A compound of the formula ##STR1## and its pharmaceutically acceptable salts are disclosed. The compound exhibits antibacterial activity, antimalarial activity, has activity as a growth promotant for ruminants and as an agent in the treatment of swine dysentery. Also provided is a process to produce the novel compound.

DESCRIPTION OF THE INVENTION 
The present invention relates to a novel polyether ionophore antibiotic of 
the formula 
##STR2## 
and its pharmaceutically acceptable salts. 
The shorthand expression Me and Et are utilized above to represent methyl 
and ethyl respectively. 
This compound agent and its salts exhibit activity as an antibacterial 
agent, for the control of swine dysentery, or antimalarial agent and as a 
growth promotant for ruminants. 
Antibiotic X-14766A is the designation given to a crystalline antibiotic 
produced by a Streptomyces organism isolated from a sample of soil 
collected at Playa Blanca, Mexico. Lyophilized tubes of the culture 
bearing the laboratory designation X-14766 were deposited with the U.S. 
Department of Agriculture, Agricultural Research Service, Northern 
Regional Research Laboratories (NRRL), Peoria, Illinois. The culture, 
given the identification number NRRL 11335 by NRRL has been made available 
to the public through NRRL. 
Antibiotic X-14766A is a polyether antibiotic and forms a variety of 
pharmaceutically acceptable salts. These salts are prepared from the free 
acid form of the antibiotic by methods well-known for compounds of the 
polyether type in the art; for example, by washing the free acid in 
solution with a suitable base or salt. Examples of such pharmaceutically 
acceptable basic substances capable of forming salts for the purpose of 
the present invention include alkali metal bases, such as sodium 
hydroxide, potassium hydroxide, lithium hydroxide and the like; alkaline 
earth metal bases, such as calcium hydroxide, barium hydroxide and the 
like; and ammonium hydroxide. Alkali metal or alkaline earth metal salts 
suitable for forming pharmaceutically acceptable salts can include anions 
such as carbonates, bicarbonates and sulfates. 
Examples of organic bases forming pharmaceutically acceptable salts with 
the polyether compounds are lower alkyl amines, primary, secondary and 
tertiary hydroxylower alkylamines such as ethylamine, isopropylamine, 
diethylamine, methyl-n-butylamine, ethanolamine and diethanolamine. 
An amine especially preferred is N-methylglucamine. Salts of 
N-methylglucamine are of special value because of their water-solubility 
which makes them amenable to parenteral use. 
MORPHOLOGICAL CHARACTERISTICS 
The representative strain of Streptomyces X-14766 has the following 
characteristics: 
Sodium chloride tolerance, hydrolysis of casein and reduction of nitrate 
were determined by the methods recommended by Gordon and Smith, J. 
Bacteriol., 66, 41-48, 1953. Starch hydrolysis was determined after growth 
on agar of Actinomyces broth (Difco) with 0.25% soluble starch, and was 
tested by flooding the plates with iodine-KI solution. Gelatin hydrolysis 
was tested according to Skerman, (A Guide to Identification of the Genera 
of Bacteria, The Williams and Wilkins Co., Baltimore, 1967) using 
Actinomyces broth (Difco) with 2% agar in place of meat infusion agar. All 
tests were run at 28.degree. C. 
The standard ISP media of Shirling and Gottlieb were used for the 
description of growth and pigmentation (color determinations were made 
after two weeks of incubation at 28.degree. C.) Carbon utilization was 
also determined by the method of Shirling and Gottlieb (Int. J. Syst. 
Bacteriol, 16, 313-340, 1966). A 24 hour old ISP-1 broth culture was 
homogenized and centrifuged to obtain a washed suspension for inoculation. 
The ability of the organism to grow at 10.degree., 28.degree., 37.degree., 
45.degree. and 50.degree. C. was investigated by inoculating broth of 
ISP-1 (Difco) medium. Cell wall analysis was performed by the method of 
Becker et al. (Applied Microbiol. 12, 421-423, 1964). 
Microscopic examination 
Strain X-14766 produces a substrate mycelium, which does not fragment into 
spores, and an aerial mycelium which eventually forms spore chains. After 
14 days of incubation at 28.degree. C., the spore chains appear spira in 
form with 15 to 20 spores per chain. Spores are spiny and range in size 
form 1.2.times.1.0 .mu.m to 1.1.times.0.73 .mu.m. The cell wall of this 
organism contains the LL-isomer of diaminopimelic acid. These 
characteristics place this organism in the genus Streptomyces (Lechevalier 
et al., Adv. Appl. Microbiol., 14, 47-72, 1971). 
