BREVUNDIMONAS SP FOR USE IN DISEASE PREVENTION AND TREATMENT

Abstract

An improved agent and treatment method for a broad variety of diseases in both animals and humans is disclosed. The agent is an inventive composition comprising a bacterial-based culture. Particularly, the inventive composition disclosed herein is an active molecule produced by a Gram-negative bacterial strain that is a member of the genus Brevundimonas. Consumption of the inventive composition by way of liquid or dry feed produces a broad range of health benefits and has proven effective in the prevention and treatment of disease. The bacterium of the disclosed composition selectively modulates Toll-like receptors (TLRs) for the prevention and treatment of disease such as coccidiosis. Specific actions include but are not limited to improvement of gut health, acceleration or enhancement of immune response using a natural immune modulator, and the promotion of animal growth.

Claims

1. A composition for the prevention and treatment of disease in animals, the composition comprising effective amounts of a feed or drink ingredient including an active molecule derived from a Gram-negative bacterium of the genus Brevundimonas.

2. The composition of claim 1 wherein the active molecule is a cell wall component.

3. The composition of claim 2 wherein the cell wall component is taken from the group consisting of a lipopolysaccharide and a lipoprotein.

4. The composition of claim 1 wherein the bacterium is selected from the group consisting of Brevundimonas vesicularis, Brevundimonas nasdae, Brevundimonas intermedia, Brevundimonas aurantiaca, Brevundimonas mediterranea, Brevundimonas albigilva, and Brevundimonas huaxiensis.

5. The composition of claim 1 wherein the composition is effective in the prevention and treatment of disease in poultry.

6. The composition of claim 1 wherein the composition is administered in an amount of between about 100.0 g dried Brevundimonas per ton of finished feed and about 300.0 g per ton of finished feed where whole bacterial cells are utilized.

7. The composition of claim 1 wherein the compound is administered in an amount of between about 0.1 g per ton of finished feed and about 3.0 g per ton of finished feed where purified LPS are utilized.

8. The composition of claim 1 wherein the compound is administered in an amount of between about 0.1 mL per liter of drinking water and about 0.3 mL per liter of drinking water when provided in drinking water.

9. A composition for the prevention and treatment of disease in animals, the composition comprising effective amounts of a feed or drink ingredient including an extracted active fraction or a purified active molecule obtained from a Gram-negative bacterium of the genus Brevundimonas.

10. The composition of claim 9 wherein the extracted active fraction or purified active molecule is a cell wall component.

11. The composition of claim 10 wherein the cell wall component is taken from the group consisting of a lipopolysaccharide and a lipoprotein.

12. The composition of claim 9 wherein the bacterium is selected from the group consisting of Brevundimonas vesicularis, Brevundimonas nasdae, Brevundimonas intermedia, Brevundimonas aurantiaca, Brevundimonas mediterranea, Brevundimonas albigilva, and Brevundimonas huaxiensis.

13. The composition of claim 9 wherein the composition is effective in the prevention and treatment of disease in poultry.

14. The composition of claim 9 wherein the composition is administered in an amount of between about 100.0 g dried Brevundimonas per ton of finished feed and about 300.0 g per ton of finished feed.

15. A selective modulator of one or more TLR pathways comprising an extracted or a purified active molecule derived from a Gram-negative bacterium of the genus Brevundimonas wherein the Gram-negative bacteria is an agonist of one or more TLR pathway.

16. The composition of claim 15 wherein the extracted or purified active molecule is a cell wall component.

17. The composition of claim 16 wherein the cell wall component is taken from the group consisting of a lipopolysaccharide and a lipoprotein.

18. The selective modulator of claim 15 wherein the bacterium is selected from the group consisting of Brevundimonas vesicularis, Brevundimonas nasdae, Brevundimonas intermedia, Brevundimonas aurantiaca, Brevundimonas mediterranea, Brevundimonas albigilva, and Brevundimonas huaxiensis.

19. The composition of claim 15 wherein the composition is effective in the prevention and treatment of disease in poultry.

20. A method for treating an animal for clinical or subclinical disease through modulation of one or more TLR pathways, the method comprising identifying a Gram-negative bacterium; extracting an active molecule from the Gram-negative bacterium; purifying the extracted molecule to form a LPS/Lipid A compound; adding a pharmaceutically effective amount of the LPS/Lipid A compound to animal feed or drink to form a treatment composition; and administering a pharmaceutically effective amount of the treatment composition to the animal at its earliest life stage.

21. The method of claim 20 wherein the Gram-negative bacterium is of the genus Brevundimonas.

22. The composition of claim 20 wherein the active molecule is a cell wall component taken from the group consisting of a lipopolysaccharide and a lipoprotein.

23. The method of claim 20 wherein the treatment composition is administered to the subject animal beginning on Day 1 of its life thereby preventing parasitic disease through immune modulation.

