IL52205A - Antibiotic bl850 ,its preparation and anticoccidial composition comprising it - Google Patents

Description

ANTIBIOTIC , ITS PREPARATION AND ANTICOCCIDIAL COMPOSITION COMPRISING IT This invention relates to a new antibiotic designated BL580A, to its production by fermentation, to methods for its recovery and concentration from crude solutions and to processes for its purification. The present invention includes within its scope the antibiotic ΒΙ-.580Δ in dilute form, as a crude concentrate and in its pure crystalline form. The effects of this new antibiotic as an anticoccidial agent together with its chemical and physical properties, differentiate it from previously described antibiotics.

Antibiotic Β1580Δ may be represented by the following structural formula which is in accordance with accepted convention in that an a-substituent is behind the plane of the paper and is represented by a bond whereas a β-substituent is in front of the plane of the paper and is represented by a -^i bond.

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 anti-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(lc¾r alkyl) amine cations (e.g. triethylamine, triethanolamine) , procaine, and the like. 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 BL5803 (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-N ' -nitro-N"-nitrosoguanidine . A viable culture of the new microorganism has been deposited with the Culture Collection Laboratory, Northern Utilization 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 NRRL 8180 are substantially the same as those of NRRL 5647 as determined by Dr. H.D. Tresner, Lederle Laboratories Division, American Cyanamid Company Pearl River, New York. The general description of the microorganism, based on diagnostic characteristics observed, is the same as that for NRRL 5647 published in United States Patent No. 3,812,249, but is reproduced below for convenience.

Observations were made of the cultural, phys-iological and morphological features of NRRL 8180 in accordance with the methods detailed by Shirling and Gottlieb, Internat. Journ. of Syst. Bacteriol., 16.' 313--340 (1966) . The underscored descriptive colors and color chip designations are taken from Jacobson, et al . , Color Harmony Manual, 3rd Edition (1948) , Container Corp of America, Chicago, Illinois. Descriptive details are recorded in Tables I through IV below.

Amount of Growth Good on yeast extract, Kuster's oatflake, tomato paste-oatmeal and potato-dextrose agars; moderate on aspargine-dextrose, Hickey and Tresner' s, inorganic salts-starch and Bennett's agars; light on Czapek's solution agar.

Aerial Mycelium Whitish to yellowish, becoming grayish in sporula-tion zones ranging from Fawn (4 ig) to Beaver (4 li) to Ashes (5 fe) . Sporulation zones becoming black and hygroscopic in older cultures.

Soluble Pigments None on most media; yellowish on yeast extract, Λ 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 adon-itol, d-galactose, d-fructose, d-raffinose, salicin, d-xylose and dextrose; poor or no utilization of d-melezi-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, 0.6-0.7^im x 0.7-0. Rum. Spores smooth as determined by electron microscopy; spore sheaths finely wrinkled.

On the basis of the general characteristics observed, microorganism NRRL 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 A strain of Streptomyces hygroscopicus as defined by H. D. Tresner and E. J. Backus, "A Broadened Concept of the Characteristics of Streptomyces hygroscopicus" , Appl. Microbiol., 4_, 243-250 (1956) and H. D. Tresner, E. J. Backus and J. A. Hayes, "Morphological Spore Types in the Streptomyces hygroscopicus-like Complex", Appl. Microbiol., 15, 637-639 (1967).

Table I Cultural Characteristics of Streptomyces hygros Incubation: 14 days Amount Aerial mycelium Soluble Medium of growth and/or spores Pigment Czapeck's solution Light Trace of whitish aerial None agar mycelium.

No sporulation.

Yeast extract agar Good Aerial mycelium whitish, Yellowish becoming Fawn (4 ig) to light Beaver (4 li) in sporulation zones. Sporulation heavy.

Kuster's oatflake Good Aerial mycelium whitish, None agar becoming Fawn (4 ig) to Beaver (4 li) in sporulation zones. Sporulation heavy.

Asparagin-dextrose Moderate Aerial mycelium whitish, None agar becoming Ashes (5 fe) to Fawn (4 ig) in sporulation areas. Sporulation moderate.

