AU667807B2 - Spray method for delivering direct feed microorganisms to poultry - Google Patents
Spray method for delivering direct feed microorganisms to poultry Download PDFInfo
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- AU667807B2 AU667807B2 AU14113/92A AU1411392A AU667807B2 AU 667807 B2 AU667807 B2 AU 667807B2 AU 14113/92 A AU14113/92 A AU 14113/92A AU 1411392 A AU1411392 A AU 1411392A AU 667807 B2 AU667807 B2 AU 667807B2
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- 244000005700 microbiome Species 0.000 title claims description 21
- 238000000034 method Methods 0.000 title claims description 17
- 239000007921 spray Substances 0.000 title description 13
- 244000144977 poultry Species 0.000 title description 8
- 241000186604 Lactobacillus reuteri Species 0.000 claims description 20
- 241000271566 Aves Species 0.000 claims description 17
- 235000013601 eggs Nutrition 0.000 claims description 16
- 229940001882 lactobacillus reuteri Drugs 0.000 claims description 14
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 8
- 239000000725 suspension Substances 0.000 claims description 5
- 241000287828 Gallus gallus Species 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims 2
- 241000736262 Microbiota Species 0.000 claims 1
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- 230000001105 regulatory effect Effects 0.000 claims 1
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- 238000011282 treatment Methods 0.000 description 12
- 241000186660 Lactobacillus Species 0.000 description 10
- 241000894007 species Species 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 9
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 8
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- 230000012447 hatching Effects 0.000 description 6
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- YPFDHNVEDLHUCE-UHFFFAOYSA-N 1,3-propanediol Substances OCCCO YPFDHNVEDLHUCE-UHFFFAOYSA-N 0.000 description 1
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- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
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- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000012248 Bacillus toyonensis Species 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
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- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
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- 230000007140 dysbiosis Effects 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Birds (AREA)
- Animal Husbandry (AREA)
- Mycology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Physiology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Fodder In General (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
-I I I r OPI DATE AOJP DATE
INTERNA
27/08/92 01/10/92 APPLN. TD 14113 92 P PCT NUMBER PCT/USq2/00667 TREATY (PCT) ig I (51) International Patent Classification 5 (I1) International Publication Number: WO 92/12723 A61K 37/00 Al (43) International Publication Date: 6 August 1992 (06.08.92) (21) International Application Number: PCT/US92/00667 (74) Agent: BARBER, Lynn, Olive Olive, P.O. Box 2049, Durham, NC 27702-2049 (US).
(22) International Filing Date: 28 January 1992 (28.01.92) (81) Designated States: AT (European patent), AU, BE (Euro- Priority data: pean patent), BR, CA, CH (European patent), DE (Eu- 646,692 28 January 1991 (28.01.91) US ropean patent), DK (European patent), ES (European patent), FI, FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, (71) Applicant: BIOGAIA BIOLOGICS AB [SE/SE]; Kungs- KP, KR, LU (European patent), MC (European patent), gatan 53, S-111 22 Stockholm NL (European patent), NO, RU, SE (European patent).
Applicntand Inventor: CASAS-PEREZ, Ivan, A. [VE/ US]; 4916 North Hills Drive, Raleigh, NC 27612 Published With international search report.
(72) Inventor: EDENS, Frank, W. 326 Northclift Drive, Raleigh, NC 27609 (US).
(u .rK .rols c Un,^.L t Pa\\.UaN i 4* 1 S678 0 7 (54) Title: SPRAY METHOD FOR DELIVERING DIRECT FEED MICROORGANISMS TO POULTRY (57) Abstract A method of establishing direct feed microorganisms such as Lactobacillus reuteri in the gastrointestinal tract of avian organisms in which the eggs or newly hatched birds of the organism arn: sprayed with the direct feed microorganism.
-2-cri ;i 1 INTERNATIONAL SEARCH REPORT International Appliestion No. PCT/US92/00667 I IP A C III-A T r r*h AS A I r .IJ IL,_I WO 92/12723 PCT/US92/00667 "SPRAY METHOD FOR DELIVERING DIRECT FEED MICROORGANISMS TO POULTRY" FIELD OF INVENTION This invention relates to a new method for delivering viable microbial cells to animals in a spray form.
