a . molecular formula : c 36 h 31 no 12 ; d . specific rotation [ α ] d 26 =- 52 ° ( c , 1 . 11 % dichloromethane ); e . ultra absorption spectra : λmax nm ε 0 . 1n hcl = 224 nm ( 27 , 700 ), 225 nm ( 21 , 900 ), 328 nm ( 16 , 400 ), 420 nm ( 4 , 230 ); λmax nm ε ch 3 oh = 222 nm ( 38 , 200 ), 240 nm ( 30 , 100 ), 255 nm ( 30 , 100 ), 321 nm ( 24 , 500 ), 384 nm ( 8 , 050 ); f . infrared absorption spectrum : ( kbr disk ), max ( cm - 1 ) 1800 , 1742 , 1690 , 1621 , 1422 , 1273 ; g . proton magnetic resonance spectrum : ( cdcl 3 ) with significant peaks as follows : ______________________________________δ # h m j ( hz ) ______________________________________13 . 47 1 s -- 8 . 20 1 d 8 . 57 . 92 1 d 8 . 57 . 58 1 s -- 7 . 19 1 s -- 7 . 09 1 s -- 4 . 78 1 d 11 . 64 . 45 1 d 11 . 64 . 02 3 s -- 3 . 99 3 s -- 3 . 48 1 d 12 . 83 . 35 1 d 12 . 82 . 21 2 d 6 . 82 . 10 1 m -- 1 . 83 3 s -- 1 . 80 3 s -- 0 . 955 6 d 6 . 48 ; ______________________________________ h . a carbon - 13 nuclear magnetic resonance spectrum : ( cdcl 3 ) with significant peaks as follows : ______________________________________δ m δ m δ m______________________________________181 . 2 s 134 . 8 s 65 . 0 t178 . 1 s 132 . 1 d 62 . 3 s 172 . 1 * s 129 . 7 s 56 . 8 q171 . 5 s 124 . 4 d 56 . 5 q165 . 8 s 120 . 8 s 42 . 9 t162 . 1 s 119 . 9 s 41 . 9 t155 . 5 s 119 . 5 s 25 . 8 q153 . 4 s 117 . 7 d 25 . 5 d150 . 8 s 107 . 3 s 22 . 4 q148 . 7 s 104 . 9 d 22 . 3 q140 . 7 s 100 . 4 d 20 . 2 q137 . 7 s 93 . 4 s______________________________________ * two superimposed resonances . in one preferred embodiment the present invention is a highly effective method for preventing , controlling or treating coccidiosis caused by eimeria species in meat - producing animals , particularly chickens , turkeys , quail , pheasants , geese and ducks , but is likewise effective for controlling coccidiosis in other meat - producing animals by orally administering thereto an anticoccidially effective amount of the compound designated ll - e19085α or a mutant thereof . in practice , it is generally found that feed or drinking water containing from about 25 ppm to 1500 ppm and preferably 50 ppm to 1430 ppm of this compound ll - e18085α or pharmaceutically acceptable salt thereof is effective for controlling , preventing or eliminating , coccidiosis in animals consuming the treated feed or drinking water . when the compounds of the invention are employed for the control of coccidiosis , the active compound is generally prepared as an animal feed premix or concentrate containing a relatively high percentage of the active ingredient . the premix or concentrate may then be blended with the animals daily ration or applied as a top dressing thereto . feed premixes or concentrates may be prepared by blending about 1 . 0 % to 25 %, and preferably about 5 . 0 % to 15 . 0 % by weight , of ll - e19085α or pharmaceutically acceptable salt of said anticoccidial or mutant thereof , or fermentation mash insolubles containing said antibacterial compound , with about 75 % to 99 . 0 % by weight of an edible carrier or diluent . diluents suitable for use in preparation of said feed supplements and premixes include : corn meal , soybean meal , cotton seed oil meal , sodium chloride , bone meal , cane molasses , corn cob grits , rice hull meal , urea , and the like . the diluent helps to assure uniform distribution of the active ingredient throughout the finished feed into which it blended . usually about one gram of premix is added per two kilograms of feed but larger or smaller amounts may be used , if desired . although administration of the compounds for controlling coccidiosis will generally be most practical when administered in or with the feed , or in the drinking water , the compounds of this invention may be administered to individual hosts in the form of pills , gels , boluses , tablets , drenches , capsules or the like . these latter types of administration are , of course , less practical than administration in or with the feed or drinking water , but can be utilized effectively for the treatment of a limited number of animals , particularly larger animals such as cattle , swine or sheep . since ll - e19085α is relatively insoluble in water , when administering said compound in water it will generally be most effective to first dissolve the active compound in a pharmaceutically acceptable organic solvent such as methanol , ethanol , acetone , dmso , or the like , containing a suitable surfactant or dispersing agent , and then disperse the liquid composition in the drinking water of the animals being treated . the new antibacterial agent ll - e19085α is produced by aerobic fermentation of microbial culture ll - e19085 which is a natural selection isolate of a culture isolated from a soil sample collected in manyara , tanzania . the culture was taxonomically characterized and identified as a new subspecies of micromonospora citrea . this new subspecies is maintained in the culture collection of the medical research division , american cyanamid company , pearl river , n . y . as culture number ll - e19085 . a viable culture of this new microorganism has been deposited with the ars culture collection , fermentation laboratory , northern regional research center , u . s . department of agriculture , 1815 north university street , peoria , ill . 