Composition for the prevention and treatment of diarrhea in livestock

A composition and method for the prevention and treatment of diarrhea in domesticated animals, comprising sterilized cells of aerobic bacteria belonging to the genus Bacillus, Brevibacterium, Corynebacterium, Escherichia, Lactobacillus, Streptococcus, or Streptomyces, a cell homogenate of said sterilized cells, a cell wall component-containing fraction of said homogenate or mixtures thereof. In particular species of Corynebacterium glutamicum and Brevibacterium lactofermentum as well as strains thereof, such as A.T.C.C. 13060 and A.T.C.C. 13869, respectively, are useful in the treatment of diarrhea in domesticated animals.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to novel compositions for the prevention and 
treatment of white diarrhea or diarrhea in livestock, poultry, pet 
animals, etc. 
2. Discussion of the Background 
White diarrhea or diarrhea in suckling pigs during the lactation period is 
currently one of the most serious problems for swine breeders. About 45 to 
70% of swine 2 to 9 weeks of age which are suckling during the lactation 
and weaning periods suffer from white diarrhea or diarrhea. The economic 
loss is extremely serious to swine breeders, resulting in a serious 
problem in the breeding industry. This tendency is also noted in advanced 
countries. In order to prevent and treat this disease, antibiotics such as 
sulfa agents or the like are generally used, but in recent years the use 
of antibiotics has been restricted due to the appearance of resistant 
bacteria and the internal persistence of the chemicals. 
For the prevention and treatment of white diarrhea/diarrhea in livestock or 
poultry, live bacterial compositions have been developed for use as agents 
other than antibiotics. In live bacterial compositions, useful live 
bacteria are directly administered to livestock or poultry, where the 
bacteria are retained in the intestine of livestock or antagonize 
enterotoxic bacteria, e.g., Escherichia coli, to eliminate the enterotoxic 
bacteria, during passage of the live bacteria through the intestine. As a 
result, the enterobacteria microflora is improved so that white diarrhea 
or diarrhea of livestock is prevented and treated. In order to exhibit 
this effect, it is necessary that the bacteria be alive. Thus, for 
purposes of enhancing physicochemical stability of the live bacterial 
composition in feed and the living animal body, there are reported 
examples of using spore forming bacteria (cf. Veterinarian and 
Stockbreeders' News, No. 695, page 343, 1979; Journal of Japanese 
Veterinary Association, 30, 645 (1977)), and an example of utilizing 
multi-drug resistant bacteria, taking into account the use in combination 
with antibiotics as feed additives (Bifidobacteria Microflora, 4, 15 
(1985). 
However, few live bacterial agents are known to have excellent stability. 
In addition, the effect is very slow since the mechanism of exhibiting the 
effect is attributted to elimination of harmful bacteria due to antagonism 
against the harmful bacteria. 
On the other hand, there has been proposed a composition for the prevention 
and treatment of white diarrhea/diarrhea in livestock or poultry 
containing as an effective ingredient the enzymatically digested product 
of cell walls of Bifidobacterium thermophilum which is considered to be an 
intestinal bacteria useful for livestock (cf. Japanese Patent Application 
Laid-Open No. 56-108717). It is also reported that among cell wall 
degradation products of bacteria belonging to the genus Bifidobacterium, a 
peptide glycan is the effective ingredient (cf. Japanese Patent 
Application Laid-Open No. 62-265231). The mechanism of its activity is 
quite dissimilar to that of conventional live bacterial agents since the 
activity is thought to be exhibited due to activation of the immune system 
of livestock thereby enhancing the ability of livestock to protect itself 
from infection. These reports are concerned with a method which comprises 
culturing useful bacteria inherently present in the intestine of 
livestock, withdrawing the effective component alone and again returning 
the effective component to the host. The Bifidobacterium used in this 
method is a strict anaerobe which requires anaerobic operations for 
incubation of bacterial cells so that the operations become complicated. 