Table I (continued) Cultural Characteristics of Streptomyces hygros Incubation: 14 days Amount Aerial mycelium Soluble Medium of growth and/or spores Pigment Hickey and Moderate Aerial mycelium whitish to None Tresner's agar yellowish, becoming Fawn (4 ig) to Beaver (4 li) in sporulation zones. Sporu- lation heavy.

Inorganic salts- Moderate Aerial mycelium whitish to None -starch agar yellowish, becoming Fawn (4 ig) to Beaver (4 li) in sporulation zones. Sporulation heavy.

Tomato-paste oatGood Aerial mycelium whitish to None meal agar yellowish, becoming Fawn (4 ig) to Beaver (4 li) in sporulation zones. Sporulation very heavy.

Bennett's agar Moderate Aerial mycelium whitish, Yellowish becoming Beaver (4 li) in light sporulation zones. Sporulation heavy.

Table I (continued) Cultural Characteristics of Streptomyces hygros Incubation: 14 days Amount Aerial mycelium Soluble Medium of growth and/or spores Pigment Potato-dextrose Good Aerial mycelium whitish to Yellowish, agar yellowish becoming Ashes light (5 fe) to Fawn (4 ig) in sporulation zones. Sporu- lation moderate.

Table II Micromorphology of Streptomyces hygroscopicu Aerial mycelium and/or Medium sporiferous structures Spore shape S Kuster ' s Aerial mycelium gives Spores are mostly 0 oatflake agar rise to spore bearing isodiametric, 0 branches which terminate cylindrical , in tightly coiled spiphalangiform rals of several turns.

Table III Miscellaneous Physiological Reactions of Streptomyces h Medium Incubation (28°C) Growth amount Organic nitrate broth 7 Days Good Organic nitrate broth 14 Days Good Gelatin 7 Days Good Peptone-iron Agar 24-48 Hours Good Purple milk 7 Days Good Yeast extract agar plus 10 Days Good (4, 7, 10 and 13%) NaCl Table IV Carbon Source Utilization Pattern of Streptomyces hygroscopicu3 NRRL 8180 Incubation: 10 Days Temperature Carbon Source Utilization* Adonitol 3 1-Arabinose 0 Dextran 3 d-Fructose 3 i-Inositol 0 Lactose 0 d-Mannitol 0 d-Melezitose 1 d-Melibiose 1 d-Raffinose 3 ■ ! 1-Rhamnose 0 Salicin 3 Sucrose 0 d-Trehalose 0 i d-Xylose 3 Dextrose 3 Negative Control 0 *3 = Good utilization 2 » Fair utilization 1 » Poor utilization 0 » No utilization It is to be understood that for the production of BL580A, 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 mutants produced from NRRL 8180 by various means, such as X-radiation, ultraviolet radiation, nitrogen mustard, phage exposure and the like.

Antibiotic BL580A is highly effective in con-trolling coccidial infections in a warm-blooded animal host. Furthermore, antibiotic BL580A is markedly less toxic than antibiotic BL580a (whose structure is set forth in Netherlands Patent No. 7,402,938). The activity of antibiotic ΒΙ.580Δ as an anticoccidial agent was demonstrated by the following in vivo tests wherein the poultry diet was used.

Viatamin-Amino Acid Premix 0.5% Trace Minerals 0.1% Sodium Chloride 0.3% Dicalcium Phosphate 1.2% Ground Limestone 0.5% Stabilized Fat .4.0% Dehydrated Alfalfa (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% Ground Yellor Corn, fine to 100% The vitamin-amino acid premix in the above poultry diet was prepared from the following formulation. The expressions of quantity relate to units per kilogram the poultry diet.

Butylated Hydroxy Toluene 125 mg. dl-Methionine 500 mg.

Vitamin A 3300 I.U.

Vitamin 1100 I.C.U.

Riboflavin 4.4 mg.

Vitamin E 2.2 I.U.

Niacin 27.5 mg.

Pantothenic Acid 8.8 mg.

Choline Chloride 500 mg.

Folic Acid 1.43 mg ..

