"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 (20) 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 (9).
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 (3) , and increase the numbers of lactobacilli and reduce coliform counts in feces (4). These findings contrast with those of others who were unable to demonstrate benefits from feeding either Lactobacillus acidophilus (8, 13) or milk cultured with Lactobacillus acidophilus or
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
(10).
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 (10). The concept underlying use of DFM's, therefore is that if sufficient numbers of an appropriate microorganism(s) are introduced into the
intestinal tract (i) at times of stress and/or disease, (ii) at birth, or (iii) after antibiotic treatment (when minimal 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 balance of beneficial microbes in the GI tract during times of stress, disease, and following antibiotic therapy (10). 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 (10), Sissons (26), 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 temperatures 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 (21). 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,
1987; and U.S. S.N. 07/539,014 filed June 15, 1990. The disclosure of these applications is incorporated herein by reference.
Lactobacillus reuteri is a species of lactic acid bacteria recognized since the turn of the century (19). Originally assigned different species names (e.g., 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 (1, 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 C02 in addition to lactic acid which is the major endproduct of homofermentative metabolism carried out by species such as Lactobacillus acidophilus (31). 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 (e.g., 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 (2, 5, 7, 28, 29, 30, 31). This unique antimicrobial activity 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 (7). It is thus very important to establish this microorganism early in
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 reuteri.
Other aspects and features of the invention will be more fully apparent from the following disclosure and 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 newly hatched birds. Cells of the selected DFM, for example, L. reuteri, are suspended in an aqueous medium such as buffered saline at a level of about loVml. 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 50 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.
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-l (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 50 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 (1.5% agar) as described in references 2, 5, and 7 is used. The percent of the colonies which is . 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 37CC 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.
Table 1 .
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. Treatment 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
sprayed 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.
REFERENCES
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