In addition, expensive raw materials such as vitamins, etc. are required 
for its growth and cell yield is poor, resulting in high costs. 
The agents for the prevention and treatment of white diarrhea/diarrhea by 
activation of the immune system of livestock are free of the problems of 
drug resistant bacteria or internal persistence of drugs, unlike 
antibiotics, and are more highly effective than live bacterial agents. 
However, such agents are less practical because of difficulty in handling 
bacteria and the poor yield of bacterial cells. 
SUMMARY OF THE INVENTION 
Accordingly, one object of the present invention is a composition for the 
prevention and treatment of white diarrhea/diarrhea in livestock, poultry, 
pet animals, etc. which can be readily produced on an industrial scale, 
can be easily handled and can provide excellent effects by activation of 
the immune system. 
These and other objects which will become apparent from the following 
specification have been achieved by the present composition which contains 
at least one of the following: (1) sterilized cells of bacteria belonging 
to the genus Bacillus, the genus Brevibacterium, the genus 
Corynebacterium, the genus Escherichia, the genus Lactobacillus, the genus 
Streptococcus or the genus Streptomyces, (2) a cell homogenate obtained by 
mechanical homogenization or enzymatic decomposition of the cells of the 
above named bacteria or (3) a cell wall component-containing fraction 
obtained by fractionating the above described cell homogenate.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
It has now been discovered that substances containing cell wall components 
of bacteria belonging to the genus Bacillus, the genus Brevibacterium, the 
genus Corynebacterium, the genus Escherichia, the genus Lactobacillus, the 
genus Streptococcus or the genus Streptomyces, which are not intestinal 
bacteria derived from livestock but which can grow under aerobic 
conditions, have the effect of preventing and treating white diarrhea or 
diarrhea of livestock, poultry, pet animals, etc. 
That is, the present invention relates to a composition for the prevention 
and treatment of white diarrhea or diarrhea in livestock, poultry, pet 
animals, etc. comprising at least one of (1) sterilized cells of bacteria 
belonging to the genus Bacillus, the genus Brevibacterium, the genus 
Corynebacterium, the genus Escherichia, the genus Lactobacillus, the genus 
Streptococcus or the genus Streptomyces, which are not intestinal bacteria 
derived from livestock but which can grow under aerobic conditions, (2) a 
cell homogenate obtained by mechanical homogenization or enzymatic 
decomposition of cells of these bacteria and (3) a cell wall 
component-containing fraction obtained by fractionating of the cell 
homogenate. 
Examples of the bacteria which can be used in the composition of the 
present invention for the prevention and treatment of white 
diarrhea/diarrhea include bacteria belonging to the genus Bacillus such as 
Bacillus subtilis ATCC 13952, etc.; bacteria belonging to the genus 
Brevibacterium such as Brevibacterium lactofermentum ATCC 13869, etc.; 
bacteria belonging to the genus Corynebacterium such as Corynebacterium 
glutamicum ATCC 13060, etc.; bacteria belonging to the genus Escherichia 
such as Escherichia coli ATCC 8739, etc.; bacteria belonging to the genus 
Lactobacillus such as Lactobacillus acidophilus ATCC 4356, etc.; bacteria 
belonging to the genus Streptococcus such as Streptococcus thermophilus 
ATCC 19987, etc.; bacteria belonging to the genus Streptomyces such as 
Streptomyces tanashiensis ATCC 15238, etc. 
For incubation of these bacteria, any nutrient sources are usable so long 
as these bacteria can assimilate them. Conventional media may be used, 
appropriately supplemented with carbohydrates such as glucose, sucrose, 
etc.; alcohols such as ethanol, glycerol, etc.; organic acids such as 
acetic acid, propionic acid, etc.; carbon sources such as soybean oil, 
etc. or a mixture thereof; nitrogen-containing inorganic or organic 
nutrient sources such as yeast extract. peptone, meat extract, corn steep 
liquor, ammonium sulfate, ammonia, etc.; inorganic nutrient sources such 
as phosphates, magnesium, iron, manganese, potassium, etc.; and vitamins 
such as biotin, thiamine, etc. Incubation may be carried out in a nutrient 
medium within a pH range of about 4.0 to 9.5 at 20.degree. to 40.degree. 