Menadione Sodium Bisulfate 1.1 mg.

Vitamin Β·^ 11 meg.

Ground Yellow Corn, fine to 5 gm.

Mixed Coccidia Infections of Eimeria tenella and Eimeria acervulina A mixed inoculum of 5000 sporulated oocysts of Eimeria acervulina and a sufficient number of oocysts of Eimeria tenella to produce 85% to 100% mortality in un-treated controls 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 the poultry diet and water during the entire test period. Two days after inoculation, medicated feed, composed of the poultry diet and several levels of BL580A, was presented to the various groups of chicks in the test. Ten days after incoulation the tests were terminated. The chicks were weighed, necropsied and their intestinal tracts examined for lesions. The results of this test appear in Table V. These results show that 100% survival of Λ 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 significatnt suppression of lesions due to Eimeria tenella and Eimeria acer-vulina when 30 ppm or 60 ppm of BL580A is administered to infected chicks in their diet.

Table V Concentration of Number of Percent Percent BL580A in Diet ppm Birds Started Survival Eimeria t 0 60 17 0 250 5 100 100 125 5 100 100 60 24 91.6 46 30 25 60 0 Mixed Coccidia Infection of Eimeria tenella, Eimeria acervulina, Eimeria necatrix, Eimeria brunetti and Eimeria maxima A commercial vaccine (Coccivac® D, Sterwin Lab-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 BL580A, was presented to the various groups of chicks in the test. Ten days after inoculation the tests were terminated and the birds were weighed, necrcpsied 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 .

Table VI Percent Bi Concentration of Number of Percent BL580A in Diet ppm Birds Started Survival tenella acervul 0 15 0 0 0 120 15 100 100 100 60 15 100 40 93 30 15 100 7 7 A 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 bor¾, 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% CaC03 0.3% Water qs 100% The flasks are incubated at a temperature from A 25°C. to 29°C, preferably 28°C. and agitated vigorously on a rotary shaker for 48 to 96 hours.

Two 100 ml. portions of this inoculum are used to inoculate 12 liters of the same sterile medium in a 20 liter bottle. This inoculum is incubated with agitation and aeration of sterile air for 36 to 64 hours at 25°C. to 29°C, preferably 28°C.

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 25°C. to 29°C, preferably 28°C.

This inoculum is used to inoculate a 4000 liter fermentation tank containing 3000 liters of a sterile medium such as the following: Corn steep liquor 0.5% Soy flour 1.0% Corn starch 4.0% CaC03 0.1% Water qs 100% This medium is fermented for 100 to 200 hours at a temperature of 27 °C. to 32°C. with agitation by an impeller and aeration at a rate of 0.4-0.8 liters of air per liter of medium per minute. Normally a defoamer such as Hodag FD82 is added at a ratio of about 1.3 gal./1000 gal. of medium.

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 ex-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 methanol 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 min-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 aount of methylene chloride, combined with silica gel, concentrat-ed to dryness and charged on a dry silica gel column.

The column is developed with 10% ethyl acetate in benzene followed by 20% ethyl acetate in benzene. The column is then allowed to drain. The column is measured into 10 equal parts (including the charge) . Core samples are removed at Rf 0.05, 0.15, 0.25, 0.35 ...etc., for the length of the entire column and eluted with an appropriate volumn of ethyl acetate : methylene chloride :-methanol (2:2:1). At places where the antibiotic overlaps, core sampling is done at every 1/8 of an Rf unit. The antibiotic is located by thin layer chromatography of the core elutates on commercially available thin layer plates (Silplate-22 distributed by Brinkmann Instrument Co., estbury, New York 11590). The respective zones were detected by charring in the presence of sul-furic acid.

The section of the column comprising Rf 0.11 to 0.35 is excised from the column and slurried in ethyl acetate :methylene chloride : methanol (2:2:1 by volume). This mixture is filtered, 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.

