Compositions and methods for controlling coccidiosis
This invention relates to compositions to control coccidiosis comprising antibiotic K-41 and its non-toxic salts as the active ingredient. Further, it relates to methods for control of coccidiosis using the above compositions. The compositions are used for prevention and treatment of coccidiosis. 
     Coccidiosis is a common widespread poultry disease caused by protozoa of the Genus Eimeria such as E. tenella, E. necatrix, E. acervulina and the like. One or more species of the protozoa infect and cause poultry to have diarrhoea and lesion of digestive organ leading to malnutrition, growth retardation and finally to death. The term `poultry` herein used includes, for example, chicken, turkey, duck and the like. The economic loss by this infection can not be overlooked. 
     Compounds heretofore used as anti-coccidial agents include sulfonamides, quinolines, anti-thiamine agents, antibiotics and the like. These known anti-coccidial agents, however, suffer from some drawbacks in anti-coccidial activity and toxicity. Further, the appearance of resistant strains is an unavoidable problem. Therefore, it is strongly desired to create a new powerful anti-coccidial agent with low toxicity, hardly producing resistant strains. Antibiotic K-41 was found to satisfy the requirement and studied to be made as an anti-coccidial agent. Several antibiotics have already been used as an effective ingredient of anti-coccidial compositions. Haney et al., U.S. Pat. No. 3,501,568 (Mar. 17, 1970), for example, teaches the use of antibiotic A3823 known as monensin to prevent the development of coccidiosis in poultry. Lasalocid is disclosed in Poultry Science 53, 1448 (1974) and salinomycin is in Poultry Science 56, 926 (1977). 
     The antibiotic K-41 is a superior anti-coccidial agent in its powerful activity and low toxicity and the compositions containing K-41 can be used to prevent and treat coccidiosis in poultry. 
     The antibiotic K-41 is a polyether produced by Streptomyces hygroscopicus (ATCC No. 31,227, FERM-P No. 1342). The preparation and properties are described in Japanese Patent Publication Specification (Examined) No. 1977/21077 and J. Antibiotics 29, 10-14 (1976). The principal physicochemical properties of its sodium salt are as follows: 
     Elemental analysis: C, 58.50; H, 8.28; O, 30.49; Na, 2.73%. 
     Molecular weight: 1039 (by osmometry in chloroform). 
     Molecular formula: C 48  H 81  O 19  Na (by  13  C NMR spectrum and elemental analysis). 
     Melting point: 196°-198° C. 
     Specific rotation: [α] D   23  +1.9±0.4 (c=1.017 methanol). 
     The LD 50  values of acute toxicity in mice are 47.4 mg/kg when intraperitoneally injected, 506.6 mg/kg orally and more than 1,000 mg/kg subcutaneously. 
     In anti-coccidial compositions, K-41 may be used singly or in combination with appropriate carriers ordinarily used in this field. The ordinary additives, vehicles, disintegrating agents, lubricants, stabilizers, flavourings, wetting agents, coloring agents, preservatives, aromatics and coating materials can also be combined therewith. 
     The composition can be prepared in the form of powders, granules, solutions, suspensions, dispersions, premixes, capsulates, emulsions, tablets and the like. If carrier is employed, a diluent ordinarily added to poultry feed may be used, for example, water, lactose, sucrose, talc, colloidal silica, soybean cake, starch, yeast, wheat, rice ban, defatted soybean, corn, wheat ban and the like. 
     The composition can be prepared by intimately dispersing or admixing K-41 as the pure compound, a salt, the mycelial cake or broth by methods such as grinding, stirring, milling or tumbling to give compositions of any desired concentration. The composition may include K-41 at a rate of about 0.001 to about 90 weight percent. 
     The anti-coccidial composition of this invention can be used by mixing with poultry feed or drinking water or administered to poultry directly, e.g. orally. Solutions, suspensions and emulsions are preferrably used with feed or drinking water and capsules and tablets are suited to oral administration. In general, the suitable rate of K-41 in poultry feed is about 0.001 to about 0.5 weight percent and favourably about 0.002 to about 0.012 weight percent. When added to drinking water, the concentration of K-41 may be about 0.0005 to about 0.03 weight percent. K-41 may be orally administered to poultry at a dosage of about 20 to about 200 mg/kg body weight, although the dosage is largely varied depending on particular circumstances such as type and severity of the coccidial infection to be treated. 
     Furthermore, the compositions may contain other known anti-coccidial agents and parasiticides, if necessary. 
     The non-toxic salts of K-41 can be also employed as the effective ingredient of the compositions as mentioned above. They are, for example, sodium, potassium, calcium, magnesium, aluminium salts and the like. 
     Tests of the efficacy of the antibiotic K-41  against Eimeria tenella and Eimeria acervulina in chicken were carried out and the results are shown below: 
     Experiment 1 
     (a) Test method 
     The chickens, 9-day old broiler chanky, were divided into groups of ten birds and infected with 50,000 sporulated oocysts of Eimeria tenella per chicken. The birds were fed with the diet containing the test antibiotic K-41 for 8 consecutive days after infection. On the 8th day, the birds were anatomized and caecal lesions were observed. The number of bloody droppings, survival ratio, relative weight gain, number of oocysts, caecal lesion score and feed conversion ratio were studied during the 8 days. 
     (b) Results 
     The results are shown in Table 1. 
     
