IE45349B1 - Antibiotic bl580 - Google Patents

Abstract

The novel antibiotic of the formula: is prepared by submerse aerobic culture of Streptomyces hygroscopicus NRRL 8180 in an aqueous nutrient medium and subsequent isolation. As an organic carboxylic acid, it forms salts with cations. The compound is active against coccidiosis; it can be administered to the poultry orally or by addition to the feed.

Description

This invention relates to a new antibiotic, designated BL580 a. The present invention includes within its scope the antibiotic Β!_.580δ in dilute form, as a crude concentrate and in its pure crystalline form.

Antibiotic Β1580Δ may be represented by the following structural formula which is in accordance with accepted convention in that an α-substituent is behind the plane of the paper and is represented by a ---- bond whereas a g-substituent is in front of the plane of the paper and is represented by a —bond. .45349 The novel antibiotic of the present invention is an organic carboxylic acid and thus is capable of forming salts with non-toxic, pharmaceutically acceptable cations. Thus, salts formed by admixture of the anti5 biotic free acid with stoichiometric amounts of cations, suitably in a neutral solvent, may be formed with cations such as the sodium ion, potassium ion, calcium ion, magnesium ion, and ammonium ion as well as the organic amine cations such as the tri'{lower alkyl)amine cations (e.g. triethylamine, triethanolamine), and procaine.

The cationic salts of antibiotic BL580A are, in general, crystalline solids, relatively insoluble in water, and soluble in most common organic solvents such as methanol, ethyl acetate, acetone, chloroform, heptane, ether, and benzene.

The new antibiotic which has been designated BL580A is formed during the cultivation under controlled conditions of a new strain of Streptomyces hygroscopicus which also produces the known antibiotics BL580a and BL580P (see United States Patent No. 3,812,249). This new strain is a mutant derived by treatment of S. hygroscopicus NRRL 5647 with N-methyl-N1-nitro-N-nitrosoguanidine. A viable culture of the new microorganism has been deposited with the Culture Collection Laboratory, Northern Utilisation Research and Development Division, United States Department of Agriculture, Peoria, Illinois, and has been added to its permanent collection. It is freely available to the public from this depository under its accession number NRRL 8180.

The cultural, physiological and morphological features of iJRHL 3180 are substantially its tame as those of NRRL 5547 as determined by cr. U.3. Tresner, Lederle Laboratories Division, American Cyaraaiid Company, Pearl River, New York. The general deccriptioa of the microorganism, based on diagnostic charaottristies observed, is the same as that for NRSL :*5C7 published in United States Patent No. 3,812,249, but is reproduced below for .'jonvex.ience.

Observations were made of cultural, physiological and morphological features of HSt”, 8180 in accordance with the methods detailed, cy Shirling and Gottlieb, Internet. Journ. of Sysf, bacterial., 16, 313-340 (1966), Tho underscored descriptive colors and color chip designations are taken from Jacobson, et al., Color Harmon’.’ Manual, 3rd Sdition (1943), Container corp, of America, Chic-’.g·:.·, Illinois. Descripci’/ε details are recorded in Tables I through IV below.

Amount of Growth Good on yeast extract, Kuster's oatflakc, tomato paste-oatmeal and potato-dextrose agars? modecate on aspargine-dextrove. Hickey and Tresner1;, inorganic ealts-starch ar.d Hannett's agars: light c-n Cs-pek's solution agar.

Aerial Mycelium Whitish tc· yellowish, becoming grayich in sporulation sones ranging from Pawn (4 ig) to Beaver (4 li) to Ashes (5 re). Hperulation zones becoming black and hygroscopic in older cultures.

Soluble Pigments None on most media; yellowish or; yeast extract, Bennett's and potato-dextrose agars and only in light amounts.

Reverse Color Generally in yellowish shades on most media.

Miscellaneous Physiological Reactions Nitrates reduced to nitrites? complete liquefaction of gelatin; no formation of melanoid pigments on peptone-iron agar? complete peptonization of purple milk in 7 days? tolerance of NaCl in growth medium 7 percent but < 10 percent. Carbon source utilization according to the method of Pridham and Gottlieb, J.Bacteriol., 56, 107-114 (1948) as follows? Good utilization of adonitol, d-galactose, d-fructose, d-raffinose, salicin, d-xylose and dextrose; poor or no utilization of d-melezii tose, d-melibiose, 1-arabinose, i-inositol, lactose, d-mannitol, 1-rhamnose, sucrose and d-trehalose. Micromorphology Aerial mycelium gives rise to spore-bearing branches , which terminate in tightly coiled spirals of several turns? spores are mostly isodiametric, cylindrical, phalangiform, 0,6-0.7pm x 0,7-0.8pm. Spores smooth as determined by electron microscopy? spore sheaths finely wrinkled.

