embodiment, the method can further comprise improving intestinal health, decreasing inflammation in the gut, and/or an improving the microbiome in the intestine of the Pig-

10012] In an embodiment, the P-mannanase can be administered orally.

[0013] In an embodiment, the composition comprising P-mannanase can further comprise animal feed. The animal feed can be mash feed. The composition can comprise P-mannanase comprises about at least 64 million units/lb.

[0014] In an embodiment, the composition comprising P-mannanase can comprise between about 1 and 100 million units. The composition can comprise P-mannanase comprises between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,

54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,

80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 million units.

The composition can comprise P-mannanase comprises about at least 64 million units per ton of feed.

[0015] In an embodiment, the P-mannanase can be from Bacillus lentus.

[0016] In an embodiment, the probiotic can comprise Bacillus strains, Pediococcus strains, Bifidobacterium strains, Lactobacillus strains, Enterococcus strains, Streptococcus strains, or a combination thereof. The probiotic can comprise a Lactic Acid Bacteria (LAB). The Lactic Acid Bacteria (LAB) can comprise Lac tobacillus, Bifidobacterium, Lactococcus Lactophaera, Leuconostoc, Melissococcus, Oenococcuss, Pediococcus, Streptococcus, Enterococcus, or a combination thereof. The probiotic comprises Bacillus subtilis strains, Pediococcus pentosaceus strains, Bacillus toyoi strains, Bifidobacterium lactis strains, or a combination thereof. The probiotic can comprise Asperigllus oryzae, Candida pintopolessi, Saccharomyces boulardii, Saccharomyces cerviseae, or a combination thereof.

[0017] In an embodiment, the composition comprising a probiotic can further comprise animal feed. The effective amount of the probiotic can be between about IxlO⁵ CFU/kg of animal feed and IxlO¹² CFU/kg of animal feed. The effective amount of the probiotic can be about IxlO⁶ CFU/kg of animal feed, IxlO⁷ CFU/kg of animal feed, IxlO⁸ CFU/kg of animal feed, IxlO⁹ CFU/kg of animal feed, IxlO¹⁰ CFU/kg of animal feed, IxlO¹¹ CFU/kg of animal feed, or IxlO¹² CFU/kg of animal feed.

[0018] In an embodiment, the composition can be a solution, suspension, gel, or lyophilisate. The composition can be formulated for oral administration. The composition can be formulated for oral administration via drinking water.

[0019] In an embodiment, the composition can further comprise excipients, solvents, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or combinations thereof. The excipients can be Dextran 40,000, sucrose, monosodium glutamate, water, optionally purified or deionized water, or combinations thereof.

[0020] In an embodiment, the method can further comprise administering a prebiotic. The prebiotic can be selected from the group consisting of Mannan-oligosacaharides (MOS), Fruto- oligosacharides (FOS), Galacto-oligosacharides (GOS), Chito-Oligosacharide (COS), Isomaltooligosaccharides (IMO), Pectic-oligosaccharides (POS), Xylo-oligosaccharides (XOS), and combinations thereof.

[0021] In an embodiment, the effective amount is sufficient to reduce the incidence of moderate to severe post-weaning E. coli diarrhea (PWD) in infected mammals.

[0022] In an embodiment, the effective amount is sufficient to reduce the fecal shedding of enterotoxigenic F4-positive and Fl 8-positive E. coli from infected mammals.

[0023] In an embodiment, the effective amount is sufficient to reduce the incidence of mortality in infected mammals.

[0024] In an embodiment, the mammal can be a pig. The pig can be a piglet. The piglets can be between about 1 day and 30 days old. The piglets can be at least about 18 days old.

[0025] In an embodiment, the F4/F18 vaccine and the composition comprising P-mannanase can be administered to the mammal on the same day. [0026] In an embodiment, the F4/F18 vaccine and the composition comprising P-mannanase can be administered to the mammal on different days. The F4/F18 vaccine can be administered prior to the composition comprising P-mannanase. The F4/F18 vaccine can be administered between about 1 to 100 days prior to administration of the composition comprising P-mannanase. The F4/F18 vaccine can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,

46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,

72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,

98, 99, or 100 prior to administration of the composition comprising P-mannanase.

[0027] In an embodiment, the F4/F18 vaccine can be administered after the composition comprising P-mannanase. The F4/F18 vaccine can be administered between about 1 to 100 days after administration of the composition comprising P-mannanase. The F4/F18 vaccine can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,

52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,

78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 after administration of the composition comprising P-mannanase.

[0028] In an embodiment, the F4/F18 vaccine and the composition comprising a probiotic can be administered to the mammal on the same day.

[0029] In an embodiment, the F4/F18 vaccine and the composition comprising a probiotic can be administered to the mammal on different days.

[0030] In an embodiment, the F4/F18 vaccine can be administered prior to the composition comprising a probiotic. The F4/F18 vaccine can be administered between about 1 to 100 days prior to administration of the composition comprising a probiotic. The F4/F18 vaccine can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 prior to administration of the composition comprising a probiotic.

[0031] In an embodiment, the F4/F18 vaccine can be administered after the composition comprising a probiotic. The F4/F18 vaccine can be administered between about 1 to 100 days after administration of the composition comprising a probiotic. The F4/F18 vaccine can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 after administration of the composition comprising a probiotic.

[0032] In an embodiment, the E. coli diarrhea may be caused by enterotoxigenic Escherichia coli (ETEC).

[0033] In an embodiment, a composition can comprise an effective amount of P-mannanase formulated for administration to vaccinated mammals. The composition can be formulated for oral administration.

[0034] In an embodiment, a composition can further comprise animal feed. The animal feed can be mash feed.

[0035] In an embodiment, the composition can comprise P-mannanase comprises about at least 64 million units per ton of feed. The P-mannanase can be from Bacillus lentus. The composition comprising P-mannanase can comprise about at least 64 million units/lb. The composition comprising P-mannanase can comprise between about 1 and 100 million units. The composition comprising P-mannanase can comprise between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,

67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,

93, 94, 95, 96, 97, 98, 99, or 100 million units.

[0036] In an embodiment, the composition can further comprise a probiotic. The probiotic can comprise Bacillus strains, Pediococcus strains, Bifidobacterium strains, Lactobacillus strains, Enterococcus strains, Streptococcus strains, or a combination thereof. The probiotic can comprise a Lactic Acid Bacteria (LAB). The Lactic Acid Bacteria (LAB) can comprise Lactobacillus, Bifidobacterium, Lactococcus Lactophaera, Leuconostoc, Melissococcus, Oenococcuss, Pediococcus, Streptococcus, Enterococcus, or a combination thereof. The probiotic can comprise Bacillus subtilis strains, Pediococcus pentosaceus strains, Bacillus toyoi strains, Bifidobacterium lactis strains, or a combination thereof. The probiotic can comprise Asperigllus oryzae, Candida pintopolessi, Saccharomyces boulardii, Saccharomyces cerviseae, or a combination thereof.

[0037] In an embodiment, the composition comprising a probiotic can further comprise animal feed. The effective amount of the probiotic can be between about IxlO⁵ CFU/kg of animal feed and IxlO¹² CFU/kg of animal feed. The effective amount of the probiotic can be about IxlO⁶ CFU/kg of animal feed, IxlO⁷ CFU/kg of animal feed, IxlO⁸ CFU/kg of animal feed, IxlO⁹ CFU/kg of animal feed, IxlO¹⁰ CFU/kg of animal feed, IxlO¹¹ CFU/kg of animal feed, or IxlO¹² CFU/kg of animal feed.

[0038] In an embodiment, the composition can be a solution, suspension, gel, or lyophilisate. [0039] In an embodiment, the composition can be formulated for oral administration.

[0040] In an embodiment, the composition can be formulated for oral administration via drinking water.

[0041] In an embodiment, the composition can further comprise excipients, solvents, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or combinations thereof. The excipients can comprise Dextran 40,000, sucrose, monosodium glutamate, water, optionally purified or deionized water, or combinations thereof.

