CA2093131A1 - Manipulation of intestinal structure and enzymes in animals - Google Patents

Abstract

A method of decreasing the reduction in digestive enzymes and altering mucosal structure in the intestine of animals is described wherein a probiotic is applied to the animals to achieve such results where the animal may benefit from such a change. Further, a method for maintaining such a benefit after weaning is demonstrated comprising in administering the probiotic to the animal prior to weaning.

Description

BACKGROUND OF THE INVENTION
Attempts have increased over the years to try to manipulate the intestinal flora of animals. It has been found that where one appropriately manipulates the intestinal microflora, there can be a positive ef~ect upon growth rate cf the animal. One of the means cf affect n~ the mlcroflora is through the use of antimicrobial agents, and while use of sucn agents in feed of animals has been well documented, the ef-ec-of changes in the microbial population of the small and larse inte~tine are not well understood.
One antimicrobial compound which has been used includes dietary copper which has been shown to be e^ ective in improving growth rates in pigs. 3rau~e, R., "Copper s 2 Growth Stimulant ln Pigs.", Ani. 21 _ -3d``CI ' ^n 3:69-7~ 'la6- .
Antibiotics have aiso been used in orde. to eiiminate pathogenic flora within the digsstive tract~ However, t;~e agriculture industry has attemoted to move away from use o-antibiotics since their ultimate effect upon humans is not well established. A widely spreading perception of the pub` ic is that antibio.tic additives in livestock are undesirable 2n~
efforts have turned away from such use and towards other m-_ns of favorably effecting the intestinal microflora.
With both consumer and manufacturer concerned about the side effects of the use of antibiotics as therapeutic agents, probiotics have received more a:tention in filling this role.
A probiotic is generally defined as a live microbial feed supplement which beneficially effects the host by improvinc its intestinal microbial balance. See, Fuller, R.
"Probiotics in Man and Animals.", J . ADD1. Bacteriol. 66:35~-378 (1989).
A major problem with probiotics is the lack of information on how such substances function. Beneficial affect upon growth is influence~ by a wide variety of preparations used, their interac:ion witn other growth-promoting substances in the die~ and poor definition of the organisms tested~ Therefore, ~:^i e th-re is data to i ~ ~,', ' ; 1 W() 93/()1)()1~ ~ U ~ J ~ 92//)55~

suggest that such preparations can be effective, the extent to which they influence animal growth in production and the mechanisms of their actions are still unclear. An understanding of this process would pro~ide a rational bas s for the level and frequency of dosing required to optimize beneficial effects. r~ith such knowledge changes in requirement and response to probiotic treatment might be anticipated with the stage of growth and development of the gastrointestinal tract and in ar.lmals subject to physiolog c21 or environmental stress which alter the microbial balance o the gut.
In many animals there is a phenomena which occurs a.tsr weaning of the animal in which it loses weight and is especially susceptibls to infsc'-o~.. Such an affoct has h=e?.
frequently studied in porcine an mals, since it is no-uncommon for there to be considerable livestock losses following weaning. This lag in the growth of pigs after weaning has become a recognized phenomena of "early weaning"
production systems. Lecce, J.G., Armstrong, W.D., Crawford, P.C. and Ducharme, G.A., "Nutrition and Management of Early Weaned Piglets: Liquid versus Dry Feeding.", J. Anim. Sci.
4~:1007-1014 ~1989).
The intestinal structure has been observed to change post weaning. Transverse sectionins cf the gastrointestinal tra^t shows that all regions of the tract are structurally simil-r and beneath the outer serous coating are layers of musculature. The innermost layer of the tract has three components: the muscularis mucosae which consists of two layers of smooth muscle, a thic~ layer of connective tissue, and a mucous membrane. The mucous membrane, luminal surface of the tract, is composed of columnar epithelial cells and all but the oesophagus. The small intestine has depressions throughout which project down in~o the connective tissue ~lamina propria) creating what is termed the crypts of Liberkhun. The small intestinai surface is further increased by folding of the mucosa ar.d su_-mucosa and bv mucosa , 2~3~
~vo~ ')2/~5~3 projections which are termed villi. ~he epithelial cells which cover the villus are continuous with those lining the crypts. weaning has been noted to have marked effects on this structure and function of the small intestinal epithelium Miller, B.G., James, P.S., Smith, N.w. and Bourne, F,J,, "Effective Weaning on the Capacity of Pig Intestir.al Villu_ t_ Digest and Absorb Nutrients.", J. A~ric. Sci. Camb., 107:579-589 (1986). Reduced height and increased complexity of tne villus structure occur post-weaning. Post weaning reductions in villus height, increases in crypt depth as crypt celi production increases results in migration onto the villus of younger enterocyte cells which may be pr_sent upon its su fac-for a shorter period of time. This limi~s the digestive and absorptive capacity of the epit:^.elial tissue. ~educt on.s i r, the digestive and absorptive ab lity of ntestinal tlssu_ as G
consequence of this may be the cause of checks in pig srG~th seen post-weaning. In addition, impaired intestinal func^~ion coupled with alterations in the composition of the gastro-intestinal microflora, i.e. particularly increased numbers of enteropathogenic strains of Escherichia coli can result in acute enteric infection after weaning.
Thus, there is a clear need for better understanding of the actions of probiotics in the intestine of animals, in order to be able to better use and apply such probiotics.
Further, there is also a need fe overcoming nesative af-^ c s on the intestinal tract of animals, and in particular porcine and other livestock animals, which occur post-weaning.
This invention relates to application of probiotics to increase digestive enzymes in the intestinal tract, to improve the intestinal epithelial structure for increased digestive and absorptive ability, and to reducing negative effects which occur in the intestinal tract post-weaning which can cause decrease in growth of animals.
Thus, it is one object of ~ is invention to provide for a means of altering digestive enz~es in animals that are benefited by such altering.

.
. . .