On the basis of the general characteristics observed, microorganism NRSL 8180 is a member of a large group of streptomycetes characterized by gray spores, spiral spore chains, smooth-walled spores and lack of melanin pigments. The hygroscopic nature of the culture along with its entire composite of morphological and physiological characteristics makes it a representative - 6 45349 strain of Streptomyces hygroscoplcus -'.tfiy.od by H.

D. Tresner and S. J. Backus, ,:A - .'.detcs Concept of the Characteristics of Streptomyces hygrotcoplcas, Appl. Microbiol., £, 243-250 (1656) and E. 3. Tresner, E. J. Backus and J. A. Hayes, Morphological pore Types in the Streptomyces hygroscopicas-like complex, Appl. Microbiol., 15, 637-639 (1967). u e (N M 4J S a Cultural Characteristics of Streptomyces hygroscopicus NRRL 8180 >1 *3 fi •H *P 0 0 0 •rl •Η 8 •H a 0, rH a 0 ο 0 β 0 8 H Ο Η X rl ϋ pH • 0 0 0 0 • 0 0 Ο M 0 M 0 0 0 Μ to Μ -Ρ · Μ -Ρ « •P 0 w -P V Λί tit fl Β) Εη β ω ΓΒ β tn β 0 Μ >< ω ω >ι 0 0 Ό 0 >10 0 0 Λ 0 a Λ 0 fl. fi N Λ υ G ε 0 0 M 0 ο SS fi N 43 β N 0 H G Ν & 0 •rl 0 •rl 0 w •rl •rl X •rl XZ3 β •Η 5? Αί (0 β to β 0 »rl Μ 0 G υ 0 0 υ ft) 0 rl CP υ 0 0 0 0 rH 0 0 rH S W 0 0 rH rH M 0 rHh 9 0 0 rH Μ 0 01 te 0 « 0 0 rH β to 0 υ Μ Π3 r-e 0 0 Λ Ρ β rH ρ ΜΗ 0 UH 0 4J ϋ 0 OJ 0 w Μ 0 3 Μ*· 0 Ofl ε ω Η 0 0 £1 Ο Μ Μ·Η 0 «Ρ ο Ρ 0 -Ρ > Η ·Η gj Οι fil S 0 043 0 •Η fl 0 Ρί υ Β « ¢4 •Pl Μ Λ 0 4J 0 ιΗ G 0 0 0 Λ 0 β Β fi 3 g 0 0 -Ρ 0 ο Η & S3 »-) ζ) s Κ 0 ·Η γ-1 σ» ω cu 0 ·Η Χ<·Η G 1 Ο Μ β. ea th 0 •Η 0 rH Λ 0 I ! Λ 0 ! i ,G •Ρ «Ρ *0 0 0 •P G 0 0 «μ G 0 0 0 •Η •Η M r-i *ri Μ H •Η ·«* rH β g Μ -Ρ r> 0 3 ρ 3 4J 0 Β 3 ω 0 •rl tne, M •p-j CP AM «.Ή ΜΗ Μ β «Η 0 0 Λ •K co 0 Λ •M 0 0 a 0 0 Η Μ S A B a X* ΙΛ &·Η 0 0 Λ •s» fi to %· fi to *α* 0 -Ρ υ α 0 β gf *-·«Η j~ S 0 >1 ω •Η G 3 3 3 ω cd & μ ο •ri rt 1^-» 0 •rl fiL*·» a •Η 0 Η G Μ •Η 'Η rH SH 0 rH 5w w Η rj Μ Η 0 £ -μ 0 (UH 0 · 0 tSH 0 . 0 0 — 0 a\ Β <0 o b C X 0 X fl X Ο < O' Α •Η Ό β γΗ Ex 0 > 0 X X •Η to Μ fi μη ε G e cn— Μ fl S 01— M 0 & σ 0 0 ο 3 ρ c 0 G 0 fi •Η 0 rH •Η M ρ λ •r] M rH •rl *** S 0 Η Α 0 g 0 o _ 0 s « 0 0 s 0 0 0 Μ •rl 0 > Η β ·!-} 0 > /H fi •rl 0 fi|0 · fl Ο M υ 0 Ρ 0 . M U 0 P 0 Μ υ S|0 0 Μ >ι Ο 0 ο 0 0 ·Η 0 0 0 ft! -rt 0 φ β|Ρ Ρ Β ES <ΛΗ1ΗΡ «; pa ra H & <Λ B.ifl fl 5 0 Ρ Β -Ρ *Ρ β 0 X! *ϊ5 0 3 μ &> 0 0 Μ 0 Ο •rl o Q 0 Ρ! © © Ό 4 0 .9 c&t G M 0 0 0 0 0 •Η O' X 0 +> 0 0 Μ Ρ r-i •μ rH •P UH Μ £» Ο 0 •P Φ 3 0 0 0 *0 •Η M 0 Ό 0 +> G 0 - 0 •(Η 3 λ: (!) ca υ μ 0 ®. •p 0 Μ Β.