[0042] In an embodiment, the composition can further comprise a prebiotic. The prebiotic can be selected from the group consisting of Mannan-oligosacaharides (MOS), Fruto- oligosacharides (FOS), Galacto-oligosacharides (GOS), Chito-Oligosacharide (COS), Isomaltooligosaccharides (IMO), Pectic-oligosaccharides (POS), Xylo-oligosaccharides (XOS), and combinations thereof. The effective amount can be sufficient to reduce the incidence of moderate to severe post- weaning E. coli diarrhea (PWD) in infected mammals. The fecal shedding of enterotoxigenic E. coli from infected mammals, optionally F4-positive and F 18- positive enterotoxigenic E. coli. The effective amount can be sufficient to reduce the incidence of mortality in infected pigs.

[0043] In an embodiment, a food additive can comprise an effective amount of P-mannanase formulated for administration to vaccinated mammals. The food additive can be formulated for oral administration.

[0044] In an embodiment, the method for the preparation of a feed composition can comprise admixing an effective amount of P-mannanase into animal feed and formulating the animal feed for administration to pigs vaccinated against diarrhea-causing E. coli.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0046] Figure 1 depicts the canonical plot for discriminant functions analysis. The discriminant function analysis allows the inventors to predict with high probability (p<0,05) what will be the expression of different biomarkers (e.g., the bubbles being different on the chart and creating subsets of data) and therefore, knowing in advance what are the benefits at intestinal level. The plot demonstrates the separation of each treatment, showing a clear and different effect from the different strategies.

[0047] Figure 2 depicts canonical plot for discriminant functions analysis at each sampling moment for control, F4/F18 vaccine alone, combination of F4/F18 vaccine with P-mannanase (HEM), and Hemicell® HT (P-mannanase) alone. The discriminant function analysis allows the inventors to predict with high probability (p<0,05) what will be the expression of different biomarkers (e.g., the bubbles being different on the chart and creating subsets of data) and therefore, knowing in advance what are the benefits at intestinal level. Here, the inventors were able to identify a different behavior of the subsets of data, showing a distinguishable response depending on the treatment.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] In order that the invention herein described may be fully understood, the following detailed description-set forth. Various embodiments of the invention are described in detail and may be further illustrated by the provided examples. Additional viable variations of the embodiments can easily be envisioned.

Definitions

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs.

[0050] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise.

[0051] “Mammal,” as used herein, refers broadly to any and all warm-blooded vertebrate animals of the class Mammalia, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. Mammals include, but are not limited to, humans, domestic and farm animals, and zoo, sports, or pet animals. Examples of mammals include but are not limited to alpacas, armadillos, capybaras, cats, camels, chimpanzees, chinchillas, cattle, dogs, gerbils, goats, gorillas, guinea pigs, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, and tapirs. Mammals include but are not limited to bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammal also includes any and all those listed on the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington D.C. Similarly, the term “subject” or “patient” includes both human and veterinary subjects and/or patients.

F4/F18 Vaccine Combinations [0052] The inventors combined F4/F18 vaccines with a second agent to provide an unexpected improvement in the treatment and/or prevention of diarrhea in pigs caused by enterotoxigenic Escherichia coli (ETEC).

[0053] The F4/F18 vaccine can comprise a combination of live non-pathogenic E. coli O8:K87 (F4ac) and live non-pathogenic E. coli 0141 :K94 (F18ac). The F4/F18 vaccine can comprise between about l.OxlO⁸ and 9xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli O8:K87 (F4ac), for example, about 1.3xl0⁸ and 9xl0⁸ CFU (colony forming Units) of live non- pathogenic E. coli O8:K87 (F4ac). The F4/F18 vaccine can comprise between about 2.0xl0⁸ and 4xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli 0141 :K94 (F18ac), for example between about 2.8xl0⁸ and 3xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli 0141 :K94 (F18ac). In the F4/F18 vaccine, the live non-pathogenic E. coli is not attenuated. The F4/F18 vaccine can be administered orally. For example, the F4/F18 vaccine is administered orally via drinking water. The F4/F18 vaccine can be provided as a lyophilisate, formulated for oral suspension. The F4/F18 vaccine can further comprise excipients, including but not limited to Dextran 40,000, sucrose, monosodium glutamate, water, optionally purified water or deionized water, or combinations thereof.

Vaccine + f>-mannanase Combination

[0054] The use of vaccines against E. coli directly delivered to piglets is one of the most interesting tools to prevent intestinal disorders during nursery and early finishing period. On the other hand, P-mannans are oligosaccharides presents in a wide range of feedstuff, which produce a mimic effect of E. coli receptor, resulting in chronic inflammation of intestine and then decrease in performances of piglets. The inventors found an unexpected improvement in the treatment and/or prevention of diarrhea in piglets caused by enterotoxigenic Escherichia coli (ETEC) by combining vaccination with P-mannanase to the animals’ feed. The P-mannanase hydrolyzes the P-mannans present in soybean and cornmeal in pig feed.

[0055] The vaccine against E. coli that cause diarrhea and the composition comprising P- mannanase can be administered to the mammal on the same day. The vaccine and the composition comprising P-mannanase can be administered to the mammal on different days. The vaccine can be a F18-E. coli vaccine, F4-E. coli vaccine, or a F4/F18 combination vaccine. The vaccine can be a vaccine against enterotoxigenic strain of Escherichia coli having the K99, K88, 987P, F41 adherence factors, or a combination thereof. The vaccine can be an attenuated vaccine or an avirulent live vaccine. The vaccine and the composition can be administered on the same day. [0056] A composition comprising P-mannanase can further comprise linseed meal solvent extracted, calcium carbonate, and mineral oil. The P-mannanase can be present in Bacillus lentus fermentation solubles. For purposes of administration, about 1 pound (lb.) of P-mannanase composition comprising at least 64 million units/lb. can be added to one ton of mash feed.

[0057] One unit of P-mannanase activity can be defined as the amount of enzyme which generates 0.72 microgram of reducing sugars per minute from a mannose-containing substrate at pH 7.5 and temperature of 104°F.

[0058] The P-mannanase can be provided by Hemicell® (P-mannanase composition).

[0059] The P-mannanase can be administered orally.

[0060] The composition comprising P-mannanase can further comprise animal feed. The animal feed can be mash feed.

[0061] The P-mannanase can be from Bacillus lentus.

[0062] The composition comprising P-mannanase can comprise about at least 64 million units/lb. The composition comprising P-mannanase can comprise between about 1 and 100 million units. The composition comprising P-mannanase can comprise between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,

58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,

84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 million units. The composition comprising P-mannanase can comprise about at least 64 million units per ton of feed.

Vaccine + Probiotic Combination

[0063] Intestinal health has arisen as a major concern in the swine industry. In fact, in 2022, zinc oxide, one of the most valuables preventive measures against diarrhea, was banned in the European Union. The inventors found an unexpected improvement in the treatment and/or prevention of diarrhea in piglets caused by enterotoxigenic Escherichia coli (ETEC) by combining vaccination with a probiotic.

[0064] The methods described herein can comprise administering a vaccine (e.g., to a nonhuman animal) against E. coli that cause diarrhea, P-mannanase, and a probiotic. The vaccine, the composition comprising P-mannanase, and a composition comprising a probiotic can be administered to the mammal on the same day. The vaccine and the compositions comprising P- mannanase and/or a probiotic can be administered to the mammal on different days. The composition comprising a probiotic be added to the animal feed, e.g., food additive, optionally the animal feed already comprises P-mannanase. The P-mannanase and the probiotic can be administered together in a single composition of separately in different compositions.

[0065] A method for the treatment and/or prevention of diarrhea caused by Escherichia coli in a pig can comprise administering a vaccine against Escherichia coli and an effective amount of composition comprising P-mannanase to the pig, optionally further comprising an effective amount of a probiotic.

[0066] The vaccine can be a F18-E. coli vaccine, F4- . coli vaccine, or a F4/F18 combination vaccine. The vaccine can be a vaccine against enterotoxigenic strain of Escherichia coli having the K99, K88, 987P, F41 adherence factors, or a combination thereof. The vaccine can be an attenuated vaccine or an avirulent live vaccine. The vaccine and the composition can be administered on the same day.

