COMPOSITIONS AND METHODS OF USE OF INTERLEUKIN-10 PEPTIDES AND ANTI-INTERLEUKIN-10 ANTIBODIES
Disclosed herein are methods of reducing a symptom of respiratory disease in a pre-weaned milk-fed mammal by orally administering an isolated antibody that specifically binds the interleukin-10 peptide or an anti-interleukin-10 antibody. Also included are methods of reducing mixing stress in human and non-human mammals by administering an isolated antibody that specifically binds the interleukin-10 peptide or an anti-interleukin-10 antibody. Further included are milk and food compositions including the interleukin-10 peptide or anti-interleukin-10 antibody.
The present disclosure is related to compositions containing interleukin-10 peptides and anti-interleukin-10 antibodies and to their methods of use in the treatment of humans and other mammals.
BACKGROUNDInterleukin-10 (IL-10) peptides and anti-IL-10 antibodies have been described in U.S. Pat. No. 8,652,457, as well as their use to treat gastrointestinal protozoan infections in animals. In particular, isolated anti-IL-10 antibodies were shown to prevent growth suppression due to Coccidiosis infection in chicks, and injection of IL-10 peptides into hens was shown to passively transfer anti-IL-10 antibodies to chicks to prevent growth suppression caused by Coccidia infection. Animal feeds and animal feed additives containing the IL-10 peptides and anti-IL-10 antibodies were described.
What is needed are additional methods for the treatment and/or prevention of additional diseases and conditions in animals.
BRIEF SUMMARYIn an aspect, a method of reducing a symptom of respiratory disease in a pre-weaned milk-fed mammal comprises orally administering to the pre-weaned milk-fed mammal an effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds an interleukin-10 peptide, wherein administration is initiated between birth and weaning, and wherein administration is performed at least once daily for a period of 7 days to 7 weeks.
In another aspect, a method of reducing mixing stress in a mammal comprises orally administering to the mammal an effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds an interleukin-10 peptide, wherein administration is initiated within four weeks of mixing the mammal with a new population of mammals, and wherein administration is performed at least once daily for a period of 7 days to 7 weeks.
In yet another aspect, a method of reducing mixing stress in a human child comprises orally administering to the human child an effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds an interleukin-10 peptide, wherein administration is initiated within four weeks of mixing the human child with a new population of human children, and wherein administration is performed at least once daily for a period of 7 days to 7 weeks.
Also included herein is a milk composition suitable for administration to a pre-weaned mammal of a genus Bos, Ovis, Capra, Bubalus, or Sus that comprises a basal milk composition and a therapeutically effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds the interleukin-10 peptide.
Further included herein is a food composition suitable for administration to a human that comprises a basal food composition and a peptide of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, or SEQ ID NO. 38; or an isolated anti-IL-10 antibody that specifically binds a peptide of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, or SEQ ID NO. 38.
The above-described and other features will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims.
DETAILED DESCRIPTIONDescribed herein are methods of reducing the incidence of disease and stress in mammals by orally administering IL-10 peptides and/or anti-IL-10 antibodies. It has been unexpectedly shown herein that oral administration of an anti-IL-10 antibody increased hipometer measurement, reduced the incidence of bovine respiratory disease, and decreased antibiotic use in pre-weaned calves. Because the IL-10 peptides and anti-IL-10 antibodies can improve respiratory immunity as well as gastrointestinal immunity, the IL-10 peptides and anti-IL-10 antibodies can be used to reduce the symptoms of a variety of stresses in mammals, such as the stress associated with mixing populations of mammals.
Young pre-weaned mammals such as dairy calves are susceptible to dairy calf pneumonia, referred to as Bovine Respiratory Disease Complex. Bovine respiratory disease complex (BRD) is a significant cause of morbidity, mortality and animal welfare concern and costs the industry between $800-900 million annually. Antibiotic treatment is costly, recurrence rates are high, the development of refractory sequelae are common, and antibiotic resistance is a concern. Thus, there is a need for improved treatment of pre-weaned mammals that are susceptible to respiratory infections, including mammals of a genus Bos (calves/cows), Ovis (lambs/sheep), Capra (kids/dairy goats), Sus (piglets/pigs) and Bubalus (calf/water buffalo), particularly milk-fed mammals.
In one embodiment, a method of reducing a symptom of respiratory disease in a pre-weaned milk-fed mammal comprises orally administering to the pre-weaned milk-fed mammal an effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds an interleukin-10 peptide, wherein administration is initiated between birth and weaning, such as within 1 to 3 days of birth, and wherein administration is performed at least once daily for a period of 7 days to 7 weeks, specifically 7 days to three, four, five or six weeks, more specifically 7 days to 2 weeks. In one embodiment, the treated mammal is a bovine that exhibits reduced evidence of respiratory disease at 56 days of age compared to a control untreated pre-weaned bovine (
As used herein, the term weaning means the practice of separating a mammal such as a calf from its source of milk. Calves are generally separated from their mothers shortly after birth and are fed whole milk or a milk replacer until weaning from their milk source, generally at 28 to 56 days of age. Calves that are raised apart from their mothers can be referred as housed calves and are generally housed in individual pens or in small groups. Nursing calves, however, can also be supplemented with milk or milk replacer. Thus, a pre-weaned mammal is a baby mammal that receives the majority of its nutrition from milk. As used herein, milk-fed means that a mammal is fed either whole milk, waste milk or milk replacer as their primary source of nutrition. Milk replacer, in the case of calves, generally includes protein such as whey protein or casein, but can also contain soy protein for example. In addition, milk replacers include fat such as animal fat or vegetable oil, essential amino acids, vitamins and minerals.
