BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to stimulation of the immune system of animals, and in particular, relates to a method of stimulating the immune system by using a probiotic microorganism.

Description of the Related Art

A number of inventions have been forthcoming from research and development on what is known as the Lactobacillus reuteri/reuterin system. L. reuteri is a species of obligately heterofermentative Lactobacillus found in the gastrointestinal (GI) tract of healthy humans, cattle, swine, poultry, rodents and all other animals examined to date, and it is reported to be the predominant hetero ermentative Lactobacillus species in these ecosystems [ 1-7] .

PCT application No. PCT/US88/01423 discloses the discovery that L. reuteri is unique among lactobacilli and bacteria in general in its ability to produce and secrete a substance shown to have potent antimicrobial activity [2,8,9]. The disclosure of this PCT patent application and all other applications, patents and publications cited herein is incorporated by reference. This substance, termed reuterin, was isolated, purified, and chemically identified using (a) Nuclear Magnetic Resonance Spectroscopy (NMR) , (b) Mass Spectroscopy (MS), (c) Infra¬ red Spectroscopy (IR), and (d) by chemical synthesis. Using these methods, reuterin was proven to be an equilibrium mixture of monomeric, hydrated monomeric and cyclic dimeric forms of 3-hydroxypropionaldehyde (3-HPA) [10-13]. Concentrations of reuterin as low as 15 to 30 μg ml ^-1 were demonstrated to inhibit growth of Gram negative and most Gram positive bacteria, yeasts, fungi, and protozoa, whereas concentrations 4 to 5 times higher are needed to kill lactic acid bacteria, including L. reuteri

itself [8,9]. This L. reuteri/reuterin system is believed to play a unique and important role in regulating and/or influencing the complex microbial ecosystem that exists in the human and animal GI tract, thereby having a beneficial influence on human and animal health.

PCT application No. PCT/US92/00708 discloses that examples of oral administration of viable, host-specific L. reuteri cells to chickens and turkeys significantly reduces the numbers of Salmonella and other undesirable microorganisms that reside in their GI tract.

This international patent application also discloses novel, commercial-scale methods for delivery and administration of viable, host-specific strains of L. reuteri to the GI tract of animals. Chicks and poults hatched and brooded commercially are unable to benefit from the maternal transfer of beneficial enteric microorganisms, such as L. reuteri. that occurs under "barn yard" conditions where their dams and other members of the flock provide all the L. reuteri cells needed to inoculate the newly hatched bird\'s GI tract immediately after hatching. This natural inoculation transfer process, now precluded in modern commercial practice, can be restored using the new technology disclosed herein. Commercial applications of this new invention will yield greater efficiency and increased productivity in the food animal industries. On the one hand, dramatic improvements in efficient production of food animals and their products have been witnessed in the past few decades. This is most evident in poultry production where the development of high-performance breeds of chickens and turkeys, improved dietary formulations, and hygienic management practices have resulted in an increasingly productive, efficient, and cost-effective industry. On the other hand, some of these very practices have yielded counter-productive consequences. For example, cost- ef ectiveness in the industry requires the placement and

grow-out of birds at such high population densities that the animals are subjected to an assortment of environmental stressors that reduce productivity. Overcrowding, servicing, holding, transporting, sub- optimal temperatures, dust, dampness, and many other stressors produce an alarm reaction that can retard an animals\' growth or initiate an actual weight loss [14-26]. It is known that exposure of neonate chicks and poult (and other animals) to these and other types of environmental and/or pathogenic stressors causes significant production losses. Young birds are particularly sensitive to these stressors, and productivity losses incurred during their neonatal period persist into adulthood [27]. Furthermore, under these conditions, overt and opportunistic pathogenic microorganisms often proliferate, retarding growth even further, increasing the likelihood of mortality. In addition, it is known that these adverse conditions contribute to consumer products highly contaminated with Salmonella, Campylobacter, and other microorganisms which are a threat to public health [28].

It is therefore an object of this invention to provide a beneficial effect on the immune system of poultry, particularly poultry under stress.

Other objects and advantages will be more fully apparent from the following disclosure and appended claims.