~v()s3/l)~ 2 ~ ~ 3 i 3 ~ 92/()553~

A further object of this invention is to provide for a method of altering the mucosal structure of the intestinal tract in animals, where such animals are benefited b~ such change.
A still further object of this invention is to cause changes to the intestinal tract and the chemical com~onerr- r-' the same in order to decrease negative effect cn the intestinal tract of animals.
A still further object of the invention is to prsvide f_-a method of improving animal health post-weaning.
Still further objects of the invention will become apparent through the following disclosure.
SUMMARY OF THE INVENTION
The invention relates to a method to alter dlges-ivr enzymes in animals where the animal can benefi~ -r^.. âu_h alteration comprising administering to the animals prob~ot`-strains of Lactobacillus and Stre~tococcus. The invention also relates to a method of altering mucosal structure in the intestinal tract of animals where the animal can benefit from such alteration through the application of such probiotic strains. The invention provides for a method of improving animal health post-weaning by applying such strains to the animal.
B~IEF DESCRIP~ION OF THE DRAWINGS
Fig. 1 is a graphic depiction showing alkaline phosphatase activity in control, tylosin, and Probios-trea;ed pigs at 7,17,42 and 80 days of age.
Fig. 2 is a graphic depiction showing sucrase activity in control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age.
Fig. 3 is a graphic depiction showing lactase activity in control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age.
Fig. 4 is a graphic depiction showing tripeptidase activity in control, tylosin, and ?robios-tceated pigs at 7,17,4~ and ~0 days of age.

~093/()()()l ~ U 9 3 ~ Cl/lJS92/0553 - Fig. 5 is a graphic depiction showing dipeptidase activity in control, tylosin, and Probios-treated pigs at 7,17,42 and 80 days of age.
~ ig. 6 is a graphic depiction showing alkaline phosphatase activity in control and Probios-treated pigs at 8,20,28 and 35 days of age.
Fig. 7 is a graphic depiction showing lactase activity in control and ?robios-treated pigs at B,20,28 and 35 days of age.
Fig. 8 is a graphic depiction showing tripeptidase activity in control and Probios-treated pigs at 8,20,28 and 35 davs of age.
Fig. 9 is a graphic depiction showing dipetidase acti~
in control and Probios-treated pigs at 8,20,28 and 35 days 5-^
age.
DETAILED DESCRIPTION OF THE INVENTION
The mode of operation of probiotics in animals, and ir.
particular how it may affect animal's intestinal tract is not well understood. As has been noted, the increased understanding of such operation would greatly assist in understanding when, how and with what strains to apply probiotics to animals.
It is the surprising finding of this invention that probiotics alter digestive enzymes, which enzymes are associated with improved capacity to digest nutrients. The inventors have also discovered that the mucosal structure of the intestinal epithelial surface in the intestinal tract of animals is changed in a manner which exposes more mature cells associated with better capacity to digest nutrients than younger cells. Where such probiotics are applied to an animal prior to weaning, it has further been found that the probiotic will increase such enzymes and mucosal strùcture associated with improved nutrient digestion and can decrease negative post-weaning effects.
~ 6 --::, . .. , `'.

~vo (,~"""~,~ 2 a 9 3 1 3 ~ I'C~ ()2/()s~

The digestive enzymes studied here included sucrase sucrose ~ -D-glucohydrolase; lactase (~ -D-galactoside galactohydrolase); dipeptidase (substrate L-leucylglycine) and tripeptidase (substra~e L-leucylglycylglycine). These enzymes are active in the final processes of di~estion prior to the absorption of nutrients into the body. of these, the carbohydrates are located in the brush border membrane of the enterocyte cell and the dipeptidase in the cytosol.
Tripeptidase activity is located primarily, although not exclusively, in the brush border region of the cell. Of thess enzymes studied, it was found that there was a significant affect on all enzvmes but tripectidase when probiotic is a?plied. ~s th_ experiment described below shows, in pigle~s in which P!obios was acplied beginnin~ at 17 days after b~ n, ths enzymes were found to be lower in the contrc` group versls the group in which Probios was added, or in which a control antibiotic was added. Further, the ty~ical pre-weaning to post-weaning decline in enzymatic activity was found to be reduced considerably by the application of Probios.
Additionally, morphology of the intestine was affected when the probiotic was applied. The villus height and ratio of villus height to crypt depth was not as greatly reduced pre- to post-weaning when the probiotic was applied. The probiotic administration to pigs influenced the pre-weaning changes in mucosal structure typically by preventing the reduction of villus height and crypt elongation between Day and 17 of age.
While not wishing to be bound by any theory, it is believed that this result of enhancement effect upon enzyme activities is a consequence of change in epithelia cell turnover, resulting in the presence upon the villus of enterocytes which are more numerous and/or functionally mature. Post-natal changes in illus structure occurring in the pig may be influenced by the composition of the endogenous microbial population. Probiotic administration, it is theori~ed, influnnces ~he balanes c m croorganisms and ths 2'~33 i .t ~( pattern of bacterial metabolism in the gastroin~estina: :ract resulting in the differenc~s in small intestinal hists og~ 2n~
enzyme activity observed in Experiment 1 Applying the probiotic prioc to weaning ha5 a fa~orable affect upon the growth rate of the animal as wel!. The growth rates of the animals in Experiment 1 increased from Day 25 o-:
when compared to the control group. In the antibiotic comparison, an increase in growth was seen as compared to the control, but not until Day 36.
The proDiotics of this invention can take a varietv of forms as long as they improve enzyme activity and structu!e of the intestine. The first preDaration here is a 2 strair version includinq Lactobacill~s acidoDhi'us (3 str2ins!; -.
ca~e~ (one s~rain), ~. ~lanta- ~r, (four s.rains! and Strer~tococcus faecium (~ strains). The five stra~n ve-s:o-.
includes L. acidoDhilus, L. c ei, two str2ins o- L. r~l-n-2-um and StrePtococcus faecium. These are de?ositeà -. Germ~any a-the Deutsche Sammlong von Mikroorganismen Und Zell Kulturen Gmbh. and will be deposited with the American Type Culture Collection to be made available to the public upon allowance of any claims.
The form of administration of the probiotic has not been found to be critical. In livestock it typically is administered in the form of granules included in feed. The granules are ordinarily applie~ at times of stress, arriva1 or change in ration of feed. A ge! composition may be used to administer the probiotic and mav supplement the addition of granules to feed. A gel is typically administered at birth, 7 to 10 days of age and at weaninq or other times o~ stress.
Since the detrimental enzyme reduction and intestinal structure changes occurring post weaning can be impacted by the probiotic, dosing prior to weaninq is most effective. I~
is known to include other compcnen s in such gel compositions and the additions are not .. . . .
., , : :

, 2~93 1 31 I'~/l.S92/()5~

critical as long as they do not interfere with the increased enzyme activity or changed intestinal structuce. Additions such as those to improve delivery include yeast, vegetable oil, silicon dioxide, titanium dioxide, vitamins, coloring agents and p~eservatives.
The amount of lactic acid bacteria included must bs sufficient to cause the desired enzymatic and structural changes to the intestine. The effective amount used in these experiments include about 107 viable counts per gram of composition.
The following is pres?nted by way of example and is not intended to limit the scope of the inven.tions.