Description

DESCRIPTION OF THE DRAWINGS

[0051] For a more complete understanding of this invention, reference should now be made to the accompanying figures in which:

[0052] FIG. 1 is a graph illustrating average pen body weight at Day 1 according to the first study;

[0053] FIG. 2 is a graph illustrating enhanced feed conversion ratio in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to a first study;

[0054] FIG. 3 is a graph illustrating feed intake comparison in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0055] FIG. 4 is a graph illustrating average pen body weight comparison in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0056] FIG. 5 is a graph illustrating reduction of mortality due to Eimeria infection in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0057] FIG. 6 is a graph illustrating reduction of an intestinal lesion score of the duodenum in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0058] FIG. 7 is a graph illustrating reduction of an intestinal lesion score of the ileum in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0059] FIG. 8 is a graph illustrating reduction of an intestinal lesion score of the ceca in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0060] FIG. 9 is a graph illustrating restoration/protection of ileum villi cell height in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0061] FIG. 10 is a graph illustrating restoration/protection of ileum crypt depth in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0062] FIG. 11 is a graph illustrating restoration/protection of ileum villi height to crypt depth ratios in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0063] FIG. 12 is a graph illustrating reduction of Eimeria in the duodenum gut in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0064] FIG. 13 is a graph illustrating reduction of Eimeria in the ileum in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0065] FIG. 14 is a graph illustrating reduction of Eimeria in the ceca in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the first study;

[0066] FIG. 15 is a graph illustrating the groups, the test material administered, the inclusion amounts, the group to which the material was fed, the status of coccidiosis challenge, and the lesion scoring dates for the second study;

[0067] FIG. 16 is a graph illustrating average pen body weight at Day 1 according to the second study;

[0068] FIG. 17 is a graph illustrating feed intake comparison in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the second study at Day 28;

[0069] FIG. 18 is a graph illustrating enhanced feed conversion ratio in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to a second study at Days 1-28;

[0070] FIG. 19 is a graph illustrating reduction of mortality due to Eimeria infection in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the second study at Days 1-28;

[0071] FIG. 20 is a graph illustrating average pen body weight gain comparison in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the second study;

[0072] FIG. 21 is a graph illustrating reduction of an intestinal lesion score of the duodenum in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the second study at Day 28;

[0073] FIG. 22 is a graph illustrating the coccidia incidence score in the small intestine in cocci-challenged subject animals fed a composition according to the disclosed inventive concept according to the second study at Day 28;

[0074] The reference “Significance (P<0.05)” in the figures refers to means within a row without a common superscript are significantly different (P<0.05), as determined by Duncan's New Multiple Range Test (MRT 1955).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0075] In the following description, various operating parameters and components are described for different constructed embodiments. These specific parameters and components are included as examples and are not meant to be limiting. Unless otherwise noted, all technical and scientific terms used herein are to be accorded their common meanings as would be understood by one having ordinary skill in the art.

[0076] The Compound Used in Treatment

[0077] The disclosed method of treatment preferably, but not absolutely, utilizes a compound generally derived from an active compound that may be found in the cell wall of a Gram-negative bacterial strain that is a member of the genus Brevundimonas.

[0078] As used herein, “modulator” refers to an activator, an inhibitor, or both. Modulation may be the result of activity by at least one Toll-like receptor (TLR), such as TLR4 or possibly TLR2 as well as other Toll-like receptors. As used herein, the term “inhibitor” refers to a molecule that reduces or attenuates the activity induced by another molecule. By way of example, a compound that might block the LPS-dependent activation of TLR4 present on the surface of immune cells in humans and animals would be regarded as an inhibitor of this particular pathway.

[0079] As used herein, the term “culture” is defined as microorganisms (either isolated or in combination) that grow in a liquid medium. Unless expressly stated otherwise, the term “biomass” refers to the microorganism cells (with the liquid culture medium removed). The “biomass” can be wet material or dried material.

[0080] Unless expressly stated otherwise, the term “supernatant” is defined as the culture medium in which the biomass is grown that contains excreted compounds from the biomass. Supernatant is obtained by growing biomass in culture medium for an appropriate length of time and then removing the microorganism cells by filtration and/or centrifugation.

[0081] Brevundimonas is a Gram-negative aerobic, non-fermenting bacterium that can grow under a variety of conditions. It is capable of metabolically utilizing several natural compounds generated by plants or algae.

[0082] Embodiments of the compound used in the treatment of disease as set forth herein include one or more LPS/Lipid A compounds produced by Gram-negative bacterial strains for use as selective modulators of the TLR signaling pathway, such as the TLR4 pathway. The disclosed inventive composition involves any combination of three fundamental steps: (1) the Gram-negative bacteria produces LPS/Lipid A compounds; (2) the LPS/Lipid compounds modulate TLR4 activity through activation or inhibition; and (3) a downstream effect results in enhanced innate and adaptive immune processes, thereby aiding in the treatment of coccidiosis, necrotic enteritis, and other conditions related to gut inflammation.

[0083] In an embodiment, the LPS/Lipid A compounds used as selective modulators of the TLR4 signaling pathway are produced from a strain of the genus Brevundimonas. The Brevundimonas strain may be a naturally occurring strain found in an algal biomass.

[0084] Accordingly, embodiments of the compound used in the treatment of disease according to the present disclosure are directed to one or more LPS/Lipid A compounds produced by a Gram-negative bacterial strain of the group Brevundimonas for use as selective modulators of the TLR signaling pathway.

[0085] The LPS/Lipid A compound employed herein may preferably be obtained from a member of the group comprising but not necessarily limited to any one of Brevundimonas vesicularis, Brevundimonas nasdae, Brevundimonas intermedia, Brevundimonas aurantiaca, Brevundimonas mediterranea, Brevundimonas albigilva, and Brevundimonas huaxiensis by any suitable method. In some examples, multiple types of LPS extraction protocols are employed to obtain an LPS compound from the bacteria, and extraction procedures may be performed more than once. Once the LPS compound is extracted and purified from the bacteria, the Lipid A fraction may be prepared by acid hydrolysis or other suitable techniques. In some examples, analysis of the structure of the LPS compound is performed using routine methods in the art, including using mass spectrometry, gas chromatography, or both.