BACKGROUND INFORMATION The terms "probiotics" is attributed to Parker who defined them as "organisms and substances which contribute to intestinal balance" when used as dietary supplements. This publication and all other publications and patents cited herein are incorporated herein by reference. Later, Fuller (11) considered this definition to be too broad since, in addition to including cell cultures and microbial metabolites, it could encompass antibiotic preparations. More recently, a number of summaries have appeared in the literature describing the scientific basis for use of probiotics as intestinal inoculants for production animals (10, 26). It has been suggested that the term "probiotics" be replaced by the term "direct feed microorganisms," or DFM's The concept of adding viable, harmless lactic acid bacteria to the gastrointestinal tract as a dietary supplement was first appreciated by Metchnikoff (16) who viewed the consumption of yoghurt by Bulgarian peasants as conferring a long span of life. Some workers have claimed that the therapeutic value derived from ingestion of such fermented milk products is related to the viable bacteria present in these products (12, 27). Since Metchnikoff's early reports, several studies have shown the ability of lactobacilli, for example, to suppress coliform growth.
Feeding viable Lactobacillus acidophilus cells to young dairy calves was shown to reduce the incidence of diarrhoea and increase the numbers of lactobacilli and reduce coliform counts in feces These findings contrast with those of others who were unable to demonstrate benefits I from feeding either Lactobacillus acidophilus 13) or milk cultured with Lactobacillus acidophilus or i 4 11111 n~"IP1~Ps -~II-~i WO 92/12723 PCT/US92/00667 -2- Lactobacillus lactis (17).
In a detailed study by Muralidhara et.al. (18), piglets given a Lactobacillus lactis concentrate for up to 8 weeks after birth showed a progressive decline in coliform counts in fecal samples. Scouring in these animals was negligible, but was evident in control pigs especially at weaning. Underdahl et al. (32) observed only mild diarrhoea lasting 2-4 days in gnotobiotic pigs inoculated with Streptococcus faecium prior to artificial Escherichia coli infection. In the same study, persistent diarrhoea occurred in pigs similarly infected with Escherichia coli, but without prophylactic treatment with the Streptococcus microorganism.
Probiotics (hereafter referred to as DFM's) are bacterial or yeast preparations that are administered orally or added to feeds. The most commonly used DFM's are strains of the lactic acid bacteria (LAB), particularly those classified in the following genera: Lactobacillus, Lactococcus, and Enterococcus. Included among these are the following species: Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus lactis, Lactococcus lactis, Lactococcus thermophilus, Lactococcus diacetylactis, and Enterococcus faecium. Besides these LAB, some species of Bacillus (Bacillus subtilis, Bacillus toyoi) and yeasts and molds (Saccharomyces cerevisiae, Aspergillus oryzae, and Torulopsis sp.) are used as DFM's It is generally held that during periods of low resistance, such as stress, undesirable microorganisms are able to proliferate in the GI tract of animals, humans included. Maintaining a normal, healthy balance of microorganisms is deemed to be critical during such stressful periods The concept underlying use of DFM's, therefore is that if sufficient numbers of an appropriate microorganism(s) are introduced into the '^rrr WO 92/12723 PCT/US92/00667 -3intestinal tract at times of stress and/or disease, i (ii) at birth, or (iii) after antibiotic treatment (when Iminimal LAB are present), the negative consequences of the microbial imbalances can be minimized or overcome. Using such preparations of live, naturally occurring microorganisms helps restore and maintain the proper i balance of beneficial microbes in the GI tract during times of stress, disease, and following antibiotic therapy I This concept, descriptions of proposed modes of action, and evidence for the efficacious uses of DFM's for all production animals are summarized in reviews by Fox Sissons and by various authors (22).
One of the major problems or limitations encountered in commercial scale application of DFM's to animals is (i) the availability of suitable delivery systems, and (ii) the ability to get the probiotic preparations to the animals as quickly as possible after birth. This is particularly true when pelletized feeds are used, as is the case in the poultry industry. The pelletization process generally includes one or more heating steps involving temperaturez high enough to pasteurize or sterilize the feed components, thereby precluding incorporation of viable microorganisms into these feeds prior to pelletization.
The present invention describes novel methods and processes for overcoming some of these problems, by delivering viable DFM's in spray form. The DFM used to develop these methods is Lactobacillus reuteri. This species was chosen because it has demonstrated efficacy as a DFM in poultry Previous patent applications have been submitted relating to unique properties of the species. These applications are: PCT/US88/01423, filed April 28, 1988 and published November 3, 1988, claiming priority from U.S. Serial No. 07/268,361 filed September 19, 1988 which is a continuation-in-part of U.S. S.N.