Measurement of growth rate occur ed -rom D' rth tc 30 cai~s of age and was co~pared with t:ne grow h r te 0~ animals e^
diets containing no growth promoting agent (negative controls), with pigs receiving an antibiotic growth-promoter used in the pig inàustry (positive controls) and the probiotic described. Aspects of the microbiology and development of the digestive track at 7, 17, 42 and 80 days of age were studied as reflected below.
PROBIOTIC PREPARATIONS
The probiotic preparations administered during this study were Probios~ brand (Pioneer Hi-Bred International, Inc., Johnston, Iowa, U.S.A.) whicn contained twelve bacterial strains, including dried Lactobacillus plantarum, Streptococcus faecium, Lactobacillus casei and Lactobacillus acidophilus and their fermentation products. The preparations used in this experiment were of two types:
Probios Oral Gel for Swine Composition: Dried fermentation products and selected strains of lactic acid bacteria (7 Lactobacillus spP. and 5 Stre~tococcus SDp. ) together with: yeast culture, vegetable oil, sucrose, silicon dioxide, ~itanium dioxide, vitamins A,D,E and a~F~D and C yellow No. 6 Lake, Polysorbate 80, TBH~

_ c 2 ~ 9 3 1 3 1~
WOs~/()t)()1' 1'(1/~ )2/

and ethoxyquin preservatives. Total lactic acid bacteria 107 viable counts/g.
~ ecommended dosage: Newborn pigs - l g at birth or a~
first handling. An additional l g given 7 to l0 dat~s late~
Weaning/starter pigs - 2 g at ~ear. ng, during ration changes and other environmental chan~es.
Probios Granules for Swine Composition: Dried fermentation products and selected strains of lactic acid bacteria (7 Lactobacillus SDp. and S
Stre~ococcus spo.) together with yeas, culture, calcium carbonate, corn cob fractions, vegetabie oil, TBHQ and ethoxyquin preservatives. Total lactic acid bacteria = lG
viable counts/g.
Recommended dosage: ~eanina and s~ar.e- pigs (u? to kg) - granules a~ a rate of l ~g~ton o. creep feed during periods of environmental changes, upon 2-rival, or ration change. Growing pigs (18 to 4; kg) - g-anules at a rate c-500 g/ton of complete feed during periods of environmental changes, upon arrival, or ration change. Finishing pigs (4;
kg to market weight) - granules at a rate of 250 g/ton of complete feed during periods of environmental changes, upon arrival, or ration change.
Sows - granules added to ration at a rate of l0 g/head/day for fiYe days prior to farro~ing. Granules at a rate of 500 g/ton of complete feed durin~ laceation.
TYLOSIN INCLUSION IN POSITIVE CONTROL DIETS
Tylosin is a macrolide antibiotic, isolated from streDtomYces fradiae (arander~ G.L. "Chemicals for Animal Health Control" Taylor and Francis Ltd. London (1986)) and active against Gram- positive organisms. At subtherapeutic levels tylosin is used extensively as a growth-promoter in pigs and poultry. In the present study tylosin was included in 'positive control' diets as Tylamix Premix l00 g/kg (Product License No 0006/4055. Elanco Products Ltd., Easingstoke, En~land.) each kilcg!am o~ which contains tylosin phosphate equivaler.t to l00 g o- ~vlcs .n _ase.

. , .

2~3l 31 I / I $') 2/ r) c .

TREATMENTS
The trial consisted of three treatments: (1) basal d:e~
containing no added growth promoter and with copper at nutritional levels; (2) basal diet and antibiotic gro~th promoter Tylosin (Elanco Products Ltd, ~asingstoke, Hantsj, a macrolide antibiotic isolated from StreDtcmvces fradlae an_ active against Gram positive organisms (~rander, Supra, as 40 mg/kg; (3) basal diet and probiotic preparation Probios~
(Pioneer Hi-Bred International Ltd, Johnston, Iowa, USA) administered as an oral gel 24 hours afeer birth (1 g), ag2ir.
at 7 days of age (1 g) and at weaning (2 g). The probiotic was included as granules in both the creep feed and follo~-or diet at 1 kg/ton. soth forms of the probio'i_ ccr.tai..a_ :^
colony-forming units/g and comD.ised a ,mixtur^ c~ m,_l- ?' strains of Lactobacillus Dlantarum, Lac;obaci`lus a_idonh :u-Lactobacillus casei and Streotococcus raecium.
EXPERIMENTAL ANIMALS
The present study was carried out on litters o~ crosâ-bred (Large White/Landrace/Duroc) piglets, and because of space limitations in the farrowing house was organized in three phases with two of the treatments represented in each phase. All four sows in each phase farrowed within 48 hours of each other and there was a period of 2 weeks between phases. Sows were paired according to farrowing date, and within each pair of sows each litter was split into two, keeping the sex ratio equal in each half. Each of the sows, therefore, had a cohort of piglets half of her own and hal-cross-fostered. Runts were included in the cross-~osterins process. Selection and allocation occurred on three occasions (i.e. the experiment was conducted in three phases). The treatments represented in a particular phase were randomly allocated to the four cohorts such that within each pair o sows one cohort was allocated to each treatment. While in ~he ~arrowing house all cohorts were penned individually, and following weaning animals were Denned in treatment groups ::ith WO93/00012 2 ~ C~/US92/0553~

the two cohorts from each treatment in each pen. At all times precautions were taken to avoid cross-contamination between treatment groups.
DIETS AND FEEDING
he creep feed and the follow-on ration were ~ormulated to provide respectively 253 and 200 g crude protein/kg (nitrogen x 6.25) with lysine contents of 0.133 and 0.11 q/kg.
The diet formulations and relevant chemical analyses are shown in Table l. ~oth diets contained copper at nutritional levels (20-70 mg/kg). Litters were offered creep feed from 7 days of age and piglets were weaned from the farrowing house to flat-dec~. housing at 21 da~s of age. All pigs were weighed at weekly intervals.