In one aspect, a pre-weaned calf is a calf that is 56 days old or younger. In the case of goats and sheep, weaning is usually based on weight, however, weaning generally occurs at 6-8 weeks of age, or longer. Weaning of water buffalo is longer than cows, generally at 90 days or more.
Symptoms of respiratory disease include elevated rectal temperature, cough, nasal discharge, ocular discharge, and/or ear droop. Diarrhea can be caused by fecal pathogens including Cryptosporidium parvum and is frequently associated with a reduced appetite, abnormal attitude, dehydration, reduced weight gain, and/or decreased fecal pH. Respiratory disease and diarrhea are the two most important diseases of preweaned calves.
Fecal pH, specifically a neutral fecal pH, may be an indication of improved digestion and gastrointestinal health. In certain aspects, fecal pH can be used as an indication of the health of the gastrointestinal tract of a calf A pH of 5.5 to 7.4 is indicative of a healthy calf, while a pH of less than 5.5 or greater than 7.4 may be indicative of digestive tract dysfunction.
In one aspect, the IL-10 peptide or anti-IL-10 antibody is administered in the form of a milk composition. As used herein, the term milk includes whole milk, waste milk, or a milk replacer composition.
It was unexpectedly found herein that oral administration of anti-IL-10 antibodies, which had previously been shown to reduce gastrointestinal parasites, can also be used to improve respiratory immunity. Administration of IL-10 peptides and/or IL-10 antibodies is expected to reduce the use of antibiotics in treated mammals, resulting in a significant cost savings. Broad spectrum antibiotics such as NUFLOR® (florfenicol), Excede® (ceftiofur), Draxxin® (tulathromycin), Baytril® 100 (enrofloxacin) and Zuprevo® (tildopirosin) are used to treat BRD associated with Mannheimia (Pasteurella) haemolytica, Pasteurella multocida, and Histophilus somni, for example. Because the immunity provided by the IL-10 peptides and/or IL-10 antibodies is expected to last past weaning, protection of post-weaned animals from BRD is expected.
Effective amount of the interleukin-10 peptide or the isolated antibody that specifically binds an interleukin-10 peptide for the treatment of mammals are 3 g/kg (dry matter) milk to 0.001 mg/Kg (dry matter) milk, specifically 1 g/Kg (dry matter) milk to 0.341 g/Kg (dry matter) milk.
In another embodiment, young and adult mammals are subject to stress related to introducing them to a new population of mammals, a process referred to herein as “mixing.” The stress associated with mixing a mammal into a new population of mammals is referred to herein as “mixing stress.” The term mammals includes both human and non-human mammals. In addition to the stress from exposing the mammal to a new population of mammals and any illness within that population, stress can be induced by moving a mammal from familiar surroundings and exposing the mammal to any combination of shipping crates, new foods, temperature changes, means of transportation, and new handlers or caregivers. Stress can result from transport, comingling, feeding changes, vaccination, animal management procedures, crowding, auction, temperature fluctuations, and the like. Stress can affect the immune system of these animals resulting in weight loss (referred to as shrink in cows), fatigue, lack of appetite, signs of depression, and even death. All mammals, including for example, animals of genus Bos (calves/cows), Ovis (lambs/sheep), Capra (kids/dairy goats), Sus (piglets/pigs) and Bubalus (calf/water buffalo), are subject to mixing stress. Additional animals that are subject to mixing stress include horses, dogs, cats, rabbits, and guinea pigs. Mixing stress can occur whether or not the new population is infected with a known illness, such as a viral or bacterial infection.
Thus, a method of reducing mixing stress in a mammal comprises orally administering to the mammal an effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds an interleukin-10 peptide, wherein administration is initiated within four weeks of mixing the mammal with a new population of mammals, and wherein administration is performed at least once daily for a period of 7 days to 7 weeks, specifically 7 days to three, four, five or six weeks, more specifically 7 days to 2 weeks. As used herein, within four weeks of mixing means that administration of the IL-10 peptide or anti-IL-10 antibody is initiated from 4 weeks prior to mixing with the new population to 4 weeks after mixing with the new population. In one embodiment, administration of the IL-10 peptide or anti-IL-10 antibody is stopped after the treatment period and is not restarted until 1 to 14 days, specifically 7 to 14 days. The IL-10 peptide or anti-IL-10 antibody can be administered in a food composition, such as a milk composition or an animal feed composition.
In one embodiment, when the mammal is a non-human mammal, the method further comprising exposing the non-human mammal to a shipping crate, new food, temperature change, a means of transportation, vaccination, castration, tail docking, branding, dehorning, a new handler, a new caregiver, or a combination thereof.
In the case of young mammals, calves for example, mixing stress can result in respiratory disease, weight loss, and behavioral changes. Mixing stress in pigs has been shown to have long-term adverse effects on immune function. In companion animals, moving of animals between shelters or adoption of an animal is often associated with an increased incidence of infectious diseases. Thus, methods of decreasing mixing stress in mammals is applicable across all species of mammals that are subject to mixing of populations.
Effective amount of the interleukin-10 peptide or the isolated antibody that specifically binds an interleukin-10 peptide for the treatment of mammals are 3 g/kg (dry matter) feed/food to 0.001 mg/Kg (dry matter) feed/food, specifically 1 g/Kg (dry matter) feed/food to 0.341 g/Kg feed/food.
In a specific aspect, human children are exposed to stress when they are mixed with a new population of children, such as in a new school or home, a new child care facility, or a new classroom. The move itself may weaken the immune system of the child and the child may be exposed to illnesses in the new population that the child was not previously exposed to. Introduction of a child into a new population of children can result in a variety of illness, including gastrointestinal and respiratory illnesses. Cryptosporidiosis, for example, is a common cause of diarrhea in child care settings. Viral and bacterial respiratory illnesses are also prevalent in childcare situations and schools. Thus, methods to reduce illness associated with mixing stress in human children are also desirable.