[0067] Probiotics refer to a group of non-pathogenic organisms that when administered in sufficient numbers are known to have beneficial effects on health of the host animal. Without wishing to be bound to a specific theory, hypothesized mechanisms of action of probiotics include: (A) reducing the pH, which is an inhospitable environment for intestinal pathogens; (B) attachment on the intestinal epithelial surfaces to prevent pathogen attachment; (C) competition for nutrients with pathogens; (D) production of inhibitory substances such as organic acids, hydrogen peroxide, and bacteriocins; and (E) stimulation of specific and nonspecific immunity such as IL and IgA.

[0068] The composition comprising probiotics can be administered to the mammal orally. The probiotics can be added to animal feed. The composition can comprise a vaccine, P-mannanase, probiotic, prebiotic, or a combination thereof. The composition can comprises a composition comprising a vaccine and P-mannanase.

[0069] The probiotic can comprise Bacillus strains, Pediococcus strains, Bifidobacterium strains, Lactobacillus strains, Enterococcus strains, Streptococcus strains, or a combination thereof.

[0070] The probiotic can comprise a Lactic Acid Bacteria (LAB). The Lactic Acid Bacteria (LAB) can comprise Lactobacillus, Bifidobacterium, Lactococcus Lactophaera, Leuconostoc, Melissococcus, Oenococcuss, Pediococcus, Streptococcus, Enterococcus, or a combination thereof. The probiotic can comprise Bacillus subtilis strains, Pediococcus pentosaceus strains, Bacillus toyoi strains, Bifidobacterium lactis strains, or a combination thereof. The probiotic can comprise Asperigllus oryzae, Candida pintopolessi, Saccharomyces boulardii, Saccharomyces cerviseae, or a combination thereof. [0071] The composition comprising a probiotic can be added to the mammal’s feed. The effective amount of the probiotic can be between about IxlO⁵ CFU/kg of animal feed and IxlO¹² CFU/kg of animal feed. The effective amount of the probiotic can be about IxlO⁶ CFU/kg of animal feed, IxlO⁷ CFU/kg of animal feed, IxlO⁸ CFU/kg of animal feed, IxlO⁹ CFU/kg of animal feed, IxlO¹⁰ CFU/kg of animal feed, IxlO¹¹ CFU/kg of animal feed, or IxlO¹² CFU/kg of animal feed.

Compositions

[0072] In addition to the F4/F18 vaccine and other agents disclosed herein, the compositions can further comprise at least one of any suitable auxiliaries including, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or combinations thereof. Pharmaceutically acceptable auxiliaries are preferred. Examples and methods of preparing such sterile solutions are well known in the art and can be found in well-known texts such as, but not limited to, REMINGTON’ S PHARMACEUTICAL SCIENCES (Gennaro, Ed., 18th Edition, Mack Publishing Co. (1990)). Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the compound.

[0073] Excipients and additives useful in the present invention can also include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, terra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination in ranges of 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin, gelatin, casein, or combinations thereof. Representative amino acid components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and combinations thereof.

[0074] Carbohydrate excipients suitable for use in the present invention include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, combinations thereof; disaccharides, such as lactose, sucrose, trehalose, cellobiose, combinations thereof; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, combinations thereof; and alditols, such as mannitol, maltitol, lactitol, sorbitol (glucitol), myoinositol, combinations thereof.

[0075] Prebiotics are non-digestible feed ingredients that are fermented in the lower gut to select for beneficial bacteria; during the fermentation, there is production of Volatile Fatty Acids (VFAs) mostly acetic, propionic and butyric acid that reduce the intestinal pH. Examples of prebiotics include but are not limited to Mannan-oligosacaharides (MOS), Fruto-oligosacharides (FOS), Galacto-oligosacharides (GOS), Chito-Oligosacharide (COS), Isomalto-oligosaccharides (IMO), Pectic-oligosaccharides (POS), and Xylo-oligosaccharides (XOS). Prebiotics have immunomodulatory properties and the potential to reduce inflammation when supplemented into pig diets. At weaning, piglets face many stressors, such as change of environment, fight for hierarchy, and change from liquid to dry feed. The methods described herein can alleviate these stressors to lower the incidence of post-weaning diarrhea (PWD).

[0076] Additionally, the compositions described herein can further comprise an additive including but not limited to polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins), polyethylene glycols, flavoring agents, anti-microbial agents, sweeteners, antioxidants, anti-static agents, surfactants (e.g., polysorbates including but not limited to “Tween® 20” and “Tween® 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and combinations thereof. These and additional known pharmaceutical excipients and/or additives suitable for use in the present invention are known in the art, e.g., as listed in REMINGTON: THE SCIENCE & PRACTICE OF PHARMACY (19th ed., Williams & Williams (1995)) and PHYSICIAN’ S DESK REFERENCE (52nd ed., Medical Economics (1998)).

Animals

[0077] The methods described herein can be used on mammals. The mammal can be a horse, cow, or pig. The mammal can be a pig. The pig can be a piglet. The piglet can be between 1 day and 30 days old. The piglet can be at least about 18 days old.

Administration Schedule

[0078] The methods described herein can comprise administering a vaccine against A. coli that cause diarrhea and P-mannanase. The vaccine and the composition comprising P-mannanase can be administered to the mammal on the same day. The vaccine and the composition comprising P-mannanase can be administered to the mammal on different days.

[0079] A method for the treatment and/or prevention of diarrhea caused by Escherichia coli in a pig can comprise administering a vaccine against Escherichia coli and an effective amount of composition comprising P-mannanase to the pig.

[0080] The vaccine can be a F l 8-E. coli vaccine, F4- . coli vaccine, or a F4/F18 combination vaccine. The vaccine can be a vaccine against enterotoxigenic strain of Escherichia coli having the K99, K88, 987P, F41 adherence factors, or a combination thereof. The vaccine can be an attenuated vaccine or an avirulent live vaccine. The vaccine and the composition can be administered on the same day.

[0081] The vaccine can be a F4/F18 vaccine and comprises a combination of live non- pathogenic E. coli O8:K87 (F4ac) and live non-pathogenic E. coli 0141 :K94 (F18ac). The vaccine can be a F4/F18 vaccine and comprises between about l.OxlO⁸ and 9xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli O8:K87 (F4ac). The vaccine can be a F4/F18 vaccine and comprises between about 1.3xl0⁸ and 9xl0⁸ CFU (colony forming Units) of live non-pathogenic E. coli O8:K87 (F4ac). The vaccine can be a F4/F18 vaccine and comprises between about 2.0xl0⁸ and 4xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli 0141 :K94 (F18ac). The vaccine can be a F4/F18 vaccine and comprises between about 2.8xl0⁸ and 3xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli 0141 :K94 (F18ac). The live non-pathogenic E. coli may not be not attenuated.

[0082] The vaccine can be administered orally. The vaccine can be administered orally via drinking water.

[0083] The vaccine can be administered prior to the composition comprising P-mannanase. The vaccine can be administered between about 1 to 100 days prior to administration of the composition comprising P-mannanase. The vaccine can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,

88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 prior to administration of the composition comprising P-mannanase.

[0084] The vaccine can be administered after the composition comprising P-mannanase. The vaccine can be administered between about 1 to 100 days after administration of the composition comprising P-mannanase. The vaccine can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,

88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 after administration of the composition comprising P-mannanase.

[0085] The vaccine and the composition comprising a probiotic can be administered to the mammal on the same day. The vaccine and the composition comprising a probiotic can be administered to the mammal on different days. [0086] The vaccine can be administered prior to the composition comprising a probiotic. The vaccine can be administered between about 1 to 100 days prior to administration of the composition comprising a probiotic. The vaccine can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 prior to administration of the composition comprising a probiotic.

[0087] The vaccine can be administered after the composition comprising a probiotic. The vaccine can be administered between about 1 to 100 days after administration of the composition comprising a probiotic. The vaccine can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 after administration of the composition comprising a probiotic.

[0088] The F4/F18 vaccine and the composition comprising P-mannanase can be administered to the mammal on the same day. The F4/F18 vaccine and the composition comprising P- mannanase can be administered to the mammal on different days.

