Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/189—Enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/205—Amine addition salts of organic acids; Inner quaternary ammonium salts, e.g. betaine, carnitine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S426/00—Food or edible material: processes, compositions, and products
  • Y10S426/805—Pet food for dog, cat, bird, or fish
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S426/00—Food or edible material: processes, compositions, and products
  • Y10S426/807—Poultry or ruminant feed

Definitions

• This invention relates to an additive for an animal feed and particularly to such an additive which improves the rate of weight gain of an animal fed with a feed in which it is incorporated. It furthermore relates to such an additive which is useful for the treatment and/or prophylaxis of coccidiosis and/or a bacterial infection including those which can result in necrotic enteritis.
• the farming of many different types of animals is important throughout the world for the production of food for human consumption. Whilst the animals are being reared, they can come into contact with a variety of infection-causing bacteria and parasites such as Eimeria, Campylobacter, Clostridium, Salmonella, E. coli and Li teria.
• Coccidiosis is a common cause of disease in intensively-reared farm livestock, particularly in poultry. Coccidiosis is caused by a protozoa, a single-celled parasite, of the subphylum Apicomplexa. Many of the species that cause the disease in domestic animals belong to the genus Eimeria. The parasites multiply in the epithelium of the intestine. In chickens, seven species of Eimeria have been identified, five of which are considered to be pathogenic. These are E. acervulina, E. maxima, E. necratrix, E. tenella and E. brunetti.
• Coccidia are ubiquitous organisms and are generally endemic wherever chickens are raised. Outbreaks of disease can vary from severe to very mild infections. Like many parasitic protozoa, the life cycle of Eimeria is relatively complex. Sexual and asexual multiplication occurs within the chickens' intestines. During this process of multiplication and development of the parasite, the host tissue is destroyed which leads to the various clinical manifestations observed in outbreaks of coccidiosis. The oocysts produced and excreted develop further outside the host where they may undergo further development and infect other chickens. Oocysts can in fact survive outside a host for a long period of time which enables them to infect other birds even after removal of the initially infected host.
• Sporulated oocysts contain four sporocysts, each containing two sporozoites. These sporozoites are released by mechanical and enzymatical action in the digestive tract of the chicken. This enables them to invade the epithelial cells in the intestine or caeca depending on the Eimera species involved.
• the symptoms displayed by an infected animal may be one or more of the following: bloody droppings, high mortality, general lethargy, emaciation, a marked drop in feed consumption, diarrhoea and a drop in egg production. It has been estimated that coccidiosis is probably responsible for around 6-10% of unwanted mortality among poultry flocks. Additionally, sub-clinical disease increases feed conversion ratios (FCRs) and reduces performance. Accordingly, the economic consequences of this disease are considerable and most undesirable.
• FCRs feed conversion ratios
• coccidiosis in poultry is routinely controlled by the use of relatively costly preventative anticoccidial drug programmes.
• Such programmes attempt to restrict coccidial infections thus limiting the effects of sub-clinical outbreaks of disease. This is usually accomplished by the continuous inclusion of anticoccidial agents in the feed from early in the life of the flock until close to slaughter for broiler birds or by controlled withdrawal for layers. When first developed, such agents were used individually. This often resulted in strains of parasites developing drug-resistance. It is presently attempted to control coccidiosis by the continual introduction of new drugs or by the use of drug programmes involving rotational use of anticoccidial agents of different biochemical structures either during the grow-out period (shuttle programmes) or at frequent intervals (rotation programmes).
• Inner salts of quaternary amine carboxylic acids function as osmoprotectants. Such salts increase the osmotic strength of cells without adversely affecting enzyme activity, and they protect enzymes from ionic or temperature inactivation (Nash et al., Aust. J. Plant Physiol. 9:47-57 (1982); Yancey et al., Science 224: 1064-1069 (1982); Rudolph et al., Archives Biochem. Biophys.
• isolated salmon liver mitochondria when exposed to osmotic stress, show increased betaine intake, but not synthesis (Bjorkoy, G., Synthesis and Accumulation of glycine betaine in Salmon (Salmo salar) and Mussels, MSc thesis, Norwegian College of Fisheries, University of Tromso, pp. 94).