In one embodiment, a method of reducing mixing stress in a human child comprises orally administering to the human child an effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds an interleukin-10 peptide, wherein administration is initiated within four weeks of mixing the human child with a new population of human children, and wherein administration is performed at least once daily for a period of 7 days to 7 weeks, specifically 7 days to three, four, five or six weeks, more specifically 7 days to 2 weeks.
In certain aspects, administration of the Il-10 peptide or anti-IL-10 antibody is stopped after the treatment period. As used herein, groups of children include school groups, such as a new school classroom, or a population of children in a childcare setting. The group of children can be as small as, for example, four to six children, to much larger classroom situations.
Reduction of mixing stress may be accompanied by a reduction in gastrointestinal and/or respiratory illness in the child, particularly illnesses associated with mixing of populations of children. Symptoms of illness include, for example, runny nose, cough, fever, listlessness, vomiting, diarrhea, earaches, sore throat, wheezing, and the like, and combinations thereof. Oral administration of an IL-10 peptide or an anti-Il-10 antibody is expected to improve the gastrointestinal and respiratory immunity of the child, thus reducing the symptoms of illness when exposed to new groups of children.
The IL-10 peptides and anti-IL-10 antibodies can be administered in the form of a pharmaceutical composition, or in the form of a food or beverage composition. Effective amount of the interleukin-10 peptide or the isolated antibody that specifically binds an interleukin-10 peptide for the treatment of mammals are depend on the level of isolation. The range of effective doses are shown in table 2.
As used herein, the term “peptide” includes the peptide as well as pharmaceutically acceptable salts of the peptide. “Amino acid residue” means the individual amino acid units incorporated into the peptides of the disclosure. As used herein, the term “amino acid” means a naturally occurring or synthetic amino acid, as well as amino acid analogs, stereoisomers, and amino acid mimetics that function similarly to the naturally occurring amino acids.
As used herein, the term “antibody”, or “immunoglobulin”, encompasses naturally occurring antibodies, such as polyclonal and monoclonal antibodies, as well as artificial or synthetic antibodies or genetically engineered forms of antibodies, including single chain (domain) antibodies (e.g., camelid antibodies, chimeric, and bifunctional antibodies, as well as fragments thereof.
The term “isolated antibody” as used herein, refers to an antibody that is at least partially purified from other naturally associated molecules, or substantially free of antibodies having different antigenic specificities. In some cases, particularly in the case of egg yolk antibodies, the antibody may comprise 50-70% or more of an isolated antibody preparation.
An IL-10 peptide of the present disclosure includes the amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, and combinations thereof (see Table 1).
SEQ ID NOs. 1-14 are amino acid sequences corresponding to peptides of the IL-10 cytokine in pre-weaned animals according to the present disclosure. SEQ ID NOs. 1-38 correspond to exemplary mammals subject to mixing stress.
The present disclosure further includes antibodies that specifically bind to the IL-10 peptides (also referred to herein as “anti-IL-10 antibody”). The antibodies of the present disclosure specifically bind to IL-10 peptides including the amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO.14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO.19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, and combinations thereof.
The present disclosure is further directed to generating antibodies that specifically bind to the IL-10 peptides. In one embodiment, an antibody is generated by administering the IL-10 peptides described above to an animal. Suitable animals to administer the IL-10 peptides for generating the antibodies include, for example, poultry. Exemplary poultry include chickens, turkeys, ducks, quail, and pheasant. Specific poultry include turkeys and chickens. Additional animals include livestock animals such as cattle, pigs, sheep, and fish.
Exemplary methods for administering the IL-10 peptides to the animal include injection and oral administration. Injection and oral administration optionally include use of an adjuvant such as, for example, Freund's Complete adjuvant and Cholera toxin. Administration optionally further includes conjugation of the IL-10 peptide to a carrier protein such as, for example, bovine gamma globulin or keyhole limpet hemocyanin.
In one embodiment, antibodies to the IL-10 peptides are generated by an animal (referred to herein as the “producer animal”). When the animal is an avian animal, as known by those skilled in the art, the antibodies generated are passed to the egg, and may specifically be concentrated in the egg yolk of the avian producer animal. Alternatively, antibodies of the present disclosure may be isolated from the animal itself such as from serum.
In one embodiment, the antibody is an avian egg yolk antibody. Egg yolks derived from a laying hen are inexpensive, convenient and can be safer to handle as compared to the hyperimmunized mammalian sera. Also, egg yolk antibodies are able to stand up to the scrutiny under modern animal protection regulations Immunoglobulin Y (IgY) is an avian immunoglobulin.
To produce avian egg yolk antibodies, the IL-10 peptides are injected into laying fowl, such as hens, preferably at various intervals, to induce an immune response. The hens may be injected intramuscularly or sub-cutaneously. The specific mode of injection is not essential. It is well known that the IgY antibodies produced by the hens in response to such an immune challenge are transferred and concentrated in the egg yolk.
Once the eggs are harvested, the eggs may be further processed to isolate the egg yolk, which itself may be further processed. The liquid egg yolk may be encapsulated or otherwise used in oral dosage forms. The egg yolk may be dried by spray or refractant drying methods, and the resulting dried powder may be encapsulated or otherwise used in oral dosage forms.