61604 and has been added to its permanent collection . it has been assigned the strain designation nrrl 18351 by such depository . observations were made of the cultural , physiological and morphological features of culture ll - e19085 using methods well known in the art . the generic assignment of ll - e19085 to the genus micromonospora was confirmed morphologically and chemically . the strain produced monospores on the vegetative hyphae . no aerial hyphae were observed . electron microscopic examination showed that the spores were warty . whole cell analysis showed that ll - e19085 containing the meso isomer as well as traces of the l isomer of diaminopimelic acid . this strain showed the presence of xylose plus traces of arabinose in its whole cell sugar hydrolysates . therefor ll - e19085 is considered to be a subspecies of micromonospora citrea . comparative data on the morphology of ll - e19085 is given in tables i and ii . physiolocial data is given in tables iii and iv . table i______________________________________isp agar solublemedium spores vegetative mycelium pigments______________________________________yeast - malt slight , strong orange ( 50 ) slight ,( isp 2 ) black to medium orange - brown - at edge yellow ( 71 *) blackoatmeal none light orange - slight ,( isp 3 ) yellow ( 70 ) to brownish vivid orange - yellow ( 66 ) inorganic none light orange - slight , salts - starch yellow ( 70 ) to brownish ( isp 4 ) vivid orange - yellow ( 66 ) glycerol - slight , light tone of slight , asparagine brownish brownish - orange brownish ( isp 5 ) at edge ( 54 ) ______________________________________ * parenthetical numbers are colors taken from kelly , k . l . and judd , d . b . color . universal language and dictionary of names , nat . bur . stand . ( u . s . ), spec . publ . 440 , 1976 , washington , d . c . and the accompanying intersociety color council , national bureau of standards centroid color charts . table ii______________________________________ actinomycete growthagar medium ( 28 ° c ., 2 weeks ) ______________________________________pablum brown vegetative hyphae . sparse spores . soluble dark brown pigment . yeast czapek &# 39 ; s brownish tan vegetative hyphae . sparse spores . slight soluble dark - brownish pigment . czapek &# 39 ; s vegetative hyphae covered / spores . black spores . slight dark pigment . yeast extract - black spores . dry solubledextrose brownish pigment . nutrient orange - brown vegetative hyphae . sparse spores . intense brown - black pigment . bennett &# 39 ; s dextrin tan vegetative hyphae . moderate black spores . soluble reddish - brown pigment . glucose asparagine orange - tan vegetative hyphae . no spores . slight soluble dark pigment . ______________________________________ table iii______________________________________ carbohydratecarbohydrate utilization______________________________________arabinose + cellulose - fructose ± glucose + inositol - mannitol - raffinose - rhamnose - sucrose ± xylose + ______________________________________ table iv______________________________________ physiologicalgordon test reaction______________________________________hydrolysis ofcasein + xanthine - hypoxanthine - tyrosine + adenine + gelatin + potato starch + esculin + physiologicalproduction ofnitrate reductase - phosphatase + urease - growth onsalicin - 5 % sodium chloride - lysozyme broth - decarboxylation ofacetate + benzoate - citrate - lactate + malate - mucate - oxalate - propionate + pyruvate + succinate - tartrate - acid fromadonitol - arabinose + cellobiose + dextrin + dulcitol - erythritol - fructose + galactose + glucose + glycerol + inositol - lactose + maltose + mannitol - mannose + α - methyl - d - glucoside + melibiose + raffinose + salicin + sorbitol - sucrose + trehalose + xylose + β - methyl - d - xyloside - growth at10 ° c . - 42 ° c . + 45 ° c . + ______________________________________ + = positive ; - = negative it is to be understood that for the production of the new antibacterial agent ll - e19085α , the present invention is not limited to this particular organism or to organisms fully answering the above growth and microscopic characteristics , which are given for illustrative purposes only . in fact it is desired and intended to include the use of mutants produced from this organism by various means such as exposure to x - radiation , ultraviolet radiation , n &# 39 ;- methyl - n - nitro - n - nitrosoguanidine , actinophages and the like , as well as the protozoacidal agents produced by these mutants . cultivation of micromonospora citrea sp . ll - e19085 may be carried out in a wide variety of liquid culture media . media which are useful for the production of antibiotic ll - e19085α 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 , cornsteep liquor , etc . ; and inorganic anions and cations , such as potassium , sodium , ammonium , calcium , sulfate , carbonate , phosphate , chloride , etc . trace elements such as boron , molybdenum , copper , etc ., are supplied as impurities of other constituents of the medium . aeration is supplied by forcing sterile air through or onto the surface of the fermenting medium . agitation is provided by a mechanical impeller . an antifoam agent may be added as needed . the growth of the organism is usually conducted at about 24 °- 37 ° c ., preferably at about 28 ° c . the invention in this application is further demonstrated by the examples set forth below . evaluation of the effectiveness of compound ll - e19085α is determined in the following manner : the vitamin - amino acid premix in the above feed composition is prepared from the following formulation . the expressions of quantity relate to the units per kilogram of the finished feed composition . 