• GB-A-2 327 345 teaches that bacterial infections in the ileum of livestock can be treated by incorporating enzymes in an animal feed which promote digestion of the feed. Such enzymes break down polysaccharides present in the cereal component of the feed into oligosaccharides which are used as a food source by host-beneficial microorganisms present in the gut. As a consequence of this proliferation, pathogenic bacteria cannot thrive due to the process of competitive exclusion. This strategy is generally more effective when the quality of food is low (Classen et al., Proc. 2 nd Eur. Symp. on Feed Enzymes, 1995, 65; Pack and
• JP-A-01 238538 and JP-A-01 132533 teach the combined use of betaine and various enzymes for improving the digestive function of animals.
• neither of these documents suggests to use such combinations for the treatment or prevention of coccidiosis or bacterial infections, or for helping to maintain the rate of growth in animals subjected to the coccidial challenge.
• Clostridium perfringens One of the most problematic diseases in livestock is necrotic enteritis caused by Clostridium perfringens. Clostridium infections are usually preceded by coccidiosis. Coccidiosis compromises an animal's immune response so that the animal is less able to respond when subsequently exposed to a bacterial infection. However, other factors such as stress, overstocking or saturation of litter can also contribute. Necrotic enteritis is usually treated by supplementing animal feed with water soluble zinc bacitracin, virginamycin or penicillin.
• protease activity is the amount of enzyme which liberates from the substrate one microgram of phenolic compound (expressed as tyrosine equivalents) in one minute under the conditions described.
• Substrate solution is usable for one week if stored in a cold room.
• Enzyme blank Equilibrate 1 ml of enzyme dilution (in 0.02 M NaCl solution) at +40°C (for about 5 minutes). Add 5 ml of precipitation reagent, stir and incubate at +40°C for exactly 30 minutes. Add 5 ml of casein substrate, stir and incubate at +40°C for exactly 30 minutes. Filter immediately with filter paper (Whatman 1 or Macherey Nagel 640 we).
• the absorbance difference between the enzyme sample and the enzyme blank should be 0.2-0.5.
• tyrosine stock solution by weighing 10 mg of L- tyrosine into a volumetric flask, dissolve in 0.02 M NaCl solution and fill to 100 ml with 0.02 M Nacl solution.
• A(X) absorbance of the enzyme sample
• One unit of xylanase activity is the amount of enzyme which liberates one ⁇ mol of reducing sugars (expressed as xylose equivalents) from the substrate in one minute under the conditions described.
• Reagents 1. 1% (w/v) xylan substrate
• the Reagent Store in a dark bottle at room temperature.
• the Reagent is stable for a maximum of 6 months.
• 1 ml of enzyme dilution (in 0.05 M sodium acetate buffer, pH 5.3) is equilibrated at +50°C.
• 1 ml of xylan substrate stir and incubate at +50°C for exactly 30 minutes.
• Add 3 ml of DNS -reagent stir and boil the reaction mixture for exactly 5 minutes. Cool the reaction mixture in a cold water bath to room temperature and measure the absorbance at 540 nm against distilled water.
• the absorbance difference between the enzyme sample and enzyme blank should be 0.3-0.5.
• A(X) absorbance of the enzyme sample
• One unit of ⁇ -amylase activity catalyses one micromole of hydrolysis of glycosidic linkages in one minute under the conditions described.
• Phadebas Amylase Test-tablet for in vitro diagnostic use (Pharmacia Diagnostics) is used.
• the tables are made in distilled water from water insoluble blue starch polymer, bovine serum albumin and buffer.
• the absorbance of the enzyme sample should be 0.3 - 0.5.
• the absorbance of sample is proportional to the ⁇ -amylase activity.
• the ⁇ -amylase activity of enzyme dilution is read from a table enclosed in the tablet kit. For each tablet batch a calibrated table is supplied.
• the ⁇ -amylase activity of the sample is calculated as follows:
• Act ⁇ -amylase activity value (expressed U/l) of enzyme dilution read from Phadebas Amylase Test table
• Df dilution factor (ml/g)
• a first aspect of the present invention provides the use of a protease and an inner salt of a quaternary amine carboxylic acid for the manufacture of an agent for the treatment and/or prophylaxis of coccidiosis, particularly when caused by Eimeria infection.
• the present invention provides the use of a protease and an inner salt of a quaternary amine carboxylic acid for the manufacture of an agent for the treatment and/or prophylaxis of a bacterial infection, e.g. an infection caused by Salmonella, Campylobacter or Listeria.
• the present invention provides the use of a protease and an inner salt of a quaternary amine carboxylic acid for the manufacture of an agent for the treatment and/or prophylaxis of necrotic enteritis resulting from inter alia Clostridium perfringens infection.
