Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
i`(l~ll'7~i
The present invention relates to a novel Antiobiotic T-42082 and a
method for the production thereof.
In search of new antibiotics, microorganisms were isolated from a
number of soil samples and separated and screened for their metabolites. As
a result, it was found, for instance, that certain microorganisms produced a
new antiobiotic, that said microorganisms belonged to the genus Streptomyces
and that by cultivating such a microorganism in an appropriate culture medium
it was possible to cause said microorganism to accumulate in the broth said
antibiotic which was found to be active against gram-positive bacteria, acid-
fast bacteria and certain fungi, such as Candida albicans and Piricularia
oryzae. The antiobiotic was isolated and, based on its physical, chemical
and biological characteristics, it was confirmed that said antibiotic was a
new polyether-type antiobiotic. The antibiotic was named Antibiotic T-42082.
In the method of the present invention there is employed an Anti-
biotic T-42082-producing strain of the genus StrePtomyces. As an example
thereof, there may be mentioned Strain T-42082 which was isolated from soil
samples collected in a mountainous area of Ito City, Shizuoka Prefecture,
Japan in April, 1973.
According to the invention, therefore, there is provided a process
for preparing Antibiotic T-42082 which comprises aerobically cultivating an
Antibiotic T-42082 producing microorganisms of Streptomyces hvgroscopicus
in a liquid nutrient medium until substantial antibiotic activity accumulates
in the resultant medium and then recovering the Antibiotic T-42082 from said
medium, either as such or in the form of a pharmaceutically acceptable salt
thereof.
The invention also provides the new Antibiotic T-42082 and its
pharmaceutically acceptable salts, said Antibiotic T-42082 having the follow-
ing properties:
(l) Melting point: 120-122C.
(2) Elemental analysis: Found ~ C, 62.73; H, 9.23; 0, 26.01;
C, 62.74; H, 9.36; 0, 27.03 t%)
(3) Molecular weight, 859 (by osmometry):
C~
.
lV91~
(4) Ultraviolet absorption spectrum; No characteristic absorptions;
(5) Infrared absorption spectrum: The dominant absorptions ~wave-
numbers) measured in KBr disc method being as follows: 2940, 1700, 1462,
1383, 1085, 972 cm 1;
~ 6) Thin-layer chromatography: silica gel, ascending method,
detected as a brown spot by a spray of 10% aqueous H2S04;
Rf values as follows:
Solvent system Rf
Ethyl acetate-benzene ~1:1) 0.52
Ethyl acetate 0.73
Benzene-acetone (9:1) 0.18
Benzene-acetone (1:1) 0.88
Chloroform-methanol (19:1) 0.87
Chloroform-ethyl acetate (2:3) 0.62
Plate: silica gel plate ~Kieselgel 60, Merck, Germany)
C7~ Color reactîons:
Color reagent
Sulfuric acid Positive ~brown)
An~line-phthalic acid Positive ~indigo blue)
~anillin-sulfuric acid Positive (violet)
Molisch Negative
Dragendorff Positive ~light orange)
Barton Negative
Molybdenic acid Negative
Benzidine Negative
Ninhydrine Negative
Alkaline potassium permanganate Negative
C8~ Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate, ethyl
ether, chloroform, benzene and carbon tetrachloride, sparingly
soluble in cyclohexane; and very sparingly soluble in water and
petroleum ether;
-- 2 --
~09117$
when prepared by the above defined process or by an obvious chemical equiva-
lent thereof.
The following are some characteristics of the strain as deposited
in various culture collections.
a) Morphological characteristics
From a well-branched vegetative mycelium extends an aerial mycelium
measuring about 1 ~, monopodially branched. At the ends of the side branches
thus formed are chains of closed spiral spores. The spore is elipsoidal to
cylindrical C0.7 - 1.0 ~ x 0.9 - 1.4 ~), the spore configuration being
verrucose Cwarty). No evidence of other special organs such as spherical
sporangium, flagella, sclerotium, etc. are observed.
b) Cultural characteristics
The characteristics displayed by the strain on various media are
set forth in Table 1. Unless otherwise specified, the observations were made
after 2 weeks' incubation at 28C.
- 2a -
1(J'i~11`7~
~ r~
P '~ 3 1 `~ n 3 ~
~ _
~ ~ ~., I ~ a ~ j !f ~,
!, ' ' ~ f : `
1091175
c) Physiological ch~racteristics
l, Temperature range for growth
10 C
+
++
24 +++
28 +++
+++
34 +++
37 +++
+
- : No growth + : Doubtful growth
: + : Growth ++ : Good growth
+++ : ~uxuriant growth
2. ~iquefaction of gelatin: Positive
3. Hydrolysis of starch: Positive
4. Peptonization of skim milk: Positive
Coagulation of skim milk : Negative
5. Production of melanoid pigment
Tyrosine-agar : Negative
P.eptone-yeaSt extract-iron-agar : Negative
6. Assimilation of carbon sources (Pridham & Gottlieb agar)
See Table 2.
-- 4 --
. . . . ~ ., .
. ': ' .
