ANTIBIOTIC-FREE COMPOSITIONS FOR THE PREVENTION OR CONTROL OF COCCIDIOSIS

Abstract

Compositions are provided that contain sporulated coccidial oocysts, wherein the compositions are free of antibiotics and comprise formalin at a concentration sufficient to inhibit microbial growth for at least 12 months while maintaining oocyst viability. Methods of preparing such compositions are also provided.

Claims

1. A composition for the prevention or control of coccidiosis comprising viable sporulated oocysts of at least one species of protozoa known to cause coccidiosis, wherein said composition is free of antibiotics and comprises formalin at a concentration of 0.006%-0.008% such that microbial growth in the composition is inhibited for at least 12 months.

2. The composition of claim 1, which comprises formalin at a concentration of about 0.007%.

3. The composition of claim 1, which comprises formalin at a concentration of 0.007%.

4. The composition of any one of claims 1-3, which comprises viable sporulated oocysts of Eimeria acervulina, Eimeria maxima and Eimeria tenella.

5. The composition of any one of claims 1-4, which further comprises a diluent.

6. The composition of claim 5, wherein the diluent comprises water.

7. The composition of claim 6, wherein the aqueous diluent comprises 0.5 phosphate buffered saline.

8. The composition of claim 6, which further comprises a buffer.

9. The composition of claim 8, wherein the buffer is selected from the group consisting of phosphate buffers, bicarbonate buffers, citric acid buffers and tris buffers.

10. The composition of claim 9, wherein the buffer controls pH between about 6.8 and about 7.8.

11. A composition for storage of sporulated coccidial oocysts, the composition comprising viable sporulated oocysts of at least one species of protozoa known to cause coccidiosis, wherein the composition is free of antibiotics and comprises formalin at a concentration of 0.01%-0.025% such that microbial growth in the composition is inhibited for at least 12 months.

12. The composition of claim 11, which comprises viable sporulated oocysts of Eimeria acervulina.

13. The composition of claim 11, which comprises viable sporulated oocysts of Eimeria maxima.

14. The composition of claim 11, which comprises viable sporulated oocysts of Eimeria tenella.

15. The composition of any one of claims 11-14, which comprises formalin at a concentration of 0.01%.

16. The composition of any one of claims 11-14, which comprises formalin at a concentration of 0.025%.

Description

EXAMPLES

[0113] The following examples are intended to provide illustrations of the application of the present invention. The following examples are not intended to completely define or otherwise limit the scope of the invention.

Example1: Inhibition of Microbial Bioburden in Eimeria Oocyst Samples by Addition of Formalin at Various Concentrations

[0114] The model chosen to examine the effect of different concentrations of formalin on the level of microbial bioburden in Eimeria oocyst samples utilized Bacillus subtilis and Penicillium chrysogenum. Cultures of each of these organisms were prepared and diluted to stocks containing either 10-100 or 1-10 cfu/mL. A low level of organisms (0-10 cfu/mL) is more typical of the amount of bioburden detected following sanitization of oocysts, so this is the target for the preservative. The preservative was added at the desired concentration after sanitization of lots of Eimeria oocysts

[0115] An aliquot of Eimeria acervulina oocysts was removed following sanitization and prior to addition of gentamicin and Amphotericin B. The oocysts were spiked with B. subtilis and P. chrysogenum at a concentration of either 1-10 cfu/mL or 10-100 cfu/mL. Formalin was added to a final concentration of either 0.01% or 0.025%, according to the treatment group descriptions in Table 1 below. Vials of each treatment group were then stored at 2-7 C., and observed monthly up to 1 year for visible growth of the organisms. The results observed with respect to visible microbial growth are summarized in Table 1.

TABLE-US-00001 TABLE 1 Inhibition of microbial growth by formalin in E. acervulina oocysts after 12 months of storage B. subtilis/ Final Formalin Observations Treatment Oocysts P. chrysogenum Concentration (Incubation at Group Added Added (cfu/mL) (%) 2-7 C.) 1 Yes 0 0 NG 2 No 10-100 0 G 3 Yes 10-100 0 G 4 No 1-10 0 G 5 Yes 1-10 0 G 6 Yes 0 0.025 NG 7 Yes 10-100 0.025 NG 8 Yes 1-10 0.025 NG 9 Yes 0 0.010 NG 10 Yes 10-100 0.010 G 11 Yes 1-10 0.010 NG G = Growth; NG = No Growth.