A two-phase system is prepared by mixing n-heptane :methanol : ethyl acetate : water (3000:1500:75:37 by volume). Celatom (Eagle-Picher Industries, Cincinnati, Ohio) , a brand of diatomaceous earth, is 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. in diameter) column. The charge is applied as a mixture of diatomaceous earth, lower phase and lyophil- lized product (40 g.:30 ml.:13.8 g.). The charged column 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 BL580A/ The invention will be described in greater de-til in conjunction with 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.

CaC03 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 is transferred to a 100 gallon tank containing 300 liters of the same sterile medium. This tertiary inoculum is aerated with sterile air at the rate of one liter of air/liter of medium/minute and agitated by an impeller operating at 173 rpm. Growth is continued for 48 hours at 28 °C. The pH at this time is 6.9 to 7.0.

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

Soy flour 1.0 g.

Corn starch 4.0 g.

CaC03 0.1 g.

Water to 100 ml.

A 3000 liter batch of fermentation medium of the above formulation in a 4000 liter tank is sterilized at 120°C. for 60 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 FD82 as a defoaming agent. Aeration is supplied at the rate of 0.6 liter of sterile air per liter of mash per minute. The mash is agitated by an impeller driven at 150 rpm. At the end of 138.5 hours of fermentation time the mash 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 ace-tate. 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 mix-ture 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 meth- ylene chloride at a rate of 40 g. /liter. A chromatographic column is prepared by packing with 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 mixed with 8-9 liters of methanol. The mixture is reduced to -10 °C. in a chill room with the aid of dry ice and maintained at -10°C. 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. A 200 g. portion of the above oil is dissolved in a minimal amount of methylene chloride. A 300 g. portion of silica gel is added and mixed thoroughly and the mixture is then concentrated in vacuo to dryness. The dried mixture is charged on the column and some sea sand is placed on the top of the column to prevent bed disturbance during elution. The plastic column is placed in a glass shell to give it support. The column is eluted with 12 liters of 10% ethyl acetate in 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 dry. The column is then purged with nitrogen. Antibiotic activity is determined by assaying the cuts vs. Streptococcus pyogenes NY5. The column is measued into 10 equal parts (includ- 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 chloride : methanol (2:2:1) and examined by thin layer chromatography using the system ethyl acetate :-chloroform (1:1) spotting 30 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 chloride :methanol (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 800 g. portion of acid washed diato-maceous 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 lyophilized 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 lyophilized providing 2.75 g. of the product BL580A primarily as the sodium salt.

Example 4 Preparation and Isolation of BL580A as the Free Acid The partial sodium salt of BL580A is prepared and isolated as described in Examp^ls 1-3. A two-phase system is prepared by mixing heptane, methanol, ethyl acetate, water (3000:1500:80:40 by volume). A glass column is packed with a mixture of 800 g. of diatomace-ous earth and 600 ml. of the lower phase 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 of 90 ml. volume are collected. Selected fractions are chromatographed on Silplate® F-22 using ethyl acetate : chloroform as de-veloper and charring for detection in order to locate the BL580A. Fractions 29-39, containing the BL580A are combined and the solvent is removed. The resulting solid is redissolved in t-butanol and lyophilized giving 4.79 g.

An 800 mg. portion of this lyophilized material is stirred in 300 ml. of a two-phase system composed of water : ether :petroleum ether (2:1:1). The pH is adjusted to 2.5 using IN HCl while stirring. The organic phase is separated and washed three times with an equal volume of water. The solvent 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 has microanalytical data as follows: C, 61.10; H, 8.9; ash, 0; and a spe- cific rotation [α]ρ = +21° ΐ 1° (C=0.9 in methanol).

The free acid of ΒΙ.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 10.47^1.

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 BL580A is shown in Figure 5 of the accompanying drawings.

A standard 13C 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 BL580A ΒΙ.580Δ 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 BL580A sodium salt and allowed to evaporate 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 BL580A.

This sodium salt of antibiotic BL580A has a melting point of 157°-161°C; C, 60.99; H, 8.47; Na, 1.95: [α]ρ5 = +6° t 1° (C=1.153 in methanol). The sodium salt of BL580A exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths: 6.27, 7.28, 9.0, 9.13, 9.23, 9.48 and 10.60u.