Macroscopic examination 
Table 1 summarizes the amount of growth, degree of sporulation, spore mass 
color and color of reverse substrate mycelium of culture X-14766 on 
various solid media. 
TABLE 1 
__________________________________________________________________________ 
Cultural Characteristics of Strain X-14766 
Amount of growth; Color of reverse-substrate 
Agar medium 
Degree of Sporulation 
Spore mass color.sup.a 
mycelium.sup.a 
__________________________________________________________________________ 
Yeast malt extract 
abundant growth; well 
2ih (dark covert gray) 
2ce (covert tan) mostly; 
(ISP-2).sup.b 
sporulated mostly; 2fe (covert 
2li (covert brown) 
gray) at edge 
Oatmeal (ISP-3).sup.b 
abundant growth; well 
3fe (silver gray) 
2ec (biscuit) 
sporulated 
Inorganic salts 
moderate growth; well 
2fe (covert gray); 
2gc (bamboo), trace of 11/2ie 
starch (ISP-4).sup.b 
sporulated; hydrolyzes 
edges of b (oyster 
(lt. olive) 
starch white) 
Glycerol asparagine 
moderate growth; 
2dc (natural string) 
2gc (bamboo) 
(ISP-5).sup.b 
moderate sporulation 
Czapek-Dox.sup.c 
moderate growth; sparse 
2dc (natural string) 
2ie (light mustard tan) 
sporulation 
__________________________________________________________________________ 
.sup.a The color scheme used was that taken from the Color Harmony Manual 
4th ed., 1958 (Container Corporation of America, Chicago). 
.sup.b Media recommended by the International Streptomyces Project 
(Shirling and Gottlieb, Int. J. System Bacteriol. 16,313-340 1966). 
.sup.c CzapekDox broth (BBL) to which 1.5% agar was added. 
TABLE 2 
______________________________________ 
Carbon Utilization by Strain X-14766 
Growth response* of: 
Carbon Source X-14766 
______________________________________ 
D-Glucose ++ 
D-Xylose + 
L-Arabinose ++ 
L-Rhamnose ++ 
D-Fructose ++ 
D-Galactose +(+) 
Raffinose - 
D-Mannitol ++ 
i-Inositol ++ 
Salicin .+-. 
Sucrose ++ 
Cellulose 
______________________________________ 
*Negative response; +, doubtful response; +, more growth than on carbon 
control but less than on glucose; +(+), growth slightly less than the 
amount on glucose, ++, positive response equal to the amount of growth on 
glucose. 
**Physiological Characteristics: Table 2 reports the results of carbon 
utilization tests by strain X14766 and Table 3 lists diagnostically 
important properties. 
TABLE 3 
______________________________________ 
Metabolic and Morphological Characteristics of Strain X-14766 
Test X-14766 
______________________________________ 
ISP-6 darkening + 
Melanin, ISP-7 variable 
Casein hydrolysis + 
Gelatin hydrolysis .+-. 
Starch hydrolysis + 
NaCl (%) tolerance -5 
Growth range temp (.degree.C.) 
28-45 
ISP-1 darkening + 
Reverse-side pigment none 
Soluble pigment none 
Antibiotic production 
X-14766A 
Nitrate reduction - 
Hygroscopic property variable 
Spore chain form/ spira/15-20 
#spores per chain 
Spore surface spiny 
______________________________________ 
A comparison of the description of strain X-14766 with those of the 
Streptomyces species described in Bergey's Manual (Buchanan and Gibbons, 
ed., Bergey's Manual of Determination Bacteriology, 8th ed., 748-829, 
1974), H. Nonomura's key for classification (J. Ferment. Technol., 52, 
78-92, 1974) and Pridham and Lyons' classification (Dev. Ind. Microbiol. 