[0089] The F4/F18 vaccine can be administered prior to the composition comprising P- mannanase. The F4/F18 vaccine can be administered between about 1 to 100 days prior to administration of the composition comprising P-mannanase. The F4/F18 vaccine can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 prior to administration of the composition comprising P-mannanase.

[0090] The F4/F18 vaccine can be administered after administering the composition comprising P-mannanase. The F4/F18 vaccine can be administered between about 1 to 100 days after administration of the composition comprising P-mannanase. The F4/F18 vaccine can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 after administration of the composition comprising P-mannanase.

[0091] The F4/F18 vaccine and the composition comprising a probiotic can be administered to the mammal on the same day. The F4/F18 vaccine and the composition comprising a probiotic can be administered to the mammal on different days.

[0092] The F4/F18 vaccine can be administered prior to the composition comprising a probiotic. The F4/F18 vaccine can be administered between about 1 to 100 days prior to administration of the composition comprising a probiotic. The F4/F18 vaccine can be administered 1, 2, 3, 4, 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,

34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,

60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 prior to administration of the composition comprising a probiotic.

[0093] The F4/F18 vaccine can be administered after the composition comprising a probiotic. The F4/F18 vaccine can be administered between about 1 to 100 days after administration of the composition comprising a probiotic. The F4/F18 vaccine can be administered 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 after administration of the composition comprising a probiotic.

EXAMPLES

EXAMPLE 1

EFFECT OF ORAL VACCINATION AGAINST ESCHERICHIA COLI COMBINED WITH A NUTRITIONAL TOOL DURING NURSERY. PERFORMANCES AND INTESTINAL INTEGRITY

Materials and Methods

[0094] Three hundred and eighty-eight piglets were individually weighed from weaning to start of finishing, and the feed conversion rate and feed consumption was calculated on a pen basis. Two preventive strategies were assayed: oral vaccination against E. coli with Coliprotect® F4/F18 (Elanco Animal Health), and feed supplementation with a probiotic. Four groups were allotted: vaccinated+ ZnO (VZn), vaccinated+probiotic (VP), probiotic (P) and ZnO (Zn).

[0095] At the end of nursery period feces samples were obtained to perform a panel of intestinal integrity by gene expression analysis of calprotectin, occluding, zonulin, IFN-y and TGF-p. The comparison of data was performed using ANOVA for physical performances and Kruskal- Wallis and Mann-Whitney’s U test for gene expression of intestinal integrity panel. To reduce intestinal integrity data, a discriminant function analysis was performed.

[0096] Results

[0097] The physical performance appears in Tables 1 and 2.

Table 1. Physical performance for prestarter feed

Table 2. Physical performance for starter feed

Starter feed

Where IW= initial weight, FW= final weight, WG= weigh gain, FP= feed per piglet (Kg), FCR= feed conversion rate (Kg/Kg), ADG= average daily gain (g/d).

[0098] As regards intestinal integrity assessment, there was a higher mRNA quantification in VP for calprotectin, occluding and IFN-y and the highest quantification for Zonulin for Zn and VP groups. The lowest quantification was observed in P groups. The discriminant functions analysis allowed to assign correctly 90% of the samples to each putative group. The Figure 2 shows the plot of the two main functions.

[0099] Discussion and Conclusion

[0100] The prevention of enteric diseases during nursery is today one of the hardest issues for veterinarians and producers. The use of vaccine and probiotics offers an improved solution for prevention of diseases, especially those related to E. coli. Oral vaccination against E. coli has been effective. The inventors observed the synergistic action of Coliprotec® F4/F18 and probiotics on intestinal integrity. In the trial, the P groups showed the lowest quantification for all the markers, indicating an intestinal healthier status. The increased quantification of calprotectin and IFN-y in VP group is consistent with a vaccination effect. In this trial, the results from vaccination with the probiotic or alone were better than ZnO group. A higher weight increase and better FCR in prestarter period suggest better intestinal function. EXAMPLE 2

STUDY OF SYNERGISTIC ACTION ON INTESTINAL INTEGRITY OF AN ORAL VACCINE AGAINST ESCHERICHIA COLI AND A B-MANNANASE ADDED TO THE FEED

Introduction

[0101] The inventors found synergistic action on intestinal integrity with the combination of an oral vaccine against Escherichia coli and a P-mannanase was added to the feed.

[0102] The use of vaccines against E. coli directly delivered to piglets is one of the most interesting tools to prevent intestinal disorders during nursery and early finishing period. On the other hand, P-mannans are oligosaccharides presents in a wide range of feedstuff, which produce a mimic effect of E. coli receptor, resulting in chronic inflammation of intestine and then decrease in performances of piglets. This work describes the results of a trial combining vaccination with a P-mannanase in feed.

Materials and Methods

[0103] Four groups of piglets were randomly allotted after weaning, receiving oral vaccination against E. coli with Coliprotect® F4/F18 (Elanco Animal Health) (COLI), one group receiving a feed supplemented with Hemicell (Elanco Animal Health), a P-mannanase (HEM), one group with vaccination and Hemicell® HT in feed (COLI+HEM), and one control untreated group (CONT). All piglets were housed in the same barn with the same managements and environmental conditions.

[0104] Samples of feces were obtained at weaning (sampling pretreatment) and subsequently at 9, 23 and 33 days after vaccination or start receiving the feed treatment. The feces were preserved in RNAlater, and freezed after 24 hours in refrigeration. The, total RNA was isolated, cDNA was synthetized using oligo-dT as primer, and qPCR was performed to detect gene expression of Calprotectin (an indicator of inflammatory cell infiltration), Ocludin and Zonulin (proteins of tight-junctions), INF-y as one of the main proinflammatory cytokines and TGF-P as the main anti-inflammatory cytokine. The housekeeper gene sed was P-actin and the relative quantification was used using the Pfaffal’s method (1). The comparison was made by Mann- Whitney’s U test, and an analysis of discriminant functions to reduce data.

Results

[0105] There was difference in the quantification for all biomarkers in the 2^nd, 3^rd and 4^th samplings. When the discriminant function analysis was performed, the separation of groups is clear at each sampling moment. The Wilks’ lambda signification was p<0.0001 for all samplings. [0106] Table 3 shows the discriminant capacity of the functions designed by the statistical software. Generally, all samples in the control group are always correctly assigned to this group. Interestingly, the wrong assignations were made between groups that share some treatment (COLI or HEM with COLI+HEM).

Table 3: Assignation of Samples to Putative Groups Using the Discriminant Functions Designed

[0107] A plot with the canonical distribution of samples is shown in Figure 2.

[0108] Results evident the separation of the groups along the time which indicate an evident effect of the treatments.

Discussion and Conclusion

[0109] The prevention of enteric diseases during nursery, avoiding the use of zinc oxide (banned in June 2022) is going to be based in combinations of tools up to get the best effect. The study of intestinal integrity allows to evaluate the effect of some of these strategies. In this case, differences in biomarkers as Calprotectin, Occludin and Zonulin, and in IFN-y were observed. Interestingly, even when the increase of this cytokine is expectable after vaccination, the increase is lower in the group COLI+HEM, which could indicate a reduction of undesirable immune stimulation derived from mannans. Anyway, the effect on all the biomarkers is evident, producing a higher separation of centroids at each subsequent sampling moment. EXAMPLE 3

HEMICELL HT® (A BETA-MANNANASE ENZYME) COMBINED WITH AN E. COLI F4/F18 VACCINATION RETAINS POST- WEANED PIGLET PERFORMANCE IN THE PRESENCE OF CHALLENGING PROTEIN SOURCES

Introduction

[0110] P-Mannans are strongly anti -nutritive polysaccharide fibres found in most vegetable feed ingredients. They belong to the hemicellulose fraction and have a backbone composed entirely of mannose, as in mannans and galactomannans, or of mannose and glucose, as in glucomannans and galacto-glucomannans. The estimated content of soluble P-mannans in common fattening diets is only 0.20-0.35%, and in vitro studies have demonstrated that as little as 0.05% soluble P- mannan in feed can elicit a strong innate immune response. This innate response is often referred to as a feed induced immune response or FUR, which suppresses growth to protect the liver and reserve energy and nutrients for high priority immune functions. Hemicell HT (Elanco) is a P-mannanase enzyme for animal feed that breaks down P-mannans and thereby prevents economic losses from the wasteful immune response to P-mannans. The objective was to compare piglet performance and antibiotic use between a Control group, fed a conventional 3- phase diet, and an Enzyme treated group, fed an adapted 3 -phase diet including a P-mannanase enzyme (Hemicell™ HT; Elanco).