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 untreated controls was given to one - day - old chicks , by direct inoculation into the crops of all chicks . the chicks were given free access to feed and water during the entire test period . two days before inoculation , medicated feed with several levels of drug was presented to the various groups of chicks . seven days after inoculation the tests were terminated and the birds were weighed , necropsied and their intestinal tracts examined for lesions . the results appear in table v below . these results show that improved survival of infected chicks is obtained when 50 to 1430 ppm or less of antibiotics ll - e19085α is administered to infected chicks in their diet . these levels also show a significant suppression of lesions due to eimeria tenella and eimeria acervulina . these levels are also shown to provide significant improvement in weight gains in the treated birds . table v__________________________________________________________________________evaluation of antibiotic ll - e19085alpha as an anticoccidial agent inchicks relative weight no % birds with reduced lesions gains untreatedtest concentration birds % e . e . uninfected birdsno compound in diet , ppm started survival tenella acervulina % ± controls__________________________________________________________________________i ll - e19085alpha 50 5 80 0 100 + 28 ll - e19085alpha 100 5 100 0 100 + 47 ll - e19085alpha 200 5 60 20 100 + 65ii ll - e19085alpha 340 5 100 0 0 + 30iii ll - e19085alpha 360 5 100 0 60 + 30 ll - e19085alpha 715 5 100 60 100 + 51 ll - e19085alpha 1430 5 100 80 100 + 36__________________________________________________________________________ a typical medium used to grow the inocula was prepared according to the following formula : this medium was sterilized and a 100 ml portion in a 500 ml flask was inoculated with mycelial scrapings from an agar slant of the culture micromonospora citrea sp ll - e19085 . the inoculated flask was then placed on a rotary shaker and agitated vigorously for approximately 48 hours at 32 ° c ., providing primary inoculum . a 100 ml portion of this primary inoculum was then used to inoculate 10 liters of the above sterile medium which was incubated at 32 ° c . with aeration for 72 hours , providing secondary inoculum . a 10 liter portion of this secondary inoculum was then used to inoculate 260 liters of the above sterile medium in a tank . this medium was incubated at 32 ° c . with agitation by an impeller driven at 180 rpm , a sterile air flow of 200 liters per minute and the addition of 50 ml of a defoaming agent for about 48 hours , providing tertiary inoculum . a 2800 liter portion of the above medium in a tank was sterilized and then inoculated with 300 liters of tertiary inoculum prepared as described in example 2 . aeration was supplied at the rate of 6 . 5 liters of sterile air per liter of mash per minute and agitation was supplied by an impeller driven at about 110 rpm . the temperature was maintained at 28 ° c . and defoaming agent was added as required . the fermentation was terminated after 129 hours . a 1500 liter portion of the whole harvest mash , prepared as described in example 3 was mixed with 15 liters of toluene for 30 minutes , then 250 lb of diatomaceous earth was added . after mixing for 15 minutes this mixture was filtered and the cake washed with 150 liters of water . the cake was slurried in a mixture of 208 liters of acetone , 416 liters of dichloromethane and 20 liters of 1 . 5n hydrochloric acid for 2 hours and then filtered . the cake was washed with about 175 liters of dichloromethane with the wash and filtrate combined . the cake was then washed with about 800 liters of water and this wash also combined with the above wash and filtrate and mixed . the dichloromethane layer was separated and washed with an equal volume of water . the dichloromethane layer was separated and concentrated to 100 liters , reextracted with fresh methylene chloride if any aqueous phase was present , and finally concentrated to about 1 - 3 liters . the dichloromethane extracts were triturated repeatedly , first with hexane : dichloromethane ( 9 : 1 ) and then with hexane along to remove the bulk of the fatty impurities giving a brown powder . several such partially purified preparations , from fermentations conducted as described in example 3 , totaling 20 g and averaging 10 - 30 % ll - e19085α , were combined and purified by reverse - phase chromatography . the column consisted of a 15 liter bed of c 18 bonded phase packing of 40 micron particle size . the charge was loaded onto the column in 500 ml of acetonitrile : tetrahydrofuran ( 1 : 1 ). the column was developed at a flow rate of 1 . 0 liter per minute with a mobile phase consisting of acetonitrile : 0 . 1m ph 4 . 5 ammonium acetate buffer ( 8 : 2 ). fractions were collected at approximately 12 minute intervals . fractions 6 and 7 were combined and evaporated , giving 2 . 7 g of pure ll - e19085α having the characteristics disclosed in the hereinabove specification .
this invention relates to a method and composition for the control of protozoal infections in warm - blooded animals by orally administering to said animals an effective amount of a new antibacterial produced in a microbiological fermentation under controlled conditions using a new subspecies of micromonospora citera and mutants thereof . the antibacterial designated ll - e19085α is a highly effective anticoccidial agent especially useful for the prophylactic , pharmaceutical , and therapeutic treatment of poultry and other animals raised for meat production .