TABLE l Components of creep feed and follow-on diet g/kg fresh weight) and dietary nutrients Creep Feed Follow-on Whëat 320 250 Barley 250 470 Dry skimmed milk 210 10 Soya-bean meal 150 150 Fishmeal 55 100 Vitamin-mineral mix 15 20 Dry matter (DM:g/kg) 921.4 915.9 Ash (g/kg DM) 52.26 59.92 Oil (g/kg DM) 20.68 18.68 Crude protein (nitrogen x 6.25: g/kg DM)245.00 233.00 Gcoss energy (MJ/kg DM) 20.24 20.24 Copper (mg/kg DM) 20.90 68.30 .
TISSUE SAMPLING
On each of days 7,17,42 and 80, one pig was randomly selected from each cohort for tissue sampling. At these times an attempt was made to balance for sex and for natural/foster mother. This process resulted in four pigs per treatment being sampled at each o~ the four ages. In order to obtain tissues that had not been subjected to mucosal shedding, animals were restrained and anaesthetized with Halothane ' , ' , ' ~ '' : ' : . .

. . :: .

2;lJ~3131 (4%)-oxygen-nitrous oxide administered via an open mask Following laparotomy the animals were killed by an ir, e~:io,.
of sodium barbitone given via the portal ~ein. The gastrointestinal tract was removed immediately and tne smaii intestine freed from the mesentery. Samples of mucosa! ~issue were isolated from 50 mm lengths of intestine taken a- _ ~e_ 0.05,0.1,0.3,0.6 and 0.8 along the small intestine.
Intestinal sections were opened longitudinally, rinser ~i~h ice-cold saline (9 g sodium chloride/l), blotted dry 2n~
placed on a chilled porcelain tile. Mucosa was scrape~ -ro~
the muscle layer using a microscope slide, wrapped in ~:n fo l and snaD frozen in liquid N~ before being stored at _,Oc ENZYME ASS.~VS
.
All assays were carried out on he~o5enates of m~c^s-`
tissue obtained by thawing approximately g tissue an`
homogenizing it in lS ml of a solution con;aining SO m;-mannitol and 2 mM-Tris-hydrochloride (pH 7.1) usinq twe v strokes of a teflon-glass homogenizer. The homogenate '~'2S
made up to 25 ml with the same buffer and portions stc!ed at -70 before being assayed.
Sucrase and lactase were assayed by the method o Dahlqvist, Arne, "Method for assay of intestinal disaccharidases" Anal~t. Biochem. 7:18-25 ~1964), and dipeptidase (substrate L-leucylglycine) and tripeptidase ~substrate L-leucylglyclglycine) by that o ~icholson, J.~.
and Kim, Y.S., "A one-step L-aminoacidoxidase assay fo-intestinal peptide hydrolase activity" Analvt. ~iochem.
63:110-117 (1975) incorporated herein by reference. In these assays the hydrolysis of the di- or tripeptide to release amino acids is linked to oxidation of the leucine by L~amino acid oxidase with the resulting formation of hydrogen peroxide. Oxidation of o-dianisidine by hydrogen peroxide following the action of peroxidase results in a specific end-point to the reaction with an absorbance at 530 nm. In using the substrates leucylglycine and leucylglycylqlycine, the ':

`~ wo (,~ ""~ 2 ~ v ~ 3 ~ J~)2/~)55:~
activities ~f the two hydrolases could be distinguished from one another. The end product o~ the tripeptidase assay, i.e.
glycylglycine, if hydrolysed by dipeptidase actlvity does not react in the assay system. hll assays were adjusted to ensure linear rates of reaction over time witil volume of homogenate added ar.d substrate concentratior..
SCANNING ELECTRON MICROSCOPY (S.E.M) Changes in overall intestinal morphology with age was assessed from sections viewed under S.E.M.. From tissues fixed in 4O ne~Jtral buffered formaldehyde smaller sections (3 mm x 3 mm) were taken and dehydrated in a series of ethanol solutions (25,50,70,90,95 and 1006 v/v), 30 minutes in each.
Tissue sections were then processe~ in the Depa-tment of ~lar.
Biology, ~niversit~ _f .~ewcasLle u?on Tvne. Each section ::-s placed in 50~ amvlacetate: 50j ethanol for 15 minutes, followed by l00~ amylacetate. rinally, tissue sections were critical-point drie~ (to avoid distortion) and carbon and go!~
'splutter-coated' before examination under S.E.~;. (Jeol S
S.E.M., Jeol (UK) Ltd., London).
LIGHT MICROSCOPY
A ~uantitative study of c~anges in intestinal morphology was performed on tissue sections viewed under the light microscope. From tissues ixed initially in 3~ glutaraldehyde smaller sections ~l mm x 2 mm) were taken under a Vickers M69 dissecting microscope and stored overnight in phosphate buf.e (pH 7.4). Tissue sections were then processed by the Department of Anatomy, Medical School, Newcastle upon Tyne.
Intestinal sections were viewed (x;0) using a Vickers V80 stereoscopic microscope and accurately drawn using a camera lucida attachment. A scale (0-50 um) was drawn (x50) from a stage micrometer, copled onto an acetate sheet and used to overlay the camera lucida drawing to allow precise measurement o~ the tissue section. Measurements taken included villus height (distance from villus tip ~o villus,'crypt junction) and crypt depth (distance from villus'crypt junction to muscularls i .. .. . ..
,. . \ . . . ~ .

~VO/)l~ ()l 2 ~ 31~ ~ A ~ /l.S9' mucosa). On average 6 measurements of each parameter we:~
made per section, and the ratio of villus height:cryp. ~?~~n calculated.
S TA T I S T ~ CA L A ~`~A L Y S ; S
. .
The data were analyzed using the G~M statistical pa-~.2qe (Baker, R.J. and ~e!der, J.A., "GL!I System Release" 3 Numerical Algorithms Group, Oxford (1978)) which allows ~^-the sequential fitting of all factors which may affect the variable being analysed. The initial analysis was carried out across all ages studied with the summed data for all sites within the intestine and was designed to test whether thera were any significan' ef'ects Oc age and treatment or the 'blocking~ factors phase, sex a.nd mothering. The latter factor was i~cludad to ident f~ ef-ects that mi _h hav~ sa-due to the fact that some pi~s were reared on a ros.er -a_h -than a natural mother. In addi-ion the interactions o- 2 the factors were fitted and the residual sums Or squa-Qs ~_s-d to calculate the error term against which all the null hypotheses wece tested. As a result of this initial analysis it was determined that the factors sex and mothering had no significant effects on any of the enzyme activities measured and the analysis was consolidated to determine the effec~s of phase, treatment and age and treatment x age interactions.
Phase was retained in the analysis as it was found to be significant in a number of cases although this may have bee.-related to the unbalanced nature of the experimental desicn (not all treatments were included in each phase) rather than any biological effect. The analysis also included splitting the age effect into pre- and post-weaning (7 ~ 17 d v. q2 - 80 d) and the treatment effect into control v. tylosin + Probios and tylosin v. Probios. An outline of the analysis of variance table is shown in Table 2.
Data analysed at each of the four ages of pig studiec were expanded to include values -or each of the five intestinal sites. The factors wera fitted in the analysis as phase, treatment, in-es~ina1 Sl-? and IrQatment x intes;ina .
;" . ''' ...