Materials & methods

[oni] A seven week feeding trial was conducted with 896 piglets - vaccinated with an E. coli F4/F18 vaccine (Coliprotec F4F18; Elanco) in two rotations of 448 piglets in 32 replicate pens of 14 pigs. Two different 3-phase diets were compared: a standard 3-phase control diet and an adapted 3-phase diet including a P-mannanase enzyme included at 300 g/tonne. The following adaptations were made:

Phase-1 (weeks 1-2): 1.14% potato protein concentrate and 1.00% Forcital (extruded soya product) were replaced with soybean meal.

Phase-2 (weeks 3-4): 0.46% potato protein concentrate and 0.68% Forcital were replaced with soybean meal.

Phase-3 (weeks 5-7): P-mannanase was formulated to replace 63 kcal/kg NE.

Standard piglet performance parameters (ADWG, ADFI, FCR) and antibiotic use were recorded. All data analyses were performed using R version 3.6.3 (R Core Team, 2020).

Results

[0112] Throughout the trial and within each phase, ADWG, ADFI and FCR were not significantly different (P > 0.05) between Control and Enzyme group. Mortality was significantly (P < 0.001) lower (-0.90 %) in the Enzyme treated group. Antimicrobial use was significantly (P < 0.01) lower (-56%) in the Enzyme treated group as compared to the Control group.

[0113] Table 4 : Overall trials results including initial body weight (BW), final BW, average daily feed intake (ADFI), average daily weight gain (ADWG), feed conversion rate (FCR), mortality and antimicrobial use (expressed as number of individual injections).

Control Enzyme P-value

Initial BW, kg 4.955 4.943 0.427

Final BW, kg 21.682 21.206 0.223

ADFI, kg/d 0.533 0.535 0.468

ADWG, kg 0.341 0.332 0.174

FCR, kg/kg 1.584 1.604 0.414

Mortality, % 1.79 0.89 < 0.001

Antimicrobial _{123 54} < ₀.01 use (# injections)

The Control group was fed a standard 3 -phase diet, whereas the Enzyme treated group received an adapted diet where expensive proteins sources were partially replaced by soybean meal in Phase- 1 and -2, and net energy was reduced by 63 kcal/kg in Phase 3. Statistical data analysis was performed using R version 3.6.3 (R Core Team, 2020).

Discussion & Conclusions

[0114] Inclusion of a P-mannanase to nursery diets with an adapted formulation by replacing expensive protein sources by soybean meal in the first two phases or reducing the NE content by 63 kcal/kg in the third phase, resulted in similar piglet performance post-weaning with reduced mortality and less antimicrobials used.

[0115] Although the invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it should be understood that certain changes and modifications may be practiced within the scope of the appended claims. Modifications of the above-described modes for carrying out the invention that would be understood in view of the foregoing disclosure or made apparent with routine practice or implementation of the invention to persons of skill in animal husbandry, vaccinations, and/or related fields are intended to be within the scope of the following claims.

[0116] All publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All such publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent, patent application publication, or patent application was specifically and individually indicated to be incorporated by reference.

[0117] While the foregoing invention has been described in connection with this preferred embodiment, it is not to be limited thereby but is to be limited solely by the scope of the claims which follow.

Claims

1. A method for the treatment and/or prevention of E. coli diarrhea in pig comprising administering a F4/F18 vaccine and an effective amount of composition comprising P- mannanase to a mammal in need thereof.

2. A method for the treatment and/or prevention of E. coli diarrhea in pig comprising administering a F4/F18 vaccine and an effective amount of a composition comprising a probiotic to a mammal in need thereof.

3. The method of claim 1 or 2, wherein the F4/F18 vaccine comprises a combination of live non-pathogenic E. coli O8:K87 (F4ac) and live non-pathogenic E. coli 0141 :K94 (F18ac).

4. The method of any one of claims 1-3, wherein the F4/F18 vaccine comprises between about l.OxlO⁸ and 9xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli O8:K87 (F4ac).

5. The method of any one of claims 1-4, wherein the F4/F18 vaccine comprises between about 1.3xlO⁸ and 9xl0⁸ CFU (colony forming Units) of live non-pathogenic E. coli O8:K87 (F4ac).

6. The method of any one of claims 1-5, wherein the F4/F18 vaccine comprises between about 2.0xl0⁸ and 4xl0⁹ CFU (colony forming Units) of live non-pathogenic E. coli O141:K94 (F18ac).

7. The method of any one of claims 1-6, wherein the F4/F18 vaccine comprises between about 2.8xl0⁸ and 3xlO⁹ CFU (colony forming Units) of live non-pathogenic E. coli O141:K94 (F18ac).

8. The method of any one of claims 1-7, wherein the live non-pathogenic E. coli is not attenuated.

9. The method of any one of claims 1-8, wherein the F4/F18 vaccine is administered orally.

10. The method of any one of claims 1-9, wherein the F4/F18 vaccine is administered orally via drinking water.

11. The method of any one of claims 1-10, wherein the P-mannanase is administered orally.

12. The method of any one of claims 1-11, wherein the composition comprising P-mannanase further comprises animal feed.

13. The method of claim 12, wherein the animal feed is mash feed.

14. The method of any one of claims 1-13, wherein the P-mannanase is from Bacillus lentus.

15. The method of any one of claims 1-14, wherein the composition comprising P-mannanase comprises about at least 64 million units/lb. The method of any one of claims 1-15, wherein the composition comprising P-mannanase comprises between about 1 and 100 million units. The method of any one of claims 1-15, wherein the composition comprising P-mannanase comprises between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,