" 1091~7~
Table 2
i-Inositol +
D-mannitol +
D-xylose +
L-arabinose +
D-glucose +
D-fructose +
Rhamnose +
Sucrose +
Raffinose +
Control (not added)
- ; no growth; +; growth
The above characteristics indicate clearly that strain
T 'l2082 belongs to the genus ~treptom~ces. Comparison of the
above characteristics with the descriptions of many Strepto-
m~ces species in S.A, Waksman, ~he Actinomycetes, Vol.2(1961),
R. HUtter, Systematik der Streptomyceten (1967~, International
Streptomyces Project (ISP) and other literature shows that
the present strain has much in common with Streptom,yces
h~groscopicus Waksman and Henrici (1948) so far as the
characteristics important for the taxonomic identification
of species are concerned. ~he inventors have relegated the
strain to the species Streptom~ces h~roscopicus and named
it StrePtom~ces h~groscopicus ~-42082.
Streptom~ces h~roscopicus ~-42082, which is exploited
according tothe present invention has been deposited at the
Institute of Microbiological ~echnology of the Agency of
Industrial ~echnology, the Institute for Fermentation, Osaka
.~
-- 5 --
.
- 1(J9117$
and American Type Culture Collection under the serial or
accession number of ~RM-P-2691, IF0-13609 and ATCC 31080,
respectively.
~ he new strain was submitted to the American ~ype
Culture Collection in 12301 Parklawn Drive, Rockville
Maryland 20852 on August 6, 1976 and identified as strePt
m~ces ~y~roscopicus ~-42082 and assigned the ATCC number
31080. ~he permanency of the deposit and the ready accesi-
bility to the deposit by the public are afforded inthe
event the patent is granted. Access to the culture is
available during the pendency of the application under
Rule 14 and 35 U.S.C, 112. All restrictions on the avail-
ability of the culture deposited to the public will be
irrevocably removed upon the granting of the patent,
Although the T-42082 strain has been described above,
it is well known that characteristics of actinomycetes are
not constant but are readily varied by spontaneous or
artificial mutation and it should be understood that the
strains of microorganisms employable according to the
present invention include all the strains which belong to
the genus Streptom~ces and which are able to elaborate
Antibiotic ~-42082.
lhe cultivation according to the present invention
is carried out by growing any of said strains in a medium
containing nutrients which may be utilized by the particular
strain. As to medium ingredients, the carbon source may
for example be glucose, starch, glycerin, dextrin, sucrose,
millet jelly, molasses or/and so forth. As the nitrogen
, . . . .
. ' ' ,
~O~il7S
source, there may be utilized meat extract, dried yeast,
yeast extract, soybean flour, corn steep liquor, wheat
embryos, cotton seed meal, ammonium sulfate, ammonium
nitr~te and so forth. If necessary, there may be added
inorganic salts such as calcium carbonate, sodium chloride,
potassium chloride, phosphates, etc. as well as organic
and inorganic materials which would assist in the growth
of the particular microorganism or in the elaboration of
Antibiotic ~-42082.
~ he cultivation may be carried out in generally the
same manner as in the production of antibiotics in general,
submerged aerobic culture in a fluid medium being particularly
desirable. ~he cultivation is carried out aerobically at
an appropriate pH level which is within the range of pH 5
to 9, preferably in the range of pH 6 to 8, and at a
temperature in the range of 15 to 40C, preferably 23 to
34C, although in many instances the cultivation is prefer-
ably carried out in the neighborhood of 28C. ~he incuba-
tion time normally ranges from 2 to 12 days, preferably 3
to 7 days.
After completion of cultivation, Antibiotic ~-42082
is separated and harvested from the broth by conventional
procedures which are normally employed for the harvest of
microbial metabolites from culture broths, either alone or
in a suitable combination ~hus, by taking advantage of
its solubility in neutral lipids, the desired antibiotic
may be separated by extraction with various organic solvents,
phasic transfer, recrystallization, chromatography on various
-- 7 --
. ~ -
lO91i`75
adsorbents and so ~orth.
~ he present antibiotic produced by the cultural methodthus described hereinbefore occurs in both the liquid phase
of the broth and the grown cells. ~herefore, the following
procedures, for example, may be exploited with advantage.
Thus, with the addition of a water-miscible solvent such
as methanol, ethanol, acetone or the like, the broth is
filtered and, after the solvent has been distilled off from
the filtrate, the aqueous solution is adjusted to pH 4-9.
~he solution is then extracted with a solvent hardly
miscible with water, such as ethyl acetate, butyl acetate,
chloroform or the like. ~hen, following removal of the
solvent by distillation, the residue is crystallized from
a solvent system such as methanol-water, ethanol-water,
acetone-water or the like, or with a solvent hardly miscible
with water, such as ethyl ether, chloroform or carbon
tetrachloride,
Alternatively, the broth is filtered together with a
filter aid and the filtrate is extracted with ethyl acetate,
chloroform or the like. The cells are also extracted with
methanol, ethanol, acetone or the like. ~he filtrate and
cell extract are pooled and, after the solvent has been
distilled off, the residue is crystallized from a solvent
such as that described. Where the broth is rich in impuri-
ties, the broth may be passed through a styrenic adsorbent
resin or chromatographed on silica gel, alumina or other
carrier material. ~hen, the eluate free from the contami-
nants is concentrated under reduced pressure and the residue
109~17S
is crystallized from said solvent.
In the above procedure, crystals of Antibiotic ~-42082
can be obtained in good yield. Antibiotic T-42082 may be
isolated as crystals of its salts, e.g sodium, potassium,
lithium, ammonium and other salts.