~ o ()~ 2 1 ~ 1 3 1 ~( 1/lS9~

site and subsequently the effect of treatment divided to consider the analysis of control v- tylosin + Probios an~
tylosin v. Probios. Intestinal site was also re-analyse~ :_ consider the effect o~ proximai (0.0~, 0. 0 and 0.30~ sl~es .~.
distal (0.60 and 0.80) sites. An outline of the analysis o-variance table is sho~n in Table 3.

Sources of variation and deare~s o~ freedom for analysis of data for enzyme activity summed across all intestinal sites for pigs at 7,17,~2 and 80 d age Source of Varia.ion Degrees of Freeds.
Total 47 Phase 2 C v . I` T ~) 1 T ~. P
Pre v. Post , Other age effects 2 (Pre v. Post) x (c ~ +
(Pre v. Post) x (T v. ?) Other treatment x age ~ fects Residual 34 Treatment: C, basal diet: T, basal diet + tylosin (40 mc/k~
P, basal diet + Probios; age: Pre, pre-weaning (7 d + li dj-Post, post-weaning (42 d + 80 d).

Sources of variation and degrees of freedom for analysis of effects of treatment and intestinal site 0?.
enzyme activity for pigs at 7,i7,42 and 80 d or aae Source of Variation Degrees of Freedo~
Total -- 59 Phase 2 C v. T + P
T v. P
Prox. v. Dist.
Other site effects 3 ~Prox v. Dist) x (C v. T ~ P) (Prox v. Dist) x (T v. ') Other treatment x site effects 6 Residual ~3 Treatment: C, basal diet: T, basal àiet + tylosir (40 m~
P, basal diet + Probios: site, ?rox, proximal sites (0.05, 0.10, 0.30): Dist. distal sites 0.~0, 0.80).

': . ' : ~ . , ' ~ ,. .

RESULTS

The Activities of Mucosal Enzvmes The mean values (+ SEM for activiti~s of alkaline phosphates shown in measurements of IU/g mucosal wet ~eigh~
(Figùre 1), sucrase (Figure 2), lactase (Figlera 3), tripeptidase (Figure 4) and dipeptidase (Figure S) at five intestinal sites in pigs is shown on treatment at 7~17~Y2 an~
80 days of age.
Table 4 summarizes the affects of treatmen; and age on each mucosal parameter studied for all inrestinal sites and pigs on each treatment at 7 ~17 ~ ~2 and 80 days of age.
TABLr 4 Summarv o ~ne affeet c' treatme.i-, a_e and treatment x age on enzvme activity in the small intes-:n-of the pig summed across all intes'inal sites Enzyme activity Treatment Age Treatment ,Y z--Sucrase NS Pre<Post*** [Pre v. Post]:C>T-[EC 3.2.1.48) [Pre v. Post]:T<P*
Lactase NSPre>Post*** [Pre v. Post]:C<T+P**
EC 3.2.1.23) [Pre v. Post]:T> D*
Dipeptidase NSPre>Post*** ;Pre v. Post]:C<T+P**
(EC 3.14.13.11) Tripeptidase NSNS NS
(EC 3.4.11.4) Treatment: C, basal diet: T, basar diet + ty!osln (40 ms~kg):
P, basal diet + Probios: age: P!e, activitv in pre-weane~ -ics (7 and 17 d of age): Post, activity in post-weaned pigs (~_ and 80 d of age): NS, not significant.
*P~0.05; **P<0.01; ***P<0.001.

Activities of alkaline phosphatase, lactase and dipeptidase were significantly greater in pre-weaned pigs compared to post-weaned animals. Conversely, sucrase activity increased between pre- and post-weaning periods. The;e was -.-significant change in tripeptidase activity pre- to pcs -weaning, There was significant effect at certain ages c ~he ~ ,. .
.

209~l 3~.
,""",. , ( "1 .s()~/~)s~

pig from treatment with Probios and antibiotic treatment on all enzymes except for dipeptidase.
7 DAYS OF AGE:
At this age alkaline phosphatase activity showed significant interactions between treatmens and site, tne increase in activity from proximal to dis;al sr,all lr,te^-~in-being significantly lower in control pigs compared with treated animals (p<0.05 for IU/gMWW and p<0.01 for speci- c activity). Other enzymes at this age did not show treatmen effect. The activities of alkaline phosphatase, tri- and dipeptides enzymes in proximal tissue wer= significantly lo~e than that in the distal intestine (p<0.00: for all excepz tripe?tidase specific activity for which ?~0.05). Con.-~s-`.
the activit~ of sucrase iias significantlv grQate~
proximal tissue (p<0.001). There were nc signi ican~ -- ^_-~of site on lactase activity.
17 DAYS OF AGE:
The activities of all enzymes studied with the exce? ion of dipeptidase were significantly lower, across all sites, in control pigs compared with treated animals (p<0.001 for sucrase IU~gMWW and lactase activity; p<0.01 for alkaline phosphatase IU/gMWW and sucrase specific activity; p<0.05 for alkaline phosphatase specific activity and tripeptidase activity)~ The activities of all enzymes studied, wi.h the exception of tri- and dipeptidase expressed as IU/gM'~' ~erQ
significantly higher in tylosin~treated pigs than in Probios-treated animals (p<0.001 for lactase activity; p<0.01 for alkaline phosphatase activity; p<0.05 for sucrase activitv and tri- and dipeptidase specific activities)~
Apart from the affects of treatment, sucrase and lactase activities were significantly higher in tissue from the proximal intestine than the distal gut (p<0.001 for IV~gM~
of both enzymes and p<0.01 for specific activity of both enzymes)~ Conversely the activities of al! the other enzvmes studied were significantly lower in the proximal intestine ,: ' . ' ' :