                                           TABLE 1                                 
__________________________________________________________________________
Test Group          Relative                                              
                          Number                                          
                               Caecal                                     
                                    Feed                                  
Concentration                                                             
        Bloody                                                            
              Survival                                                    
                    Weight                                                
                          of   Lesion                                     
                                    Conversion                            
in feed (%)                                                               
        Droppings                                                         
              Ratio (%)                                                   
                    Gain (%).sup.(2)                                      
                          Oocyst.sup.(3)                                  
                               Score.sup.(4)                              
                                    Ratio.sup.(5)                         
__________________________________________________________________________
X-41 0.012                                                                
        --    100   59.2  0    0    2.14                                  
0.008   --    100   76.4  0    0    2.03                                  
0.004   --    100   96.9  1.9 × 10.sup.4                            
                               10   1.65                                  
Control                                                                   
(Untreated)                                                               
Infected                                                                  
        ++    100   79.1  5.6 × 10.sup.5                            
                               26   2.25                                  
Uninfected                                                                
        --    100   100.0 0    0    1.70                                  
__________________________________________________________________________
 Notes:                                                                   
 .sup.(1) Total count of bloody droppings per group on the 5th and 6th day
 after the infection are classified as follows:                           
 0: --, 1-5: +, 5-20: ++, more than 20: +++,                              
 .sup.(2) Ratio of the increased body weight of infected chickens to that 
 of uninfected chickens                                                   
 .sup.(3) The count of oocysts existing in one gram of feces of chickens o
 the 8th day after the infection (O.P.G.)                                 
 .sup.(4) The degree of pathological change in the caeca. Survival chicken
 were anatomized on the 8th day after the infection and the pathological  
 change of the oaecas was macroscopically degrees The degree of change wer
 classified into 5 orders from 0 (no change) to 4 (seriousness). The figur
 in the table means total of the score for one group of 10 chickens. (K.  
 Tsunoda and T. Ishii; Method for the examination on the chicken          
 coccidiosis, page 18 (1971).)                                            
 .sup.(5) The ratio of the feed intake (kg) to body weight gain (kg) durin
 8 days.                                                                  
 
    
     Experiment 2 
     (a) Test method 
     The chickens, 9-day old broiler chanky, were divided into groups of ten birds and infected with 500,000 sporulated oocysts of Eimeria acervulina per chicken. The birds were fed with diet containing K-41 for 5 consecutive days after infection. Discharge of mucous droppings, survival ratio, relative weight gain, number of oocyst and feed conversion ratio were observed during the five days. 
     (b) Results 
     The results are shown in Table 2. 
     
                       TABLE 2                                                     
______________________________________                                    
Test                       Rela-                                          
Group    Discharge Sur-    tive         Feed                              
Concentra-                                                                
         of Mucous vival   Weight                                         
                                 Number Conver-                           
tion in  Drop-     Ratio   Gain  of     sion                              
feed (%) pings.sup.(1)                                                    
                   (%)     (%).sup.(2)                                    
                                 Oocyst.sup.(3)                           
                                        Ratio.sup.(4)                     
______________________________________                                    
K-41 0.012                                                                
         --        100     48.4  4.4 × 10.sup.4                     
                                        2.18                              
0.008    --        100     73.8  3.5 × 10.sup.5                     
                                        1.69                              
0.004    --        100     83.1  2.4 × 10.sup.6                     
                                        1.91                              
Control                                                                   
(Untreated)                                                               
Infected +++       100     53.2  1.1 × 10.sup.7                     
                                        2.27                              
Uninfected                                                                
         --        100     100.0 0      1.88                              
______________________________________                                    
 Notes:                                                                   
 .sup.(1) The degrees of mucous droppings on the 5th and 6th days were    
 graded as follows:                                                       
 --: regular droppings, +: slightly mucous droppings, ++: medium mucous   
 droppings, +++: seriously mucous droppings                               
 .sup.(2) See (2) in the notes of TABLE 1                                 
 .sup.(3) The count of oocysts (O.P.G.) on the 5th day after the infection
 .sup.(4) See (5) in the notes of TABLE 1                                 
 