Μ 0 p M 0 Μ 0 0 0 0 0 Α0 ν tn ft) 3 CP ω CP U (S BS 0 ¢55 0 0349 Cultural Characteristics of Streptomyces hygroscopicus NRRi. 8130 - 9 ~ 4S34S Q) k •P d k Φ Dd tn Λ4 M te © © rk CO d color ϋ (11 03 Oi ϋ M a o u Oi k tr Oi ϋ o 4J & k +> cn □ •H •H k 0) 4J U d k m. xs u rk d k 4J rk U M $ K Φ JJ H C §< i-ι 6> O-ri tn n< □ tn •ri ω r4 Sj Φ 0 υ a >(« % r-i 0 «J'S. •Η Ό M S3 ¢) 0 < Oi >1 d Ό ? rk O •k 4J d •9 ϋ a H Λ JJ JJ & §s o Cn S w o u •k a* o ϋ rk co d O k k +> « tn c Oi MJ GJ Λ ϋ c XJ 0 w -A •k Λ « O d d © rk M8 0) H pl φ t ►4 XJ cd •H g Ο X» HXi rk &l 0-H >»H «y X! Φ 0X3 •k CL·— -ρ κδΓεή •k ..1 X3 cn & ii rk 01 d 0 J3fal 0 >»X3 O g 03 «Ρ h’sd o 0 Ή rk CM k rk 0 cn ¢3 k d , Cn &» d © □ ,β 0 o «y n d te ω ο Ό •ri O jj B ra d K 3 O k ·Η O P & d 03 rk Strepfcomyces hyqrose.opicus NRRL 8180 J. JL Miscellaneous Physiological Reactions of Streptomyces hygroscopicus NRRL 8180 Cable IV Carbon Source utilisation Pattern og Streptomycea hygroscopicua NRRL· 8100 Incubation: 10 Days Temperature: ?8“C Carbon Source Utilization* Adonitol l-Arafci.'esa Dextran d-Pructoae i-Inoaitoi Lactose d-Mannitol d-Meiezitose d-Melibiose d-Raffinoee 1-Rhamnr - .-λ Salicin Sucrose d-Trehaioaa <2-Xyie?e Dextrose Negative Control *3 « Good utilization 2 » Fair utilization 1 » Poor utilization 0 No utilization 43349 It is to be understood that for the production of 31580Δ, the present invention is not limited to this particular microorganism or to microorganisms fully answering the growth and microscopic characteristics of NRRL 8180. In fact, it is desired and intended to include the use of antibiotic BL580A-producing mutants derived from NRRL 8180 by various means, such as X-radiation, ultraviolet radiation, nitrogen mustard and phage exposure.

Antibiotic BL580a is effective in controlling coccidial infections in a warm-blooded animal host, and it is markedly less toxic than antibiotic BL580a (whose structure is set forth in Netherlands Patent No. 7,402,938).

Thus, the present invention provides a method of treating and preventing coccidiosis in poultry, which comprises administering orally to said poultry an anti-coccidaily-effective amount of antibiotic BL580 or a pharmacologically acceptable cationic salt thereof.

