Methods and compositions for the control of coccidiosis

Abstract

Methods are provided for the sporulation, sterilization and storage of coccidial oocyst which are characterized by an absence of the highly toxic chemical potassium dichromate. Also provided are compositions containing sporulated oocysts which are free of potassium dichromate.

What is claimed is:  
     
         1 . A composition for the prevention or control of coccidiosis comprising viable wild type sporulated oocysts of at least one species of protozoa known to cause coccidiosis, wherein said composition is sterile and contains at least about 10,000 oocysts per milliliter and less than about 0.8% by weight of alkali metal dichromate.  
     
     
         2 . A composition as set forth in  claim 1  wherein the composition contains less than about 0.6% by weight of alkali metal dichromate.  
     
     
         3 . A composition as set forth in  claim 2  wherein the composition contains less than about 0.4% by weight of alkali metal dichromate.  
     
     
         4 . A composition as set forth in  claim 3  wherein the composition contains less than about 0.2% by weight of alkali metal dichromate.  
     
     
         5 . A composition as set forth in  claim 4  wherein the composition contains less than about 0.1% by weight of alkali metal dichromate.  
     
     
         6 . A composition as set forth in  claim 1  wherein said composition is characterized as substantially free of alkali metal dichromate.  
     
     
         7 . A composition as set forth in  claim 1  wherein said composition contains less than about 0.3% by weight of dichromate ion.  
     
     
         8 . A composition as set forth in  claim 7  wherein said composition contains less than about 0.15% by weight of hexavalent chromium.  
     
     
         9 . A composition for the prevention or control of coccidiosis comprising viable wild type sporulated oocysts of at least one species of protozoa known to cause coccidiosis, wherein said composition is sterile and contains at least about 300 oocysts per milliliter and less than about 0.002% by weight of alkali metal dichromate.  
     
     
         10 . A composition for the prevention or control of coccidiosis comprising viable wild type sporulated oocysts of at least one species of protozoa known to cause coccidiosis, wherein said composition is sterile and contains less than about 5.0×10 −3  μg of alkali metal dichromate per oocyst.  
     
     
         11 . A composition as set forth in  claim 10  wherein said composition is sterile and contains less than about 3.8×10 −3  μg of alkali metal dichromate per oocyst.  
     
     
         12 . A composition as set forth in of  claim 11  wherein said composition is sterile and contains less than about 1.3×10 −3  μg of alkali metal dichromate per oocyst.  
     
     
         13 . A composition as set forth in of  claim 12  wherein said composition is sterile and contains less than about 6.3×10 −5  μg of alkali metal dichromate per oocyst.  
     
     
         14 . A composition as set forth in  claim 1 , further comprising a diluent.  
     
     
         15 . A composition as set forth in  claim 14 , wherein the diluent comprises water.  
     
     
         16 . A composition as set forth in  claim 15 , wherein the aqueous diluent comprises 0.5× phosphate buffered saline.  
     
     
         17 . A composition as set forth in  claim 16  further comprising a buffer.  
     
     
         18 . A composition as set forth in  claim 17 , wherein said buffer is selected from the group consisting of phosphate buffer, bicarbonate buffer, citric acid and tris buffers.  
     
     
         19 . A composition as set forth in  claim 17 , wherein said buffer controls pH between about 7.0 and about 7.8  
     
     
         20 . A composition as set forth in  claim 14 , further comprising a bactericide.  
     
     
         21 . A composition as set forth in  claim 20 , wherein said bactericide is selected from the group consisting of potassium perchlorate, sodium hypochlorite, hydrochlorous acid, sodium hydroxide and antibiotics.  
     
     
         22 . A composition as set forth in of  claim 21 , wherein said composition contains from about 0.1 to about 0.75 wt % potassium perchlorate, and/or from about 0.001 to about 0.01 wt % sodium hypochlorite, and/or from about 1 to about 5 ppm hydrochlorous acid, and/or from about 0.5 to about 1.5 mM sodium hydroxide and/or from about 20 to about 30 μg/ml gentamicin, in the final composition.  
     
     
         23 . A composition as set forth in  claim 14 , further comprising a composition that ameliorates a decline in post-challenge performance.  
     