The following are the physical and chemical properties
of Antibiotic T-42082 and of its sodium salt, which are
obtainable according to the procedures set forth herein-
after in Example 1.
~I) The free form of Antibiotic T-42082
(1) Color and appearance: Colorless needles
(2) Melting point: 120-122C
(3) Elemental analysis: ~ound 0 C, 62.73; H, 9.23; 0,26.01;
~ C, 62.74; H, 9.36; 0, 27.03(%)
(4) Molecular weight: 859 (by osmometry)
(5) Ultraviolet absorption spectrum: No characteristic
absorptions
(6) Infrared absorption spectrum: The spectrum (KBr) is
given in ~ig l. ~he dominant absorptions (wave-numbers)
are as follows: 2940, 1700, 1462, 1383, 1085, 972 cm 1
(7) ~hin-layer chromatography: silica gel, ascending
method, detected as a brown spot by a spray of 10 %
aqueous H2S04. The Rf values are given in ~able 3.
. . ' " "
' ,
lO9il75
~able ~
Solvent system Rf ___
Free form Sodium salt
.
Ethyl acetate-benzene (1:1) 0 52 0.51
Ethyl acetate 0.73 0.73
Benzene-acetone (9:1) 0.18 0.18
Benzene-acetone (1:1) 0 88 0.89
Chloroform-methanol(19:1) 0.87 0.87
Chloroform-ethyl acetate~2:3) 0.62 0.62
Plate: silica gel plate (Kieselgel 60, ~lerck, Germany)
(8) Color reactions: Refer to ~able 4.
~able 4
Color rea~ent ~ree form Sodium salt
Sulfuric acid Positive(brown) Positive (brown)
Aniline-phthalic acid Positive(indigo Positive(indigo
blue) blue)
Vanillin-sulfuric acid Positive (violet) Positive(violet)
Molisch Negative Negative
Dragendorff Positive(light Positive(light
orange) orange)
Barton ~egative Negative
Molybdenic acid Negative Negative
Benzidine Negative Negative
Ninhydrin Negative ~egative
Alkaline potassium ~egative ~egative
permanganate
(9) Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate,
ethyl ether, chloroform, benzene and carbon tetrachloride;
-- 10 --
1091~7~
sparingly soluble in cyclohexane; and very hardly soluble
in water and petroleum ether,
~ he sodium salt of Antibiotic ~-42082
(1) Color and appearance: Colorless needles
(2) Melting point: 180-182C(with brownish discoloration)
(3) Elemental analysis: Found ~ C, 62,05, H, 8.99;
Na, 2.08; ~ C, 62 27; H, 9.01; ~a, 2.79(%)
(4) Moleculax weight: 871, 878(by osmometry)
(5) Optical rotation (a)D5-4.5+0.5 (in chloroform, c=l.O)
(6) Ultraviolet absorption spectrum: No characteristic
absorption at and over 210m~.
(7) Infrared absorption spectrum: ~he spectrum(KBr) is
given in Fig.2. ~he dominant absorptions (wave-numbers)
are as follows: 2935, 1610, 1461, 1382, 1080, 972 cm 1
(8) NMR: ~he nuclear magnetic resonance spectrum is
reproduced in Fig.3 which attests to the presence of 3
methoxy groups.
(9) Thin-layer chromatography: Refer to Table 3.
(10) Color reactions: Refer to Table 4.
(11) Solubilities:
Soluble in methanol, ethanol, acetone, ethyl acetate,
ethyl ether, chloroform, benzene and carbon tetra-
chloride; sparingly soluble in cyclohexane; and very
hardly soluble in water and petroleum ether.
The biological characteristics of Antibiotic ~-42082
sodium salt are given in ~able 5.
-- 11 --
.. . .
. .
' . . ~ .
~091~7S
Table
Minimal inhibitory
Assay organismconcentration(mcg/m~)
. .
Bacillus subtilis PCI 219 0.31
Bacillus subtilis A~CC 6633 0~31
Bacillus cereus IF0 3466 < 0.1
Bacillus megaterium IF0 121080.2 - 0.31
Bacillus pumilus IF0 3813 0.2
Staphylococcus aureus 209P 0.2
Escherichia coli I~0 3044 > 100
Proteus vulgaris I~0 3045 ~ 100
Pseudomonas aeruginosa I~0 3080> 100
Mycobacterium sp. A~CC 607 10
Mycobacterium smegmatis I~0 3083 0.4
Candida albicans IF0 0583 10
Aspergillus niger I~0 4066 > 50
Penicillium chrysogenum I~0 4626 > 50
Piricularia oryzae P-18 10
The acute oral toxicity (LD50) of Antibiotic T-42082
sodium salt in mice is about 2000 mg/kg, with the ~D50 value
in mice by the intraperitoneal route being 125-250 mg/kg.
~ he above physical and chemical properties suggest
that Antibiotic ~-42082 is a polyether antibiotic compound
containing three methoxy groups.