Alternatively, a procedure of partial purification or fractionation may be carried out to remove the majority of the non-aqueous bio-molecules and granules and optionally the majority of other proteins in the egg yolk Exemplary purification techniques include the use of PEG, dextran sulfate or a natural gum, such as sodium alginate, carrageenan and xanthan gum, to coprecipitate the undesired substances, and the use of an aqueous buffer or water to obtain an aqueous phase rich with antibodies.
In a specific embodiment, the yolk is separated from the egg white, and then washed with distilled water to remove as much albumen as possible. The vitelline membrane encasing the yolk is punctured, and the separated yolk fraction is then diluted with an effective amount of an aqueous buffer or water to form a suspension of the egg yolk. The collected egg yolk may be diluted with an aqueous buffer solution or distilled water in a ratio of about 1:2 to about 1:40 v/v, and more specifically, in a ratio of about 1:5 to about 1:30 v/v. For efficient recovery of yolk antibodies, pH is about 5-7. Desirably, the temperature in this step is within about 0° C. to about 60° C. The suspension of the egg yolk is gently agitated to form a homogenous mixture, and then allowed to stand for a period of time sufficient to form the aqueous and non-aqueous phases. The water insoluble materials, including non-aqueous bio-molecules such as lipoproteins, phospholipids, sterols and the like, are then removed from the aqueous yolk suspension by centrifugation. The resulting antibody-containing supernatant may then be separated from the viscous precipitant by decanting, suctioning, or other like methods known in the art.
Optionally, the yolk supernatant is further treated with a high concentration of a non-denaturing salt to induce precipitation of the antibodies. Examples of the salts useful for precipitation of the yolk antibodies include, but are not limited to, NaCl, Na2SO4, (NH4)2SO4, KCl, CaCl2, and MgSO4. Specific salts include Na2SO4 and (NH4)2SO4. The salt concentration for precipitating antibodies depends on the type of the salt. In one embodiment, the salt is present in an amount of higher than 15% and lower than 35% by weight, specifically between 20% and 30% by weight of the salt, on the basis of the final volume of the yolk supernatant.
Alternatively, the antibodies may be purified or isolated using any conventional technique such as by immunoaffinity purification.
In one embodiment, egg yolk antibodies are prepared by the following method. Laying hens are inoculated with IL-10 peptide. Optionally, an adjuvant is administered in conjunction with the IL-10 peptide to enhance the immunization. An adjuvant useful for this purpose is a water-in-oil emulsion adjuvant such as complete Freund's adjuvant. The IL-10 peptide causes the hens to produce anti-IL-10 antibodies which are passively transferred into the egg yolk of eggs laid by the hens.
Egg yolks or whole eggs containing the anti-IL-10 antibody can be collected and homogenized to form an emulsion. The resulting emulsion can be dried to form a powder containing the anti-IL-10 antibody. This powder can then be formulated in a manner appropriate to the administration route and then administered to the desired animals using methods known in the art. The preparation is preferably administered orally, such as in an oral dosage form or in a supplement to the animal's diet.
The antibodies that specifically bind to IL-10 peptides may be isolated and purified from animal serum or egg using a suitable method known in the art. Such methods include affinity chromatography, as well as other suitable methods for antibody isolation and purification known in the art and described in U.S. Pat. No. 6,608,172 and De Meulenaer et al., “Isolation and Purification of Chicken Egg Yolk Immunoglobulins: A Review,” Food and Agricultural Immunology, Vol. 13(4), 2001, hereby incorporated by reference to the extent that they are consistent herewith. In one particularly suitable embodiment, the production animal is an avian animal such as a chicken, turkey, duck, or quail, and the antibody is isolated from the egg yolk of the egg of the avian animal.
In one embodiment, the egg yolk or serum including the antibodies are further dried to form a powder including the antibodies. The whole egg, egg yolk or parts of the egg may be spray dried. Serum may be separated from whole blood according to methods known by those skilled in the art. Spray drying of egg and serum may be performed using known spray drying methods and commercially available spray drying equipment. Dry egg and serum powders may also be prepared by lyophilization or vacuum drum drying. The dried egg, egg yolk or serum powder may then be introduced into animal feeds as a feed additive to transfer antibodies to an animal.
In another aspect, isolated antibodies can include antibodies in serum, or antibodies that have been purified to varying degrees. Such antibodies may include polyclonal antibodies, camelid antibodies, monoclonal antibodies, humanized or chimeric antibodies, anti-idiotypic antibodies, single chain antibodies, Fab fragments, fragments produced from a Fab expression library, epitope-binding fragments of the above, and the like. Production of antibodies is well-known in the art.
In yet another aspect, an antibody is isolated from the colostrum of an animal such as from bovine colostrum.
The methods disclosed herein can be achieved using food/feed additives including the IL-10 peptides, or isolated antibodies which specifically bind to IL-10 peptides.
As used herein, the term “feed” broadly refers to a material, liquid or solid, that is used for nourishing an animal, and for sustaining normal or accelerated growth of an animal including newborns or young and developing animals. The term includes a compound, preparation, mixture, or composition suitable for intake by an animal. Specifically, the feed is suitable for herbivorous mammals such as cattle, horses, sheep and goats; for fish; or for companion animals. A feed composition comprises a basal food composition and one or more feed additives. The term “basal food composition” refers to a food composition combinable with additives such as the peptides and antibodies described herein. Basal animal food compositions may include components such as proteins, grains, flavor compositions, vitamins, minerals, preservatives, and the like. Basal food compositions can be suitable for ingestion by a target animal. The term “feed additive” as used herein refers to components included in small quantities for the purpose of fortifying basic feed with nutrients, stimulants, medicine, or to promote feed intake or alter metabolism. Feed additives include pre-mixes of biological compositions, or in the present disclosure, pre-mixes of IL-10 peptide or isolated antibody that specifically binds to IL-10 peptide.