45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,

69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,

93, 94, 95, 96, 97, 98, 99, or 100 million units. The method of any one of claims 1-17, wherein the composition comprising P-mannanase comprises about at least 64 million units per ton of feed. The method of any one of claims 1-18, wherein the method further comprises administering a probiotic. The method of any one of claims 1-19, wherein the probiotic comprises Bacillus strains, Pediococcus strains, Bifidobacterium strains, Lactobacillus strains, Enterococcus strains, Streptococcus strains, or a combination thereof. The method of any one of claims 1-20, wherein the probiotic comprises a Lactic Acid Bacteria (LAB). The method of claim 21, wherein the Lactic Acid Bacteria (LAB) comprises Lactobacillus, Bifidobacterium, Lactococcus Lactophaera, Leuconostoc, Melissococcus, Oenococcuss, Pediococcus, Streptococcus, Enterococcus, or a combination thereof. The method of any one of claims 1-22, wherein the probiotic comprises Bacillus subtilis strains, Pediococcus pentosaceus strains, Bacillus toyoi strains, Bifidobacterium lactis strains, or a combination thereof. The method of any one of claims 1-23, wherein the probiotic comprises Asperigllus oryzae, Candida pintopolessi, Saccharomyces boulardii, Saccharomyces cerviseae, or a combination thereof. The method of any one of claims 1-24, wherein the composition comprising a probiotic further comprises animal feed. The method of any one of claims 1-25, wherein the effective amount of the probiotic is between about IxlO⁵ CFU/kg of animal feed and IxlO¹² CFU/kg of animal feed. The method of any one of claims 1-26, wherein the effective amount of the probiotic is about IxlO⁶ CFU/kg of animal feed, IxlO⁷ CFU/kg of animal feed, IxlO⁸ CFU/kg of animal feed, IxlO⁹ CFU/kg of animal feed, IxlO¹⁰ CFU/kg of animal feed, IxlO¹¹ CFU/kg of animal feed, or IxlO¹² CFU/kg of animal feed. The method of any one of claims 1-27, wherein the composition is a solution, suspension, gel, or lyophilisate. The method of any one of claims 1-28, wherein the composition is formulated for oral administration. The method of any one of claims 1-29, wherein the composition is formulated for oral administration via drinking water. The method of any one of claims 1-30, wherein the composition further comprises excipients, solvents, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or combinations thereof. The method of claim 31, wherein the excipients comprise Dextran 40,000, sucrose, monosodium glutamate, water, optionally purified or deionized water, or combinations thereof. The method of any one of claims 1-32, wherein the method further comprises administering a prebiotic. The method of claim 33, wherein the prebiotic is selected from the group consisting of Mannan-oligosacaharides (MOS), Fruto-oligosacharides (FOS), Galacto-oligosacharides (GOS), Chito-Oligosacharide (COS), Isomalto-oligosaccharides (IMO), Pectic- oligosaccharides (POS), Xylo-oligosaccharides (XOS), and combinations thereof. The method of any one of claims 1-34, wherein the effective amount is sufficient to reduce the incidence of moderate to severe post-weaning E. coli diarrhea (PWD) in infected mammals. The method of any one of claims 1-35, wherein the effective amount is sufficient to reduce the fecal shedding of enterotoxigenic F4-positive and Fl 8-positive E. coli from infected mammals. The method of any one of claims 1-36, wherein the effective amount is sufficient to reduce the incidence of mortality in infected mammals. The method of any one of claims 1-37, wherein the mammal is a pig. The method of claim 38, wherein the pig is a piglet. The method of claim 39, wherein the piglets are between about 1 day and 30 days old. The method of claim 39 or 40, wherein the piglets are at least about 18 days old. The method of any one of claims 1-41, wherein the F4/F18 vaccine and the composition comprising P-mannanase are administered to the mammal on the same day. The method of any one of claims 1-42, wherein the F4/F18 vaccine and the composition comprising P-mannanase are administered to the mammal on different days. The method of any one of claims 1-43, wherein the F4/F18 vaccine is administered prior to the composition comprising P-mannanase. The method of any one of claims 1-44, wherein the F4/F18 vaccine is administered between about 1 to 100 days prior to administration of the composition comprising P- mannanase. The method of any one of claims 1-45, wherein the F4/F18 vaccine is administered 1, 2, 3,

4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,

30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,

54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,

78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 prior to administration of the composition comprising P-mannanase. The method of any one of claims 1-46, wherein the F4/F18 vaccine is administered after the composition comprising P-mannanase. The method of any one of claims 1-47, wherein the F4/F18 vaccine is administered between about 1 to 100 days after administration of the composition comprising P- mannanase. The method of any one of claims 1-48, wherein the F4/F18 vaccine is administered 1, 2, 3,

78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 after administration of the composition comprising P-mannanase. The method of any one of claims 1-49, wherein the F4/F18 vaccine and the composition comprising a probiotic is administered to the mammal on the same day. The method of any one of claims 1-50, wherein the F4/F18 vaccine and the composition comprising a probiotic is administered to the mammal on different days. The method of any one of claims 1-51, wherein the F4/F18 vaccine is administered prior to the composition comprising a probiotic. The method of any one of claims 1-52, wherein the F4/F18 vaccine is administered between about 1 to 100 days prior to administration of the composition comprising a probiotic. The method of any one of claims 1-53, wherein the F4/F18 vaccine is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,90 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 prior to administration of the composition comprising a probiotic. The method of any one of claims 1-54, wherein the F4/F18 vaccine is administered after the composition comprising a probiotic. The method of any one of claims 1-55, wherein the F4/F18 vaccine is administered between about 1 to 100 days after administration of the composition comprising a probiotic. The method of any one of claims 1-56, wherein the F4/F18 vaccine is administered 1, 2, 3,

78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 after administration of the composition comprising a probiotic. The method of any one of claims 1-57, wherein the E. coll diarrhea is caused by enterotoxigenic Escherichia coli (ETEC). The method of any one of claims 1-58, wherein the method comprises administering a composition comprising the vaccine, P-mannanase, probiotic, prebiotic, or a combination thereof. The method of any one of claims 1-59, wherein the method comprises administering a composition comprising the vaccine and P-mannanase. A method for the treatment and/or prevention of diarrhea caused by Escherichia coli in a pig comprising administering a vaccine against Escherichia coli and a composition comprising an effective amount of P-mannanase to the pig. The method of claim 61, wherein the vaccine is a F18-E. coli vaccine, F4-E. coli vaccine, or a F4/F18 combination vaccine. The method of claim 61, wherein the vaccine is against enterotoxigenic strain of Escherichia coli having the K99, K88, 987P, F41 adherence factors, or a combination thereof. The method of any one of claims 61-63, wherein the vaccine is an attenuated vaccine or an avirulent live vaccine. The method of any one of claims 61-64, wherein the vaccine and the composition are administered on the same day. The method of any one of claims 61-62, wherein the vaccine is a F4/F18 vaccine and comprises a combination of live non-pathogenic E. coli O8:K87 (F4ac) and live non- pathogenic E. coli 0141 :K94 (F18ac). The method of any one of claims 61-65, wherein the vaccine is a F4/F18 vaccine and comprises between about l.OxlO⁸ and 9xl0⁹ CFU (colony forming Units) of live non- pathogenic E. coli O8:K87 (F4ac). The method of any one of claims 61-65, wherein the vaccine is a F4/F18 vaccine and comprises between about 1.3xlO⁸ and 9xl0⁸ CFU (colony forming Units) of live non- pathogenic E. coli O8:K87 (F4ac). The method of any one of claims 61-65, wherein the vaccine is a F4/F18 vaccine and comprises between about 2.0xl0⁸ and 4xl0⁹ CFU (colony forming Units) of live non- pathogenic E. coli 0141 :K94 (F18ac). The method of any one of claims 61-65, wherein the vaccine is a F4/F18 vaccine and comprises between about 2.8xl0⁸ and 3xl0⁹ CFU (colony forming Units) of live non- pathogenic E. coli 0141 :K94 (F18ac). The method of any one of claims 61-70, wherein the live non-pathogenic E. coli is not attenuated. The method of any one of claims 61-71, wherein the vaccine is administered orally. The method of any one of claims 61-72, wherein the vaccine is administered orally via drinking water. The method of any one of claims 61-73, wherein the P-mannanase is administered orally. The method of any one of claims 61-74, wherein the composition comprising P-mannanase further comprises animal feed. The method of claim 75, wherein the animal feed is mash feed. The method of any one of claims 61-76, wherein the P-mannanase is from Bacillus lentus. The method of any one of claims 61-77, wherein the effective amount of P-mannanase comprises about at least 64 million units/lb. The method of any one of claims 61-78, wherein the effective amount of P-mannanase comprises between about 1 and 100 million units. The method of any one of claims 61-79, wherein the effective amount of P-mannanase comprises between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 million units. The method of any one of claims 61-80, wherein the composition comprising P-mannanase comprises about at least 64 million units per ton of feed. The method of any one of claims 61-81, wherein the method further comprises administering a probiotic. The method of any one of claims 61-82, wherein the probiotic comprises Bacillus strains, Pediococcus strains, Bifidobacterium strains, Lactobacillus strains, Enterococcus strains, Streptococcus strains, or a combination thereof. The method of any one of claims 61-83, wherein the probiotic comprises a Lactic Acid Bacteria (LAB). The method of claim 84, wherein the Lactic Acid Bacteria (LAB) comprises Lactobacillus, Bifidobacterium, Lactococcus Lactophaera, Leuconostoc, Melissococcus, Oenococcuss, Pediococcus, Streptococcus, Enterococcus, or a combination thereof. The method of any one of claims 61-85, wherein the probiotic comprises Bacillus subtilis strains, Pediococcus pentosaceus strains, Bacillus toyoi strains, Bifidobacterium lactis strains, or a combination thereof. The method of any one of claims 61-86, wherein the probiotic comprises Asperigllus oryzae, Candida pintopolessi, Saccharomyces boulardii, Saccharomyces cerviseae, or a combination thereof. The method of any one of claims 61-87, wherein the composition comprising a probiotic further comprises animal feed. The method of any one of claims 61-88, wherein the effective amount of the probiotic is between about IxlO⁵ CFU/kg of animal feed and IxlO¹² CFU/kg of animal feed. The method of any one of claims 61-89, wherein the effective amount of the probiotic is about IxlO⁶ CFU/kg of animal feed, IxlO⁷ CFU/kg of animal feed, IxlO⁸ CFU/kg of animal feed, IxlO⁹ CFU/kg of animal feed, IxlO¹⁰ CFU/kg of animal feed, IxlO¹¹ CFU/kg of animal feed, or IxlO¹² CFU/kg of animal feed. The method of any one of claims 61-90, wherein the composition is a solution, suspension, gel, or lyophilisate. The method of any one of claims 61-91, wherein the composition is formulated for oral administration. The method of any one of claims 61-92, wherein the composition is formulated for oral administration via drinking water. The method of any one of claims 61-93, wherein the composition further comprises excipients, solvents, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or combinations thereof. The method of claim 94, wherein the excipients comprise Dextran 40,000, sucrose, monosodium glutamate, water, optionally purified or deionized water, or combinations thereof. The method of any one of claims 61-95, wherein the method further comprises administering a prebiotic. The method of claim 96, wherein the prebiotic is selected from the group consisting of Mannan-oligosacaharides (MOS), Fruto-oligosacharides (FOS), Galacto-oligosacharides (GOS), Chito-Oligosacharide (COS), Isomalto-oligosaccharides (IMO), Pectic- oligosaccharides (POS), Xylo-oligosaccharides (XOS), and combinations thereof. The method of any one of claims 61-97, wherein the effective amount is sufficient to reduce the incidence of moderate to severe post-weaning E. coli diarrhea (PWD) in infected mammals. The method of any one of claims 61-98, wherein the effective amount is sufficient to reduce the fecal shedding of enterotoxigenic E. coli from infected mammals, optionally F4-positive and Fl 8-positive enterotoxigenic E. coli. The method of any one of claims 61-99, wherein the effective amount is sufficient to reduce the incidence of mortality in infected pigs. The method of any one of claims 61-100, wherein the method comprises administering a composition comprising the vaccine, P-mannanase, probiotic, prebiotic, or a combination thereof. The method of any one of claims 61-101, wherein the pig is a piglet. The method of claim 102, wherein the piglets are between about 1 day and 30 days old. The method of claim 102 or 103, wherein the piglets are at least about 18 days old. The method of any one of claims 61-104, wherein the vaccine and the composition comprising P-mannanase are administered to the mammal on the same day. The method of any one of claims 61-105, wherein the vaccine and the composition comprising P-mannanase are administered to the mammal on different days. The method of any one of claims 61-106, wherein the vaccine is administered prior to the composition comprising P-mannanase. The method of any one of claims 61-107, wherein the vaccine is administered between about 1 to 100 days prior to administration of the composition comprising P-mannanase. The method of any one of claims 61-108, wherein the vaccine is administered 1, 2, 3, 4, 5,