When the physical and chemical properties of Anti-
biotic ~-42082 are compared with those of known polyether
antibiotics, it is found that Antibiotic X-537A ~The Journal
of American Chemical Society 7~, 5295 (1951)~, Salinomycin
- 12 -
-` lV91175
(Japanese Patent Application Laid Open No.25392/1972),
Antibiotic K-178(Zeitschrift f~r Allgemeine Mikrobiologie
4 236(1964)~, Allgemeine Mikrobiologie 4, 269(1964)~,
Dianemycin (The Journal of Antibiotics 22, 161(1969)),
Antibiotic A-130A (Japanese Patent Publication No.4558/1973),
etc. have characteristic ultraviolet absorption spectra,
whereas Antibiotic ~-42082 has no such characteristic
absorption spectrum. Antibiotic X-206 ~Chemical Communica-
tions (1971), 927~ has no methoxy group. Monensin ~he
Journal of American Chemical Society 89, 5737(1967)),
Nigericin ~Biochemical and Biophysical Research Communica-
tions 33, 29(1968)~ and Grisorixin ~Chemical Communications,
1421(1970)~ contain one methoxy group each. Antibiotic
A-28695 A(Japanese Patent Application Laid Open ~o.68795/
1973) and Antibiotic A-218(Japanese Patent Application Laid
Open No.80793/1973) each have four methoxy groups, and
Antibiotic A-2C4A~he Journal of American Chemical Society
95, 3399(1973)) and Antibiotic K-41 (Japanese Patent Appli-
cation Laid Open No.14692/1974) contain five methoxy groups.
Therefore, the aforementioned antibiotics are clearly
distinct from Antibiotic ~-42082 which has three methoxy
groups. As an antibiotic having three methoxy groups like
Antibiotic ~-42082, there may be mentioned Antibiotic A-
28695 B(Japanese Patent Application Laid Open ~o.68795/I973)
but in view of their differences in melting point, optical
rotation, infrared absorption spectrum and other properties,
these antibiotics are considered to be dissimilar substances.
Based on the above findings, A~tibiotic ~-42082 according
-` 10~:~17S
to the present invention is considered to be a novel compound.
Figure 1 is an infrared absorption spectrum (KBr) of
the free form of Antibiotic T-42082.
Figure 2 is an infrared absorption spectrum (KBr) of
Antibiotic T-42082 sodium salt; and
Figure 3 is a nuclear magnetic resonance spectrum of
Antibiotic T-42082 sodium salt.
As described hereinbefore, Antibiotic T-42082 according
to the present invention inhibits growth of gram-positive bacteria,
acid-fast bacteria and certain fungi.
Therefore, the present antibiotic is of value in the
treatment of infections with such microorganisms. For example,
it can be utilized as a cream or ointment for topical application
containing 0.3 to 1.0 % of the same in the prevention or
treatment of fester of a wound caused by Staphylococcus aureus.
For the purpose of the prevention or treatment of intestine
infection by Staphylococcus aureus, it can also be administered
as tablets, capsules, etc. at a normal dose level of 20 to 200
mg/kg daily or as injections at a daily dose of 1 to 20 mg/kg
per adult human. Since the present antibiotic inhibits growth
of Piricularia oryzae, it may also be employed as an agricultural
aid.
In addition, Antibiotic T-42082 is useful for the pre-
vention and treatment of coccidiosis.
Coccidiosis is an infectious disease caused by para-
sitic protozoa in domesticated fowls and animals, manifestations
of which include diarrhea and nutritional disturbances.
- 14 -
~09~1`75
htrl
poultry, ducks, turkeys, quails, rabbits, goats, sheep and
cattle, for instance, often succumb to the disease and
sustain serious damages,
Various drugs have been employed for the prevention
or treatment of coccidiosis, but they have such drawbacks
as inadequate potency, serious side effects or/and liability
for the emergence of drug-resistance protozoas.
Antibiotic ~-42082, of this invention, has been found
to display positive effects in the prevention and treatment
of coccidiosis, overcoming the drawbacks of the anticoccidial
drugs thus far available.
~ he anticoccidial drug is produced by processing Anti-
biotic T-42082, in the absence or presence of a solid or
liquid diluent, into powders, dusts granules, tablets,
liquids, capsules and so on, or by adding to feed, drinking
water or the like with or without previous dispersion in a
diluent. The said diluent may be any diluent which per se
is physiologically harmless, although it is preferably a
substance that, by itself, may be a feedstuff or a feed
ingredient.
As a solid diluent, there may be mentioned barley
flour, wheat flour, rye flour, corn flour, soybean flour,
soybean cake, rapeseed cake, wheat bran, rice bran, extracted
rice bran, sweet potato flour, potato flour, soybean curd
residue, starch, lactose, sucrose, glucose, fructose, yeast,
spent yeast, fish meal, talc, acid clay, clay and so forth.
As examples of said liquid diluent, there may be mentioned
water, physiological saline, organic solvents which are
lU~il75
physiologically harmless and so forth
In addition to said diluent, there may also be com-
pounded appropriate auxiliary agents such as an emulsifier,
dispersing agent, suspending agent, wetting agent, thickener,
gelling agent, solubilizer and so forth in appropriate
proportions The resultant composition may be further
supplemented with a preservative, fungicide, antibiotic,
enzyme preparation, lactobacillus preparation and so forth,
Moreover, pyrimethamine, sulfa drugs, vitamin preparations,
etc may be further incorporated.