• the present invention provides an additive for an animal feed comprising a protease and an inner salt of a quaternary amine carboxylic acid.
• the agent or additive further comprises a xylanase.
• Figure 1 shows a graphical representation of the effects of supplementing the feed of healthy broiler chickens with betaine; a protease; and with a combination of betaine and a protease.
• Figure 2 shows a graphical representation of the effects of supplementing the feed of broiler chicken challenged with Eimeria maxima with betaine; a protease; and with a combination of betaine and a protease.
• Figure 3 is a gel chromatogram showing the presence of C. perfringens toxin gene in the ileum of chickens fed with variously supplemented feeds and challenged with various pathogens.
• the present invention provides for the use of a protease and an inner salt of a quaternary amine carboxylic acid for the manufacture of an agent for the treatment and/or prophylaxis of coccidiosis, bacterial infection or necrotic enteritis resulting from inter alia Clostridium perfringens infection.
• the advantage of using feeds containing a combination of betaine and a protease for rearing animals is that the amount of antimicrobial drugs which have previously been routinely incorporated into feeds can be reduced, or in some cases omitted entirely. In countries where such drugs are banned, it represents a new approach to the control of bacterial diseases.
• the present invention also has benefits for human health. Its use reduces the selection pressure for antibiotic-resistant strains of bacteria, by allowing antibiotics to be removed from animal feed. Accordingly, more antibiotic- susceptible strains will be present in the gut of the animal, thereby ensuring a more likely lethal effect in the event of antibiotics being used on the equivalent human condition.
• the feed additive according to the present invention can be prepared in a number of ways. For instance, it can be prepared simply by mixing the different appropriate compounds to produce the additive. This can then be either mixed directly with a feed, or more conventionally impregnated onto a cereal-based carrier material such as milled wheat, maize or soya flour. Such an impregnated carrier also constitutes a feed additive in accordance with the fourth aspect of the present invention.
• the feed additive may be mixed directly with the animal feed, or alternatively mixed with one or more other feed additives such as a vitamin feed additive, a mineral feed additive or an amino acid feed additive.
• the resulting feed additive including several different types of components can then be mixed in an appropriate amount with the feed.
• the feed additive provided by the present invention can be formulated as a pre- mix together with any other enzymes which it is desired to include.
• the pre-mix can be added to the raw materials before feed manufacture, during feed manufacture or as a final step once the feed is otherwise ready for use. It is possible to add the combination of the inner salt and protease directly to a feed material pre-formed as pellets or as a mash.
• the feed should comprise at least 25% by weight of a cereal, and preferably at least 35% by weight of the cereal.
• the cereal may be any one or more of wheat, maize, rye, barley, oats, triticale, rice, and sorghum. It is particularly preferred that the cereal is maize or wheat. If the cereal is maize, then the additive or agent preferably further comprises an ⁇ -amylase.
• the cereal component of a cereal-based diet constitutes a source of protein
• sources of supplementary protein such as those derived from fishmeal, meatmeal or vegetables.
• sources of supplementary protein may constitute up to 50% by weight of the animal feed.
• Sources of vegetable protein include at least one of full fat soybean, rapeseed, canola, soybean meal, rapeseed meal and canola meal.
• An inner salt of a quaternary amine carboxylic acid has the general structural formula R'R 2 R 3 N + -L-C00 ⁇ wherein the substituents R 1 , R 2 and R 3 independently represent any organic radicals, preferably alkyl groups, more preferably alkyl groups having 1-6 carbon atoms, and most preferably are all methyl: the linking group -L- represents any organic linking group such as alkoxyalkyl or alkylene, preferably C C 6 alkylene, and most preferably methylene.
• Examples of the inner salt of a quaternary amine carboxylic acid include betaine (Me 3 N + CH 2 COO ), MeaN ⁇ CHMeCOO " Et 3 N T CH 2 COO ⁇ Me 2 EfN T CH 2 COO ⁇
• the inner salt is preferably betaine which is commercially available from Finnfeeds under the trade name Betafin ® .
• the inner salt is preferably included in the feed at the rate of 0.01 - 20 g per kg of feed, more preferably 0.1 - 10 g/kg, and most preferably 0.5 to 2 g/kg.
• the protease used can be any form of protease, however the use of a subtilisin derived from the Bacillus subtilis with an activity of not less than 40.000 U/g is preferred. Such subtilisins are described for example in US 4.760.025 A.