[0086] The one or more LPS/Lipid A compounds derived from Gram-negative bacterial strains of the genus Brevundimonas, may selectively modulate the TLR4 signaling pathway to alter inflammatory responses and to improve immune health in a variety of uses and applications. In an embodiment, the LPS/Lipid A compound derived from species of the genus Brevundimonas may be incorporated within a feed ingredient to improve gut health of poultry.

[0087] The disclosed LPS/Lipid A compound derived from a species of the genus Brevundimonas may be used to improve the health of poultry or other animals through a variety of mechanisms. For example, the LPS/Lipid A compound may protect against internal inflammation in poultry by negatively regulating inflammatory mediators via the downregulation of TLR4 expression and the downstream inhibition of NF-kappa B activation in a typical inflammatory cascade. In another example, the LPS/Lipid A compound may inhibit the activation of TLR4 in poultry by interfering with cysteine residue-mediated receptor dimerization. In yet another example, the LPS/Lipid A compound may inhibit the ability of non-infectious and infectious stimuli to interact with TLR4 and trigger a pro-inflammatory response, thereby improving poultry gut integrity. In a further example, the LPS/Lipid A compound may modulate TLR4 through either ligand-dependent or ligand-independent activation. As another example, the LPS/Lipid A compound may act in concert with other TLR agonists to provide a heightened immune response, while reducing the metabolic costs to the host.

[0088] The treatment compound is dried biomass containing Gram-negative bacteria of the genus Brevundimonas, or compounds derived therefrom, provided in drinking water or as animal feed. In animal feed, once the biomass and feed additive are combined to the preferred premix level, the combined batch is poured or administered evenly into a ribbon mixer containing finished feed. The combined batch is preferably provided in an amount of between about 100.0 g dried Brevundimonas per ton of finished feed and about 150.0 g per ton of finished feed, is more preferably provided in an amount of between about 120.0 g per ton of finished feed and 130.0 g per ton of finished feed and is most preferably though not exclusively provided in an amount of about 125.0 g dried Brevundimonas per ton of feed with good efficacy without being wasteful. As an alternative, the combined batch may be provided in an amount of between about 100.0 g dried Brevundimonas per ton of finished feed and about 300.0 g per ton of finished feed, is more preferably provided in an amount of between about 150.0 g per ton of finished feed and 250.0 g per ton of finished feed, and is most preferably though not exclusively provided as an alternative in an amount of about 200.0 g dried Brevundimonas per ton of feed with good efficacy without being wasteful. As an active fraction or isolated molecule, the amounts administered are less than the amounts needed for whole cells in that the amount required for disease prevention and treatment depend on the potency of the material. For example, where purified LPS is used in feed the ratio is between about 0.1 g per ton of finished feed and about 3.0 g per ton of finished feed with the preferred range being between about 0.16 g per ton of finished feed and about 2.5 g per ton of finished feed. Where the active is administered in water, where a supernatant is the source from an algal culture, the amount is between about 0.1 and 0.3 mL per liter of drinking water with 0.2 mL per liter being most preferred. Where a semi-pure LPS fraction is utilized, the amount is between about 1.0 and 2.0 mg/L with the preferred amount being 1.5 mg/L. Where a purified LPS is utilized, the amount is between about 0.001-0.03 mg/L with between about 0.002-0.02 g/L being most preferred.

[0089] Investigations

[0090] Investigations were undertaken to determine the response and efficacy of a feed ingredients containing Brevundimonas biomass incorporated at a specific amount into a commercial-type corn-soybean diet and fed to floor-pen raised broilers challenged with Eimeria to cause coccidiosis. The investigations were undertaken over a 28-42 day period, from Day 0 to Day 28 or Day 0 to Day 42. Twenty investigations were performed under a wide range of test conditions and configurations designed to represent the broad range of conditions expected in commercial poultry houses. Several of the investigations were performed by administering biomass from the isolated active Brevundimonas bacterial strain. All other investigations involved administering culture biomass containing the active bacterial strain as the test material.

[0091] Investigations were conducted in both battery cages and floor pens, using both mash and pelleted feed rations using appropriate numbers of birds and replicates to validate statistically significant findings (P<0.05). In most investigations, a disease challenge involving three species of Eimeria (E. tenella, E. maxima, and E. acervulina) was applied, but the timing of the challenge was varied to validate efficacy under different infection scenarios. Investigation endpoints included performance parameters (e.g., feed intake, body weight gain, FCR), disease endpoints (e.g., mortality, lesion score, intestinal morphology, parasite enumeration). While a majority of investigations were conducted over the full 42-day broiler grow-out period, several investigations were truncated to 28-days in order to streamline the product development cycle after validating that the results from 28-day investigations are fully representative and predictive of results for full-length investigations. Two specific studies selected from the full research program are discussed here.

[0092] First Study—Treatment Method

[0093] Chicks were obtained within twelve hours of hatching from fecal contaminated flocks at a commercial hatchery on Day 0 (hatch and placement day). Three-hundred mixed-sex broiler chicks (50:50 sex ratio) were randomly assigned on Day 0 by individual weights to each of several test group pens, each with replicates. Only antibiotic-free birds were sourced, and no coccidiosis vaccine was administered at the hatchery or at any time during the study. Chicks were evaluated upon receipt for signs of disease or other complications that could affect study outcome. Weak birds were humanely sacrificed. Birds were not replaced during the study.

[0094] Following examination, chicks were weighed and allocated to pens for the various treatment groups using a randomized block design. Weight distribution across the treatment groups was assessed prior to feeding by comparing the individual test groups' standard deviations of the mean against that of the control group. Weight distribution across the groups was considered acceptable for this study when differences between control and test groups were within one standard deviation.