In one embodiment, the methods disclosed herein are achieved using animal feed additive including IL-10 peptides including the amino acid sequence of any one of SEQ ID NOs. 1-34, and combinations thereof.
In another embodiment, the methods of the present disclosure utilize an animal feed additive including isolated antibodies that specifically bind to the IL-10 peptide including the amino acid sequence of SEQ ID NOs. 1-34, and combinations thereof.
The IL-10 peptides or isolated antibodies which specifically bind to IL-10 peptides may be added to an animal feed as a feed additive or mixed into an animal feed by a method known in the art for mixing feed additives and animal feed. In one embodiment, the IL-10 peptide or isolated antibody which specifically binds to the IL-10 peptide is directly added to the animal feed or mixed with the animal feed just prior to feeding the animal. In another embodiment, since feeds may be pelleted or extruded, the IL-10 peptide or isolated antibody which specifically binds to the IL-10 peptide may be coated on the surface of feed (pellet) after the feed has been pelleted or extruded (post pelleted application) in order to maintain functional properties of the IL-10 peptide or isolated antibody which specifically binds to the IL-10 peptide. The addition of the IL-10 peptide or isolated antibody which specifically binds to the IL-10 peptide post pelleting can be aided by mixing the IL-10 peptide or isolated antibody which specifically binds to the IL-10 peptide in water, oil, or another suitable carrier and spraying it on the pellets as they exit the pellet die.
The amount of the IL-10 peptide or isolated antibody that specifically binds to IL-10 peptide added and/or mixed with the animal feed depends on the feeding regimen and the type of feed for the animal, and may be determined by those skilled in the art. Typically, the amounts of IL-10 peptides and/or isolated antibodies to IL-10 peptide to be used in an animal feed are summarized in Table 2 below. Antibody prepared using other sources may be calculated as equivalents using Table 2.
The doses shown are based on the amount of epitope specific antibody in total IgY (1 to 10%), the amount of IgY in egg (5-10 mg/Kg of feed(dry matter)), antibody losses due to drying storage and gastrointestinal degradation.
An animal feed may further include optional ingredients including vitamins, minerals, antibiotics, lipids, carbohydrates, proteins, antioxidants, and amino acids.
Exemplary vitamins include Vitamin A, Vitamin B, Vitamin D, Vitamin E, and Vitamin K. Exemplary minerals include calcium, phosphorus, sodium, potassium, magnesium, chlorine, cobalt, iodine, iron, manganese, copper, molybdenum, zinc and selenium. Common mineral supplements used in poultry feed, for example, include limestone, bone meal, oyster shell, sodium chloride, dicalcium phosphate, manganese sulphate, potassium iodide, and superphosphate.
In some embodiments, one or more antibiotics may be included in the animal feed along with the feed additive. Exemplary antibiotics include penicillin, streptomycin, tetracyclines, zinc bacitracin and aureomycin.
Exemplary lipids include oil seeds, oils and lipids derived from plants or animals. Sources of oilseeds, oils and lipids include corn, soybean, cotton, lupin, peanut, sunflower, canola, sesame seed oil, olive oil, copra and coconut oil, palm kernels and palm oil, casein, butterfat, lard, fish oils, linseed and oil, tuna oil, tallow and yellow grease, and mixtures thereof.
Exemplary carbohydrates include starch, cellulose, pentosans, other complex carbohydrates, corn, milo, barley, rye, oats, wheat, wheat middlings, and various grain-by-products.
Exemplary sources of protein include protein obtained from meat meal or fish meal, liquid or powdered egg, fish solubles, whey, milk protein, rice, milo, millet, corn, oats, barley, wheat, rye, wheat bran and/or middlings, soybeans, sesame seeds, peas and beans, sunflower seeds, wheat germ, alfalfa seed, flaxseed, yeast, earthworms, and fish.
Exemplary amino acids include arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, tyrosine ethyl HCl, alanine, aspartic acid, sodium glutamate, glycine, proline, serine, cystein ethyl HCl, and analogs, and salts thereof.
Exemplary antioxidants include beta-carotene, Vitamin E, Vitamin C, and tocopherol, or synthetic antioxidants.
Specifically, the animal feed including the feed additive of either IL-10 peptide or isolated anti-IL-10 antibody is a feed for a non-human mammal such as a cow, horse, goat or sheep.
In one aspect, included herein is a milk composition suitable for administration to a pre-weaned mammal of a genus Bos, Ovis, Capra, Bubalus or Sus containing a basal milk composition and a therapeutically effective amount of an IL-10 peptide or anti-IL-10 antibody. Basal milk compositions include whole milk, waste milk, or a milk replacer composition. As used herein, a therapeutically effective amount of an IL-10 peptide or anti-IL-10 antibody is an amount effective to reduce the incidence of respiratory or gastrointestinal disease in the animal when administered daily for a period of 7 days to 7 weeks. Exemplary therapeutically effective amounts of interleukin-10 peptide or the isolated antibody that specifically binds an interleukin-10 peptide include 3 g/kg (dry matter) milk to 0.001 mg/Kg (dry matter) milk, specifically 1 g/Kg (dry matter) milk to 0.341 g/Kg (dry matter) milk. Exemplary peptides for inclusion in the milk compositions include the peptides of any one of SEQ ID Nos. 1-14, or combinations thereof. Exemplary anti-IL-10 antibodies for inclusion in milk compositions include antibodies that specifically bind a peptide of any one of SEQ ID NOs. 1-14, or any combination of antibodies.