2û93131 ~,'() ') 3/~ ()2/~155:~

compared to the distal (p<0.001 for all activities except tripeptidase IU/gMWW for which p<0.05).
The magnitudes of change in activit~ between proximai ar~
distal regions was significantly lower in control pigs co~pared with treated animals for sucrase, tripeptidase an~
dipeptidase (p<0.001 for tripeptidase specific activity;
p<O.01 for sucrase IU/gMWW, tripeptidase IU/gMWW and dipeptidase specific activity; p<0.05 for sucrase specifi-activity and dipeptidase IU/gMWW). Sucrase activity increasQ~
between the proximal and distal small intestine in control animals while decreasing in the tylosin- and Probios-treated groups. Conversely, tripeptidase activity decreased bet~sen these two regions in control animals while increasing in tylosin- and Probios-treated animals. The increase in tylosin-treated pigs was greater than in Probios-treate~
p<0.05). Dipeptidase activity in control and treated pigs increased between the proximal and distal small intestine, th-increase in specific activity of this enzyme for tylosin-treated being (p<0.01) greater than for Probios-treated animals. There were no significant interactions between site and treatment for alkaline phosphatase or lactase activities.
42 DAYS OF AGE:
Activities in tylosin-treated animals at this age were all significantly lower than in Probios-treated (p<0.001 fo!
sucrase IU/gMWW; p<0.01 for sucrase specific activity; D<O.O-for alkaline phosphatase IU/gMWW). At this age, across all sites, there were no significant treatment affects on alkalir.e phosphatase specific activity, tri- or dipeptidase activities.
Alkaline phosphatase IU/gMWW and sucrase activity were not significantly different between control and treated pigs.
Lactase activities in control pigs were significantly (p<0.05) greater than in treated animals.
Site was found to have no effect on the levels of alkaline phosphatase activity or tripeptidase IU/gMWW
Sucrase activity and tripeptidase specific activity in 2 ~ ~ 3 1 3 .~ x~

proximal tissue were significantly lowec than in the dlstal intestine (p<0.00l). Conversely, proximal activity was significantly greater than distal for lactase (p~0.00l) and dipeptidase IU/gM~1 (p<o.os)~ DipeptidaSe specific activ ty did not differ significantly between proximal and distal regions. The magnitude of increase in tripeptldase spec fic activity between the proximal and distal intestine was not significantly different for control pigs when compared with treated animals. However, this increase in tylosin-treated animals was significantly (p<0.05) lower than in the PrGbios-treated group.
80 DAYS OF AGE:
Sucrase and dipeptidase activities were significantl.
lower across proximal sites studied than distal ones ( D<~_ . 00 ;^r sucrase activity; p<0.0l for dipeptidase IU/gM~ and p<0.05 for dipeptidase specific activity)~ Conversely, lactase activity was significantly greater in proximal ~ ssue compared with that in the distal intestine (p<0.00l). The.re were no significant differences between the proximal anà
distal small intestine in levels of alkaline phosphatase or tripeptidase activity.
For all enzymes studied, except the dipeptidase, activities at 17 days of age in control pigs, across all sites, were shown statistically to be lower than those in other treatment groups and to be greater in the tylosin- han Probios-treated animals ~although not for the tripeptidase when expressed on a mucosal wet weight basis)~ Specific activity of the dipeptidase in 17 day old pigs was greater in tylosin- than Probios-treated animals but in control and Probios groups the activity was similar. Although statistically there was no treatment effect on activity of the dipeptidase (IU/gMWW) it was greater in tylosin- than Probios-treated pigs and lowest in cont-ols. The inclusion of either an antibiotic or probiotic in the diet influenced the pre- to post-weaning changes in enzyme a^tivity.

.. . . . .
.. ' ''' ~ .~
. . ..
, .

~0 ~ r ~ r~ s~)2/oss~4 2 ~
Effects of Treatment and Aqe on Intestinal Structure Mean values (+ SE) of villus height, crypt depth and villus height:crypt depth ratio at 0.10 and 0.60 intestinal sites for pigs on each treatment at 7,17,42 and 80 days of age are given in Table 5 below.

Villus heigha Crypt depth V-illus:Crypt (um) (um) ratio A~e Treatment mean SE mean SE mean SE
0.10 smal! intestinal site C 793 (4)33296 (4) 55 2.95 (4) 0.43 7 Days T 676 (4)8,263 (4) 40 2.66 (4) 0.32 P 664 (3)11~304 (3) '~08 3.10 (3) 1.37 C 582 (3)43510 (3) 116 1.31 (3) 0.30 17 Days T 566 (3)9'302 (3) 32 2.18 (3) C.ll ? 709 (4)99217 (4) 15 3.25 (4) 0.54 C 397 (4)38400 (4) 30 1.10 (4) 0.11 42 Days T 331 (3)15310 (3) 47 1.18 (3) 0.32 - P 396 (3)35307 (3) 71 1.55 (3) 0.53 C 413 (1) -363 ll) - 1.14 (1) 80 Days T 422 (3)23365 (3) 15 1.17 (3) 0.14 P 455 (3)21395 (3) 26 1.15 (3) 0.03 0.60 small intestinal site C 844 (3)87306 (3) 59 3.07 (3) 0.74 7 Days T 794 (2)89345 (2) 42 2.31 (2) 0.05 P 914 (4)12a312 (4) 68 3.31 (4) 0.72 C 535 (4)82265 (4) 36 2.13 (4) 0.39 17 Days T 746 (4)108307 (4) 46 2.52 (4) 0.30 P 583 (4)85251 (4) ~9 2.85 (4) 0.70 C 504 (3)58325 (3) 11 1.57 (3) 0.26 42 Days T 385 (3)72497 (3) 119 0.82 (3) 0.15 P 275 (1) -404 (1) - 0.68 (1) C 343 (2)18410 (2) 10 0.75 (2) 0.13 80 Days T 406 (3)30366 (3) 24 1.27 (3) 0.18 p 531 (2)4315 (2) 20 1.72 (2) 0.24 Treatments~ C - control; T - tyi^sin, P - Probios.

.