[0095] All birds received nutritionally adequate diets as pellets (crumbled pellets for Days 1-14). Birds were fed their respective treatment diets ad libitum from day of hatch to 28 days of age. (The typical average market age is 42 days.) Birds were raised on built-up litter to further mimic stress conditions typically experienced in poultry production.

[0096] All diets were offered ad libitum without restrictions to full-fed consumption, except for an 8-hour fasting period prior to cocci-challenge on Day 7 when all birds assigned to challenge test groups received oocyst-inoculated feed containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Dietary requirements for protein, lysine, methionine, methionine+cystine, arginine, threonine, tryptophan, total phosphorus, available phosphorus, total calcium, dietary sodium, and dietary choline were met by adjusting the concentrations of corn and soybean meal ingredients, as well as other minor ingredients commonly used in poultry production.

[0097] Throughout the study, birds were observed at least three times daily for overall health, behavior, and evidence of toxicity. Pens were monitored for environmental conditions, including temperature, lighting, water, feed, litter condition, and unanticipated house conditions/events. Pens were checked daily for mortality. Examinations were performed on all broilers found dead or moribund. Mortalities were recorded (date and weight) and examined (both internal and external body mass).

[0098] Cocci-Challenge—On Day 7, all birds assigned to challenged test groups received oocyst-inoculated feed containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Adequate feed was precisely weighed and provided to birds to consume at the rate of 100% fill-capacity on average. Prior to the challenge, all birds were starved for eight hours. Inoculated feed was provided to the birds. Following a specific time, all remaining inoculated feed was removed and weighed to assure equal consumption per pen and per bird. The quantity of feed (both placed and withdrawn) was recorded on each pen's feed record.

[0099] First Study Evaluation

[0100] Differences between groups were evaluated at P<0.05, employing Treatment x Replicate RCB (Randomized Complete Block) design. Particularly and as noted, “Significance (P<0.05)” refers to means for groups not sharing common letter designations in the figures which indicates significant difference (P<0.05) between said groups, as determined by Duncan's New Multiple Range Test (MRT 1955).

[0101] The relevant evaluation results from the first study are set forth in the following chart and are referenced below by individual graphically represented figures:

TABLE-US-00001 Feed Conversion % Avg Pen Lesion Lesion Lesion Ratio Mortality Body Feed Intake Score Score Score Corrected Days 1- Weight (g) (g/bird/day) Duodenum Ileum Ceca Means Days 1-28 28 Day 28 Day 28 Day 28 Day 28 Day 28 1. No Tx, No 1.527 1.852 1418.47 88.502 0.950 0.067 0.075 Challenge 2. No Tx, 1.595 17.593 1365.56 98.530 2.483 2.525 2.492 Cocci 3. Coban, 1.527 1.852 1430.88 89.499 0.525 0.433 0.583 Cocci 4. Brev, 1.536 3.241 1446.26 91.538 0.450 0.500 0.483 Cocci Significance (P < 0.05) 1. No Tx, No a a a a b a a Challenge 2. No Tx, b b b b c c c Cocci 3. Coban, a a a a ab b b Cocci 4. Brev, a a a a a b b Cocci

[0102] Benefits of Disclosed Inventive Composition in Animal Growth Performance

[0103] The disclosed inventive composition offers several benefits not seen in known feed compositions. The results from the 20 studies performed demonstrate that the actives of the disclosed inventive composition consistently improve the efficiency with which broiler chickens convert feed into body mass gains in the presence of a disease challenge. More specifically as illustrated in the representative study described above in conjunction with the attached figures, Eimeria-challenged birds ingesting the actives of the disclosed inventive composition consistently show statistically significant improvements in FCR compared to non-treated, cocci-challenged birds. In addition to deliberate Eimeria inoculation early in life, studies were conducted on built-up litter providing ongoing exposure to Eimeria and other environmental pathogens during the course of the studies. Despite this, the FCR status of birds fed the disclosed inventive composition was consistently found to be not statistically different from unchallenged birds and challenged birds treated with leading ionophore products commonly used in poultry production, such as Salinomycin, Coban® (Monensin, USP), Elanco Animal Health, and Maxiban® (Narasin and nicarbazin), Elanco Animal Health. These improvements in FCR are reliably shown to be due to increases in body weight and not due to increased feed intake.

[0104] Day 1 Body Weight Evaluation—Referring to FIG. 1, Average Body Weight at Day 1 is illustrated. Body weights are not statistically different among groups at this stage, ranging from a high weight of 59.096 g for birds assigned to the unchallenged and untreated groups to 59.821 g for birds assigned to the challenged and untreated groups:

TABLE-US-00002 Avg Pen Body Weight Means (g) Day 1 1. No Tx, No Challenge 59.096 2. No Tx, Cocci 59.821 3. Coban, Cocci 59.407 4. Brev, Cocci 59.429 Significance (P <0.05) 1. No Tx, No Challenge a 2. No Tx, Cocci a 3. Coban, Cocci a 4. Brev, Cocci a

[0105] Feed Conversion Rate—As set forth in FIG. 2 which is a graph illustrating enhanced feed conversion ratio in cocci-challenged subject animals fed a composition according to the disclosed inventive concept, mortality-corrected FCR was determined from day of hatch through Day 28 for control birds receiving no Eimeria challenge and no treatment (first bar) and birds challenged with Eimeria on Day 7 but untreated (second bar), challenged and treated with Coban® (third bar), or challenged and treated with the immune modulator of the disclosed composition (fourth bar). Growth performance as measured by FCR for the group fed the disclosed inventive composition is dramatically improved compared to the challenged/untreated group and not statistically different from the unchallenged/untreated control group or the challenged/Coban® treated group.