The invention also provides a composition for the treatment and prevention of coccidiosis in poultry, which comprises a poultry diet comprising edible feedstuffs and an anticocci daily-effective amount of antibiotic BL5804 or a pharmacologically acceptable cationic salt thereof.

It is preferred that the antibiotic be administered orally to poultry at a concentration in the diet of from 125 to 250 ppm.

The activity of antibiotic BL5804 as an anticoccidial agent was demonstrated by the following in vivo tests wherein the following poultry diet was used.

Vitamin-amino Acid Premix 0.5% Trace Minerals 0.1% Sodium Chloride 0.3% Dicalciuiji Phosphate Ground Limestone 30 Stabilized Fat 4.0% Dehydrated Alfafa (17% protein) 2.0% Corn Gluten Meal (41% protein) 5.0% Menhaden Fish Meal (60% protein) 5.0% Soybean Oil Meal (44% protein) 30.0% 35 Ground Yellow Corn, fine to 100% The vitamin-amino acid premix in the above poultry prepared from the following formulation. The expressions of quantity 34S 1 relate to units per kilogram of the poultry diet. Butylated Hydroxy ‘folvovt- 125 mg. dl-Methionine 500 mg. Vitamin A 3300 I.U. 5 Vitamin 1100 I.C.U Riboflavin 4.4 mg. Vitamin E 2.2 I.U. Niacin 27.5 mg. Par.tot ifeuic Acid 8.8 mg. 10 Choline Chloride 500 mg. Polic Acid 1.43 mg.. Menadione Sodium Bisulfate 1.1 mg. Vitamin «·,·, 11 meg. Ground Yellow Corn, fine to 5 gm.

Mixed Coocidia Infections of Elmeria tanella and yimeria acervullna Λ mixed inoculum of 5000 sporulated oocysts of Eimeria afiervulina and a sufficient number of oocysts of Eimeri-a tenella no produce 85% to 100% mortality in un20 treated controls was given tc groups of seven-day-old chicks, by direct in·«aviation into the crops of all chicks. The chicks were givan free access to the poultry diet and water during the entire test period. Two days after inoculation, medicated seed, composed of the poultry diet and several levels of BL580t, was presented to the various groups of chicks in the test. Ten days after incoulation the tests were terminated. The chicks were weighed, subject to necropsy and their intestinal tracts examined for lesions. The results of this test appear in Table V. These results show that 100» survival of infected chicks was obtained when 125 ppm or 250 ppm of BL580A was administered to infected chicks in their diet. These results also show a significant suppression of lesions due to Eimeria tenella and Eimeria acer5 vulina when 30 ppm or 60 ppm of ΒΙ.580Δ is administered to infected chicks in their diet. - 16 45349 > Mixed Coccidia Infection of Eimeria tenella, Eimeria. acervulina, Eimeria necatrix, Eimeria brunetti and Eimeria maxima A commercial vaccine (* Cocci vac D, Sterwin Lab5 oratories, Opalika, Alabama) containing a mixture of at least five species of Eimeria coccidia, was administered to chicks at 70 times the normal immunizing dose.

The vaccine was given to groups of seven-day-old chicks, by direct inoculation into the crops of all chicks. The chicks were given free access to water and the above poultry diet during the entire test period. Two days after inoculation medicated feed, composed of the poultry diet and several levels of ΒΕ580Δ, was presented to the various groups of chicks in the test. Ten days after inoculation the tests were terminated and the birds were weighed, neercpsied and their intestinal tracts examined for lesions. The results of this test appear in Table VI. These results show that 100% survival of infected chicks is obtained when 120 ppm of BL580A is administered to infected chicks in their diet. This level, also shows a significant suppression of lesions due to Eimeria tenella, Eimera acervulina, Eimeria necatrix, Eimeria brunetti and Eimeria maxima.

‘ *Coccivac is a trade mark Fermentation Process Cultivation of the microorganism Streptomyces hygroscopicus NRRL 8180 may be carried out in a wide variety of liquid culture media. Media which are useful for the production of antibiotic BL580A include an assimilable source of carbon such as starch, sugar, molasses, glycerol, etc.; an assimilable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc.; and inorganic anions and cations such as potassium, sodium, calcium, sulfate, phosphate, chloride, etc. Trace elements such as borin, molybdenum, copper, etc. are supplied as impurities of other constituents of the media. Aeration in tanks and bottles is provided by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller.