     
         24 . A composition as set forth in  claim 23 , wherein said composition is selected from the group consisting of Alum, Freund's adjuvant, calcium phosphate, beryllium hydroxide, dimethyl dioctadecyl ammonium bromide, saponins, polyanions, Quil A, inulin, lipopolysaccharide endotoxins, liposomes, lysolecithins, zymosan, propionibacteria, mycobacteria, interleukin-1, interleukin-2, interleukin-4, interleukin-6, interleukin-12, interferon-α, interferon-γ, and granulocyte-colony stimulating factor.  
     
     
         25 . A composition as set forth in  claim 23 , wherein said composition is selected from the group consisting of cytokines, growth factors, chemokines, mitogens and adjuvants.  
     
     
         26 . A composition as set forth in  claim 25 , wherein said composition comprises  Propionibacterium acnes.    
     
     
         27 . A composition as set forth in  claim 26 , wherein said composition contains at least about 3.0 milligrams (dry weight) of  P. acnes  per milliliter.  
     
     
         28 . A composition as set forth in  claim 26 , wherein said composition contains at least about 30 milligrams (dry weight) of  P. acnes  per milliliter.  
     
     
         29 . A composition as set forth in  claim 1  comprising: 
 viable sporulated oocysts of at least one species of protozoa known to cause coccidiosis,  
 a diluent,  
 a buffer, and  
 a bactericide,  
 wherein said composition contains about 10,000 oocysts pers milliliter and less than about 0.8% weight to volume of alkali metal dichromate.  
 
     
     
         30 . A composition as set forth in  claim 29 , further comprising a composition that ameliorates a decline in post-challenge performance.  
     
     
         31 . A method for producing a composition for the prevention or control of coccidiosis comprising: 
 collecting manure from host animals wherein said manure contains oocysts known to cause coccidiosis;    diluting said manure in an aqueous medium to create a slurry;    separating unwanted fecal matter from said slurry and collecting the aqueous fraction containing oocysts;    subjecting said aqueous fraction to solid/liquid phase centrifugal-based separation and collecting the solid phase;    combining a dense aqueous liquid with said collected solid phase wherein said dense liquid has a density greater than about 1.09 g/ml and wherein the oocysts are buoyant;    subjecting the combination of said dense aqueous liquid and collected solid phase to centrifugation and collecting the dense liquid fraction containing oocysts;    diluting said dense liquid fraction to a specific gravity wherein the oocysts are no longer buoyant;    separating oocyst solids from said diluted liquid fraction by centrifugal-based separation and re-collecting the solid phase.    
     
     
         32 . A method as set forth in  claim 31  further comprising: 
 diluting said re-collected solid phase in an aqueous sporulation medium;  
 sporulating said oocysts while in contact with said sporulation medium;  
 separating sporulated oocysts from said sporulation medium;  
 sterilizing said sporulated oocysts; and  
 diluting said sporulated oocysts to form a vaccine composition.  
 
     
     
         33 . A method of separating oocysts from a liquid suspension by the use of a hydrocyclone.  
     
     
         34 . A method as set forth in  claim 33  wherein the oocysts are collected in the underflow from the hydrocyclone.  
     
     
         35 . A method for isolating oocysts comprising: 
 collecting manure from host animals wherein said manure contains oocysts known to cause coccidiosis;    diluting said manure in an aqueous medium to create a slurry;    separating unwanted fecal matter from said slurry and collecting the aqueous fraction containing oocysts;    subjecting said aqueous fraction to solid/liquid phase centrifugal-based separation by means of a hydrocyclone.    
     
     
         36 . A method for isolating oocysts comprising: 
 collecting manure from host animals wherein said manure contains oocysts known to cause coccidiosis;    diluting said manure in an aqueous medium to create a slurry;    separating unwanted fecal matter from said slurry and collecting the aqueous fraction containing oocysts;    subjecting said aqueous fraction to solid/liquid phase centrifugal-based separation and collecting the solid phase;    combining a dense aqueous liquid with said collected solid phase wherein said dense liquid has a density greater than about 1.09 g/ml and wherein the oocysts are buoyant;    subjecting the combination of said dense aqueous liquid and collected solid phase to centrifugation and collecting the dense liquid fraction containing oocysts;    diluting said dense liquid fraction to a specific gravity wherein the oocysts are no longer buoyant;    separating oocyst solids from said liquid phase by means of a hydrocyclone and re-collecting the solid phase.    
     
     
         37 . A method as set forth in  claim 31  wherein said animals comprise the class Aves.  
     