6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,

55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,

79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 prior to administration of the composition comprising P-mannanase. The method of any one of claims 61-109, wherein the vaccine is administered after the composition comprising P-mannanase. The method of any one of claims 61-110, wherein the vaccine is administered between about 1 to 100 days after administration of the composition comprising P-mannanase. The method of any one of claims 61-111, wherein the vaccine is administered 1, 2, 3, 4, 5,

79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 after administration of the composition comprising P-mannanase. The method of any one of claims 61-112, wherein the vaccine and the composition comprising a probiotic is administered to the mammal on the same day. The method of any one of claims 61-113, wherein the vaccine and the composition comprising a probiotic is administered to the mammal on different days. The method of any one of claims 61-114, wherein the vaccine is administered prior to the composition comprising a probiotic. The method of any one of claims 61-115, wherein the vaccine is administered between about 1 to 100 days prior to administration of the composition comprising a probiotic. The method of any one of claims 61-116, wherein the vaccine is administered 1, 2, 3, 4, 5,

79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 prior to administration of the composition comprising a probiotic. The method of any one of claims 61-117, wherein the vaccine is administered after the composition comprising a probiotic. The method of any one of claims 61-118, wherein the vaccine is administered between about 1 to 100 days after administration of the composition comprising a probiotic. The method of any one of claims 61-119, wherein the vaccine is administered 1, 2, 3, 4, 5,

79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 after administration of the composition comprising a probiotic. The method of any one of claims 61-120, wherein the E. coli diarrhea is caused by an enterotoxigenic Escherichia coli (ETEC) strain. The method of any one of claims 1-121, wherein the method further comprises improving intestinal health, decreasing inflammation in the gut, and/or an improving the microbiome in the intestine of the pig. A composition comprising an effective amount of P-mannanase formulated for administration to vaccinated mammals. The composition of claim 123, wherein the composition is formulated for oral administration. The composition of claim 123 or 124, wherein the composition further comprises animal feed. The composition of claim 125, wherein the animal feed is mash feed. The composition of claim 125, wherein the composition comprising P-mannanase comprises about at least 64 million units per ton of feed. The composition of any one of claims 123-127, wherein the P-mannanase is from Bacillus lentus. The composition of any one of claims 123-128, wherein the composition comprising P- mannanase comprises about at least 64 million units/lb. The composition of any one of claims 123-129, wherein the composition comprising P- mannanase comprises between about 1 and 100 million units. The composition of any one of claims 123-130, wherein the composition comprising P- mannanase comprises between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,

66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 million units. The composition of any one of claims 123-131, wherein the composition further comprises a probiotic. The composition of claim 132, wherein the probiotic comprises Bacillus strains, Pediococcus strains, Bifidobacterium strains, Lactobacillus strains, Enterococcus strains, Streptococcus strains, or a combination thereof. The composition of claim 133, wherein the probiotic comprises a Lactic Acid Bacteria (LAB). The composition of claim 134, wherein the Lactic Acid Bacteria (LAB) comprises Lactobacillus, Bifidobacterium, Lactococcus Lactophaera, Leuconostoc, Melissococcus, Oenococcuss, Pediococcus, Streptococcus, Enterococcus, or a combination thereof. The composition of claim 132, wherein the probiotic comprises Bacillus subtilis strains, Pediococcus pentosaceus strains, Bacillus toyoi strains, Bifidobacterium lactis strains, or a combination thereof. The composition of claim 132, wherein the probiotic comprises Asperigllus oryzae, Candida pintopolessi, Saccharomyces boulardii, Saccharomyces cerviseae, or a combination thereof. The composition of any one of claims 123-137, wherein the composition comprising a probiotic further comprises animal feed. The composition of any one of claims 123-138, wherein the effective amount of the probiotic is between about IxlO⁵ CFU/kg of animal feed and IxlO¹² CFU/kg of animal feed. The composition of any one of claims 123-139, wherein the effective amount of the probiotic is about IxlO⁶ CFU/kg of animal feed, IxlO⁷ CFU/kg of animal feed, IxlO⁸ CFU/kg of animal feed, IxlO⁹ CFU/kg of animal feed, IxlO¹⁰ CFU/kg of animal feed, IxlO¹¹ CFU/kg of animal feed, or IxlO¹² CFU/kg of animal feed. The composition of any one of claims 123-140, wherein the composition is a solution, suspension, gel, or lyophilisate. The composition of any one of claims 123-141, wherein the composition is formulated for oral administration. The composition of any one of claims 123-142, wherein the composition is formulated for oral administration via drinking water. The composition of any one of claims 123-143, wherein the composition further comprises excipients, solvents, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or combinations thereof. The composition of claim 144, wherein the excipients comprise Dextran 40,000, sucrose, monosodium glutamate, water, optionally purified or deionized water, or combinations thereof. The composition of any one of claims 123-145, wherein the composition further comprises a prebiotic. The composition of claim 146, wherein the prebiotic is selected from the group consisting of Mannan-oligosacaharides (MOS), Fruto-oligosacharides (FOS), Galacto- oligosacharides (GOS), Chito-Oligosacharide (COS), Isomalto-oligosaccharides (IMO), Pectic-oligosaccharides (POS), Xylo-oligosaccharides (XOS), and combinations thereof. The composition of any one of claims 123-147, wherein the effective amount is sufficient to reduce the incidence of moderate to severe post-weaning E. coli diarrhea (PWD) in infected mammals. The composition of any one of claims 123-148, wherein the effective amount is sufficient to reduce the fecal shedding of enterotoxigenic E. coli from infected mammals, optionally F4-positive and Fl 8-positive enterotoxigenic E. coli. The composition of any one of claims 123-149, wherein the effective amount is sufficient to reduce the incidence of mortality in infected pigs. The composition of any one of claims 123-150, wherein the pig is a piglet. The composition of claim 151, wherein the piglets are between about 1 day and 30 days old. The composition of claim 151 or 152, wherein the piglets are at least about 18 days old. A food additive comprising an effective amount of P-mannanase formulated for administration to vaccinated mammals. The food additive of claim 154, wherein the composition is formulated for oral administration. The composition of claim 154 or 155, wherein the P-mannanase is from Bacillus lentus. The food additive of any one of claims 154-156, wherein the food additive comprises between about 1 and 100 million units of P-mannanase. The food additive of any one of claims 154-157, wherein the food additive comprises between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,