An antifoaming agent such as one percent octadecanol in lard oil may be added as needed.

Inoculum Preparation Shaker flask inoculum of Streptomyces hygroscopicus NRRL 8180 is prepared by inoculating 100 ml. portions of sterile liquid in 500 ml. flasks with scrapings or washings of spores from an agar slant of the culture. The following medium is ordinarily used: Soy flour 1.0% Glucose 2.0% Corn steep liquor 0.5% CaCO3 0.3% Water qs 100% The flasks are incubated at a temperature from 45348 “C. to 29*C., preferably 28eC. and agitated vigorously on a rotary shaker for 48 to 96. '-ura.

Two 100 ml. portions of this inoculum are used tc inoculate 12 liters of the same sterile medium in a 20 liter bottle, Thi3 inoculum is incubated with agitation and aeration of sterile air for 36 to 54 hours at 25eC. to 29°C., preferably 28CC.

This inoculum is used to inoculate 300 liters of the .same sterile medium in a tank fermentor. This inoculum is incubated with agitation and aeration of sterile air for 36 to 64 hours at 255C. to 29“c., preferably 28 °C.

This inoculum is used to inoculate ε 4000 liter fermentation tank containing 3000 liters of a sterile medium such as the following: Cor;· steep liquor 0.5% Soy flour 3,0*. Corn starch 4.0% CaCOg 0.1% Water qs 100% This mr.diu-t is fermented for 10C to 200 hours at a temperature of 27°C·. to 32°c. with agitation by an impeller and aeration at a rate ”£ 0.4-0,3 liters of air per liter of medium per minute. Normally a defoamer such as Hodag FD62 ic added. at a ratio of about 1.3 gal,/1000 gal. of medium.

Preferred Purification Procedure After the fermentation is completed, the fermented mash containing antibiotic BL580A is combined with about one-half its volume of ethyl acetate and stirred for 2-3 hours. An approximate 8% portion of diatomaceous earth is added and the mixture is filtered through a plate and frame filter press. The cake is washed on the press with ethyl acetate. The ethyl acetate ex5 tracts are collected and concentrated in a still to a syrup.

The above syrup is stirred with twice its volume of heptane and stored at 4°C. overnight. The supernatant is recovered by decantation and concentrated to a gummy residue.

The gummy concentrate is treated with 10 liters of methanoi and chilled with the aid of dry ice for several hours. The mixture is filtered through sintered glass with diatomaceous earth precoat and washed with cold methanol. The methanol solution is concentrated to dryness in vacuo.

A chromatographic column is prepared with activated carbon at a ratio of about one liter of carbon per 50 g. of charge. The dried residue is dissolved in methylene chloride at a ratio of 40 g./liter and charged on the column. The methylene chloride eluate is collected as one cut and concentrated to dryness. The residue is mixed with methanol and stored in a chill room with dry ice to reduce the temperature to -10°C. for 15 min25 utes. After 15 minutes the solidified oil is filtered off and the methanol soluble material is concentrated to dryness in vacuo giving an oil.

This oil is dissolved in a minimum amount of methylene chloride, combined with silica gel, concentrat30 ed to dryness and charged on a dry silica gel column.

The column is developed with 10% ethyl acetste ia benzene followed fay 20% ethyl acetate bansene. The column is then allowed tc drain. The column is measured into 10 equal parts (including tha charge). Core samples are removed at Kf 0.05, 0,13, 0.25, 0.35 ...etc., for the length of the- entire column and eluted with an appropriate volume of ethyl acetatesmethylene chloride:nethanol )2:2:1). Kt places where the antibiotic overlaps, coru sampling is done as every 1/9 of an Rf unit. The antibiotic is located by thin layer chromatography cf the core eluates on commercially available thin layer plates (Si.Iplace-22 distributed by Brinkmann Instrument Co., Westbury, Kew York 11590). The respective zones were detected fay charring in the presence of sulfuric acid.