The proper dosage of the anticoccidial drug varies
with the species and breed of domesticated fowl or animal,
its age, the route of dosi~g, symptoms and other factors
~or the prevention of coccidiosis in poultry, for example,
the present drug is desirably given in such amounts that
the poultry will receive about 4.0 to 12 5 mg/kg/day of
Antibiotic ~-42082. For the treatment of the same disease,
the poultry preferably takes about 10 to 30 mg of the anti-
biotic per kg body weight daily, ~o achieve this dose
level, the drug may be added to the feed in such a propor-
tion that the concentration of Antibiotic T-42082 will be
somewhere between about 50 and 300 ppm.
The toxicity of Antibiotic ~-42082 in animals is low.
For example, the acute toxicity of Antibiotic ~-42082 sodium
salt in mice (ID50) is about 2000 mg/kg by the oral route,
or 125 to 250 mg/kg by the intraperitoneal route. ~he
oral toxicity (LD50) in poultry is 1100 mg (423-2860 mg)/kg.
An outstanding characteristic of the anticoccidial
10~11`7~
drug according to this invention is that it is by far safer
than any known anticoccidial drug of the polyether type.
The experimental data shown are in the Experimental
Description which follows Example 1 are illustrative of the
superior effectiveness of the anticoccidial drug.
Example 1
A culture medium prepared by adding 0.5 % of precip-
itated calcium carbonate to an aqueous solution (pH 7.0)
containing 2 % of glucose, 3 % of soluble starchJ 1 % of
raw soybean flour, 1 % of corn steep liquor, 0.5 % of peptone
and 0.3 % of sodium chloride was inoculated with Strepto
myces hygroscopicus T-42082 (FERM P 2691, IFO 13609, ATCC
31080). The inoculated medium was incubated under shaking
at 28C for 48 hours. One liter of the resultant culture
fluid was transferred to a 100-liter tank containing 30 -~
liters of a culture medium prepared by adding 0.5 % of
precipitated calcium carbonate to an aqueous solution
~pH 7.0~ containing 5 % of dextrin, 3 % of raw soybean flour
and 0.1 % of peptone and aerobic stirred culture was carried
out at 28C with sparging at the rate of 30 liters/min. for
140 hours. -
To the resultant broth was added an equal volume
of acetone and after 30 minutes' stirring, the cells were
separated from the broth. The cells were resuspended in
acetone, the volume of which was equal to that of the broth,
and following 30 minutes stirring, the filtrate was separated
and pooled with the filtrate previously obtained. The
- 17 -
lO9i~:`75
filtrate was concentrated under reduced pressure until the
acetone was distilled off and, then, extracted with ethyl
acetate, followed by drying over anhydrous sodium sulfate.
~he ethyl acetate solution was then passed through a column
of 400 m~ activated carbon, which was washed with ethyl
acetate. ~he active fractions were pooled and the solvent
was distilled off. The oily residue was dissolved in
benzene and the solution was passed through a column of
500 m~ silica gel. ~lution was carried out with benzene,
benzene-ethyl acetate (9:1), benzene-ethyl acetate (8:2),
benzene-ethyl acetate (1:1) and ethyl acetate in the order
mentioned. ~he active fractions were pooled and the solvent
was distilled off, whereupon 12.5 g crude powders of Anti-
biotic ~-42082 were obtained. ~he powders were dissolved
in 80 /0 acetone water and, after being adjusted to pH 9.0
with lN-sodium hydroxide, the solution was concentrated
under reduced pressure to remove the acetone. By the above
-procedure were obtained crystals of Antibiotic T-42082
sodium salt. ~he crystals were collected and recrystallized
from acetone water. By this procedure were obtained 4 g
colorless needles of Antibiotic ~-42082 sodium salt. ~he
recrystallization mother fluid was adjusted to pH 5.0 with
lN-hydrochloric acid and concentrated under reduced pressure
to remove the acetone. The resultant crude crystals were
collected and recrystallized from acetone water. ~he pro-
cedure described above provided 2.5 g colorless needles of
free Antibiotic T-42082.
- 18 -
1(~91175
EXPERIMEN~AL
The basal diet composition used in the ~ollowing
experiment is a nutritionally balanced feed and it i5 known
that chicks show quite good growth on it. By way of example,
the chicks grown on this diet in a breeding battery at room
temperature, 26+1C. (40C. within the battery) on an ad
libitum basis for both feed and drinking water will have
body weights in excess of 90 grams on the 9th day after
hatching. ~he test of anticoccidial effect was performed
with 3 or 5 healthy chicks having even body weights in a
stainless steel cage at 26+1C, under 24 hour lighting,
with free access to feed and water and under conditions
precluding microbial infections.
The term "healthy control!! as used hereinafter means
"uninfected, untreated control". All parts are by weight.
EXPERIMENTA~ 1
~ rom the caecal contents of a chick, fresh oocysts
were collected on the 8th day after infection with Eimeria
tenella. After sporulation, the oocysts were artificially
threshed by the method of Doran and Vettering (Journal of
Protozoology 14, 657-662(1967)) to obtain the sporozoides.