SUMMARY OF THE INVENTION Production losses in poultry can be reduced by altering the neonate birds\' gastrointestinal icroflora/itiicrobiota by oral administrations of viable, host-specific strains of Lactobacillus reuteri. Described herein is a new method of using Lactobacillus reuteri/reuterin to improve production efficiency in the poultry industry. This method has been tested under both laboratory and commercial, field-test conditions and found to reduce stress-associated morbidities and mortalities in both chickens and turkeys. Approximately 10 ⁵ viable L.

reuteri cells vectored on whey-based particles are mixed with pelletized commercial feed just prior to consumption by the animals. This treatment ensures colonization of the bird\'s GI tract with L. reuteri. This treatment, initiated at hatching and continued thereafter for at least 6 weeks, has been shown in laboratory and large- scale, commercial field trials to consistently (a) stimulate increased body weight growth by approximately 5 to 10% in comparison to untreated animals, (b) increase feed conversion efficiency, and (c) decrease neonate mortalities (caused by Salmonella tvphimurium challenges or spontaneously by arizonosis or unknown et.iological agents) by 50 to 75% in comparison to untreated flocks. Specific examples of these benefits are cited below. Other aspects and features of the invention will be more fully apparent from the following disclosure and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows two regression analysis plots of the average ileal villi height vs. chicken weight, with the upper plot being for control chickens and the lower plot being the Lactobacillus reuteri-treated chickens.

Figure 2 is a bar-graph showing the effect of Lactobacillus reuteri treatment on anti-Salmonella IgM antibody response in 21 day old chickens (C = controls; L.r = Lactobacillus reuteri) orally challenged at day 1 with 10 ⁵ CFU Salmonella typhimurium.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF The present invention provides a method of stimulating the immune system of animals such a poultry. The method comprises providing the animal with sufficient viable Lactobacillus reuteri cells over a sufficient time period to provide a beneficial effect on the immune system of poultry.

In particular, the invention is a method of providing a beneficial effect on the immune system of poultry,

comprising providing Lactobacillus reuteri to said poultry beginning close to hatch in a manner effective to moderate T-cell activity, to increase splenic natural killer cell activity in stressed poultry, and to increase anti- Salmonella IgM antibody titer of previously Salmonella challenged poultry.

The Lactobacillus reuteri is preferably given to the poultry orally as part of the feed or a feed additive or in the water. The amount of Lactobacillus reuteri used is preferably about 10 ⁴ to about 10 ⁷ CFU (most preferably about 10 ⁵ ) per g of feed or water as the medium of delivery to the poultry. Preferably the administration of the L_ _j _ reuteri microorganisms begins at least on the first day after hatch. In a preferred embodiment, administration is before hatch, according to PCT application No. PCT/US92/00654. Administration may also be in the form of a spray treatment with a liquid suspension being the medium of delivery of the microorganisms, according to PCT application No.

Salmonella challenge normally occurs naturally in a poultry houses where previous occupants were in ected with Salmonella.

The features and advantages of the present invention will be more clearly understood by reference to the following examples, which are not to be construed as limiting the invention.

EXAMPLES The ability of orally administered, viable L. reuteri cells to ameliorate morbidity and mortality losses can be demonstrated in both chickens and turkeys. However, because many of the key immunological reagents (e.g. , appropriate monoclonal antibodies, etc. ) that are needed to characterize immunocytes are available only for chickens, the following experimental test system was developed using newly hatched chickens rather than turkeys. Mixed-sex Arbor Acre broiler chicks obtained

within 6 hr post-hatch are placed in custom-designed floor brooders or Petersime batteries, as appropriate, equipped with heaters, pen dividers, and deck lights. Feed, water, and light are provided continuously for a 21 day experimental period. The birds are allowed to consume ad libitum a commercial type broiler diet containing 21% protein and 3100 kcal kg ^-1 . No antibiotics are included in the diets. The following treatments are used:

Normal Brooding, Control birds: Birds are placed in pens held at 90 to 95°C for 8 days, at 85°C for the next 6 days and then at 80°C thereafter.

Stressed (Cold) birds: Pen heaters are turned off for the first hr after placement to decrease the temperature to 65°C, and then turned on for 2 hr for the temperature to again reach 90°C. This cycle is repeated for the first 2 days with normal brooding resumed thereafter.

Stressed (Cold + Salmonella-challenged) birds: The birds are subjected to the same cold stress as above, but are also administered 10 ⁴ to 10 ⁶ CFU S. typhimuriu via gavage into the crop. A nalidixic acid-resistant (Nal ^r ), novobiocin resistant (Nov ^r ) strain of £5. typhimurium is used.