C. for 12 hours to 5 days under aerobic conditions. 
The cells obtained by the incubation are isolated from the medium and 
sterilized by a heat treatment, for example. The sterilized cells may be 
used as they are. However, it is preferred to use homogenized cells. The 
cells to be homogenized may be either vial cells or sterilized cells. The 
method of homogenization may be a mechanical method or a method utilizing 
an enzyme. As an example of the mechanical method, the cells are 
homogenized using, e.g., a French press, etc. under a pressure of about 
800 to 2000 kg/cm.sup.2. Alternatively, the cells may also be homogenized 
with an ultrasonic homogenizer. In the case of homogenizing the bacterial 
cells using an enzyme, the cultured cells or mechanical homogenate of the 
cultured cells are suspended in physiological saline, etc. and an enzyme 
capable of lysing the cell wall is added to the suspension to degrade the 
cell wall. The enzyme used for this purpose may be any enzyme capable of 
degrading the cell wall. Representative examples of the enzyme include 
lysozyme, protease, etc. Known methods and conditions for treatment with 
the enzyme may be used. In either the mechanical method or the enzyme 
method, it is preferred that the degree of cell homogenization be about 
20%, more preferably about 60%. The degree of homogenization can be 
determined in terms of the degree of reduction in turbidity of the 
suspension at a wavelength of 660 nm. In order to increase the degree of 
homogenization, it is preferred to use the mechanical method and the 
enzyme method in combination. 
The cell homogenate may also be fractionated and the isolated fraction 
containing cell wall components may be used. Fractionation may be effected 
by simply centrifuging the cell homogenate to remove insoluble materials. 
Furthermore, known methods for fractionating proteins, by molecular 
weight, for example, ultrafiltration, gel filtration, etc. may also be 
utilized. 
The composition of the present invention, that is, the prepared cells, 
homogenates thereof or fractions containing the cell wall components, may 
be given orally to livestock, poultry, pet animals, etc. in the form of a 
liquid, generally an aqueous liquid; or if necessary and desired, the 
composition may be dried to a powdery form, which is added to feed for 
livestock, poultry, pets, etc. The composition may be administered at any 
time, however the effect of the composition for the prevention and 
treatment of white diarrhea/diarrhea during lactation period is enhanced 
when the composition is given on consecutive days for 1 to 2 weeks after 
birth. A daily dose is about 10 mg to about 50 g, preferably about 100 mg 
to about 2 g, on a dry weight basis. During the weaning period or in 
poultry, the composition may be added to feed in about 0.01 to 5%, 
preferably about 0.05 to 1%, on dry weight basis. 
In the present invention, livestock includes swine, cow, horse, goat, 
sheep, etc.; poultry includes fowls such as chicken, etc.; and pets 
include dogs, cats, etc. 
Other features of the invention will become apparent in the course of the 
following descriptions of exemplary embodiments which are given for 
illustration of the invention and are not intended to be limiting thereof. 
EXAMPLES 
Example 1 
(1) Preparation of bacterial cells 
In a flask of a 500 ml volume was charged 50 ml of medium (pH 7) containing 
1.0 g/dl of glucose, 1.0 g/dl of yeast extract, 1.0 g/dl of peptone, 0.5 
g/dl of (NH.sub.4).sub.2 SO.sub.4, 0.3 g/dl of K.sub.2 HPO.sub.4, 0.1 g/dl 
of KH.sub.2 PO.sub.4 and 0.05 g/dl of MgSO.sub.4.7H.sub.2 O followed by 
sterilization at 115.degree. C. for 15 minutes. Bacillus subtilis ATCC 
13952, Brevibacterium lactofermentum ATCC 13869, Corynebacterium 
glutamicum ATCC 13060, Escherichia coli ATCC 8739, Lactobacillus 
acidophilus ATCC 4356, Streptococcus thermophilus ATCC 19987 and 
Streptomyces tanashiensis ATCC 15238 were precultured in bouillon agar 
medium at 30.degree. C. for one day. A loopful of each culture, 
respectively, was inoculated into separate sterilized medium followed by 
shake culture at 30.degree. C. for 24 hours. After completion of the 
culture, each medium was centrifuged to collect the cells. The respective 
cells were suspended in physiological saline of the same volume as that of 
the medium followed by a heat treatment at 100.degree. C. for 10 minutes. 