10, 183-221, 1969), showed that no known species are identical to X-14766 
based on the following combination of criteria: gray spore mass color, 
spiral spore chain form, spiny spore surface, chromogenic reaction on ISP 
media, 1, 6 and 7, and carbon utilization characteristics. Streptomyces 
malachitorectus and S. malachitofuscus resemble X-14766 based on carbon 
utilization data. Culture X-14766 is closest to S. malachitofuscus because 
of a further similarity in a negative nitrate reduction. Slight 
differences in spore ornamentation (spiny for X-14766 and spiny/hairy for 
S. malachitofuscus) and the property of production of a unique antibiotic, 
X-14766A, justifies our considering X-14766 as a variety of S. 
malachitofuscus, which we named S. malachitofuscus subsp. downeyi. 
The Streptomyces X-14766 described herein includes all strains of 
Streptomyces which form a compound as claimed in the present application 
and which cannot be definitely differentiated from the strain NRRL 11335 
and its subcultures including mutants and variants. The claimed compound 
is described herein and after this identification is known, it is easy to 
differentiate the strains producing this compound from others. 
Streptomyces X-14766 when grown under suitable conditions, produces an 
antibiotic X-14766A. A fermentation broth containing Streptomyces X-14766 
is prepared by inoculating spores or mycelia of the organism producing the 
antibiotic into a suitable medium and then cultivating under aerobic 
conditions. For the production of the antibiotic, cultivation on a solid 
medium is possible but for production in large quantities, cultivation in 
a liquid medium is preferable. The temperature of cultivation may be 
varied over a wide range, 20.degree.-35.degree. C., within which the 
organism may grow but a temperature of 26.degree.-30.degree. C. and a 
substantially neutral pH are preferred. In the submerged aerobic 
fermentation of the organism for the production of antibiotic X-14766A, 
the medium may contain as the source for carbon, a commercially available 
glyceride oil or a carbohydrate such as glycerol, glucose, maltose, 
lactose, dextrin, starch, etc. in pure or crude states and as the source 
of nitrogen, an organic material such as soybean meal, distillers' 
solubles, peanut meal, cotton seed meal, meat extract, peptone, fish meal, 
yeast extract, corn steep liquor, etc. and when desired inorganic sources 
of nitrogen such as nitrates and ammonium salts and mineral salts such as 
ammonium sulfate, magnesium sulfate and the like. It also may contain 
sodium chloride, potassium chloride, potassium phosphate and the like and 
buffering agents such as sodium citrate, calcium carbonate or phosphates 
and trace amounts of heavy metal salts. In aerated submerged culturing 
procedures, an anti-foam agent such as liquid paraffin, fatty oils or 
silicone compounds is used. More than one kind of carbon source, nitrogen 
source or anti-foam source may be used for production of antibiotic 
X-14766A. 
Antibiotic X-14766A has a toxicity (LD.sub.50) in mice of 350 mg/kg (po) 
and 5.75 mg/kg (ip). 
The antibiotic activity of the antibiotic X-14766A is shown by the 
following table: 
TABLE 4 
______________________________________ 
Minimum Inhibitory 
Concentration 
Organism (.mu.g/ml) 
______________________________________ 
Staphylococcus aureus 
ATCC 6538P 0.2 
Sarcina lutea ATCC 9341 0.2 
Bacillus sp. E 
ATCC 27859 0.04 
Bacillus subtilis 
Nrrl 558 0.2 
Bacillus megaterium 
ATCC 8011 0.08 
Bacillus sp. TA 
ATCC 27860 0.08 
Mycobacterium phlei 
ATCC 355 0.2 
Streptomyces cellulosae 
ATCC 3313 0.2 
______________________________________ 
As is indicated above, antibiotic X-14766A and its salts possess the 
property of adversely affecting the growth of certain Gram-positive 
bacteria. It is useful in wash solutions for sanitary purposes as in the 
washing of hands and the cleaning of equipment, floors or furnishings of 
contaminated rooms or laboratories. It is useful also for suppressing the 
growth of sensitive organisms in plate assays and other microbiological 
media. 
Antibiotic X-14766A has also exhibited antimalarial activity with an 
ED.sub.50 of 2.5 mg/kg against Plasmodium bergei in mice. 
In vitro activity against anaerobic bacteria has also been exhibited by the 
antibiotic as shown below. 