L. reuteri-treated birds in every case are identical to the corresponding control animals except that their feed is supplemental with 0.5% whey pellets coated with viable L. reuteri cells to yield a final concentration of 5 x 10 ⁵ CFU g ^-1 feed.

Experimental groups generally consists of 6 pens with 16 birds per pen. A total of 24 pens are used allowing for 4 treatment-sets per experiment. Mortalities are monitored daily, and 60 birds from each treatment group are selected randomly for body weight determinations at 0, 7, 14, and 21 days of age. A minimum of 6 to 8 birds from each treatment group is used, after sacrifice by cervical dislocation, for each experimental determination.

Example 1 Oral L. reuteri administration stimulates development of deeper crypts and longer villi in the ileum of 3-day old chicks. This was concluded on the basis of the standard morphometric analyses conducted by Dr. B. Black (Dept. Zoology, NC State Univ., Raleigh, NC) . The results of these analyses are presented in Table 1. Table 1

¹ Units are μmj all values are the average + S.E.M. measurements from the indicated number of samples

² Value is significantly different from control (p <0.025)

The analyses were conducted on 8 control (however, only 7 samples were available for the duodenum controls) and 8 L. reuteri-treated chicks. Intestinal samples were removed from each chick, cut into segments of 3-5 mm, split open, fixed in Carnoy\'s fixative, and store in 70% alcohol. The samples were subsequently dehydrated, cleared in xylene, and embedded in paraffin. Serial sections 5 μm thick were made by cutting cross sections of

the embedded tissue, perpendicular to the lumenal surface. Sections were stained with Feulgen reaction and counterstained with 0.05% fast green. Morphometric measurements were conducted using an AO microscope equipped with an eyepiece micrometer scale. The results summarized in Table 1 show that there are no differences in mucosal height, villus height, epithelial height or crypt depth in tissues obtained from the duodenal and jejunal regions of 3-day old control or L. reuteri-treated chicks. In the ileal region, however, significant differences are observed between the control and the L. reuteri-treated birds, The L. reuteri-treated chicks have on the average approximately 12% longer villi and 20% deeper crypts (p <0.025) in the ileal region of the GI tract. A regression analyses of these data (Figure 1) indicates a high correlation (R ² value =0.697) between chick weight and ileal villus height in the L. reuteri- treated birds. In the mouse, this stimulation of ileal villus and crypt tissue development is quantitatively related to mucosal T-cell mediated immune reactions [29]. Thus, there is a clear indication that L. reuteri cells colonizing the avian gut are able to modulate T-cell activities in these animals.

Additional confirmation that L. reuteri colonization of the chicken gut stimulates T-cell activity is obtained from histochemical analyses of frozen tissue sections obtained from control and L. reuteri-treated chicks. These studies, supervised by Dr. D. Weinstock (College of Veterinary Medicine, NC State University, Raleigh, NC) , involve indirect immunoperoxidase staining of these frozen sections using avian monoclonal antibodies to lymphocyte subsets as described by Lillehoj and Weinstock [34]. Studies indicate higher numbers of mammalian homologies of CD-4 antigen-bearing T-cells in the ileum lamina propria tissue of L. reuteri-treated chicks than in control chicks.

Example 2 Additional evidence that oral administration of L. reuteri influences the T-cell immune response in these animals is presented in Table 2. Table 2

Treatments Age PHA response % of (days) ( m) Control

Experiment 1 (Wattle)

Stressed-brooding -C* 20 34.8+2.93

Stressed-brooding +L.r. .20 22.6+2.93 ² -35% Experiment 2 (Toe Web)

Stressed-brooding -C* 5 7.9+1.19 Stressed-brooding +L.r. 5 3.5+1.19 ² -56%

Normal-brooding -C* 5 8.0+1.19

Normal-brooding +L.r. 5 3.1+1.19 ² -61% Experiment 3 (Wattle)

Stressed-brooding -C* 20 25.7+3.98 Stressed-brooding +L.r. 20 13.9±3.98 ² -46%

Normal-brooding -C* 20 32.1+3.98 ^\'