[0106] Feed Intake—As set forth in FIG. 3 which is a graph illustrating feed intake comparison in subject animals fed a composition according to the disclosed inventive concept, feed intake was measured from day of hatch through Day 28 for control birds receiving no Eimeria challenge and no treatment (first bar) and birds challenged with Eimeria on Day 7 but untreated (second bar), challenged and treated with Coban® (third bar), or challenged and treated with the immune modulator of the disclosed composition (fourth bar). As expected, feed intake increased significantly in challenged/untreated birds. In contrast, feed intake of challenged birds receiving the immune modulating actives of the disclosed composition was not significantly different than that of the unchallenged/untreated control group or the challenged/Coban® treated group.

[0107] Body Weight—As set forth in FIG. 4 which is a graph illustrating average pen body weight comparison in subject animals fed a composition according to the disclosed inventive concept, average pen body weight was measured from day of hatch through Day 28 for control birds receiving no Eimeria challenge and no treatment (first bar) and birds challenged with Eimeria on Day 7 but untreated (second bar), challenged and treated with Coban® (third bar), or challenged and treated with the immune modulator of the disclosed composition (fourth bar). As expected, body weight was significantly decreased in challenged/untreated birds compared to the other groups. In contrast, body weight of challenged birds receiving the immune modulating actives of the disclosed composition was not significantly different than that of the unchallenged/untreated control group or the challenged/Coban® treated group.

[0108] Mortality—Across all 20 studies, actives of the disclosed composition consistently reduced mortality in Eimeria-challenged broiler chickens, resulting in mortality levels not statistically different from those observed in unchallenged control groups and challenged birds treated with leading ionophore products commonly used in poultry production.

[0109] As set forth in FIG. 5 which is a graph illustrating reduction of mortality due to Eimeria infection in subject animals fed a composition according to the disclosed inventive concept, the percent mortality was calculated from day of hatch through Day 28 for control birds receiving no Eimeria challenge and no treatment (first bar) and birds challenged with Eimeria on Day 7 but untreated (second bar), challenged and treated with Coban® (third bar), or challenged and treated with the immune modulator of the disclosed composition (fourth bar). Mortality in the challenged/untreated group was approximately 17% indicating that the Eimeria challenge resulted in significant disease. In contrast, mortality of challenged birds receiving the immune modulating actives of the disclosed composition was held to a level not significantly different than that of the unchallenged/untreated control group or the challenged/Coban® treated group.

[0110] Protection of Gut

[0111] Lesion Scores—Another primary benefit of actives of the disclosed composition is reduced presence and severity of coccidia lesions and damage to the intestinal lining in Eimeria-challenged broiler chickens. Lesion scores in the duodenum, ileum, and ceca are consistently decreased in birds ingesting actives of the disclosed composition, to the same extent as unchallenged control groups and challenged birds treated with leading ionophore products commonly used in poultry production. Lesion score data for the duodenum are provided in FIG. 6. Similar trends were observed in the ileum and ceca as set forth in FIGS. 7 and 8 respectively.

[0112] As set forth in FIG. 6 which is a graph illustrating reduction of intestinal lesion score in subject animals fed a composition according to the disclosed inventive concept, intestinal lesion score (Johnson and Reid method) was determined in the duodenum on Day 28 for control birds receiving no Eimeria challenge and no treatment (first bar) and birds challenged with Eimeria on Day 7 but untreated (second bar), challenged and treated with Coban® (third bar), or challenged and treated with the immune modulator of the disclosed composition (fourth bar). As expected, the challenged/untreated group exhibited significantly more lesions due to the Eimeria challenge when compared to the unchallenged control. The challenged/composition-fed group showed no such increase in lesion score. In fact, the lesion score of the challenged/composition-fed group was numerically and statistically lower than that of the unchallenged control group and not significantly different compared to the challenged birds receiving Coban®. Similar lesion score results were found in the ileum as illustrated in FIG. 7 and in the ceca as illustrated in FIG. 8.

[0113] Intestinal Morphology—The immune modulating actives of the disclosed composition also demonstrate positive effects on gut morphology, including restoring or protecting villi height and crypt depth in the ileum following a cocci challenge (FIGS. 9 and 10).

[0114] As set forth in FIG. 9 which is a graph illustrating restoration/protection of ileum cell villi height in subject animals fed a composition according to the disclosed inventive concept, cell villi height in the ileum was measured on Day 28 for control birds receiving no Eimeria challenge and no treatment (first bar) and birds challenged with Eimeria on Day 7 but untreated (second bar), challenged and treated with Coban® (third bar), or challenged and treated with the immune modulator of the disclosed composition (fourth bar). The detrimental effects of coccidiosis are clearly seen in the challenged/untreated group which had significantly reduced villi height compared to the unchallenged control group. In contrast, there is no significant difference between the cell villi height measured in birds treated with the disclosed composition as compared to the unchallenged control or in challenged birds treated with Coban®.

[0115] As set forth in FIG. 10 which is a graph illustrating restoration/protection of ileum crypt depth in subject animals fed a composition according to the disclosed inventive concept, ileum crypt depth was measured on Day 28 for control birds receiving no Eimeria challenge and no treatment (first bar) and birds challenged with Eimeria on Day 7 but untreated (second bar), challenged and treated with Coban® (third bar), or challenged and treated with the immune modulator of the disclosed composition (fourth bar). Crypt depth in birds fed the actives of the disclosed composition was significantly improved compared to the challenged/untreated group and was not statistically different than for challenged birds treated with Coban®.