• the protease is included in the feed in such an amount that the feed has a protease activity corresponding to 100 U to 100,000 U per kg of feed, preferably 500 U to 10,000 U per kg of feed.
• Suitable proteases include but are not limited to the following commercially available proteases: Novo NEUTRASE (TM) (commercially available from Novo Nordisk); PURAFECT (TM) (commercially available from Genencor International, Inc); SAVINASE (TM) (commercially available from Novo Nordisk); MAXACAL (TM) (commercially available from Gist-Brocades); DURAZYM (TM) (commercially available from Novo Nordisk); and MAXAPEM (TM) (commercially available from Gist Brocades).
• Novo NEUTRASE TM
• PURAFECT TM
• SAVINASE TM
• MAXACAL TM
• DURAZYM TM
• MAXAPEM MAXAPEM
• the agent or additive additionally comprises a xylanase.
• Xylanases can be derived from fungal sources such as Trichoderma, Aspergillus, Humicola, Neocallimastix or Thermomyces. It is preferred that the xylanase is the low pi xylanase and/or the high pi xylanase obtainable from Trichoderma longibrachiatum such as described in Example 22 of WO 92/06209. Alternatively, the xylanase can also be obtained from a bacterium such as Bacillus, Streptomyces, Microtetraspora, Clostridium or Ruminococcus.
• the xylanase may be obtained from a host which has been subjected to genetic manipulation such as by the inclusion of an appropriate gene within a host bacterial or fungal strain.
• the preferred activity of the xylanase should be about 3000 U/g.
• Xylanase from Trichoderma longibrachiatum with a minimum activity of 3000 U/g is commercially available from Finnfeeds International under the trade name AVIZYME ® .
• the xylanase is included in the feed in such an amount that the feed has a xylanase activity corresponding to 100 U to 100,000 U per kg of feed, preferably an activity of 200 U to 1000 U per kg of feed.
• ⁇ -amylase may also be present in the agent or additive, particularly in the case that the agent or additive is to be included in a maize-containing feed.
• ⁇ -amylase from Bacillus subtilis with a minimum activity of 4000 U/g.
• ⁇ -amylase from Bacillus subtilis with a minimum activity of 4000 U/g is commercially available from Finnfeeds International under the trade name AVIZYME* .
• the ⁇ -amylase is included in the feed in such an amount that the feed has an ⁇ -amylase activity corresponding to 10 U to 100,000 U per kg of feed, preferably an activity of 100 U to 4,000 U per kg of feed.
• the inner salt, protease, and optionally xylanase and/or ⁇ -amylase can be mixed together prior to use but can also be applied separately to the feed.
• a combination of protease, xylanase and ⁇ -amylase is commercially available under the trade name AVIZYME 1510 from Finnfeeds International.
• a further combination of these enzymes is commercially available as a dry premix under the trade name AVIZYME ® 1500. It should be noted that it is generally not advisable to store a protease together with other proteins for an extended period of time as it tends to degrade the latter.
• the feed additive of the present invention can be used for a wide variety of animals, but use of the invention is particularly preferred in domestic animals and farm livestock.
• Animals which may in particular benefit from the invention include poultry (such as chickens, turkeys, ducks and geese), ruminants (such as cattle, horses and sheep), swine (pigs), rodents (such as rabbits) and fish.
• poultry such as chickens, turkeys, ducks and geese
• ruminants such as cattle, horses and sheep
• rodents such as rabbits
• fish such as rabbits
• the invention is particularly useful in respect of broiler chickens.
• efficacious levels of the additive of the present invention may be administered to alleviate the adverse effects of any coccidiosis- inducing pathogen, and especially any Eimeria species including for example, the Eimeria species necatrix, galloparvonis, meleagrimitis, innocua, meleagridis, subrotunda, dispersa, truncata, acervulina, brunetti, maxima, mitis, praecox and tenella. More particularly, the additive promotes weight gain in an animal infected with Eimeria.
• efficacious levels of the additive of the present invention may be administered to alleviate the adverse effects in poultry due to infection by Eimeria species, especially, the Eimeria species E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella.
• efficacious levels of the additive of the present invention may be administered to alleviate the adverse effects induced in cattle when infected with a member of the Eimeria species, especially, the Eimeria species zuernii, bovis (smithii), ellipsoidlis.
• efficacious levels of the additive of the present invention may be administered to alleviate the adverse effects induced in sheep when infected with a member of the Eimeria species including the Eimeria species arloingi A (ovina), weybridgensis (ai ongis B), crandallis, ahsata, ovinoidalis, gilruthi.