Normal-brooding +L.r. 20 14.4+3.98 ² -55%

C*= ^* controls; L.r.=~Lactobacillus reuteri treated ² =P <0.01; six to eight birds were analyzed in each treatment group

A standard delayed-type hypersensitivity (DTH) test was conducted on control (C) vs. L. reuteri-treated (L.r. ) chicks at 5 and 20 days of age. Wattles or toe webs, as indicated, were injected with physiological saline (controls) or 0.1 ml of a 1 mg ml ^-1 solution of phytohemagglutinin, a T-cell mitogen. These test were conducted using standard methods as described by Brown- Borg et al. [30]. The data obtained show that the L. reuteri-treated birds exhibit a very significantly depressed response to this mitogen. Thus, while the L.

reuteri treatment stimulates a localized T-cell response in the ileal region of the GI tract, it suppresses a systemic DTH T-cell response. The mechanism(s) underlying this interesting differential effect on T-cell responses is not understood at this time. Clearly however, it is an important clue as to how L. reuteri cells are able to exert their beneficial effects on the host animals.

Example 3 The data presented in Table 3 indicate that stressed (cold plus S. typhimurium) chicks are less able than their non-stressed counterparts to recruit peritoneal macrophages. Table 3

Treatments Average No. (x 10 ⁶ )

Macrophage recruited

Normal brooding-controls 49.6

Stressed brooding-controls 14.8 Stressed brooding-L. reuteri- treated 26,8

Chicks were injected with Sephadex on day 14, sacrificed and assayed for total peritoneal macrophages present on day 16. Four chicks were analyzed per treatment.

It can also be seen from these data that if the birds are stressed, the L.-reuteri-treated chicks are better able to recruit these macrophages than their non-treated counterparts. Here again may reside a clue as to how L. reuteri colonization of the gut can confer the observed benefits to the host animal. These macrophage assays were carried-out under the supervision of Dr. M. Quershi (Dept. Poultry Science, NC State University, Raleigh, NC) using standard methods published by Quershi and co-workers [31,32].

Example 4 The spleens of 8 day old control and L. reuteri- treated chicks were analyzed for natural killer (NK) cell activity using the methods described by Lillejoh and Chai [33]. The data presented in Table 4 show that L. .reuteri- treated chickens have higher splenic NK cell activity than their non-treated counterparts. Table 4

Treatments Average % of Splenic

NK Activity

Normal brooding-controls 0.5

Normal brooding-L.reuteri 0.7 Stressed brooding-controls 1.3

Stressed brooding-L. reuteri 6.2

Spleens were removed from 8-day old chicks and assayed for NK activity. Six birds were analyzed per treatment.

Here again, there are indications that the efficacy of the L. reuteri treatment may be greater under stress- brooding conditions than under normal-brooding conditions. These findings are consistent with the mortality and morbidity studies described above which show that L. reuteri administrations are most effective when applied to animals reared under stressful conditions.

Example 5

Evidence that L. reuteri administrations also influence the humoral immune response in chickens has been obtained. The data summarized in Figure 2 were obtained using chicks that had been subjected to combined cold and

Salmonella (day 1 oral inoculation) stress (see above description of model system) . Serum samples obtained from

21 day old control and L. reuteri-treated chicks (L.r) have higher titers of anti-Salmonella IgM antibodies than

control (C) chicks. Anti-Salmonella IgG antibody titers are the same for both groups of chicks (data not shown) .

BEST MODE FOR CARRYING OUT THE INVENTION

Preferably the method of the invention of providing a beneficial effect on the immune system of poultry, comprises providing Lactobacillus reuteri to the poultry beginning close to hatch in a manner effective to moderate

T-cell activity, to increase splenic natural killer cell activity in stressed poultry, and to increase anti- Salmonella IgM antibody titer of previously Salmonella challenged poultry. Preferably the L. reuteri is provided for a period of at least three weeks, and most preferably for six weeks, and at a level of about 10 ⁴ to 10 ⁷ microorganisms per gram of a delivery medium. INDUSTRIAL APPLICABILITY

Use of the method of the invention provides a method of stimulating the immune system of the poultry and thus enhancing the poultry\'s resistance to pathogens. In particular, the invention provides a means for the poultry industry to decrease the detrimental effects on poultry survival and weight gain due to Salmonella infection, and to decrease the public health risk caused by the unchecked spread of Salmonella.

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.

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