In another aspect, included herein is a food composition suitable for administration to humans such as human children containing a basal food composition and an IL-10 peptide or anti-IL-10 antibody. A food composition suitable for administration to a human comprises a peptide of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, or SEQ ID NO. 38; or an isolated anti-IL-10 antibody that specifically binds a peptide of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, or SEQ ID NO. 38. Exemplary basal food compositions include milk, juice, formula, and solid foods such as snack food consumable by humans including human children.
In one embodiment, the methods involve injecting or orally administering an IL-10 peptide to an animal, thereby producing antibodies within the animal that specifically bind to the IL-10 peptide. IL-10 cytokine production is associated with down regulation of inflammation, and the IL-10 cytokine functions as an essential immunoregulator of the intestinal tract. The antibody to IL-10 peptide prevents the IL-10 cytokine from down regulating the immune system, thereby allowing the immune system to eliminate the pathogen.
In some embodiments, the methods involve injecting or orally administering an antibody to the IL-10 peptide to an animal. The term “animal”, as used herein to describe animals administered an IL-10 peptide or isolated antibody to the IL-10 peptide in accordance with the present disclosure.
The invention is further illustrated by the following non-limiting examples.
EXAMPLES Example 1 Anti-IL-10 Antibody ProductionIn this Example, the concentration of anti-IL-10 antibody production contained within the egg yolk of IL-10 peptide-administered producer hens was determined by using Enzyme-linked immunosorbent assay (ELISA) techniques.
Specifically, IL-10 peptide SEQ ID NO: 2 was conjugated to hen ovalbumin (OVA, Sigma, St. Louis, Mo.) for ELISA using a glutaraldehyde procedure. A 96-well Nunc™ immunosorbent F-series microplate (Sigma, St. Louis, Mo.) was coated with 100 μg/plate of peptide-specific OVA conjugate in sodium carbonate coating buffer having a pH of 9.6. The plate was allowed to coat overnight (100 μl/well) at 4° C. Dry egg yolk samples containing antibody to IL-10 Peptide SEQ ID NO. 2 were diluted 1:10 in acidic PBS having a pH of 4 and allowed to incubate overnight at 4° C. After overnight incubation, the antibody was extracted using centrifugation and used as a source of antibody to determine specificity for the peptide conjugated to the IL-10 peptide. The plate coated with OVA-peptide conjugate was washed 6 times with PBS/0.5% Tween solution, blocked with non-protein blocking buffer (200 μl/well, Pierce Scientific, Rockford, Ill.), and allowed to incubate at room temperature for at least 1 hour. The plate was washed 6 times and then samples of either adjuvant only injected control or egg antibody (isolated as described above) were added at a concentration of 100 μl/well in duplicate at 10× serial dilutions starting at 1:20. Primary antibodies were incubated for 1 hour, the plate was washed 6 times, and then secondary antibody (HRP-conjugated goat anti-chicken antibody, Bethyl Labs, Montgomery, Tex.) was diluted in blocking buffer 1:5000 and added at a concentration of 100 μl/well. Secondary antibody was incubated for 30 minutes, the plate was washed 6 times, and then substrate solution containing 19.74 ml 0.05M sodium acetate, 100 μl 20 mg/mL 3,3′,5,5′ Tetramethyl Benzidine (TMB), 128 μl 0.5M H2O2 was added at a concentration of 125 μl/well and allowed to incubate until sufficient color development during the linear phase of development (blue color indicates primary antibody presence). A stop solution (0.5M sulfuric acid) was added to produce a yellow stable color and the plate was read at 450 nm on a Biotek EL800 plate reader. Triplicate optical densities were averaged and blocking buffer background was subtracted to produce a final optical density. The optical density of antibody to IL-10 peptide SEQ ID NO. 9 and FCA control were compared to determine specificity and dose level used in the final chick experiment (see Table 3).
The study was a double-blinded, randomized, prospective clinical trial. There were 134 calves randomly assigned to a treatment group (72) and a control group (62) of primarily Holstein calves. Calves were enrolled in the study on five separate days within a 2-week period. On enrollment day, 23-28 calves (depending on the number of calves that arrived on the farm) were randomly assigned to a group designated by color (green=anti IL-10 IgY treatment, purple=anti IL-10 IgY treatment, orange=egg powder control group). Calf feeders, health screeners and investigators were blinded to the color code until the study was completed. Calves in this study originated from 12 surrounding dairy farms, where colostrum and newborn calf care were administered. Calves arrived between 1 and 3 days of age. On enrollment day, the data collected included body weight (electronic scale), hipometer (stall-side indirect measure of body weight), packed cell volume (PCV) and serum total protein concentration (indicator of dehydration and adequate passive transfer of immunoglobulins, respectively), and fecal pH. Calves were identified by group with color-coded zip ties on eartags and color-coded Duct Tape® on their individual hutches. The calves were fed in milk either 0.96 g of egg yolk powder containing anti IL-10 antibodies (treatment) or 0.96 g control egg yolk powder without anti-IL-10 antibodies (control egg yolk powder from hens receiving a sham inoculation) added to their milk daily (divided into 2 feedings) from Day 0 (day after enrollment) through Day 10. Attitude, appetite and health scores were recorded daily for 14 days using The Calf Health Scorer App. Fecal pH and fecal pathogen testing (C. parvum RT-PCR, Coronarvirus RT-PCR, Rotavirus RT-PCR and Salmonella culture) were performed on Day 5 and Day 14 of the trial. Hydration status (PCV and serum total protein) and hipometer measurements were recorded on Day 7 and Day 14. Growth (average daily gain and hipometer) measurements along with health score were performed on Day 56 (for Calf Health Score) or when calves were moved out of the hutches. At this time, antibiotic administration data (by farm personnel) was also recorded.