Each suspension was again centrifuged to collect the cells. 
For control, Bifidobacterium thermophilum ATCC 25525 (derived from swine 
intestine) and Clostridium butylicum (derived from human intestine) were 
used as strict anaerobes. In a flask of a 300 ml volume was charged 280 ml 
of medium (pH 7.0) containing 1.0 g/dl of glucose, 1.0 g/dl of yeast 
extract, 1.0 g/dl of peptone, 0.02 g/dl of MgSO.sub.4.7H.sub.2 O, 0.05 
g/dl of Tween 80, 0.2 g/dl of ammonium acetate, 10% tomato juice filtrate 
and 1.0 g/dl of CaCO.sub.3 followed by sterilization at 115.degree. C. for 
15 minutes. The control bacteria were subjected to stab culture in the 
same agar medium and then inoculated into the sterilized medium followed 
by culturing at 37.degree. C. for 2 days under anaerobic conditions. After 
completion of the culture, each medium was centrifuged to collect the 
cells. The respective cells were suspended in physiological saline of the 
same volume as that of the medium. The suspension was heat treated at 
100.degree. C. for 10 minutes and again centrifuged to collect the cells. 
The weight of the wet cake obtained per 100 ml of medium after the culture 
is shown in Table 1. 
TABLE 1 
______________________________________ 
Weight of Wet Cake 
g/100 ml 
______________________________________ 
Bacillus subtilis ATCC 13952 
4.2 
Brevibacterium lactofermentum ATCC 13869 
5.6 
Corynebacterium glutamicum ATCC 13060 
5.3 
Escherichia coli ATCC 8739 
4.5 
Lactobacillus acidophilus ATCC 4356 
0.8 
Streptococcus thermophilus ATCC 19987 
0.5 
Streptomyces tanashiensis ATCC 15238 
2.1 
Bifidobacterium thermophilus ATCC 25525 
0.2 
Clostridium butylicum 0.3 
______________________________________ 
(2) The respective cells (wet cakes) prepared in (1) were suspended in 25 
mM phosphate buffer (pH 7.0) at 10 wt %, respectively. The cell suspension 
was put into a stainless steel bottle (50 ml volume) and treated for 15 
minutes at an oscillation frequency of 20 kHz and an output of 200 W using 
a ultrasonic homogenizer (Model UR-200 P, manufactured by Tomy Seiko Co., 
Ltd.). After treatment, the homogenate was further centrifuged to obtain a 
fraction containing the cell wall components. 
(3) Enzyme degradation product 
To 25 mM phosphate buffer (pH 7.0) containing as a solid content 10 wt % of 
the mechanical cell homogenate prepared in (2) were added 0.01 wt % of 
albumen lysozyme (manufactured by Sigma Co.) and 0.02 wt % of actinase 
(manufactured by Kaken Pharmaceutical Co., Ltd., 7000 units). The mixture 
was treated at 37.degree. C. for 12 hours and then heat-treated at 
100.degree. C. for 2 minutes to inactivate the enzyme. 