The section of the column comprising Rf 0.11 to 0.35 is excised from the column and slurried in ethyl acetate tmc-thylene cbloridetmethanol (2:2:1 by volume). This mixture is filcerea, washed with additional solvent mixture and concentrated in vacuo to dryness. The residue is dissolved in t-butanol, filtered and freeze dried to give a fluffy solid. Λ two-phase system is prepared by mixing nheptane .-methanols ethyl ace tats: water (3000; 1500:75:37 by volume). Ccl&to© 'faagIe--Picher Industries, Cincinnati, Ohio), a brand of diatomaceous earth, ie mixed with the lower phase of this system at a ratio of about 800 g./600 ml, of lower phase and packed in increments into a (7.5 cm. m diameter) column. The charge is applied as a mixture of diatomaceous earth, fewer phase and lyophil- 23 45349 lized product {40 g.:30 ml.:13.8 g.). The charged col umn is developed with upper phase and fractions of 25 ml. are collected. The activity is detected by thin layer chromatography on selected fractions using a gelplate, chloroform:ethyl acetate (1:1) as developer and charring for detection. Fractions 90-150 are combined and concentrated giving antibiotic Βίι580Δ/ The invention is illustrated by the following specific Examples.

Example 1 Inoculum Preparation A typical medium used to grow the primary inoculum is prepared, according to the following formula: Soy flour 1.0 g. Glucose 2.0 g. Corn steep liquor 0.5 g. CaCG-β 0.3 g. Water to 100 ml. The washed or scraped spores from an agar alant of Streptomyces hygroscopicus NRRL 8180 are used to inoculate two 500 ml. flasks each containing 100 ml. of the above medium which has been sterilized. The flasks are placed on a rotary shaker and agitated vigorously for 72 hours at 28°C.

The resulting flask inoculum is transferred to a 5 gallon glass bottle containing 12 liters of the same sterile medium. This secondary inoculum is aerated with sterile air while growth is carried out for 48 hours at 28°C.

The resulting secondary inoculum Ϊ3 transferred to a 100 gallon tank containing ?00 liter of the same sterile medium. This tertiary ir ’'•alum is aerated with sterile air at the rate of one it ter of air/liter of medium/minute and agitated by an impeller operating at 173 rpm. Growth is continued for 48 hours ..t 28°C. The pH at this time is 6.9 to 7.0.

Example 2 Fermentation A fermentation medium is prepared according to 10 the following formula.' Corn steep liquor 0.5 g.

Soy flour 1.0 g.

• Corn sf.r .. 4,0 g, CacO3 0,1 g.

Water to 100 ml.

A 3v-,r liter batch of fermentation medium of the above formulation in a 4000 liter tank is sterilized at 120eC, for 30 minutes. The pH of the medium after sterilization is 6.4 to 6.5. This medium is inoculated with 300 liters of tertiary inoculum prepared as described in Example 1·· The fermentation is carried out at 28°-30*C., using 4.0 liters of Hodag FT02 as a defoaming agent. Aeration is supolied at the rate of 0.6 liter of sterile air per liter cf mash per minute. The mash is agitated by an impeller driven at ISO rpm. At the end of 138.5 hours of fermentation time the maeh is harvested.

Example 3 Isolation and Purification A 2550 liter portion of fermented mash prepared as described in Example 2, having a pH of 7.4 is combined with 1275 liters of ethyl acetate and stirred for 2.5 hours. An 8% (by weight) portion of diatomaceous earth is added. The mixture is filtered in several portions, with stirring, through a pair Of frame presses. The aqueous-ethyl acetate filtrates are pooled providing 3250 liters which is allowed to separate, providing 1000 liters of ethyl acetate extract. After each portion of mash-ethyl acetate-diatomaceous earth is filtered through a press, the pad is washed On the press with ethyl acetate. The ethyl acetate washings are combined and separated giving 535 liters of ethyl acetate washings. The 1000 liters of ethyl acetate extracts and 535 liters of ethyl acetate washings are combined and concentrated in a 400 gallon still to 225 liters. This 225 liters is further concentrated in a 50 gallon still to 20 liters. This 20 liters is further concentrated in a glass still to a syrup.

The syrup is stored at 4°C. for 48 hours and then stirred with twice its volume of heptane. The mixture is allowed to stand at 4°c. overnight. The supernatant is recovered by decantation and concentrated to a gummy residue.

A 10 liter portion of methanol is added to the gummy residue and the mixture is chilled with the aid of dry ice for several hours. The mixture is filtered through sintered glass containing a diatomaceous earth precoat and washed with cold methanol. The combined filtrate and washings are concentrated to dryness in vacuo providing 1353.5 g. of residue.