07/102,830 filed September 22, 1987 which is a continuation-in-part of U.S. S.N. 07/046,027 filed May 1, 3UI^I I WO 92/12723 PCT/US92/00667 I -4i 1987; and U.S. S.N. 07/539,014 filed June 15, 1990. The Sdisclosure of these applications is incorporated herein by i reference.
Lactobacillus reuteri is a species of lactic acid bacteria recognized since the turn of the century (19).
Originally assigned different species names Lactobacillus fermentum biotype II), it obtained distinct species status in 1980 and is registered in the 1988 edition of Bergey's manual (14, 15). It is found in foods, particularly dairy products and meats, but exists primarily in the GI tract of healthy animals, including humans 6, 7, 14, 15, 23, 24, 25, 33).
Lactobacillus reuteri is the dominant heterofermentative Lactobacillus inhabiting the GI tract (23, 24, 25). It is a typical heterofermenter, converting sugars into acetic acid, ethanol, and CO 2 in addition to lactic acid which is the major endproduct of homofermentative metabolism carried out by species such as Lactobacillus acidophilus It utilizes the phosphoketolase pathway for conversion of glucose to endproducts. When glycerol, an alternate hydrogen acceptor, is present in the culture medium together with glucose or other utilizable carbon and energy sources lactose), acetate rather than ethanol accumulates, and the glycerol is reduced to 1,3-propanediol via the metabolic intermediate, 3-hydroxypropionaldehyde (3-HPA).
3-HPA has been shown to have potent antimicrobial activity, and Lactobacillus reuteri appears to be unique among microorganisms examined to date in its ability to secrete this substance, termed reuterin, into the surrounding medium 5, 7, 28, 29, 30, 31). This unique antimicrobial a-tivity may play a role in competitive survival of this species in the gastrointestinal ecosystem, and/or its ability to regulate growth and activities of other microorganisms in this ecosystem It is thus very important to establish this microorganism early in WO 92/12723 PCT/US92/00667 animals. It is therefore an object of the invention to provide a method for delivering DFM's, such as Lactobacillus, to avian species. Other objects and advantages will be more fully apparent from the following disclosure and appended claims.
SUMMARY OF INVENTION Just before hatching and on or about the expected day of hatch, poultry eggs, or hatching or hatched birds are sprayed with a suspension of a DFM, such as Lactobacillus I 10 reuteri.
II Other aspects and features of the invention will be Smore fully apparent from the following disclosure and i appended claims.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF The present invention provides a method of delivering DFM's to avian embryos or newly hatched birds so that the DFM's are established in the gastrointestinal tract of the I newly hatched birds.
Cells of the selected DFM, for example, L. reuteri, are suspended in an aqueous medium such as buffered saline I 6 at a level of about 106/ml. The suspension is placed in any type of sprayer, for example, a hand-held atomizer or spray-bottle. The eggs are sprayed at a level of about ml suspension per 300 eggs on the day before hatch. The spray treatment is repeated the day of hatch. In the second spray treatment some of the eggs will be hatching or will have hatched. In this case, the spray treatment is of the newly hatched birds.
The features and advantages of the present invention will be more clearly understood by reference to the following example, which is not to be construed as limiting the invention.
I--i WO 92/12723 PcF-/US92/00667 -6-
EXAMPLE
Eggs of turkeys (Nicholas) are incubated at standard incubation conditions for turkeys 27 days (until one day before the eggs are due to hatch). A mixture of equal amounts of two Lactobacillus reuteri strains, strain T-1 (isolated from turkeys) and strain 11284 (isolated from chickens) is prepared in a buffered saline solution. Each of these strains has been deposited at the American Type Culture Collection in Rockville, Maryland. The saline solution may be at any concentration of solutes and contain any components which are normally used for suspending living microorganisms. With a hand-held sprayer, about ml per 300 eggs is sprayed on to the eggs at day 27 (the day prior to hatch). On the next day, the eggs, and any hatching or hatched birds, are again sprayed (50 ml/300 eggs or birds). Three to five hours later the birds are taken from the hatchery to a pen.