The protocol deviations in the study were minor. The unequal group sizes are based on randomization using 3 colors assigned on the day that calves arrived. One calf died after enrollment and was removed from the study. Two missing data points on calf health scores and on enrollment fecal pH's, respectively were handled by leaving those calves out of the analysis of those specific data sets but not other parameters where the data collection was complete.
Experimental procedure: In this trial, 0.96 g egg yolk powder containing anti IL-10 IgY antibodies was added to the milk of calves in the treatment group while 0.96 egg yolk powder without anti IL-10 IgY antibodies was added to the milk of calves in the control group for 11 days. The dose of egg yolk powder administered in the milk was 1.2 gm/kg of milk dry matter, using an assumption of 12.5% dry matter for whole milk. The egg product used is defined by American Association of Feed Control Officials (AAFCO) 9.74.
Results: Average total health score and days of diarrhea were not significantly different between the two groups. There was a higher score for the diarrhea average score from Days 0 through Day 4 in the treated group (p=0.03). Rotavirus was significantly increased in the treatment group on Day 5 (p=0.02), but at Day 14 it was no longer significant. There was no significant difference in C. parvum oocyst shedding between the two groups. However, on Day 5 there was a trend (p=0.06) for increased C. parvum oocyst shedding in the treatment group, whereas the trend was reversed on Day 14 when there was decreased shedding of C. parvum oocysts in the treatment group. The mean cycle threshold value for C. parvum fecal PCR detection was higher in the treatment group than the control group on Day 14, another indicator of reduced C. parvum oocyst shedding in anti IL-10 antibody treated calves. Fecal pH was significantly higher in the treatment group at Day 14 (p=0.004). The differences in fecal pH from enrollment day to Day 5, enrollment day to Day 14 and Day 5 to Day 14 were all significantly higher in the treatment group. A more alkaline fecal pH is associated with improved digestion and overall gastrointestinal health.
Average daily gain was significantly increased in the control group (p=0.04) but the hipometer measurement from Day 0 to Day 56 was significantly higher in the treated group (p=0.02). The latter finding is indicative of a larger frame size (width between the greater trochanter of the left and right femurs) in treated calves, even though average daily gain was greater in the control calves.
The prevalence of respiratory disease on Day 56, as determined by the calculated total respiratory score, was significantly higher in the control group (21%) versus the treated group (8%) (p=0.04). In addition, use of antibiotics by farm personnel for respiratory disease (investigators excluded antibiotic treatment for joint or navel infections) was significantly higher in the control group (20.7%) versus the treated group (7.2%) (p=0.04).
General discussion and conclusions: Dairy calf health is significantly affected by both enteric disease (most severely in the preweaning period) and by pneumonia (most commonly in the postweaning period). Antibiotic therapy is commonly used to treat both enteric and respiratory pathogens, leading to issues regarding antibiotic resistance, withdrawal times and residues. Cryptosporidium parvum is an Apicomplexan protozoan that infects intestinal cells and destroys villi, causing a malabsorptive diarrhea. The shedding prevalence of C. parvum is reported in the United States as 8 to 49% in calves and 6 to 13% in adult cows. Within-farm prevalence can be high as 70% on some farms, and both shedding and intensity of shedding have been significantly associated with diarrhea. C. parvum and Rotavirus have been found to be the most common enteric pathogens in diarrheic feces and most calves with diarrhea have more than one pathogen isolated from their feces. Oral IgY products have been successful in treating gastrointestinal infections, including bovine rotavirus, coronavirus, enterotoxigenic E. coli, and Salmonella spp. Although orally administered IgY is proteolytically digested to Fab and Fc fragments, both show neutralizing activity in the GI tract and can be detected in feces. Previous studies have shown that IL-10 expression is increased in calves infected with C. parvum. In addition, IL-10 knockout mice have been found to be significantly resistant to C. parvum infection. The present study was conducted to investigate the effects of feeding anti-IL-10 egg yolk antibodies on fecal pathogen shedding, health, and weight gain in dairy calves. The results showed a trend in reduced C. parvum shedding when evaluating the cycle threshold values at day 14 (p=0.08) and a significant increase in fecal pH in the treatment group. Fecal pH is an indicator of colonic acidity, as nutrients that escape digestion in the small intestine ferment in the large bowel. It has been shown that decreased fecal pH in dairy calves is associated with elevated lactate production and reduced volatile fatty acid concentrations in the colon (VFAs). Absorption of VFAs from the large intestine is critical for promoting sodium and water reabsorption in the gut. Without being held to theory, we associate the significant increase in fecal pH and the trend towards reducing fecal shedding of C. parvum in the treatment group as indicators of improved gastrointestinal health. The increased shedding of rotavirus could be related to increased pass-through of the virus without attachment to the small intestinal villi.
An unanticipated benefit to the treatment group was a decrease in respiratory disease and less antibiotic usage than the control group during the trial (day 0-day 56) (
In summary, the results presented herein suggest that improved gastrointestinal health is linked to improved respiratory immunity utilizing the common mucosal immune system hypothesis. From this study, there is evidence of improved gastrointestinal tract health in enteric pH data; there is a trend for reduced C. parvum oocyst shedding, and improved frame size in neonatal calves given anti IL-10 antibody for 11 days. With improved digestive function, there appear to be combined benefits of improved enteric and respiratory mucosal immunity. The significant reduction in respiratory disease in dairy calves at weaning has the potential to decrease respiratory disease in post weaning group housing. Respiratory disease in this age group has significant negative long term economic and welfare consequences of recurring disease, poor growth, delayed breeding, reduced milk production in the first lactation and early culling.