On the other hand, using Eagle ~DEM(Gibco, U.S.A.), BK cells
were allowed to multiply well on a glass slip sealed in a
Rayton tube. Then, 3x104/0.3~ of the sporozoides previously
prepared, 0.2 m~ of a test sample (Antibiotic ~-42082 as
dissolved in methanol to a concentration of 1 mg/m~ and,
then, diluted 10-fold with the medium to 10 5 mcg/m~) and
1 5 m~ of medium were injected. Cultivation was carried
- 19 -
109117~
out at 41C for 3 da~s, ~he sporozoides which had entered
the BK cells and the grown and multiplied schizonts were
stained and counted under a microscope, The results are
shown in Table No.l,
_able No.l
Level of addition of Grown schizonts
sample, mcg/m~(-log) 1 2-
O x x
4 ++ _ +++ ++ _ +++
+++ +++
Inoculated control +++ +++
Untreated control
Scoring scheme:
he exfoliation and denaturation of cells (cytotoxicity):
x The exfoliation of cells from the glass surface as
well as a denaturation of cells are clearly in evidence.
2. Inhibition of growth of schizonts: - - +++
- : when the specimen (24x9mm) was examined under a
microscops ~x400(40xlO)), no grown schizont at all
in two rows.
+ : About 5 schizonts in two rows under the microscope.
+ : About one schizonts per field,
++ : About 5 schizonts per field.
+++ : About 10 schizonts per field.
- 20 -
i~)9il~75
''.
EXPERIMEN~A~ 2
~est materials and testing procedure:
(1) Test chicks : White ~eghorn, male, 10 days old at the
start of the experiment.
(2) Coccidium : Eimeria tenel1a; The inoculum size per
bird: 5xlO sporulated oocysts/0.2 -
0.5 m~.
~ est compound: Antibiotic ~-42082
(4) Levels of addition of the test drug: ~he test compound
was added to portions of a compound mash for newly
hatched chicks, ~eed EP-l-A(composition hereinafter in
Example 1), which contained no anticoccidial agent, at
the varying levels of 30, 60, 90 and 120 ppm.
(5) ~esting procedure: The test chicks were bred in
quarters protected against coccidial infections and
after it had been confirmed that they had no other
disease, either, but were healthy, each chick was
weighed. ~hey were classed into groups of three birds
in such a manner that the body weight distributions of
the groups would be substantially identical. ~wo of
the groups were used as "infected control" and "healthy
control", respectively.
After the grouping, each test group of chicks were
placed on the diet containing the test drug, while the
"infected control" and "healthy control" birds were kept
on the drugfree diet.
After 24 hours, excepting the birds in the healthy
control group, all the birds were orally inoculated with
- 21 -
, . .
.
,
10~117S
imeria tenella at a rate of 5x104 sporulated oocysts
per bird.
(6) Scoring scheme: ~ill the end of the test period, each
chick was weighed every morning before feeding and, at
the same time, the drops of blood (h~emorrhage) in its
droppings were counted, ~he chicks were also investi-
gated for deaths. Eight days following the oocyst
inoculation, all the test chicks were autopsied and the
apparent pathological changes of the caeca and the caecal
contents were microscopically examined to evaluate the
effectiveness of the drug.
(7) ~otes to the description in the table.
(a) Relative weight gain =
Percent weight gain for test group
x 100
Percent weight gain for healthy
control group
(b) Pathological change of the caecum:
~ he ~est Procedures for Coccidiosis in Poultry (See
Kiyoshi ~sunoda and Toshio Ishii. ~he Research Society
of Poultry Diseases (1971), p.20)
~he results of post-mortem examinations were scored
according to the following scheme.
he caecum is completely normal.
(+) : ~he caecum retains its normal shape. The contents
are slightly fluid, with a tinge of yellow, The
mucous membrane of the caecum is slightly swollen
and whitish.
- 22 -
10911~75
(+~he caecum is substantially normal in shape. The
; entire mucous membrane is swollen No haemorrhage
is noted in the contents The viscous fluid shows
a yellowish discoloration. Within the mucous
membrane, a few white dot-like necrotic lesions and
blood patches are in evidence.
(+++) : ~he atrophy and deformation of the caecum are
obvious. The caecum extends slightly longer than
the rectum. ~he contents include nothing normal
and, in many cases, the caecum is full of blood
clots or grayish white cheesy denaturation products
~he caecal wall is considerably thickened and
brittle, sometimes left with dot-like patches of
blood. The lesion extends to the base of the caecum
but not to the rectum.
(++++): ~he atrophy and deformation of the caecum are pro-
nounced. Generally the caecum looks like a sausage,
as long as the rectum or shorter. The lesion
extends to about one-third or one-quater o~ the
rectum. Other findings are similar to those
described in (3).
In the above evaluation, where one side of the caecum
had been affected more seriously than the other side, the
findings on the more seriously affected side were recorded.
- 23 -
- .: - ' ' ' ' ,
' ~
lO9il75
. .. . _ __ .
~rl X ~ C~ ~ ~ ~ ~ O ~ C~ ~
0,bD~ ~; O O' O' ~ ~:' .D O; ~D ~
0 _ _ __ _ _ __ __ _
.