` ""'- 2 ~ 3 ~ i 3 1 ~ /ls()2/n55~

Initial analysis of villus height:crypt depth indicat~d a significant interaction between treatment and msthe~ing, the latter was therefore included as a "blocking" 'actor in subsequent analysis. There was a signifl~ant '~ect of treatment on villus height or crypt depth at particular ages of the animal.
Tylosin or Probios administration to pigs during the present experiment was associa~-d with prPventicn of reductio..
in villus height and the increase in cryp- depth at the 0.10 intestinal site seen between 7 and 17 days of age in control animals. At the 0.50 site only antibiotic-tre2~ed pigs showed no obvious loss in villus heish.t betweer. and :, days of aae All animals had reduced villus height fro.- 17 t^ 42 days o-age at the 0.10 site. At the 0.50 site, ~..erP ~-e-P r.c -~2rl~P' changes in ~.~illus heiqht or crypt dept:. i. ^ont _`' pias between 17 and 42 days of age, howeve-, r-_ucti^.-.s and increases respectively in these parameters were --?parent in both tylosin- and Probios-treated animals.
Probiotic or antibiotic administration to pigs altered the pattern of epithelial structural changes preweaning particularly in the proximal intestine. These changes reflect the theory that there is a slower rate of epithelial cell turnover in these treated animals, and therefore an increased population of functionally mature enterocytes. .~mong potential benefits conferred by such chan~--s in in:estinal functions, a result of treatment may be reflectec in improved digestion and increased growth rate post weaninc. Probiotics also afford the advantage of avoiding consumer concern associated with antibiotic treatment.

This experiment further demonstrates the effects of probiotic feeding on animals pre- and post-weaning.
The methodology used was essentially the sa.~e as in Experiment 1, with the pcimary d~fference that the probiotic preparation contained strains of only four Lactobacillus SD.
and one Stre~tococcus SD..
.

. ~ . ,, . . :

VO93/0~)~)l2 2 ~ 9 3 ! 3 ~ ~'C~/lS92/05534 ~- Creep and grower diets varied somewhat from the first experiment. An antibiotic group was not included, and instead a control treatment and a Probios treatment were the only ones included. They were varied as follows:

Components of Creep and Grower Diets Creep Grower (9o fresh weight) (Qo fresr. weight) Complan 25.0 s2rley ~8.0 Wheat 24.0 Wheat 25.0 sarley 24.0 Sc~a bean mea __.C
Soya bean meal15.0 Fishmeal i0.0 Fishmeal 10.5 Vitamin/mineral mix '.0 Vitamin/mineral mix 1.5 Calculated dietary nutrients ~fresh weight basis):
Protein (%) 23.40 20.40 D.E. (MJ/kg) 15.20 13.10 Av. Lysine (%)1.39 1.07 Cu (mg/kg) 30.40 36.50 Oil ~%) 7.81 l.91 Fibre ~%) 2.73 3.84 Salt (~) 0,57 0.45 Methionine &
Cysteine ~%) 0.a6 0.69 Threonine (%) 0.72 0.54 Ca (~) l.i9 1.13 p (%) 0,77 0.71 wo ,~,~",~ 2 ~ ~ 3 1 3 1 1'(''1 /l,~.'j~/~)55.~1 -There were four phases. All pigs were subject to the normal unit regimen and were weaned at 21 days of age with ~Ae exception of those animals born in the ~irst and second phases which were weaned at Day 28. Samples were taken at 8 days ^f age, when the piglet was established on the sow and had not yet taken creep feed; 20 days of age, when the piglet had creep feed available but was not yet weaned; 28 days of age, when the piglet had been weaned for one wee~ and was no~ fu :
adapted to the grower diet; and 35 days of age, where the piglet had recovered from weaning stress and was adapted tc the grower diet.
RESULTS
The five strain probiotic has the effect o- creater recovery of both pe?tidasa enzymes studied. The activ~ties --all enzymes fell between 20 and 2~ days in the animals ~ea.^--at 21 days and by 3ay 35 there was evidence of a recove-~ -.
activity of both dipeptidase enzymes which was greater in treated animals. The fall in enzymes after weaning in the animals weaned at 28 days was not as great as in 21 day weaned animals. The significant effect of treatment seen in the first experiment was not repeated here, however, there was the recovery affect noted with treatment and a significant increase in dipeptidase was seen in the second experiment at Day ~. There was a ceduction in weight seen with control animals past weaning not apparent in Probios-treated an ma's.
The table shows that between 8 and 20 days of age, compared with controlled pigs, there was a smaller r?!uction in villus height in Probios-treated animals. Between 20 and 28 days of age, the loss in villus neight and therefore the change in villus height:crypt depth ratio was greater in Probios treated pigs than control animals.
At the 0.6 site the effect of treatment was apparent at 28 and 35 days of age. This observation was made on crypt depth determinations in one pig oer treatment. The probiotic treatment did not have a significant effect upon the magnituà-', ' .; . . ' :

2 ~ ~1 31~ ,"~
of pre- to post-weaning changes in structural paramerers studied with the five strain species, however, the re~ -r -^, in villus height and villus height:crypt depth ratio ~Gr~,/een 8 and 20 days of a~e seen in control pigs appeared to na - ~ee-increased by Probios administration, although cr~pt e_-nga-~n . between these two ages was not distinctly affeeted ~;~
treatment. conversely, between 20 and 28 days of age ~he i~s~
in villus height seen in control pigs was not 2~ evic~
- Probios-treated animals.