[0116] As set forth in FIG. 11 which is a graph illustrating restoration/protection of ileum villi height to crypt depth ratios in subject animals fed a composition according to the disclosed inventive concept, which were measured on Day 28 for control birds receiving no Eimeria challenge and no treatment (first bar) and birds challenged with Eimeria on Day 7 but untreated (second bar), challenged and treated with Coban® (third bar), or challenged and treated with the immune modulator of the disclosed composition (fourth bar). Crypt depth ratios in birds fed the actives of the disclosed composition was significantly improved compared to the challenged/untreated group and was only slightly greater than for challenged birds treated with Coban®.

[0117] Secondary Benefits of the Inventive Composition

[0118] Reduction of Oocysts

[0119] Multiple studies demonstrate that the actives of the disclosed composition reduce E. acervulina in the loop of the small intestine area, E. maxima in the jejunum, and E. tenella in the ceca.

[0120] As set forth in FIGS. 12, 13, and 14 which are graphs illustrating reduction of Eimeria in the broiler gut in subject animals fed a composition according to the disclosed inventive concept, coccidia count scores in the duodenum, the ileum, and the ceca respectively are shown confirming trends for all intestinal regions. The coccidia count score was determined on Day 28 for control birds receiving no Eimeria challenge and no treatment (first bar) and birds challenged with Eimeria on Day 7 but untreated (second bar), challenged and treated with Coban® (third bar), or challenged and treated with the immune modulator of the disclosed composition (fourth bar). Actives of the disclosed composition provided a significant reduction in the coccidia score relative to challenge/untreated birds but did not reduce the score to levels comparable with either the unchallenged control or the challenged birds treated with Coban®.

[0121] Second Study

[0122] A second study was undertaken to determine the response and efficacy of a dried bacterial biomass feed ingredient incorporated at a specific amount into a commercial-type corn-soybean diet and fed to floor-pen raised broilers. The second study was undertaken over a 28-day period, from Day 1 to Day 28.

[0123] The animals were raised under a disease challenge environment (cocci-challenge+built-up litter). For the study and as set forth in FIG. 15, six groups were established, including a first group that received no feed additive ingredient, a second group treated with the anti-coccidia drug Coban® (Elanco), a third group fed a Variovorax-based biomass, a fourth group fed a Brevundimonas-based biomass, a fifth group fed a Sphingomonas-based biomass, and a sixth group fed a 50:50 mixture of a Variovorax and Brevundimonas-based biomass. The feed included corn and soybean meal rations with normal nutritional formulations. No coccidiostat (except for treatment with Coban® provided to the second group) or ABF (antibiotic free products) were administered during the study. No coccidiosis vaccine was administered at the hatchery or during the course of the study.

[0124] Second Study—Treatment Method

[0125] A total of 1,800 mixed sex broiler chicks were obtained within twelve hours of hatching from fecal contaminated flocks at a commercial hatchery on Day 0 (hatch and placement day). A number of mixed-sex broiler chicks (50:50 sex ratio) were randomly assigned on Day 0 by individual weights to one of several test group pens, each with replicates. Only antibiotic-free birds were sourced, and no coccidiosis vaccine was administered at the hatchery or at any time during the study. Chicks were evaluated upon receipt for signs of disease or other complications that could affect study outcome. Weak birds were humanely sacrificed. Birds were not replaced during the study.

[0126] Following examination, chicks were weighed and allocated to pens for the various treatment groups using a randomized block design. Weight distribution across the treatment groups was assessed prior to feeding by comparing the individual test groups' standard deviations of the mean against that of the control group. Weight distribution across the groups was considered acceptable for this study when differences between control and test groups were within one standard deviation.

[0127] All birds received nutritionally adequate diets as pellets (crumbled pellets for Days 1-14). Birds were fed their respective treatment diets ad libitum from day of hatch to 28 days of age. (The typical average market age is 42 days.) Birds were raised on built-up litter to further mimic stress conditions typically experienced in poultry production.

[0128] All diets were offered ad libitum without restrictions to full-fed consumption, except for an 8-hour fasting period prior to cocci-challenge on Day 7 when all birds assigned to the challenge test groups received oocyst-inoculated feed containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Dietary requirements for protein, lysine, methionine, methionine+cystine, arginine, threonine, tryptophan, total phosphorus, available phosphorus, total calcium, dietary sodium, and dietary choline were met by adjusting the concentrations of corn and soybean meal ingredients, as well as other minor ingredients commonly used in poultry production.

[0129] Throughout the study, birds were observed at least three times daily for overall health, behavior, and evidence of toxicity. Pens were monitored for environmental conditions, including temperature, lighting, water, feed, litter condition, and unanticipated house conditions/events. Pens were checked daily for mortality. Examinations were performed on all broilers found dead or moribund. Mortalities were recorded (date and weight) and examined (both internal and external body mass).

[0130] Cocci-Challenge—On Day 7, all birds assigned to the challenge test groups received oocyst-inoculated feed containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Adequate feed was precisely weighed and provided to birds to consume at the rate of 100% fill-capacity on average. Prior to the challenge, all birds were starved for eight hours. Inoculated feed was provided to the birds. Following a specific time, all remaining inoculated feed was removed and weighed to assure equal consumption per pen and per bird. The quantity of feed (both placed and withdrawn) was recorded on each pen's feed record.

[0131] Second Study Evaluation

[0132] Differences between groups were evaluated at P<0.05, employing Treatment x Replicate RCB (Randomized Complete Block) design. Particularly and as noted, “Significance (P<0.05)” refers to means within a column without a common superscript are significantly different (P<0.05), as determined by Duncan's New Multiple Range Test (MRT 1955).