The 1353.5 g. of residue is dissolved in meth26 45349 ylane chloride at a rata of 40 y,/liter. A chromatographic column is prepared by pec.-11-.7 witn 27.07 liters of 20 x 40 mesh granular carbon. The residue in methylene chloride is passed through this column at a flow rate of 375-400 ml. per minute. The methylene chloride eluate is collected as one cut and concentrated to dryness giving 1053 g. of residue, The residue is thoroughly mixau with 2-9 liters of methanol. The mixture is reduced to -10eC. in a chill rooi?. with the aid of dry ice and maintained at -10°G. for 15 minutes. Any solidified oil is removed by filtration and the methanol filtrate is concentrated to dryness in vacuo giving 781.4 g. as an oil.

A dry pack chromatographic column is prepared by packing 4 kg. of silica gel onto a 12 circumference plastic column. 1 200 g. portion of the above oil is dissolved in ?. minimal amount of methylene chloride. A 300 g. portion of silica gel is add-sd and mixed thoroughly and the mixture is then concentrated in vacuo to dryness.

The dried mixture is charged on the and some sea sand is placed on tha top of the eoiuiua to prevent bed disturbance during elution. The plastic coluifin is placed in a glass shell co give it support. Ths column is eluted with 12 liters o.i ICi ethyl acetate ia benzene.

The column is allowed to run dry and then eluted with 7.6 liters of 20% ethyl acetate in benzene. Cuts are collected and the column is allowed to run dryc '.ΓΉ',ϊ column is then purged with nitrogen. Antibiotic activity is determined by assaying the cuts vs. Strap tcce-acus pyogenes NY5. The column is measured into 10 equal parts (includ- 27 ing the charge). Core samples are removed at Rf 0.05, 0.15, 0.25, 0.35.....etc., for the length of the entire column. At places where the antibiotic bands overlapped, core sampling is done at every 1/8 of an Rf unit. Each core--sample is eluted With 10 ml. of ethyl acetate:methylene chloridemethanol (2:2:1) and examined by thin layer chromatography using the system ethyl acetate: chloroform (1:1) spotting 30χ of sample and charring with sulfuric acid. The section of column Rf 0.11 to Rf 0.35 is removed and slurried in ethyl acetate:methylene chloridemethanol (2:2:1). The mixture is filtered and washed with the same solvent mixture and concentrated to dryness in vacuo. The residue is dissolved in t-butanol, filtered and lyophilized giving 26.7 g.

A two-phase system is prepared by mixing n-heptane:methanol:ethyl acetate:water [3000:1500:75:37 (by volume)]. An 300 g. portion of acid washed diatomaceous earth is mixed with 600 ml. of the lower phase of this solvent system and packed in increments into a 7.5 cubic inch glass column. The charge, comprising 40 g. of diatomaceous earth, 30 ml. of lower phase solvent and 13.8 g. of the above lyophilised material, is applied as a mixture. The charged column is developed with the upper phase of the solvent system and 25 ml. fractions are collected. The desired compound is located by assaying fraction samples as above with thin layer chromatography. Fractions 90-150 are combined and lyophilised providing 2.75 g. of the product BL580A primarily as the sodium salt. 4S349 Example 4 Preparation and Isolation of B15'f ms the Free Acid The partial sodium saiu ·-1 Π158ΰΔ is prepared and isolated as describad in Examples i-3. a two-phase system is prepared by mixing heptane, methanol, ethyl acetate, water (300C:1500:8C:4G by volume). Λ glass column is packed with a mixture of 800 g. of diatomaceous earth and 600 ml. of the lewer jhtte of the above system. The charge is applied as a mixture of 28 g. of diatomaceous earth, 21 ml, of lower phase and 10.9 g, of the BL580A partial sodium salt. The column is developed with upper phase. Fractions cf 30 nil. volume are collected. Selected fractions are chromatographed on Silplate® F-22 using ethyl acetate:chloroform as developer and charring for detection in order to locate the BXi58CA. Free lions 29-39, containing the ΒΙ·580Δ are combined and the solvent is removed. The resulting solid is redissolved in t-butanoi and lyophilized giving 4.73 g, An 800 mg- portion cf this lyophilized material is stirred in 300 ml. of a two-phase system composed cf water:ether:petrcleum etner (2:1:1). Ths pH is adjusted to 2.5 using IK HC1 while stirring. The organic phase is separated and washed three ti.'ias with an equal volume of water. The scivo.it extract is concentrated in vacuo to a residue. The residue is dissolved in t-butanol and then lyophilized giving 657 mg. of BL580A as the free acid.