About 5-10 hours post-hatch, the total number of lactobacilli found in the bird's cecum is determined for each treatment as colony-forming units per excised and homogenized cecum. Solid Lactobacillus selection medium agar) as described in references 2, 5, and 7 is used.
The percent of the colonies which is L. reuteri is determined as described in international patent application PCT/US88/01423 but using L. plantarum as the indicator organism. In this test, colonies of lactobacilli on the LBS agar medium are overlaid with 10 ml of 1% liquified agar containing 0.5 M glycerol and a L. plantarum inoculum.
After anaerobic (Gas-Pack System) incubation at 37 0 C for 24 hours, zones of growth inhibition are seen around colonies that produce reuterin from glycerol. These colonies are thus identified and enumerated as L. reuteri.
As seen in Table 1, the spray treatment does not affect survival or hatchability.
WO 92/12723 PCT/US92/00667 -7- Ji Table 1.
i Treatment live survivors embryos at at day 7 Untreated embryos 96 81 L. reuteri sprayed (106 CFU) 97 79 As seen in Table 2, colonization of the ceca by L.
reuteri at hatch is increased by the spray treatments.
Table 2.
Yreatment CFU per cecum Untreated embryos 3.3 x 102 L. reuteri sprayed 7.4 x 105 Although not shown in the table, such colonization increased in the same manner with birds in which the second spray treatment had been of the egg and those in which the second treatment had been of the newly hatched bird.
Similar results are obtained with chickens treated by the method of the invention.
Although the preferred method of the invention combines two spray treatments as described, either spray treatment may be used separately to provide somewhat lower levels of colonization (results not shown).
While the invention has been described with reference to specific embodiments thereof, it will be appreciated that numerous variations, modifications, and embodiments are possible, and accordingly all such variations, modifications, and embodiments are to be regarded as being within the spirit and scope of the invention.
BEST MODE FOR CARRYING OUT THE INVENTION Just before hatching and on or about the expected day of hatch, poultry eggs, or hatching or hatched birds are
II
WO 92/12723 PCT/US92/00667 -8sprayed with a suspension of a DFM, saline, such as Lactobacillus reuteri, preferably suspended in buffered saline.
INDUSTRIAL APPLICABILITY Poultry hatched from eggs sprayed with Lactobacillus reuteri or birds sprayed with these microorganisms have increased resistance to microbial pathogens and gain weight faster, thus resulting in savings to the growers and consumers.
WO 92/12723 PCT/US92/00667 -9-
REFERENCES
I 1. Axelsson L, Lindgren SE. 1987. Characterization and DNA homology of Lactobacillus reuteri strains isolated from pig intestine. J. Appl. Bacteriol., 62:433-440.
2. Axelsson L, Chung TC, Dobrogosz WJ, Lindgren SE.
1989. Production of a broad spectrum antimicrobial substance by Lactobacillus reuteri. Microbial Ecol. Health iDis., 2:131-136.
3. Becihman TL, Chambers JV, Cunningham MD. 1977.
Influence of Lactobacillus acidophilus on performance of i young diary calves. J. Dairy Sci., 60:74(abs).
4. Bruce BB, Gilliland SE, Bush LJ, Staliy TE. 1979.
Influence of feeding ce ls of Lactobacillus acidophilus on the fecal flora of young calves. Oklahoma Anim. Sci. Res.
Rep., 207.
Chung TC, Axelsson L, Lindgren Se, Dobrogosz WJ.
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Claims (4)
1. A method of establishing a direct feed microorganism in the gastrointestinal tract of an avian organism, comprising spraying the organism with a suspension of the direct feed microorganism on the day of hatch, wherein the form of said organism when sprayed may be as an embryo within an egg or as a newly hatched organism.