(4) Effect of immunological activation using mouse spleen cells 
RPMI 1640 medium was supplemented with 10% immobilized fetal calf serum and 
5.times.10.sup.-5 M 2-mercaptoethanol, and was then filtered and 
sterilized. Spleen cells prepared from the spleen of a DBA/2 strain female 
mouse 10 weeks old were suspended in the sterilized medium at 
2.5.times.10.sup.6 cells/ml. The cells and enzyme degradation product 
prepared in (1) and (3), or cells of Kluyvera citophilia IFO 8193 or 
Beijerinckia indica ATCC 9037 and the enzyme degradation product prepared 
in a manner similar to (1) and (3) were added to the suspension, 
respectively, in the respective concentrations followed by culturing at 
37.degree. C. for 4 days in a 5% CO.sub.2 incubator. After culturing, the 
supernatant was diluted 1000-fold with 0.01M Tris-hydrochloride buffer (pH 
8.1) containing 1% bovine serum albumin. Thereafter, the total amount of 
mouse IgM produced in each medium was measured by ELISA to determine the 
effect of immunological activation of each system. As a standard substance 
for assaying the effect of immunological activation, lipopolysaccharide 
(LPS) derived from pathogenic Escherichia coli was used. The results are 
shown in FIGS. 1-12. 
FIGS. 1-12 show the relationship between the sterilized cells from each 
genus, enzyme degradation products thereof and mouse IgM antibody 
production. In the figure, the abscissa indicates the addition 
concentration (.mu.g/ml) and the ordinate indicates the concentration 
(.mu.g/ml) of total mouse IgM produced in the culture broth of spleen 
culture cells. Broken lines and solid lines show where the sterilized 
cells were added and where the enzyme degradation products were added, 
respectively. 
As is clear from FIGS. 1-12, the antibody productivity of Bacillus subtilis 
ATCC 13952, Brevibacterium lactofermentum ATCC 13869, Corynebacterium 
glutamicum ATCC 13060, Escherichia coli ATCC 8739, Lactobacillus 
acidophilus ATCC 4356, Streptococcus thermophilus ATCC 19987 and 
Streptomyces tanashiensis ATCC 15238 according to the present invention is 
higher than that of Bifidobacterium thermophilum or Clostridium butylicum 
which are strict anaerobes derived from the intestine of livestock, and 
higher than that of Kluyvera citophilia or Beijerinckia indica which are 
aerobes, in the control groups. 
In addition, it can be seen that the antibody productivity of the enzyme 
degradation products according to the present invention is higher than the 
antibody productivity of the sterilized cells. The enzyme degradation 
products all show a high activity exceeding the maximum value (150 g/ml) 
of the antibody production in LPS, indicating that the enzyme degradation 
products possess excellent immune activation properties. In contrast, no 
activity greater than LPS was noted in the control groups. As is also 
clear from FIGS. 1-12, there is no expectation that antibody production in 
these control groups could be increased, even if the additive 
concentration is increased. 
(5) Effect on white diarrhea or diarrhea in piglets during lactation 
Sixty three (63) piglets during lactation were divided into 9 groups, each 
group being 7. One group was used as the control (non-administered) group. 
In the other eight groups, a suspension daily prepared by suspending 200 
mg of dry powders of the enzyme degradation products of cells prepared as 
in (4) in 3 ml of water was orally administered once a day for 7 
consecutive days from birth. 
For 20 days from Day 8 to Day 27 after birth, the piglets were observed to 
determine whether white diarrhea or diarrhea had occurred. As shown in 
Table 2, the incidence of white diarrhea or diarrhea was low and the 
average body weight increase was high in all of the groups administered 
the enzyme degradation products of the bacteria of the present invention, 
as compared to the control group and to the groups administered the enzyme 
degradation products of Bifidobacterium thermophilum. 