TABLE 5 
______________________________________ 
Minimum Inhibitory 
Concentration* 
Organism (.mu.g/ml) 
______________________________________ 
Bacteroides fragilis 
ATCC 12290 0.195 
Clostridium histolyticum 
503-86 0.0098 
Clostridium septicum 
503-34 0.0098 
______________________________________ 
*Two-fold serial broth dilution tests were carried out using trypticase 
soy broth (TSB, BBL) as the test medium. All tests were incubated at 
37.degree. C. overnight under anaerobic conditions. 
Antibiotic X-14766A also exhibited in vitro anticoccidial activity against 
Eimeria tenella at 0.1 ppm. 
Testing for activity against Treponema hyodysenteriae, a cause of swine 
dysentery consisted of inoculation of blood agar plates containing a 
series of two and fourfold dilutions of antibiotic X-14766A and 
ipronidazole, an agent used in the treatment of swine dysentery, with 
tenfold dilutions of each of the T. hyodysenteriae strains (H 78, H 140, H 
179). After 48 hours of incubation at 42.degree. C. in an anerobic 
atmosphere, Minimum Inhibitory Concentrations were recorded as the lowest 
concentrations of compound which completely inhibited the most dilute 
inoculum of each T. hyodysenteriae strain. The results which show 
X-14766A's superior activity are as follows: 
TABLE 6 
______________________________________ 
Method of 
Antibiotic X-14766A 
Ipronidazole 
T.hyo T.hyo Strains (MIC) 
Inoculation 
B78 B140 B179 B78 B140 B179 
______________________________________ 
Steers &lt;0.04 &lt;0.04 &lt;0.04 0.16 0.16 0.16 
Steers &lt;0.04 &lt;0.04 &lt;0.04 0.16 0.16 0.31 
Steers 0.02 0.04 0.02 0.31 0.31 0.31 
Syringe 0.02 0.02 -- 0.63 0.63 -- 
Syringe 0.04 0.04 -- 0.63 0.63 -- 
Syringe 0.04 0.04 -- 0.63 0.63 -- 
______________________________________ 
Administration of antibiotic X-14766A hereafter "Antibiotic" or "Antibiotic 
Compound" prevents and treats ketosis as well as improves feed utilization 
in ruminants or swine. The causative mechanism of ketosis is a deficient 
production of propionate compounds. A presently recommended treatment is 
administration of propionate acid or feeds which preferentially produce 
propionates. It is obvious that encouraging propionate production from 
ordinary feeds will reduce incidence of ketosis. 
It has been found that antibiotic X-14766A increases the efficiency of feed 
utilization in ruminant animals when it is administered orally to the 
animals. The easiest way to administer the antibiotic is by mixing it in 
the animal's feed. 
However, the antibiotic can be usefully administered in other ways. For 
example, it can be incorporated into tablets, drenches, boluses, or 
capsules, and dosed to the animals. Formulation of the antibiotic compound 
in such dosage forms can be accomplished by means of methods well known in 
the veterinary pharmaceutical art. 
Capsules are readily produced by filling gelatin capsules with any desired 
form of the desired antibiotic. If desired, the antibiotic can be diluted 
with an inert powdered diluent, such as a sugar, starch, or purified 
crystalline cellulose in order to increase its volume for convenience in 
filling capsules. 
Tablets of the antibiotic are made by conventional pharmaceutical 
processes. Manufacture of tablets is a well-known and highly advanced art. 
In addition to the active ingredient, a tablet usually contains a base, a 
disintegrator, an absorbent, a binder, and a lubricant. Typical bases 
include lactose, fine icing sugar, sodium chloride, starch and mannitol. 
Starch is also a good disintegrator as is alginic acid. Surface active 
agents such as sodium lauryl sulfate and dioctyl sodium sulphosuccinate 
are also sometimes used. Commonly used absorbents again include starch and 
lactose while magnesium carbonate is also useful for oily substance. 
Frequently used binders are gelatin, gums, starch, dextrin and various 
cellulose derivatives. Among the commonly used lubricants are magnesium 
stearate, talc, paraffin wax, various metallic soaps, and polyethylene 
glycol. 
The administration of the antibiotic compound may be as a slow-pay-out 
bolus. Such boluses are made as tablets except that a means to delay the 
dissolution of the antibiotic is provided. Boluses are made to release for 
lengthy periods. The slow dissolution is assisted by choosing a highly 
water-insoluble form of the antibiotic. A substance such as iron filing is 
added to raise the density of the bolus and keep it static on the bottom 
of the rumen. 