    
     (c) Conclusion 
     It is obvious from the above experimental results that K-41 has excellent anti-coccidial activity against Eimeria acervulina. 
     Experiment 3 
     (a) Test method 
     The chickens, 9-day old white leghorns, were divided into groups of 10 birds and infected with 50,000 sporulated oocysts of Eimeria tenella. The birds were fed with diet containing K-41 or monensin (product of Eli Lilly &amp; Co., Ltd.) for 8 consecutive days after infection. On the 8th day, the birds were anatomized and caecal lesions were observed. The number of bloody discharge, survival ratio, relative weight gain, number of oocyst and feed conversion ratio were studied during the 8 days. 
     (b) Results 
     The results are shown in Table 3. 
     
                       TABLE 3                                                     
______________________________________                                    
Test                                                                      
Group                  Rela-                                              
Concen-        Sur-    tive                Feed                           
tration                                                                   
       Bloody  vival   Weight                                             
                             Number Caecal Con-                           
in feed                                                                   
       Drop-   Ratio   Gain  of     Lesion version                        
(%)    pings   (%)     (%).sup.(2)                                        
                             Oocyst.sup.(3)                               
                                    Score.sup.(4)                         
                                           Ratio.sup.(5)                  
______________________________________                                    
K-41                                                                      
0.012  --      100     96.2  5.0 × 10.sup.4                         
                                    10     1.99                           
Monensin                                                                  
0.012  +       100     82.3  2.8 × 10.sup.5                         
                                    10     2.23                           
Control                                                                   
Infected                                                                  
       +++      80     43.0  1.5 × 10.sup.6                         
                                    40     2.41                           
Un-                                                                       
infected                                                                  
       --      100     100   0       0     2.05                           
______________________________________                                    
 NOtes:                                                                   
 .sup.(1), .sup.(2), .sup.(3), .sup.(4), (5) See the notes in the         
 corresponding number in Table 1                                          
 
    
     (c) Conclusion 
     It is obvious from the above results that K-41 shows the superior anti-coccidial activity against Eimeria tenella in chickens to monensin, an anti-coccidial agent on the market. 
     As clearly shown above, K-41 has a powerful anti-coccidial activity. Thus, K-41 can be used to prevent and treat coccidiosis. 
    
    
     The following examples are given solely for the purpose of illustration and are not be construed as limitative of the present invention, many variations of which are possible. In these examples all the percents are given by weight. 
     EXAMPLE 1 
     Ten percent of K-41 is admixed with 90 percent of lactose to give a 10-fold trituration. The mixture is diluted with feed to a concentration of 0.001 to 0.05% of the effective ingredient in the feed before use. 
     EXAMPLE 2 
     Ten percent of K-41 is admixed with 90 percent of sucrose or starch to give a 10-fold trituration. The mixture is diluted with feed to a concentration of 0.001 to 0.05% of the effective ingredient in the feed before use. 
     EXAMPLE 3 
     Ten percent of K-41 is admixed with 90 percent of defatted soybean to give a 10-fold trituration. The mixture is diluted with feed to a concentration of 0.001 to 0.05% of the effective ingredient in feed before use. 
     EXAMPLE 4 
     Twenty-five percent of K-41 is admixed with 75 percent of wheat flour to give a powder. The powder is diluted with feed to a concentration of 0.001 to 0.05% of the effective ingredient in the feed before use. 
     EXAMPLE 5 
     Forty-five percent of K-41, 12 percent of sucrose, 15 percent of starch, 25 percent of talc, 2 percent of magnesium stearate and 1 percent of stearic acid are admixed. 
     The mixture is made to granules and pressed into tablets. 
     EXAMPLE 6 
     The percent of sodium salt of K-41 is admixed with 90 percent of defatted soybean to give a 10-fold trituration. The mixture is diluted with feed to a concentration of 0.001 to 0.05% of the effective ingredient in feed before use. 
     EXAMPLE 7 
     One percent of sodium salt of K-41 is admixed with 99 percent of lactose to give a 100-fold trituration. The mixture is diluted with water to a concentration of 0.005 to 0.03% of the effective ingredient.