[0133] The relevant evaluation results from the second study are set forth in the following chart and are referenced below by individual graphically represented figures:

TABLE-US-00003 Coccidia Feed Incidence Conversion Avg Pen Lesion Score Ratio % Body Feed Intake Score Small Corrected Mortality Weight (g) (g/bird/day) Duodenum Intestine Means Days 1-28 Days 1-28 Day 28 Day 28 Day 28 Day 28 1. No Tx, Cocci 1.498 15.000 1294.183 76.196 2.075 1.683 2. Coban, Cocci 1.403 3.750 1443.182 75.493 0.317 0.325 3. VP, Cocci 1.461 10.833 1339.628 75.446 1.508 1.300 4. Brev, Cocci 1.409 5.417 1433.982 76.005 1.033 0.958 5. SG, Cocci 1.456 7.917 1375.182 75.772 1.325 1.200 6. VP + Brev, Cocci 1.435 5.833 1405.136 75.826 0.942 1.017 Significance (P < 0.05) 1. No Tx, Cocci d c d a e d 2. Coban, Cocci a a a a a a 3. VP, Cocci c b c a d c 4. Brev, Cocci ab a a a b b 5. SG, Cocci c ab bc a c c 6. VP + Brev, Cocci bc a ab a b b

[0134] As noted above, the test subjects were divided into six groups. Members of the first group received no feed additive ingredient in its food regimen. Members of the second group were given feed that included the anti-coccidia drug Coban® (Elanco) according to the manufacturer's instructions. Members of the third group were given feed containing a Variovorax-based biomass. Members of the fourth group were given feed containing a Brevundimonas-based biomass. Members of the fifth group were given feed containing a Sphingomonas-based biomass. Members of the sixth group were given feed containing a 50:50 mixture of a Variovorax and Brevundimonas-based biomass.

[0135] All data points were analyzed at the 5% level of probability, including composite weighted average of entire pen, feed:gain and mortality. Birds were evaluated in terms of physical live performance and digestive health. The six groups were evaluated over a 28-day period for body weight, feed conversion ratio (FCR) (corrected for mortality), mortality, body weight gain, and average weight gain.

[0136] Day 1 Body Weight Evaluation—Referring to FIG. 16, Average Body Weight at Day 1 is illustrated. Body weights are not statistically different among groups at this stage, ranging from a high weight of 54.727 g for Group 1 to a low weight of 54.339 g for Group 3:

TABLE-US-00004 Avg Pen Body Weight (g) Means Day 1 1. No Tx, Cocci 54.727 2. Coban, Cocci 54.595 3. VP, Cocci 54.339 4. Brev, Cocci 54.521 5. SG, Cocci 54.402 6. VP + Brev, Cocci 54.369 Significance (P <0.05) 1. No Tx, Cocci a 2. Coban, Cocci a 3. VP, Cocci a 4. Brev, Cocci a 5. SG, Cocci a 6. VP + Brev, Cocci a

[0137] Feed Intake—The graph set forth in FIG. 17 illustrates a feed intake comparison in subject animals fed a composition according to the disclosed inventive concept. Feed intake was measured at Day 28 for Group 1 (first bar), for Group 2 (second bar), for Group 3 (third bar), for Group 4 (fourth bar), for Group 5 (fifth bar), and for Group 6 (sixth bar). Feed intake was highest in challenged/untreated birds. However, there were no statistical differences in feed intake for any of the groups.

[0138] Feed Conversion Rates Corrected—FCR was recorded on Days 1-28 as shown in FIG. 18. Group 2, which received feed that included Coban®, had the lowest FCR, while Group 4, which received feed containing a Brevundimonas-based biomass had the second lowest rate. Starting on Day 8 and continuing throughout the study duration, improvements in FCR for Group 4 were not statistically different from Group 2, demonstrating that the Brevundimonas-based biomass in feed performed as well as a long-standing, commercially available coccidiosis treatment, such as Coban®. Groups 2 and 4 had statistically lower FCR compared to Group 1, which received no feed additive ingredient in its food regimen.

[0139] Mortality Percentage—Mortality rates were recorded on Days 1-28 as illustrated in FIG. 19. Mortality rates were lowest for members of the second group, which received feed that included Coban® and for members of the fourth group, which received feed containing a Brevundimonas-based biomass as well as for Group 6, which received feed containing a 50:50 mixture of a Variovorax and Brevundimonas-based biomass.

[0140] Average Pen Body Weight—Average pen body weight was recorded on Day 28 and is set forth in FIG. 20. Weight gain was most pronounced for the second group, which received feed that included Coban® but was followed closely by weight gain in members of the fourth group, which received feed containing a Brevundimonas-based biomass. Members of Group 6, which received feed containing a 50:50 mixture of a Variovorax and Brevundimonas-based biomass also showed noteworthy weight gain.

[0141] Lesion Scores—Gross necropsy and lesion scoring were performed. Birds were selected, sacrificed, weighed, and examined for the presence and degree of duodenal and coccidia lesions. Damage scores were assessed and recorded.

[0142] With respect to the duodenum lesion scores for Day 28, the lowest score was evident with the second group, which received Coban®, followed by groups 4 given feed containing a Brevundimonas-based biomass and 6 given feed containing a 50:50 mixture of a Variovorax and Brevundimonas-based biomass. These results are illustrated in FIG. 21.

[0143] With respect to the coccidia lesion incidences score of the small intestine for Day 28, the lowest score was again experienced by the second group, a statistically significant reduction compared to all other groups, followed by the fourth and sixth groups. These results are illustrated in FIG. 22.