72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,

96, 97, 98, 99, or 100 million units of P-mannanase. The food additive of any one of claims 154-158, wherein the food additive further comprises a probiotic. The food additive of claim 159, wherein the probiotic comprises Bacillus strains, Pediococcus strains, Bifidobacterium strains, Lactobacillus strains, Enterococcus strains, Streptococcus strains, or a combination thereof. The food additive of claim 159, wherein the probiotic comprises a Lactic Acid Bacteria (LAB). The food additive of claim 161, wherein the Lactic Acid Bacteria (LAB) comprises Lactobacillus, Bifidobacterium, Lactococcus Lactophaera, Leuconostoc, Melissococcus, Oenococcuss, Pediococcus, Streptococcus, Enterococcus, or a combination thereof. The food additive of claim 159, wherein the probiotic comprises Bacillus subtilis strains, Pediococcus pentosaceus strains, Bacillus toyoi strains, Bifidobacterium lactis strains, or a combination thereof. The food additive of claim 159, wherein the probiotic comprises Asperigllus oryzae, Candida pintopolessi, Saccharomyces boulardii, Saccharomyces cerviseae, or a combination thereof. The food additive of any one of claims 154-164, wherein the food additive is in the form of a solution, suspension, gel, or lyophilisate. The food additive of any one of claims 154-165, wherein the food additive further comprises excipients, solvents, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or combinations thereof. The food additive of claim 166, wherein the excipients comprise Dextran 40,000, sucrose, monosodium glutamate, water, optionally purified or deionized water, or combinations thereof. The food additive of any one of claims 154-167, wherein the food additive further comprises a prebiotic. The food additive of claim 168, wherein the prebiotic is selected from the group consisting of Mannan-oligosacaharides (MOS), Fruto-oligosacharides (FOS), Galacto- oligosacharides (GOS), Chito-Oligosacharide (COS), Isomalto-oligosaccharides (IMO), Pectic-oligosaccharides (POS), Xylo-oligosaccharides (XOS), and combinations thereof. The food additive of any one of claims 154-169, wherein the effective amount is sufficient to reduce the incidence of moderate to severe post-weaning E. coli diarrhea (PWD) in infected mammals. The food additive of any one of claims 154-170, wherein the effective amount is sufficient to reduce the fecal shedding of enterotoxigenic E. coli from infected mammals, optionally F4-positive and Fl 8-positive enterotoxigenic E. coli. The food additive of any one of claims 154-171, wherein the effective amount is sufficient to reduce the incidence of mortality in infected pigs. The food additive of any one of claims 154-172, wherein the food additive is formulated to be added to animal feed, optionally pig feed. The food additive of claim 173, wherein the pig is a piglet. The food additive of claim 172 or 173, wherein the piglets are between about 1 day and 30 days old. The food additive of claim 174 or 175, wherein the piglets are at least about 18 days old. A method for the preparation of a feed composition comprising admixing an effective amount of P-mannanase into animal feed and formulating the animal feed for administration to pigs vaccinated against diarrhea-causing E. coli. The method of claim 177, wherein the method further comprises admixing a probiotic. The method of claim 177 or 178, wherein the animal feed is mash feed. The method of any one of claims 177-179, wherein the method comprises adding at least 64 million units of P-mannanase per ton of feed. The method of any one of claims 177-180, wherein the P-mannanase is from Bacillus lentus. The method of any one of claims 177-181, wherein the method comprises adding between about 1 and 100 million units of P-mannanase per ton. The method of any one of claims 177-182, wherein the method comprises adding between about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,

50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,

74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,

98, 99, or 100 million units of P-mannanase per ton. The method of any one of claims 177-183, wherein the method further comprises adding an effective amount of a probiotic. The method of claim 184, wherein the probiotic comprises Bacillus strains, Pediococcus strains, Bifidobacterium strains, Lactobacillus strains, Enterococcus strains, Streptococcus strains, or a combination thereof. The method of claim 184, wherein the probiotic comprises a Lactic Acid Bacteria (LAB). The method of claim 186, wherein the Lactic Acid Bacteria (LAB) comprises Lactobacillus, Bifidobacterium, Lactococcus Lactophaera, Leuconostoc, Melissococcus, Oenococcuss, Pediococcus, Streptococcus, Enterococcus, or a combination thereof. The method of claim 184, wherein the probiotic comprises Bacillus subtilis strains, Pediococcus pentosaceus strains, Bacillus toyoi strains, Bifidobacterium lactis strains, or a combination thereof. The method of claim 184, wherein the probiotic comprises Asperigllus oryzae, Candida pintopolessi, Saccharomyces boulardii, Saccharomyces cerviseae, or a combination thereof. The method of any one of claims 177-189, wherein the effective amount of the probiotic is added at between about IxlO⁵ CFU/kg of animal feed and IxlO¹² CFU/kg of animal feed. The method of any one of claims 177-190, wherein the effective amount of the probiotic is added at about IxlO⁶ CFU/kg of animal feed, IxlO⁷ CFU/kg of animal feed, IxlO⁸ CFU/kg of animal feed, IxlO⁹ CFU/kg of animal feed, IxlO¹⁰ CFU/kg of animal feed, IxlO¹¹ CFU/kg of animal feed, or IxlO¹² CFU/kg of animal feed. The method of any one of claims 177-191, wherein the P-mannanase is a solution, suspension, gel, or lyophilisate. The method of any one of claims 177-192, wherein the method further comprises adding excipients, solvents, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, or combinations thereof. The method of claim 193, wherein the excipients comprise Dextran 40,000, sucrose, monosodium glutamate, water, optionally purified or deionized water, or combinations thereof. The method of any one of claims 177-194, wherein the method further comprises adding a prebiotic. The method of claim 195, wherein the prebiotic is selected from the group consisting of Mannan-oligosacaharides (MOS), Fruto-oligosacharides (FOS), Galacto-oligosacharides (GOS), Chito-Oligosacharide (COS), Isomalto-oligosaccharides (IMO), Pectic- oligosaccharides (POS), Xylo-oligosaccharides (XOS), and combinations thereof. The method of any one of claims 177-196, wherein the effective amount is sufficient to reduce the incidence of moderate to severe post-weaning E. coli diarrhea (PWD) in infected mammals. The method of any one of claims 177-197, wherein the effective amount is sufficient to reduce the fecal shedding of enterotoxigenic E. coli from infected mammals, optionally F4-positive and Fl 8-positive enterotoxigenic E. coli. The method of any one of claims 177-198, wherein the effective amount is sufficient to reduce the incidence of mortality in infected pigs. The method of any one of claims 177-199, wherein the pig is a piglet. The method of claim 200, wherein the piglets are between about 1 day and 30 days old. The method of claim 200 or 201, wherein the piglets are at least about 18 days old.