     
         38 . A method as set forth in  claim 37  wherein said slurry is created by mixing manure and water in relative proportions of from about 0.5 gallons to about 5 gallons of domestic water per the amount of manure obtained in about 3 days from about six animals comprising the class Aves.  
     
     
         39 . A method as set forth in  claim 38  wherein said animals are chickens.  
     
     
         40 . A method as set forth in  claim 31  wherein said separation of unwanted fecal matter comprises sieving.  
     
     
         41 . A method as set forth in  claim 40  wherein said sieving is by the use of multiple-tier shaker screens.  
     
     
         42 . A method as set forth in  claim 41  wherein said shaker screens comprise a 50-mesh screen and a 250-mesh screen.  
     
     
         43 . A method as set forth in  claim 31  wherein said method is carried out at a temperature between about 4° C. and about 30° C.  
     
     
         44 . A method as set forth in  claim 43  wherein said sieving is carried out at a temperature between about 22° C. and about 28° C.  
     
     
         45 . A method as set forth in  claim 44  wherein said sieving is carried out at about 25° C.  
     
     
         46 . A method as set forth in  claim 31  wherein each of said centrifugal-based separations comprises the use of a centrifuge or a hydrocylcone.  
     
     
         47 . A method as set forth in  claim 46  wherein said centrifugal-based separation comprises the use of a hydrocyclone.  
     
     
         48 . A method as set forth in  claim 47  wherein said centrifugal-based separation comprises the use of a centrifuge.  
     
     
         49 . A method as set forth in  claim 48  wherein said centrifuge is a bottle centrifuge.  
     
     
         50 . A method as set forth in  claim 48  wherein said centrifuge is a continuous centrifuge.  
     
     
         51 . A method as set forth in  claim 31  wherein said centrifugation is a bottle centrifuge.  
     
     
         52 . A method as set forth in  claim 31  wherein said dense aqueous liquid comprises a solution of corn syrup or sodium chloride.  
     
     
         53 . A method as set forth in  claim 31  wherein said aqueous solution has a density from about 1.07 g/ml to about 1.20 g/ml.  
     
     
         54 . A method as set forth in  claim 53  wherein said aqueous solution has a density from about 1.08 g/ml to about 1.14 g/ml.  
     
     
         55 . A method as set forth in  claim 54  wherein said aqueous solution has a density from about 1.09 g/ml to about 1.10 g/ml.  
     
     
         56 . A method for inducing sporulation of oocysts comprising: 
 introducing into an aqueous sporulation medium oocysts of at least one species of protozoa known to cause coccidiosis;    incubating said oocysts in said aqueous sporulation medium, thereby causing sporulation of oocysts; and    introducing an oxidizing agent into said medium at a rate sufficient to maintain the average dissolved oxygen content during sporulation at at least 30%.    
     
     
         57 . A method as set forth in  claim 56  wherein said dissolved oxygen content is substantially maintained at at between about 30% and about 80% of saturation throughout the period of sporulation.  
     
     
         58 . A method as set forth in  claim 57  wherein said dissolved oxygen content of the medium is substantially maintained at between about 40% and about 60% of saturation throughout the period of sporulation.  
     
     
         59 . A method as set forth in  claim 58  wherein said dissolved oxygen content of the medium is substantially maintained at about 50% of saturation.  
     
     
         60 . A method as set forth in  claim 56  wherein the alkali metal dichromate content of said sporulation medium is less than about 0.8% by weight during incubation of oocysts.  
     
     
         61 . A method as set forth in  claim 56  comprising addition to said sporulation medium of an oxidizing agent having a standard reduction potential of at least about 0.5 V.  
     
     
         62 . A method as set forth in  claim 61  comprising addition of both molecular oxygen and another oxidizing agent.  
     
     
         63 . A method as set forth in  claim 62  wherein said oxidizing agent has a standard reduction potential of at least about 0.5 V.  
     
     
         64 . A method as set forth in  claim 63  wherein said oxidizing agent is selected from the group consisting of an alkali metal hypochlorite, an alkali metal chlorite, an alkali metal chlorate, an alkali metal perchlorate, and an alkali metal permanganate.  
     
     
         65 . A method as set forth in  claim 64  wherein said oxidizing agent comprises hypochlorite ions.  
     