Dissolution of the antibiotic is delayed by use of a matrix of insoluble 
materials in which the drug is imbedded. For example, substances such as 
vegetable waxes, purified mineral waxes, and water-insoluble polymeric 
materials are useful. 
Drenches of the antibiotic are prepared most easily by choosing a 
water-soluble form of the antibiotic. If an insoluble form is desired for 
some reason, a suspension may be made. Alternatively, a drench may be 
formulated as a solution in a physiologically acceptable solvent such as a 
polyethylene glycol. 
Suspensions of insoluble forms of the antibiotic can be prepared in 
nonsolvents such as vegetable oils such as peanut, corn, or sesame oil, in 
a glycol such as propylene glycol or a polyether glycol; or in water, 
depending on the form of the antibiotic chosen. 
Suitable physiologically acceptable adjuvants are necessary in order to 
keep the antibiotic suspended. The adjuvants can be chosen from among the 
thickeners, such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, 
and the alginates. Many classes of surfactants serve to suspend the 
antibiotic. For example, lecithin, alkylphenol polyethylene oxide adducts, 
naphthalenesulfonates, alkylbenzesulfonates, and the polyoxyethylene 
sorbitan esters are useful for making suspensions in liquid nonsolvents. 
In addition many substances which affect the hydrophilicity, density, and 
surface tension of the liquid can assist in making suspensions in 
individual cases. For example, silicone anti-foams, glycols, sorbitol, and 
sugars can be useful suspending agents. 
The suspendable antibiotic may be offered to the grower as a suspension, or 
as a dry mixture of the antibiotic and adjuvants to be diluted before use. 
The antibiotic may also be administered in the drinking water of the 
ruminants. Incorporation into drinking water is performed by adding a 
water-soluble or water-suspendable form of the antibiotic to the water in 
the proper amount. Formulation of the antibiotic for addition to drinking 
water follows the same principles as formulation of drenches. 
The most practical way to treat animals with the antibiotic compound is by 
the formulation of the compound into the feed supply. Any type of feed may 
be medicated with the antibiotic compounds, including common dry feeds, 
liquid feeds, and pelleted feeds. 
The methods of formulating drugs into animal feeds are well-known. It is 
usual to make a concentrated drug premix as a raw material for medicated 
feeds. For example, typical drug premixes may contain from about one to 
about 400 grams of drug per pound of premix. The wide range results from 
the wide range of concentration of drug which may be desired in the final 
feed. Premixes may be either liquid or solid. 
The formulation of ruminant feeds containing the proper amounts of 
antibiotic for useful treatment is well understood. It is necessary only 
to calculate the amount of compound which it is desired to administer to 
each animal, to take into account the amount of feed per day which the 
animal eats and the concentration of antibiotic compound in the premix to 
be used, and calculate the proper concentration of antibiotic compound, or 
of premix, in the feed. 
All of the methods of formulating, mixing and pelleting feeds which are 
normally used in the ruminant feed art are entirely appropriate for 
manufacturing feeds containing the antibiotic compound. 
As has been shown, oral administration of the antibiotic beneficially 
alters the production of propionates relative to the production of 
acetates in the rumen. It may therefore be postulated that the same 
treatment would also benefit monogastric animals which ferment fibrous 
vegetable matter in the cecum since it would be expected that a beneficial 
change in the propionate/acetate ration would occur upon oral 
administration of the instant antibiotic. Horses, swine and rabbits are 
exemplary animals which digest a part of their food by cecal fermentation. 
Determination of volatile fatty acid production 
A bovine, surgically modified with a rumen fistula, is used as a source of 
rumen fluid. The integrity of the rumen is maintained by a rumen cannula 
(Bar Diamond Labs, Parma, Idaho) which is opened in order to obtain rumen 
fluid samples. The animal is fed twice daily an 80% concentrate (AHRES 
ration #39):20% roughage ration. The rumen fluid is obtained prior to the 
A.M. feeding. The rumen fluid is strained through 4 layers of cheesecloth 
into a 1 gallon Nalgene container and is kept under anaerobe quality 
CO.sub.2. One thousand mls of the strained rumen fluid are added to 2000 
mls of an ice cold buffer based upon that specified by Cheng et al., J. 