TABLE 2 
______________________________________ 
Total Days Body Weight 
Total with White Increase 
Administered 
Days Diarrhea or on Day 27 
Bacteria Observed.sup.1 
Diarrhea.sup.2 
(kg/pig) 
______________________________________ 
Control (none) 
.sup. 132.sup.3 
84 4.2 
Bacillus subtilis 
140 32 6.5 
ATCC 13952 
Brevibacterium 
140 25 6.8 
lactofermentum 
ATCC 13869 
Corynebacterium 
140 28 6.5 
glutamicum 
ATCC 13060 
Escherichia coli 
140 23 6.3 
ATCC 8739 
Lactobacillus 
140 46 5.9 
acidoiphilus 
ATCC 4356 
Streptococcus 
140 36 6.1 
thermoiphilus 
ATCC 19987 
Streptomyces 
.sup. 124.sup.4 
50 5.0 
tanashiensis 
ATCC 15238 
Bifidobacterium 
140 58 4.8 
thermophilum 
ATCC 25525 
______________________________________ 
.sup.1 The number (7) of animals tested .times. the number (20 days) of 
days observed = total days observed 
.sup.2 Total days when white diarrhea or diarrhea was observed during the 
total number of days of observation 
.sup.3,4 One pig each was dead. 
(6) Twenty eight (28) piglets during lactation were divided into 4 groups, 
each group being 7. One group was used as a control (non-administered) 
group. In the other three groups (administered groups), a suspension daily 
prepared by suspending 200 mg of dry powders of the heat-treated cells of 
Brevibacterium lactofermentum ATCC 13869 and its mechanical homogenate, 
also its enzyme degradation products and the fraction thereof prepared as 
in (1), (2) and (3) in 3 ml of water was orally administered once a day 
for 7 consecutive days from birth. 
For 20 days from Day 8 to Day 27 of the birth, the piglets were observed to 
determine if white diarrhea or diarrhea had occurred. As shown in Table 3, 
the incidence of white diarrhea or diarrhea was low and the average body 
weight increase was high in all of the groups in the present invention, as 
compared to the control group. 
In the administered groups, the effect of preventing white diarrhea or 
diarrhea was higher in the groups administered with the mechanical 
homogenate and its enzyme degradation products, than in the group 
administered with the heat-treated cells. 
TABLE 3 
______________________________________ 
Total Days Body Weight 
Total with White Increase 
Administered Days Diarrhea or 
on Day 27 
Bacteria Observed.sup.1 
Diarrhea.sup.2 
(kg/pig) 
______________________________________ 
Control (none) 
.sup. 132.sup.3 
84 4.2 
Heat-treated cells 
140 46 5.4 
Mechanical 140 30 6.7 
homogenate of cells 
Enzyme degradation 
140 25 6.8 
products of cells 
______________________________________ 
.sup.1 The number (7) of animals tested .times. the number (20 days) of 
days observed = total days observed 
.sup.2 Total days when white diarrhea or diarrhea was observed during the 
total number of days of observation 
.sup.3 One pig was dead. 
(7) Feeding test of calves by addition to feed 
Twelve (12) male calves of the Holstein species, one week old after birth, 
were divided into 3 groups, one group being a control (non-administered) 
group. In the other two groups, assorted feed for calves during weaning 
prepared by adding 0.1 wt. % and 1 wt. % of dry powders of the enzyme 
degradation products of Brevibacterium lactofermentum ATCC 13869 prepared 
according to (4) was administered (administered groups) and the calves 
were suckled (lactated) for 3 weeks. During the test period, the incidence 
of diarrhea and soft feces was observed and the body weight increase was 
measured. 
As shown in Table 4, the incidence of diarrhea and soft feces was low and 
the body weight increase was good in the administered groups, as compared 
to the control group. Further in the administered groups, the 1% 
administered group showed somewhat better results than in the 0.1% 
administered group. 
TABLE 4 
______________________________________ 
Average Body 
Incidence of Diarrhea 
Weight Increase 
of Soft Feces (%)* 
(kg/calf) 
______________________________________ 
Control 82 10 
1% Administered 
46 16 
Group 
0.1% Administered 
58 15 
Group 
______________________________________ 
##STR1## 
Obviously, numerous modifications and variations of the present invention 
are possible in light of the above teachings. It is therefore to be 
understood that within the scope of the appended claims, the invention may 
be practiced otherwise than as specifically described herein.