• efficacious levels of the additive of the present invention may be administered to alleviate the adverse effects induced in goats when infected with a member of the Eimeria species including, for example, immunization with the Eimeria species arloingi, faurei, caproina, ninakohlyakimovae, christenseni.
• efficacious levels of the additive of the present invention may be administered to alleviate the adverse effects induced in pigs when infected with a member of the Eimeria species including, for example, immunization with the Eimeria species debliecki, scabra, penninuta; and, including adverse effects induced by a member of the Isospora species, for example, Isospora suis.
• efficacious levels of the additive of the present invention may be administered to alleviate the adverse effects in poultry due to infection by bacteria such as Clostridium, Salmonella, Campylobacter, E. coli, Listeria and especially Clostridium perfringens, Salmonella enteritidis and Campylobacter jejuni.
• bacteria such as Clostridium, Salmonella, Campylobacter, E. coli, Listeria and especially Clostridium perfringens, Salmonella enteritidis and Campylobacter jejuni.
• the nutritional additive can accordingly be used for the preparation of an agent for the treatment and/or prophylaxis of necrotic enteritis.
• Controlled delivery may be accomplished by selecting appropriate macromolecules such as polyesters, polyaminoacids, polypyrrolidone, ethylene vinylacetate, methylcellulose, carboxymethyl-cellulose or protamine sulfate and combining these according to well-established procedures in order to control release.
• the duration of action of the inner salt and protease may also be controlled by incorporating these agents into particles of polymeric materials such as polyesters, polyaminoacids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers.
• the inner salt and protease may be formulated in microcapsules.
• Various materials and methods for making and using microcapsules are disclosed in Remington's Pharmaceutical Sciences, (16th edition, A. Oslow, ed., Mack, Easton, Pa. 1980).
• the present invention further embodies the use of other compositions containing the inner salt and protease which may be co-administered with any desired composition, including any vaccines, nutrients or medicaments.
• Example illustrates the effects when a feed additive of the present invention is incorporated in the feed of healthy animals as well as for the manufacture of an agent for the treatment of Eimeria infection.
• Eimeria maxima challenge was made by inoculating E. acervulina and E. maxima oocysts to the crop of the chicks in a 2 ml volume of tap water.
• the dose per bird was 100,000 oocysts of E. acervulina and 50,000 oocysts of E. maxima.
• E. acervulina infects the duodenum and the early jejunum
• E. maxima prefers the mid-small intestine: from distal jejunum to mid ileum.
• the birds were kept in cages between days 0 - 14 and in open pens with a litter of wood shavings between days 14 - 21.
• the feed provided to the birds was corn-soy based with no coccidiostats.
• the food was cold-pressed into small friable pellets having a diameter of about 5mm.
• Starter feeds were used on days 0 - 14 and finisher feeds on days 14 - 21.
• the additives were sprayed on to feed pellets where appropriate by way of an aqueous solution of betaine (at a dose of lg per kg of feed) and an aqueous solution of AVIZYME ® 1510 which comprises about 300 U/g of xylanase, 4,000 U/g of protease and 400 U/g of ⁇ -amylase available from Finnfeeds International at a dose of 1 g/kg of feed.
• the nutritional composition of these feeds was as follows:
• Table 1 shows that the use of a combination of betaine and the protease results in an increased weight gain compared to when these compounds are added individually to the feed.
• Example 1 The activity of a combination of betaine and the protease used in Example 1 on Clostridium perfringens infection was investigated in this Example.
• the feed used was the Finisher Feed as described in Example 1.
• the intensive band in lane 3 of the gel shown in Figure 3 indicates the presence of the ⁇ -toxin gene of endogenous C. peifringens in the animal.
• the absence of such bands in lanes 2 and 4 shows that the Eimeria challenge is an essential prerequisite for C. perfifngens infection to take hold.
• the Eimeria challenge reduces the normal levels of protease in the intestine which then diminishes the nutrient uptake in the upper regions of the intestine and results in increased protein levels (and thus improved growth conditions for C. perfringens) in the ileum.
• a further aspect which might be of significance is the effect of the Eimeria challenge on the immune system of the host animal.

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• 1999
  • 1999-12-09 GB GB9929152A patent/GB2358135A/en not_active Withdrawn
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• 2000
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  • 2000-12-08 DK DK00987375T patent/DK1237569T3/da active
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• 2002
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• 2006
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