     
         66 . A method as set forth in  claim 64  wherein a sufficient amount of an alkali metal hypochlorite is added to achieve an alkali metal hypochlorite weight percent from about 0.001 weight percent to about 0.1 weight percent of the sporulation medium and oocysts combined, wherein said alkali metal hypochlorite is from about 1.0% to about 10.0% by volume.  
     
     
         67 . A method as set forth in  claim 56  further comprising: 
 separating sporulated oocysts from said sporulation medium;  
 sterilizing sporulated oocysts by contacting said sporulated oocysts with a chemical disinfectant; and  
 storing said sporulated oocysts in a sterile diluent, said diluent containing less than about 0.8% by weight alkali metal dichromate.  
 
     
     
         68 . A method as set forth in  claim 56  wherein said medium contains less than about 0.3% by weight dichromate ion during incubation of said oocysts.  
     
     
         69 . A method as set forth in  claim 56  wherein said medium contains less than about 0.15% by weight hexavalent chromium during incubation of said oocysts.  
     
     
         70 . A method as set forth in  claim 56  wherein said dissolved oxygen content is established by bubbling an oxygen-containing gas through said sporulation medium.  
     
     
         71 . A method as set forth in  claim 70  wherein said oxygen-containing gas consists essentially of air.  
     
     
         72 . A method as set forth in  claim 70  wherein said gas comprises commercially pure oxygen.  
     
     
         73 . A method as set forth in  claim 56  further comprising maintaining the temperature from a temperature that substantially avoids freezing to about 45° C.  
     
     
         74 . A method as set forth in  claim 73  wherein temperature is maintained from about 15° C. to about 40° C.  
     
     
         75 . A method as set forth in  claim 74  wherein temperature is maintained from about 20° C. to about 30° C.  
     
     
         76 . A method as set forth in  claim 75  wherein temperature is maintained at about 28° C.  
     
     
         77 . A method as set forth in  claim 56  further comprising incubating the oocysts under said conditions from about 72 hours to about 120 hours.  
     
     
         78 . A method as set forth in  claim 77  wherein the oocysts incubate from about 72 hours to about 96 hours.  
     
     
         79 . A method as set forth in  claim 78  wherein the oocysts incubate for about 72 hours.  
     
     
         80 . A method as set forth in  claim 56  further comprising controlling the pH of the sporulation medium.  
     
     
         81 . A method as set forth in  claim 79  wherein the pH is controlled by the introduction of an acid or base to the sporulation medium.  
     
     
         82 . A method as set forth in  claim 81  wherein the pH of the sporulation medium is controlled by alternatively adding sodium hydroxide and sulfuric acid to the sporulation medium.  
     
     
         83 . A method as set forth in  claim 82  wherein the pH of the sporulation medium is controlled from about 7.2 to about 7.5.  
     
     
         84 . A method as set forth in  claim 83  wherein the pH of the sporulation medium is controlled at about from 7.35 to about 7.45.  
     
     
         85 . A method as set forth in  claim 67  wherein said sporulated oocysts are separated from said sporulation medium by filtration or by centrifugal-based separation.  
     
     
         86 . A method as set forth in  claim 85  wherein said sporulated oocysts are separated by filtration.  
     
     
         87 . A method as set forth in  claim 86  wherein said sporulated oocysts are separated from the sporulation medium by tangential flow filtration.  
     
     
         88 . A method as set forth in  claim 67  wherein said sterilization is achieved by adding a chemical disinfectant to sporulated oocysts separated from said sporulation medium.  
     
     
         89 . A method as set forth in  claim 88  wherein said sterilization substantially eliminates microorganisms.  
     
     
         90 . A method as set forth in  claim 89  wherein said microorganisms are selected from the group comprising infectious bursal disease virus and chicken anemia virus.  
     
     
         91 . A method as set forth in  claim 88  wherein said sterilization is by a chemical disinfectant other than an alkali metal dichromate.  
     
     
         92 . A method as set forth in  claim 88  wherein said chemical disinfectant comprises a solution of an alkali metal hypochlorite.  
     
     
         93 . A method as set forth in  claim 92  wherein said chemical disinfectant comprises a solution of sodium hypochlorite.  
     
     
         94 . A method as set forth in  claim 93  wherein said solution used is at a concentration from about 1% to about 20% by volume of active chlorine.  
     
     
         95 . A method as set forth in  claim 94  wherein said is at a concentration from about 5% to about 15% by volume of active chlorine.  
     
     
         96 . A method as set forth in  claim 95  wherein said solution is at a concentration of about 10% by volume of active chlorine.  
     