Treatment Age Villus height Crypt depth Villus~
(um) (u~) .ra_:_ ; (days) Mean Sr Mean Sr~ Mean __ . . .
0.10 small in~estinal site Control 8 636 (5) 9117 (5) 10 5.88 .. _ 384 (8) 22186 (8` 13 2.18 ~
. 28 294 (6) 11194 (6) 13 1.55 ~~l .39 28* 386 (2) 37198 t2) 6 1.94 (2) C.13 35 302 (4) 35213 (4) 23 1.48 (~) 0.22 35* 330 (2) 44314 (2) 30 1.04 (2) Q.04 Probios 8 555 (9) 80129 (9) 12 4.52 (9) 0.71 446 (8) 46199 (8) 19 2.28 (8) 0.13 28 242 (5) 25223 (5) 28 1.15 (~) C.14 2B* 319 (1) ~171 (1) - 1.87 (1) 337 (3) 22194 (3) 24 1.78 (3~ C.'1 35* 350 (2) 27263 (2) 1 1.23 (_~ Q.Q3 0.60 small intestinal site -- .
Control 8 753 (8)98 99 (8) 5 7.75 (8! 1.05 20 519 (7) 3 160 (7) 3 3.75 (~ ~.33 28 288 (4)19 166 (4) 22 1.81 (~` 0 . 1 4 28* 447 (2) 3 248 (2) 16 1.82 (2j Q.10 35 153 (1) - 162 (1) - 0,94 (1) 35~ 342 (3) 8 271 (3) 26 1.29 (3) 0. 04 Probios 8 781 (6)91 108 (6~ 9 7.73 (6) 1.~1 20 381 (8) 3 179 (8) 12 2.18 (8) Q.1 28 369 (4)30 245 (4) 36 1.60 (4!
28* 411 (3) 53 223 (3) 17 1.94 '3! 0-4-35 288 (1) - 223 (1) - 1.29 (1~ -35~ 329 (3) 36 266 (3) 15 1.22 (~i O.C
Pigs ~weaned at 28 days of age - 2~ -... .
~; ,, ,~ ,, . . ::.
.~

~ o ".~"~1""~ 2 0 ~ ~ ~ 3 ~ C~ 92/()55W

The decline between 20 and 28 days in villus height at the 0.10 intestinal site was not apparent with animals weanec at 28 days. The large loss in villus height at the 0.06 site seen between 20 and 28 days in pigs weaned at 21 d~as did nc~
occur in pigs weaned at 28 days.
In the first experiment improvements seen in weight gai-were not apparent until Day 26 and Day 36 for the Probios and tylosin groups respectively and it is possible that the animals in the latter experiment would have shown a greate!
correlation across the board of treatment if measurements nad continued: It is of interest that in this experiment there W25 a reduction in weight associated with weaning in the control group that was not apparent in the Probios-treated animals and the probiotic group had a hiaher bodv-weiaht at Day 34. An effect of treatment upon recovery from the reductions in growth rate and enzyme activity seen at ~eanina was apparent in both animal trials carried out in this study.
Thus, the experiments show that Probios is correlated with effects in alleviating post-weaning detrimental changes in intestinal structure and enzyme reduction.
The application of Probios has been associated with increased activity of digestive enzymes which can include alkaline phosphatase, sucrase, lactase and tripeptidase. The effect varies across intestinal sites.
Reduction in villus height:crypt depth also àemonserates affects of Probios administration. The redirection occurrina typically post weaning in enzyme activity and villus heiqht and accompanying increase in crypt depth is decreased with probiotic use.
Therefore, it can be seen that the invention accomplishes at least all of its objectives.

~;

.

Claims

We claim:
A method of stimulating activity of digestive enzymes in animals benefited by such alteration comprising administering to the animal a probiotic composition.

A method according to Claim 1 wherein the probiotic composition used comprises Lactobacillus and Streptococcus.

The method of Claim 2 wherein the composition used comprises Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantorium and Streptococcus faecium.

The method according to Claim 3 wherein the probiotic composition used comprises Lactobacillus strains identified by.
ATCC number , , , , , , , and Streotococcus strains identified by ATCC number , , , , .

A method according to Claim 3 wherein the probiotic composition comprises Lactobacillus strains identified by ATCC
number , , , , and Streptococcus strain identified by ATCC number A method according to Claim 1 wherein the digestive enzyme is selected from the group consisting of sucrase, lactase, alkaline phosphatase, dipeptidase and tripeptidase and combinations thereof.

A method of altering intestinal mucosal structure in the intestine of an animal benefited by such alteration comprising administering to the animal a probiotic composition.

A method according to Claim 7 wherein the probiotic is a composition used comprises Lactobacillus and Streptococcus.

The method of Claim 8 wherein the composition comprises Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantorium and Streptococcus faecium.
A method according to Claim 9 wherein the probiotic composition used comprises Lactobacillus strains identified by ATCC number , , , , , , , and Streptococcus strains identified by ATCC number , , , , .

A method according to Claim 9 wherein the probiotic composition comprises Lactobacillus strains identified by ATCC
number , , , , and Streptococcus strain identified by ATCC number A method according to Claim 7 whereby an increase in villus-height-to-crypt-depth ratio is obtained.

A method according to Claim 7 whereby a reduction in the decrease in villus height to crypt depth ratio occurring post weaning is obtained.

A method according to Claim 13 wherein the probiotic is administered to the animal prior to weaning.
A method of altering animal intestinal structure and enzymatic processing of ingested nutrients post-weaning in an animal benefited by such alteration comprising administering a probiotic composition to the animal.

The method of Claim 15 wherein the probiotic composition used comprises Lactobacillus and Streptococcus.

The method of Claim 16 wherein the composition comprises Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantorium and Streptococcus faecium.

A method according to Claim 17 wherein the probiotic composition used comprises Lactobacillus strains identified by ATCC number , , , , , , , and Streptococcus strains identified by ATCC number , , , , .

A method according to Claim 17 wherein the probiotic composition comprises Lactobacillus strains identified by ATCC
number , , , , and Streptococcus strain identified by ATCC number The method of Claim 15 wherein the probiotic is administered prior to weaning.

The method of Claim 16 wherein the probiotic is administered to a porcine animal.

A method according to Claim 21 wherein the probiotic comprises a composition of lactic acid bacteria and is administered in doses of about 10 viable counts of lactic acid bacteria per gram of composition.

A method according to Claim 22 wherein the probiotic composition is administered in the form of a gel.

A method according to Claim 23 wherein the compound administered to newborn animals, at about 7 to 10 days of age and at weaning or times of stress.
A method according to Claim 22 wherein the probiotic composition is administered in the form of granules in feed.

A method according to Claim 25 wherein the probiotic composition is administered to the animals at times of stress, arrival or change in ration of feed.

A method of altering animal intestinal structure and chemical composition post weaning, comprising administering to an animal benefited by such alteration prior co weaning a probiotic composition comprising Lactobacillus and Streptococcus.

A method according to Claim 27 wherein the composition comprises Lactobacillus strains identified b ATCC number , , , , , , , and Streptococcus strains identified by ATCC number , , , , .

A method according to Claim 27 wherein the probiotic composition comprises Lactobacillus strains identified by ATCC
number , , , , and Streptococcus strain identified by ATCC number A method according to Claim 27 comprising stimulating digestive enzymes in the animal and reducing post-weaning decrease in villus height to crypt depth ratio.