2. A method according to claim 1 wherein the microorganism is Lactobacillus reuteri.
3. A method according to claim 2 wherein the avian organism is a chicken.
4. A method according to claim 2 wherein the avian organism is a turkey. A method according to claim 1 further comprising spraying eggs of the organism with the direct feed microorganism one day prior to hatch. INTERNATIONAL SEARCH REPORT International App!ict-ion No. PCT/US92/00667 I. CLASSIFICATION OF SUBJECT MATTER (if several classifiration symbols apply, indicate all) 3 According to International Patent Classification (IPC) or to both National Classification and IPC :PC A61K 37/00 US CL 424/93R, 93D, 93J, 93C II I lI rll cEADI fU n I Minimum Documentation Searched' Classification System Classification Symbols U.S. 424/93R, 93D, 93J, 93C Documentation Searched other than Minimum Documentation to the extent that such Documents are included in the Fields Searched III. DOCUMENTS CONSIDERED TO BE RELEVANT"1 Category' Citation of Document, 16 with indication, where appropriate, of the relevant passages 17 Relevant to Claim No. Ia y Regulatory and Protective Role of the Normal Microflora, issued 1988, Dobrogosz et al, "Lactobacillus reuteri and the Enteric Microbiota", pages 283-292, see entire document. y US, A, 4,335,107 (Snoeyenbos et al) 15 June 1982, see entire document. y US, A, 4,518,696 (Gehrman et al) 21 May 1985, see entire document. y US, A, 4,689,226 (Nurmi et al) 25 August 1987, see 1-9 entire document. 'Special categones of cited documents: 1 later document published after the international filing document defining the general state of the art which is date or priority date and not in conflict with the not considered to be of particular relevance application but cited to understand the principle or earlier document but published on or after the theory underlying the invention international filing date document of particular relevance: the claimed document which may throw doubts on prionry claim(s) invention cannot be considered novel or cannot i. or which is cited to establish the publication date of considered to involve an inventive step another citation or other special reason las specified) document of particular relevance: the claimed document refernng to an oral disclosure, use, exhibition invention cannot be considered to is volve an or other means inventive step when the document is combined with m one or more other such documents, such combindtion "P document published prior to the international filing date being obvious to a person skilled in the art but later than the pnonty date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search 2 Date of Mailing of this International Search Report 02 APRIL 1992 1 1 MAY 199 International Searching Authority' inature of Authorized Officq SA/US -Susan M. Weber Form PCT/ISA/210 (second sheet(May 1986) B
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US64669291A | 1991-01-28 | 1991-01-28 | |
US646692 | 1991-01-28 | ||
PCT/US1992/000667 WO1992012723A1 (en) | 1991-01-28 | 1992-01-28 | Spray method for delivering direct feed microorganisms to poultry |
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AU1411392A AU1411392A (en) | 1992-08-27 |
AU667807B2 true AU667807B2 (en) | 1996-04-18 |
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AU14113/92A Expired - Fee Related AU667807B2 (en) | 1991-01-28 | 1992-01-28 | Spray method for delivering direct feed microorganisms to poultry |
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AU (1) | AU667807B2 (en) |
CA (1) | CA2100866C (en) |
FI (1) | FI933361A0 (en) |
MX (1) | MX9200355A (en) |
WO (1) | WO1992012723A1 (en) |
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JP3028214B2 (en) * | 1997-06-03 | 2000-04-04 | カルピス株式会社 | How to administer live birds |
GB9803424D0 (en) * | 1998-02-18 | 1998-04-15 | Pfizer Ltd | Performance enhancement |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4335107A (en) * | 1978-06-05 | 1982-06-15 | Snoeyenbos Glenn H | Mixture to protect poultry from salmonella |
FI59925C (en) * | 1980-01-11 | 1981-11-10 | Esko Viljo Nurmi | FOERFARANDE FOER FRAMSTAELLNING AV ETT BAKTERIEPREPARAT ANVAENDBART FOER FOERHINDRANDE AV SALMONELLAINFEKTION HOS FJAEDERFAE |
US4518696A (en) * | 1983-01-11 | 1985-05-21 | Chr. Hansen's Laboratory, Inc. | Stabilized liquid bacterial suspension for oral administration to animals |
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1992
- 1992-01-28 MX MX9200355A patent/MX9200355A/en unknown
- 1992-01-28 AU AU14113/92A patent/AU667807B2/en not_active Expired - Fee Related
- 1992-01-28 CA CA002100866A patent/CA2100866C/en not_active Expired - Fee Related
- 1992-01-28 WO PCT/US1992/000667 patent/WO1992012723A1/en not_active Application Discontinuation
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1993
- 1993-07-27 FI FI933361A patent/FI933361A0/en not_active Application Discontinuation
Non-Patent Citations (3)
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FI933361A0 (en) | 1993-07-27 |
WO1992012723A1 (en) | 1992-08-06 |
CA2100866C (en) | 2002-11-19 |
CA2100866A1 (en) | 1992-08-06 |
MX9200355A (en) | 1994-03-31 |
AU1411392A (en) | 1992-08-27 |
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