     
         97 . A method as set forth in  claim 93  wherein said sporulated oocysts are treated with said sodium hypochlorite from about 5 to about 25 minutes.  
     
     
         98 . A method as set forth in  claim 97  wherein said sporulated oocysts are treated with sodium hypochlorite from about 8 to about 20 minutes.  
     
     
         99 . A method as set forth in  claim 98  wherein said sporulated oocysts are treated with sodium hypochlorite for about 10 minutes.  
     
     
         100 . A method as set forth in 92 further comprising substantially separating said sodium hypochlorite from the sporulated oocysts by filtration.  
     
     
         101 . A method as set forth in  claim 100  wherein said filtration is by means of tangential flow filtration.  
     
     
         102 . A method for inducing sporulation of oocysts comprising: 
 introducing into an aqueous sporulation medium oocysts of at least one species of protozoa known to cause coccidiosis;    incubating said oocysts in said aqueous sporulation medium, thereby causing sporulation of oocysts; and    introducing an oxidizing agent having a standard reduction potential of at least about 0.5 V into said medium at a rate sufficient to maintain the oxidation potential of said medium equivalent to the oxidation potential of a medium containing dissolved molecular oxygen in concentration of at least 30% of saturation during sporulation;    said medium containing less than about 0.8% by weight alkali metal dichromate during incubation of said oocysts.    
     
     
         103 . A method for monitoring sporulation of oocysts comprising: 
 incubating viable oocysts in an aqueous sporulation medium; and    during incubation, monitoring said medium to detect a change in at least one of the following parameters: 
 (i) dissolved oxygen content;  
 (ii) pH;  
 (iii) rate of introduction of oxidizing agent into said medium;  
 (iv) flow rate of acid or base into said medium.  
   
     
     
         104 . A method as set forth in  claim 103  wherein dissolved oxygen content of said medium is controlled by addition of molecular oxygen thereto, and monitoring sporulation comprises detecting a change in oxygen consumption as indicated by detection of a change in oxygen flow to the medium and/or a permanent or transient change in dissolved oxygen content.  
     
     
         105 . A method as set forth in  claim 103  wherein pH of said medium is controlled by addition of acid or base thereto, and monitoring sporulation comprises detecting an increase in acid consumption as indicated by an increase in acid flow to the medium and/or a permanent or transient increase in pH.  
     
     
         106 . A method as set forth in  claim 103  wherein the end point of sporulation is determined from substantial cessation of oxygen consumption or generation of alkalinity in the sporulation medium.  
     
     
         107 . A method as set forth in  claim 106  wherein the end point is indicated by the substantial cessation of change in at least one of said parameters.  
     
     
         108 . A method as set forth in  claim 106  wherein said sporulated oocysts are maintained is said medium under sporulation conditions for at least another 48 hours after the indicated end point of sporulation.  
     
     
         109 . A method as set forth in  claim 103 , wherein said change in dissolved oxygen content is a decrease.  
     
     
         110 . A method as set forth in  claim 103 , wherein said change in pH is an increase.  
     
     
         111 . A method as set forth in  claim 110 , wherein said increase in pH is at least 0.5 pH units.  
     
     
         112 . A method as set forth in  claim 111 , wherein said increase in pH is at least 0.25 pH units.  
     
     
         113 . A kit for the prevention or control of coccidiosis comprising, 
 a composition containing, sterile, viable, sporulated oocysts of at least one species of protozoa known to cause coccidiosis, said composition containing 0.8% by weight of alkali metal dichromate; and    instructions for administration of said composition to an animal.    
     
     
         114 . A kit as set forth in  claim 113  containing less than about 0.3% by weight of dichromate ion.  
     
     
         115 . A kit as set forth in  claim 113  containing less than about 0.15% by weight of hexavalent chromium.  
     
     
         116 . A kit as set forth in  claim 113  characterized as substantially free of alkali metal dichromate.  
     
     
         117 . A kit as set forth in claim of  113 , wherein said composition comprises the composition of  claim 1 .  
     
     
         118 . A kit as set forth in  claim 113 , further comprising: 
 a diluent, said diluent characterized as substantially free of alkali metal dichromate; and    instructions for mixing said diluent with said composition to form a mixture.    
     
     
         119 . A kit according to  claim 118 , wherein said diluent comprises a sterile diluent.  
     