o.~ + _ K~ _ ___ __ _ _ h
h + _ _ _ _ _ o
~0~ + ~ ~ ~ ~ ~ ~q
h ~ ~ ~ _ . _ -- h
¢ ~ ~ o ~ ~ O
O ~ Ll\ \ ~ \ ~ ~ ~ ~ r(~ ~ h 1~1
_ _ . _ _ ~ .,_1
h O o o O O ~1 O o O O
C~ __ _ ~ ,
o.~ c~ O o o o ~ o o o o o a~ h
0 ~0 ~
~J~o ~ ~) O O O O O O O O O O 0 o
o _ _ -- s~
~i ~ ~ O O O O O O ~ O O O a) ~
a)~:i . _ _ _ _ h
~ h~ O O O O O O O O O O ~ 0
,D0~ ~) O O O O ~ cs~ r-l' O O O I~
~ O h ~u~ O O O O ~ O ~ O O O 'hl ~
O h ~ 0 ._ ~1 _ bD
~ .q, ~ O O O O O ~D N~ O O O ~ ~
~' ~ ,~
~ ~U ~ O
/ ~ /~ ~ ~0 ~0 ~o ~ ~ ~ p o
,, ~/~ H ~, h h h h h h
-- 24 --
109117S
EXPERIMEN~A~ ~
Heretofore, clopidol, amplolium, decoquinate, etc,
have been employed as anticoccidial drugs and, more recently,
lobenidine has been introduced However, continued administ-
ration of the same anticoccidial drug induced the emergence
of drug-resistant strains. ~he following experiment was
carried out with a wild strain resistant to the conventional
anticoccidial drugs.
~ he oocysts, predominantly of Eimeria tenella, obtained
from a wild strain were used to inoculate chicks at a rate
of lOx105 sporulated oocysts per bird. Other test materials
are the same as those used in Experimental 2. ~he battery
test procedure was repeated.
.
iU9~5
l ~4 O .~ J. J. it 01 ~ u\o
,0 ,W"~ ~ co ~; ~ D o C~ a) ;~; r;
~ D .~
.~ ___ .
0 l O O O O ~ O O
'~4 _ O O_ O O ~ 00
~' ~'o + .~ .~ O O O O O O O
0 ~ J O r-J O O O O O O O
~1 ~ + ___
a) 0 ~ +
~ ~, ++ O .~ ~ N~ O O O ~ ~U
\ 4 h-. _ _
~ 0 h
~ ~ ~ ~ ~ ~ K~ ~
~'h \ N~ ~ ~ K~ N~ N~ ~ ~ ~i
i
4~ U~ o~ O O O O O O O O O
o ~ C' O O O O O O O O O
o' o ~ ~ O O O O O O O O O
~i ~; ~ U\ O O O O O O O O ~
.~ ~ O rt~Lr\
C' O ,~ J' ~ O O O ~ ~-
E~ ~ .~ . . .
o." ~9 o ~ o o
h ~g O O ,~ O O O ,~
~ L~ O .~ O N~ O O O O' O'
O h ~ H
~;~ J O O O' ~`J' O O O
_ ._ _ . .
~ ^ U~ O O
~o,,~ ~D ~ O O O O O
c~ h _
.~ ~ .~ ~.~ ~.~
0 " oo .~ C) ~0 ~ h
h ~ O ~ ~ ~ c) ~
~ ~ ~ ,~io ,a~O
~ ._ .H
-
-- 26 --
~9~1'75
EXPERIMENTAL 4
The same test materials and testing procedure as
those described in Experimental 2 were employed.
Notes to the description in the table:
(a) Average lesion index (degree)
~ he testing procedure used for coccidiosis in poultry
was in conformity with the method of J. Johnson and W. M.
Reid (Experimental Parasitology 28, 33(1970)).
The results of post-mortem examinations performed on
the 6th to 7th day following the infection were recorded
according to the following scheme,
- : The small intestine was completely normal (no patho-
- logical change at all).
+ : ~ed small dots of blood appeared on the serous membrane
in the middle of the small intestine.
Though the small intestine shows neither swelling nor
thickening, it sometimes contains an orange-colored
viscous fluid.
++ : A number of red small patches of blood appear on
the mucous membrane of the small intestine. It is
also full of orange-colored viscous fluid. The
swelling of the small intestine is slight, the
intestinal wall being slightly thickened.
+++ : The intestinal wall is swollen and thickened, The
surface of the mucous membrane become loose and the
intestine is full of blood clots and viscous fluids
++++ : The small intestine is swollen all over and contains
a great many blood clots. Moreover, owing to the
- 27 -
lO9il~5
digestion of red blood cells, the small intestine
assumes a peculiar shade and gives off a rank odor,
The intestinal wall is exceedingly swollen. Dead
-chicks are included in this category.
(b) OPG: Oocysts per gram of feces
~ he mark " - " in the OPG column means that the value
is less than log 3 (not measurable).
Table No.4
Infective Test material: Relative Average OPG
oocysts Antibiotic weight patho- (log)
4T-42082 gain logical - 1 --- ~
(lOxlO / index 4 5 1 6 (
bird)(ppm) (degree) _ _ days
Eimeria125 108.3 _ _ _ _ /
acervulina 62.5 98.4 ++ _ 5. 5.4 /
_ . .
O 90.2 ++++6.6 6. 6.4 /
.... _ , . ,
Eimeria125 97.4 + / / 4.8 4.2
maxima62.5 94.9 + ~ ~ 5. 5.5
0__ 78.8 ++ / ~ 5.2
EXPERIMENTAL ~
~he test materials ? testing procedure, and scheme of
evaluation are similar to those used in Experimental 2 and
4,
- 28 -
10~3~175
Table No,5
Infectlve Test material: Relative Average I OPG
oocystsAntibiotic weight pathologica _ (lo 3)
(lOxlO4/bird) ~-42082 gain i7dex 4 5 lays
. . .,.