A standard infrared absorption spectrum of the sodium salt of BL580A prepared in a KBr pellet is shown in Figure 1 of the accompanying drawings. 13 A standard C nuclear magnetic resonance spectrum of the sodium salt of BL580A is shown in Figure 2 of the accompanying drawings.

A standard proton magnetic resonance spectrum of the sodium salt of BL580A is shown in Figure 3 of the accompanying drawings.

Example 6 Preparation and Isolation of the p_-Bromophenacyl Ester of BL580A The partial sodium salt of BL580A is prepared and isolated as described in Examples 1-3. A one gram portion of this BL580A sodium salt, 834 mg. of p_-bromo-phenacyl bromide, 600 mg. of lithium carbonate and 20 ml. of dry dimethylformamide are placed in a flask and allowed to react at 37°C. for 16 hours. Four volumes of chloroform are added and the resulting suspension is filtered. The filtrate is concentrated iri vacuo to remove the solvent giving a syrupy residue. A diatomaceous earth partition column is prepared using a system composed of heptane :ethyl acetate :methanol :water (2000:25:-1000:17). A 120 g. portion of diatomaceous earth and 90 ml. of lower phase are used for the column. The syrupy residue, 12 g. of diatomaceous earth and 9 ml. of lower phase are applied to the top of the column. The column is developed with upper phase. Fractions of 10 ml. each are collected. The activity in the fractions is located by thin layer chromatography. Fractions 22-41 are combined and concentrated in vacuo to a residue. The residue is dissolved in 50 ml. of methanol and filtered. The filtrate is warmed on a steam bath, 10 ml. of water is added and the mixture is allowed to cool slowly to 4°C. The resulting crystals are collected giving 566 mg. of BL580A as the p-bromophenacyl ester.

The p_-bromophenacyl ester of BL580A has micro-analytical data as follows: C, 58.80; H, 7.80; Br, 8.64; a specific rotation [a]^5 = +63° 2° (CHC13 at 0.51%).

The p_-bromophenacyl ester of BL580A exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths: 2.95; 5.88; 6.30; 8.20; 8.45; 8.60; 9.00; 9.15 (braod) ; 9.37 (broad); 9.62; 10.06; and 10.37.

The molecular weight of the p_-bromophenacyl ester of BL580A monohydrate is 1114 0.3% as determined by X-ray diffraction.

A standard infrared absorption spectrum of the p_-bromophenacyl ester of BL580A prepared in a KBr pellet is shown in Figure 7 of the accompanying drawings.

A standard ultraviolet absorption spectrum of the £-bromophenacyl ester of BL580A prepared at a concentration of 33.84 meg. /ml. in methanol is shown in Figure 8 of the accompanying drawings.

A standard proton magnetic resonance spectrum of the p_-bromophenacyl ester of BL580A is shown in Figure 9 of the accompanying drawings.

Claims (6)

1. A compound selected from the group consisting of antibiotic BL580A of the formula: and the pharmacologically acceptable cationic thereof.

2. A process for the production of antibiotAic BL580A which comprises cultivating Streptomyces hygro-scopicus NRRL 8180 or mutants thereof in an aqueous nutrieint medium containing assimilable sources of carbohydrate, nitrogen, and inorganic salts under submerged aerobic conditions until substantial antibiotic acitivity is imparted to said medium, and then recovering antibiotic BL580A therefrom.

3. A method of treating and prevent coccidiosis in poultry which comprises administering orally to said poultry an anticoccidally-effective amount of a compound selected from the group consisting of antibiotic BL580A and the pharmacologically acceptable cationic salts thereof.

4. The method of Claim 3 wherein the antibiotic is administered orally to poultry at a concentration in the diet of from about 125 ppm. to about 250 ppm.

5. A composition of matter for the treatment and prevention of coccidiosis in poultry which comprises a poultry diet comprising edible feedstuffs and an anticoccidally-effective amount of a compound selected from the group consisting of antibiotic BL580A and the pharmacologically acceptable cationic salts thereof.

6. A composition according to Claim 5 wherein the antibiotic is present in the diet at a concentration of from about 125 ppm.