     
         120 . A composition for the storage of sporulated oocysts comprising an aqueous diluent and a bactericide, said composition characterized as substantially free of alkali metal dichromate wherein said composition is characterized as having: 
 a diluent comprising 0.5× phosphate buffered saline;    a pH from about 5.0 to about 8.0; and    wherein said bactericide is selected from the group consisting of an alkali metal perchlorate, an alkali metal hypochlorite, hydrochlorous acid, sodium hydroxide and antibiotics.    
     
     
         121 . A composition as set forth in  claim 120  having a pH from about 7.0 to about 7.5.  
     
     
         122 . A composition as set forth in  claim 120  wherein said bactericide comprises gentamicin.  
     
     
         123 . A composition as set forth in  claim 120  further comprising an oxidizing agent.  
     
     
         124 . A composition as set forth in  claim 120  characterized in that an oocyst population in contact with said composition remains at least about 60% viable for 13 weeks at about 25° C.  
     
     
         125 . A composition as set forth in  claim 120  characterized in that an oocyst population in contact with said composition remains at least about 60% viable for 26 weeks at about 5° C.  
     
     
         126 . A composition as set forth in  claim 120  characterized in that an oocyst population in contact with said composition decrease in viability no more than about 20% over a period of at least about 13 weeks at about 25° C.  
     
     
         127 . A composition as set forth in  claim 120  characterized in that an oocyst population in contact with said composition decrease in viability no more than about 20% over a period of at least about 26 weeks at about 5° C.  
     
     
         128 . A composition as set forth in  claim 120  further comprising a dye.  
     
     
         129 . A composition for the storage of sporulated oocysts comprising: 
 5× PBS; and    about 30 μg/ml gentamicin,    said composition characterized as substantially free of alkali metal dichromate, and wherein said composition is characterized in that oocysts in contact with said composition decrease in viability no more than about 20% over a period of at least about 26 weeks at about 5° C.    
     
     
         130 . A method for storing sporulated oocysts comprising contacting said sporulated oocysts with the composition of  claim 120 .  
     
     
         131 . A method as set forth in  claim 130  further comprising storing said sporulated oocysts in contact with the composition of  claim 120  at either about 25° C. or about 5° C.  
     
     
         132 . A method as set forth in  claim 130  wherein said population of sporulated oocysts is maintained at least 60% viable for 13 weeks at about 25° C.  
     
     
         133 . A method as set forth in  claim 130  wherein said population of sporulated oocysts is maintained at least 60% viability for 26 weeks at about 5° C.  
     
     
         134 . A method as set forth in  claim 130  wherein said method prevents a decrease in oocyst viability of greater than 20% over a period of at least 13 weeks at about 25° C.  
     
     
         135 . A method as set forth in  claim 130  wherein said method prevents a decrease in viability of greater than 20% in a population of sporulated oocysts over a period of at least 26 weeks at about 5° C.  
     
     
         136 . A composition for the prevention or control of coccidiosis comprising viable wild type sporulated oocysts of at least one species of protozoa known to cause coccidiosis, said composition having been made by the method of  claim 56 , wherein said composition is characterized as substantially free of alkali metal dichromate.  
     
     
         137 . A composition as set forth in  claim 136  wherein said composition comprises viable wild type sporulated oocysts from a species of Eimeria selected from the group consisting of  Eimeria acervulina, Eimeria maxima,  and  Eimeria tenella.    
     
     
         138 . A composition as set forth in  claim 137  wherein said composition comprises viable wild type sporulated oocysts of  Eimeria acervulina, Eimeria maxima,  and  Eimeria tenella.    
     
     
         139 . A composition as set forth in  claim 137  wherein said vaccine comprises viable wild type sporulated oocysts of  Eimeria acervulina, Eimeria maxima,  and  Eimeria tenella  in a ratio defined by the minimum immunizing dose and amount determined by storage half-life determinations.  
     
     
         140 . A composition as set forth in  claim 137  wherein said composition comprising at least about 1.25×10 4  viable wild type sporulated oocysts per milliliter.  
     
     
         141 . The composition as set forth in  claim 137  wherein said composition is characterized as substantially free of added bactericide.  
     
     
         142 . A composition as set forth in  claim 137  comprising a composition which ameliorates a decrease in post-challenge performance.  
     
     
         143 . A composition as set forth in  claim 142  wherein said composition comprises  Proprionibacterium acnes.