[0144] Results—in General

[0145] The disclosed inventive feed additive containing a Brevundimonas-based biomass consistently promoted improved growth performance in broilers receiving a coccidiosis challenge that was not statistically different from that observed for birds given feed that included the anti-coccidia drug Coban®. This outcome holds true over all assessed growth performance characteristics including body weight, feed conversion ratio (FCR), mortality, body weight gain and average weight gain. The positive results obtained with the inventive composition were so observed without risk of drug resistance developing as is often the risk with the use of anti-coccidia drugs.

[0146] I. Results—Agents and Method for Improving Gut Health

[0147] The disclosed inventive composition demonstrates improved live performance and digestive health parameters for weight gain, feed efficiency, mortality, intestinal villi cell height, crypt depth, villi height to crypt depth ratio, and intestinal coccidiosis across all age ranges. The disclosed inventive composition is effective in ameliorating the physical effects of environmental stress on live performance that are typically experienced in poultry production.

[0148] The disclosed inventive composition demonstrates a positive impact on the gut health of boilers through improved gut morphology under disease stress by improving nutrient uptake and eventual bird growth and overall improved gut integrity. The treatment method and compound may have benefits that go beyond the poultry industry to other animals and possibly to humans.

[0149] II. Results—Use of Brevundimonas Microbes as an Alternative Treatment for Coccidiosis

[0150] In an additional study which considered the use of Brevundimonas microbes as an alternative treatment for coccidiosis, analysis of tissue from broiler chickens provided a feed mixture that included the disclosed compound demonstrated a significant maturing of innate immune pathways when compared with sacrificed birds fed a conventional feed mixture without the disclosed compound. Also, in general, kinomic analysis of tissue from the birds provided a feed mixture that included the disclosed compound demonstrated significant enhancement of adaptive immune pathways.

[0151] Furthermore, analysis of the broiler tissues from chickens fed the treatment compound according to the regimen set forth above, as well as those not fed the treatment compound, verified that the treated chickens demonstrated an increase in adaptive immune response indicating a more immunologically competent or developed immune system. Significantly, the analysis indicated that treatment with the disclosed compound resulted in a more adaptive-biased immune system in the supplemented birds and further demonstrated an alteration of immunometabolism in gut, liver, and muscle tissue by priming the animal for a more rapid and robust immune response compared to birds receiving a standard, unfortified diet. Comparative analysis of tissues of chickens fed the treatment compound and those not fed the treatment compound verified that the treated chickens demonstrated an altered immune response consistent with the effects of TLR4 modulation.

[0152] Advantageously, studies showed that the identified immunomodulatory adaptation does not come at the cost of impaired growth and development, rather metabolic efficiency was facilitated in the latter part of life. By following the method and treatment of the disclosed inventive concept, a more rapid, adaptive immune response that is comparable to a response usually seen in an older bird is possible in young birds.

[0153] III. Results—Natural Feed Composition Containing Brevundimonas for the Promotion of Animal Growth

[0154] In general, analysis of the results supports the conclusion that inclusion of the innovative compound as part of a conventional diet leads to a significant increase in growth and weight gain when compared to the control flock.

[0155] Moreover, following treatment with the disclosed compound on selected birds, samples of both treated and non-treated birds were examined by gross necropsy which included internal examination. Kinomic analysis of tissues collected from sacrificed birds fed the dietary mixture of the inventive composition and conventional feed confirmed that the biomass alters multiple growth-related pathways as proposed, thus initiating pathway activation.

[0156] Further analysis demonstrates that the birds consuming the supplemented diet converted feed into body mass more efficiently than the birds fed the control diet. Birds receiving the diet supplemented with the inventive composition had statistically significant decrease in FCR compared to the untreated control group in the given studies. Perhaps most significantly, while there were no differences in overall carcass yield, the total breast weight, as well as the Pectoralis major (breast fillets) and Pectoralis minor (tender) weights, were increased in the group fed the diet supplemented with the inventive composition disclosed herein.

[0157] IV. Results—Enduring Effects on Coccidiosis Prevention and Treatment Via Animal Feed

[0158] The use of the innovative compound in the treatment of coccidiosis-challenged poultry demonstrates an enduring effect on the coccidiosis prevention and treatment by delivery through animal feed whereby the positive effects of treatment persist well beyond the time of product withdrawal unlike the loss of efficacy noted immediately after withdrawal of conventional anticoccidial products. Overall, analysis of birds fed the test material for the first 14 days of life demonstrated changes in the immune system consistent with a bolstered innate immune response thereby providing enduring effects over the life of the animal.

[0159] Notably, a significant decrease in the presence and degree of coccidia lesions and damage to the intestinal lining typically experienced following coccidiosis infection was noted.

[0160] Upon examination of the sacrificed birds, it was found that the average lesion scores throughout the intestine of poultry treated with the disclosed composition were consistently lower than the scores of sacrificed untreated disease-challenged birds. Ileum villi cell height, crypt depth, and the villus height to crypt depth ratio generally showed improvement. Particularly, ileum villi cell height and crypt depths in birds fed the inventive formulations disclosed herein were competitive with coccidiosis-challenged birds treated with conventional antibiotics.

[0161] The average oocyst count of the duodenum, mid-gut, and cecum of sample poultry given feed or water having the disclosed inventive composition were lower than the scores of sacrificed untreated disease-challenged birds. The groups treated with the inventive composition had improved mortality compared to challenged, untreated groups.

[0162] The improvement of the overall health of disease-challenged poultry as a result of being given feed or water containing the disclosed inventive composition was achieved without the use of antibiotics. Overall the inventive composition demonstrates a cost-effective and practical approach to the treatment of disease states in animals.