Dair. Sci., 38, 1225 (1955). The composition of this buffer is as follows: 
______________________________________ 
Na.sub.2 HPO.sub.4 
0.316 g/l MgSO.sub.4 0.112 
KH.sub.2 PO.sub.4 
0.152 CaCl.sub.2 0.038 
NaHCO.sub.3 2.260 FeSO.sub.4 . 7H.sub.2 O 
0.008 
NaCl 0.375 ZnSO.sub.4 . 7H.sub.2 O 
0.004 
KCl 0.375 CuSO.sub.4 . 5H.sub.2 O 
0.002 
______________________________________ 
The buffered rumen fluid is held in a 4 liter separatory funnel. In order 
to help maintain the anaerobic character of the rumen fluid and the 
homogeneity of the buffered rumen fluid, anaerobe quality CO.sub.2 is 
bubbled constantly through the fluid in a separatory funnel beginning 
approximately 1/2" above the separatory funnel stopcock. 
Two hundred and fifty ml Erlenmeyer flasks are used for individual 
fermentations. Each flask to which a compound will be added contains one 
gram of a finely ground 80% concentrate:20% alfalfa hay ration. Flasks 
which are to be used as drug-free controls contain 1.07 grams of the 
finely ground ration. One ml of test compound dissolved in an appropriate 
solvent is added to each flask and allowed to sit for 1/2 to 1 hour. Each 
compound is examined in duplicate flasks at a final concentration of 50 
ppm. Solvent without test compound is added to drug-free control 
fermentation flasks. Monensin at 10 and 50 ppm is used as a positive 
control in all fermentations. 
Eighty grams of buffered rumen fluid are added to each flask containing 
test compound and 85.93 grams are added to control flasks. Flasks to which 
all components have been added are stoppered with a gas collection 
apparatus and left sitting at room temperature until all flasks have been 
completed. Six ml samples are withdrawn from all control flasks as the 0 
time samples. The incubation period and the collection of gas evolved 
during fermentation is initiated 10 minutes after the flasks have been 
placed in a 38.degree. C. water bath. Flasks are incubated with shaking 
(90 oscillatons per minute) for 4 hours. 
The volume of gas produced by each fermentation is measured at 1/2 hour 
intervals. The manometric apparatus for collection of gas and measurement 
of the volume evolved has been described by Trei et al., J. Anim. Sci., 
30, 825 (1970). 
Rumen fluid is poured into 25.times.150 mm glass tubes and left in an ice 
bath for approximately 15 minutes to permit settling of particulate 
matter. The 6 ml quantity of rumen fluid is then added to a 2 ml quantity 
of 25% (W/V) metaphosphoric acid (J. T. Baker) in 13 ml polycarbonate 
centrifuge tubes (Autoclear, IEC). Each tube is stoppered and thoroughly 
mixed. Tubes are left in an ice bath for 30 minutes and then centrifuged 
at 16,000 rpms for 10 minutes in an 874 angle head in an IEC B20 
centrifuge. A 1 ml quantity of the internal standard (0.25% 2-methyl 
valeric acid, Aldrich Chemical Company) is then added to a 4 ml quantity 
of the supernate. The resulting mixture is filtered through a 0.22 micron 
Millipore filter using a Swinnex filter and a 5 ml syringe. The filtrate 
is sealed in one ml glass vials with Teflon lined rubber crimp septa. 
Each vial, representing each of the individual fermentations, is analyzed 
for volatile fatty acids. 
Each vial is analyzed with three consecutive injections. Concentrations of 
acetate, propionate, i-butyrate, n-butyrate, i-valerate and n-valerate are 
calculated by comparison with analyses of a standard solution of VFA's 
using an internal standardization method. 
The results are stated in the following table: 
TABLE 1 
______________________________________ 
Effect of miscellaneous ionophores on gas and 
VFA production in in vitro rumen fermentations. 
Percent Production 
Compound of Control Fermantations 
Concen- Rate of 
tration Total Gas 
Compound (ppm) VFA(%) Production 
C.sub.3 /C.sub.2 + nC.sub.4 
______________________________________ 
X-14766A 50 150.1 90 0.501 
10 138.7 89 0.488 
Monensin 50 105.3 89 0.565 
10 110.7 85 0.575 
Narasin 50 106 86 00.583 
10 102.5 88 0.581 
Salino 50 146.9 83 0.497 
10 145.5 90 0.512 
Control 0 100 100 0.349 
______________________________________