Example 3 Second Field TrialThe proposed second field trial will investigate a dose response to anti IL-10 antibody feeding (0, 0.5, 1 and 2 times the dose used in trial 1 and a farm control group) and will prolong inclusion in the diet for 14 days, when the risk of neonatal diarrhea is greatly reduced. Parameters of health monitoring, gastrointestinal function, fecal pathogen shedding, growth and antibiotic usage will be similar to trial 1 but, to validate the finding of reduced respiratory disease prevalence in trial 1, health and respiratory disease parameters will be monitored twice weekly for the entire 56-day clinical trial. Thoracic ultrasound and respiratory pathogen detection by deep nasopharyngeal swab sampling will confirm that respiratory disease is present and help define the benefit of feeding anti IL-10 antibody.
The use of the terms “a” and “an” and “the” and similar referents (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms first, second etc. as used herein are not meant to denote any particular ordering, but simply for convenience to denote a plurality of, for example, layers. The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The endpoints of all ranges are included within the range and independently combinable. All methods described herein can be performed in a suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as used herein.
While the invention has been described with reference to an exemplary embodiment, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
Claims
1. A method of reducing a symptom of respiratory disease in a pre-weaned milk-fed mammal, comprising
- orally administering to the pre-weaned milk-fed mammal an effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds an interleukin-10 peptide,
- wherein administration is initiated between birth and weaning, and
- wherein administration is performed at least once daily for a period of 7 days to 7 weeks.
2. The method of claim 1, wherein administration of the Il-10 peptide or anti-IL-10 antibody is stopped after the 7 day to 7 week period and is not restarted for 1 to 14 days.
3. The method of claim 1, wherein the pre-weaned milk-fed mammal is of a genus Bos, Ovis, Capra, Bubalus, or Sus.
4. The method of claim 3, wherein the pre-weaned milk-fed mammal is a bovine 56 days of age or younger and the respiratory disease is bovine respiratory disease complex.
5. The method of claim 4, wherein the symptom of respiratory disease is elevated rectal temperature, cough, nasal discharge, ocular discharge, ear droop, diarrhea, increased fecal pathogens including Cryptosporidium parvum, reduced appetite, abnormal attitude, dehydration, reduced weight gain, decreased fecal pH, or a combination thereof.
6. The method of claim 1, wherein the isolated antibody that specifically binds the interleukin-10 peptide is an egg yolk antibody.
7. The method of claim 1, wherein the interleukin-10 peptide or the isolated antibody that specifically binds the interleukin-10 peptide is administered in a milk composition.
8. The method of claim 1, wherein the interleukin-10 peptide is SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14.
9. A method of reducing mixing stress in a mammal, comprising
- orally administering to the mammal an effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds an interleukin-10 peptide,
- wherein administration is initiated within four weeks of mixing the mammal with a new population of mammals, and
- wherein administration is performed at least once daily for a period of 7 days to 7 weeks.
10. The method of claim 9, wherein administration of the Il-10 peptide or an isolated anti-IL-10 antibody is stopped after the 7 day to 7 week period and is not restarted for 1 to 14 days.
11. The method of claim 9, wherein the isolated antibody that specifically binds the interleukin-10 peptide is an egg yolk antibody.
12. The method of claim 9, wherein the interleukin-10 peptide or the isolated antibody that specifically binds the interleukin-10 peptide is administered in a food composition.
13. The method of claim 9, wherein the mammal is a non-human mammal.
14. The method of claim 13, further comprising exposing the non-human mammal to a shipping crate, new food, vaccination, branding, tail docking, castration, dehorning, temperature change, a means of transportation, a new handler, a new caregiver, or a combination thereof.
15. The method of claim 13, wherein the interleukin-10 peptide is any one of SEQ ID NOs. 1-34.
16. A method of reducing mixing stress in a human child, comprising
- orally administering to the human child an effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds an interleukin-10 peptide,
- wherein administration is initiated within four weeks of mixing the human child with a new population of human children, and
- wherein administration is performed at least once daily for a period of 7 days to 7 weeks.
17. A milk or formula composition suitable for administration to a pre-weaned mammal of a genus Bos, Ovis, Capra, Bubalus or Sus comprising a basal milk composition and a therapeutically effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds the interleukin-10 peptide.
18. The milk composition of claim 17, wherein the basal milk composition comprises whole milk, waste milk, or a milk replacer composition.
19. The milk composition of claim 17, wherein the therapeutically effective amount of an interleukin-10 peptide or an isolated antibody that specifically binds the interleukin-10 peptide is 3 g/kg to 0.0001 mg/kg of the milk composition as a dry matter weight.
20. The milk composition of claim 17, wherein the peptide is of any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14. or the isolated anti-IL-10 antibody specifically binds a peptide of and one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, or SEQ ID NO. 14.
21. The milk composition of claim 17, wherein the isolated antibody that specifically binds the interleukin-10 peptide is an egg yolk antibody.
22. A food composition suitable for administration to a human, comprising a basal food composition and a peptide of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, or SEQ ID NO. 38; or an isolated anti-IL-10 antibody that specifically binds a peptide of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, or SEQ ID NO. 38.
23. The food composition of claim 22, wherein the basal food composition is milk, juice, formula, or a solid food consumable by humans.
24. The food composition of claim 22, wherein the isolated antibody that specifically binds the interleukin-10 peptide is an egg yolk antibody.
Type: Application
Filed: Mar 27, 2015
Publication Date: Sep 29, 2016
Inventors: MARK ERIC COOK (MADISON, WI), JORDAN MARSHALL SAND (MADISON, WI), SHEILA MARY McGUIRK (MADISON, WI), JANE ELLEN RIEMAN (DAVIS, IL), SARAH MARIE RAABIS (MADISON, WI)
Application Number: 14/670,636