The free acid of BL580A feas microanalytical data aa follows: C, 61.10; H, 8,9; a uh, 0; md a spe1 cific rotation [α]ρ25= +21° ί 1° (C=0.9 in methanol).

The free acid of Βϊ,580Δ exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths: 2.95; 5.88; 8.35; 8.60; 9.00; 9,12; 9.43; 9.62; 10.05; and 10.47ji.

A standard infrared absorption spectrum of the free acid of BL580A prepared in a KBr pellet is shown in Figure 4 of the accompanying drawings.

A standard proton magnetic resonance spectrum of the free acid of ΒΒ580Δ is shown in Figure 5 of the accompanying drawings.

A standard C nuclear magnetic resonance spectrum of the free acid of BL580A is shown in Figure 6 of the accompanying drawings.

Example 5 Preparation of the Sodium Salt of ΒΒ580Δ ΒΒ580Δ free acid (1 g.) is dissolved in 300 ml of ether-petroleum ether (1:1). This solution is added to 200 ml.’of water to give a two-phase system. The pH is adjusted to 10.0 by the addition of 0.1N NaOH while stirring, after which the organic phase is separated and concentrated in vacuo to a residue. The residue is dissolved in 10 ml. of ether and 20 ml. of petroleum ether (30°-70°C.) is added. The resulting solution is seeded with a crystal of ΒΒ580Δ sodium salt and allowed to evap· orate slowly at 4°C. until a crystalline solid forms.

The crystals are collected on a filter, washed with cold petroleum ether and air dried to yield 323 mg. of the sodium salt of ΒΒ580Δ.

This sodium salt of antibiotic ΒΙ580Δ has a melting point of 157° - 16l°C,: C- 60.93; H, 8.47; Ha, 1.95: £°ΰ[)Ζ5 = + 6° + 1° (C = 1.153 in methanol). The sodium salt of BL 580δ exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths: 6.27, SA . 9.0, 9.13, 9.23, 9.48 end 19.60μ.

A standard infrared absorption spectrum of the sodium salt of BL 580δ prepared in a KBr pellet is shown in Figure 1 of the accompanying adrawings.

A standard JC nue’ear magnetic resonance spectrum of the 10 sodium salt of EL589a S shown in Figure 2 of the accompanying drawings.

A standard proton magnetic resonance spectrum of the sodium salt of BLEaOfi shown in Figure 3 of the accompanying drawi ngs,

Claims (8)

1. Antibiotic BL580A of the formula: and the pharmacologically acceptable cationic salts thereof.

2. A process for the production of anitaiutics CLSCOa, which comprises cultivating Streptomyces hygroscopicus NRRL 8133 :.· cn antibiotic BL580a-producing mutant there.if in a;· acucous nutrie!-it medium containing assimilable sources of carbohydrate, nitrogen, and inorganic 5 salts under submerged aerobic conditions until substantial antibiotics activity is imparted to said medium, and then recovering antibiotic BLSBCf, therefrom

3. A method :»f treating and preventing enecioiosis in poultry, which comprises administering orally ro said poultry an anti cocci dally10 effective amount of antibiotic Bl580a or a pnaraacologically acceptable cationic salt thereof.

4. A method according to .'''aim 3, wherein tho antibiotic is administered orally to poultry at a concentration in the diet of from 125 ppm tc 250 ppm. 15

5. A composition tor the treatment and prevention of coccldiosis in poultry, which cor-pr.-TS a poultry diet comprising edible feedstuffs and an anticoccido· iy-effective amount of antibiotic BL580a or a pharmacologically acceptable cationic salt thereof.

6. A precess for tne production of sr.cibiotic Β1580Δ, according to 20 Claim 2 and substantially as -Jcscrlbeu ir. Examples 1-- herein.

7. A process for the pr.-.duction of antibiotic BlSSOa, according to Claim 2 and substantially a:· described in Exot-g ' ·;’ ~- f ‘ r herein.

8. The sodium salt of antibiotic fi.5S3c.