Eimeria 120 100.3 _ _ _ _
acervulina90 100.4 _ _ _ _ _
60 _ 92.6 ++__ 5.1 6.0 4,4
_ _ 0 90.2 ++++ _ 6.5 6.9 6.6
Example
: ~en parts of Antibiotic ~-42082, previously milled
to sizes not exceeding 149 ~, was mixed with 90 parts of dry
extracted soybean flour to prepare a 10 k powder of Anti-
biotic ~-42082. l.Ox103 parts of this powder was compounded
with l.Ox106 parts of Feed KP-l-A (composition hereinafter)
(for young broilers) to obtain an avian anticoccidial diet
for broilers ~he concentration of Antibiotic T-42082:
100 ppm).
- 29 -
1091175
Formula of KP-l-A
(for young broilers)
. .
Ingredient Compounding
ratio, weight %
Yellow corn 55.
Wheat bran 5.
Soybean cake 18.0
Fish meal 8.0
Fish soluble 3.0
Alfalfa meal 3.
Tallow complex 5.7
Calcium carbonate 0.9
~ribasic calcium phosphate o.7
Sodium chloride 0.25
A-Food E beads * o.o5
B-Feed S * 0,1
Neo-Mine feed C~ o.o5
Vitamin B12 - T * o.o5
Mixture of active agent and
extracted soybean flour 0.2
.. ~ --- _ .
otal 100.0
: ~rade names (Distributor: ~akeda Chemical Industries,
~td.(Japan))
ExamPle~_ ?
25 parts of Antibiotic ~-42082, previously milled to
sizes not exceeding 149 ~ in diameter, was blended well
with 75 parts of dry extracted soybean flour to prepare a
- 30 -
10911 ~5
powder containing 25 % of Antibiotic ~-42082. 4.0xlO
parts of this powder was compounded with l.Ox106 parts of
~eed KP-l-A to obtain an anticoccidial diet for poultry
~he concentration of Antibiotic ~-42082: 100 ppm).
Example
0.5x103 parts of the 10 % powder obtained in Example
1 was compounded with l.Ox106 parts of Feed KP-l-A to prepare
an anticoccidial diet for poultry ~he concentration of
Antibiotic ~-42082: 50 ppm)
Example 4
10 parts of Antibiotic T-42082, previously milled to
sizes not exceeding 149 ~, was blended with 2 parts of
hydroxypropyl-cellulose and 88 parts of lactose. ~he mixture
was kneaded with 20 parts of water and granulated in a
beater-granulator. ~he wet granules thus obtained were
dried at 35C overnight, whereby 10 k granules 150 to 1,000
in diameter are obtained. l.Ox103 parts of this granular
product was compounded with l.Ox106 parts of ~eed KP-l-B
(composition hereinafter)(for grown broilers) to prepare an
anticoccidial diet for broilers ~he concentration of
Antibiotic ~-42082: 100 ppm~.
- 31 -
,
lO~il`7S
Formula of KP-l-B
(for grown broilers)
. .
Ingredient _ --' Y
Yellow corn 58,0
Wheat bran 5.o
Soybean cake 14,0
Fish meal 6,8
Fish soluble 3.o
Alfalfa meal 3.o
Tallow complex 8.0
Calcium carbonate 0,9
Tribasic calcium phosphate 0,68
Sodium chloride 0.25
A-Feed E beads o~o5
B-Feed S 0.1
Mixture of active agent 0,1
with lactose, etc, 0,12
Total 100.0
Example ~
25 parts of Antibiotic 'r-42082, previously milled
to sizes not exceeding 149 11, were blended with 2 parts of
hydroxypropyl-cellulose and 88 parts of lactose. The
mixture was kneaded with 20 parts of water and granulated
in a beater-granulator. The resultant wet granules were
dried at 55C overnight to obtain 25 % dry granules 150 to
1,000 11 in diameter. 0,4x103 parts of the granules were
-- 32 --
.'" --' -. ' ' ' ' ' " ' , ''
:- ' .
" iO9~1`7S
compounded with l,Ox106 parts of Feed KP-l-B to prepare an
anticoccidial diet for poultry (~he concentration of Anti-
biotic ~-42082 : 100 ppm).
Example 6
2.0x103 parts of the 10 % granules obtained in Example
4 were compounded with 1,Ox106 parts of ~eed KP-l-B to
prepare an anticoccidial diet for poultry (~he concentration
of Antibiotic ~-42082: 200 ppm),
Example 7
1.2x103 parts of the 25 % granules obtained in Example
5 were compounded with l.Ox106 parts of Feed KP-l-B to
prepare an anticoccidial diet for poultry (The concentration
of Antibiotic ~-42082: 300 ppm).
By a procedure similar to that described in ~xample 4,
the 10 /0 granules obtained in Example 4 were compounded with
feed in various ratios to prepare diets containing Anti-
biotic T-42082 in various concentrations.
Amount of the Amount of Concentration of
10 % granules ~eed KP-l-B Antibiotic
obtained in ~-42082
Ex 4
~parts) (parts) (ppm)
0.6 x 103 1.0 x 1o6 60
0.9 x 103 1~0 x 106 go
1.2 ~ 103 1.0 x 1o6 120
1.8 x 103 1.0 x 1o6 180
2.4 x 103 1.0 x 1o6 240
.