[0116] The results demonstrated that when formalin was added to oocysts at a concentration of 0.025%, visible growth of B. subtilis and P. chrysogenum, at both 1-10 and 10-100 cfu/mL, was inhibited when stored for up to 12 months at refrigerated temperatures. Formalin added at a concentration of 0.010% inhibited visible growth of 1-10 cfu/mL B. subtilis and P. chrysogenum, but not 10-100 cfu/mL.

[0117] Thus, overall, these studies demonstrated that 0.025% formalin was found to be the most effective concentration for inhibiting microbial bioburden at either 1-10 or 10-100 cfu/mL, while the lower concentration of 0.01% formalin was similarly effective for inhibiting microbial bioburden at 1-10 cfu/mL.

Example2: Inhibition of Microbial Bioburden in a Coccidiosis Vaccine Preparation by Addition of Formalin

[0118] In this example, an antibiotic-free version of the ADVENT coccidiosis vaccine, containing E. acervulina, E. maxima and E. tenella oocysts but lacking gentamicin and amphotericin B (referred to herein as AF ADVENT) was prepared and then was spiked with B. subtilis and P. chrysogenum at either 1-10 or 10-100 CFU/ml. Formalin was then added to a final concentration of either 0.0028% or 0.0071%, according to the treatment group description in Table 2 below. Vials of each treatment group were then stored at 2-7 C., and observed monthly up to 1 year for visible growth of the organisms. The results observed with respect to visible microbial growth are summarized in Table 2.

TABLE-US-00002 TABLE 2 Inhibition of microbial growth by formalin in AF ADVENT after 12 months of storage B. subtilis/ Final Formalin Observations Treatment Vaccine P. chrysogenum Concentration (Incubation at Group Added Added (cfu/mL) (%) 2-7 C.) 1 Yes 0 0 NG 2 No 10-100 0 G 3 Yes 10-100 0 G 4 No 1-10 0 G 5 Yes 1-10 0 G 6 Yes 0 0.0071 NG 7 Yes 10-100 0.0071 G 8 Yes 1-10 0.0071 NG 9 Yes 0 0.0028 NG 10 Yes 10-100 0.0028 G 11 Yes 1-10 0.0028 G G = Growth; NG = No Growth.

[0119] All treatment groups were observed for the presence of macroscopic growth after twelve months of incubation. Visible growth was observed when B. subtilis and P. chrysogenum were added to the vaccine containing 0.0028% formalin. At a final formalin concentration of 0.0071% however, visible growth was only observed when the bioburden was 10-100 cfu/mL B. subtilis and P. chrysogenum, but was not observed at a bioburden of 1-10 cfu/mL. Since a bioburden of 1-10 cfu/mL is typical of the amount of bioburden detected following sanitization of oocysts, these results demonstrate that a final formalin concentration of 0.0071% in the final vaccine preparation is effective for inhibition of microbial bioburden.

Example3: Effect of Formalin on Oocyst Viability

[0120] The effect of formalin on oocyst viability was investigated using oocyst lots of each Eimeria species (E. acervulina, E. maxima and E. tenella) and on vaccine. Viability was determined initially prior to the addition of formalin, and then after 1 year of storage at 2-7 C. The results are summarized in Table 3 below.

TABLE-US-00003 TABLE 3 Viability of Eimeria Oocysts lots and Vaccine after Storage for 12 Months % Viable Oocysts after Storage for 12 Months Formalin E. acervulina E. maxima E. tenella Vaccine Serial Concentration Initial 12 mo Initial 12 mo Initial 12 mo Initial 12 mo 0.000 85 48 69 32 93 92 Not Applicable 0.010 59 47 95 0.025 27 41 86 0.0000 Not Applicable 90 81 0.0028 83 0.0071 84

[0121] The results demonstrated that following 1 year in storage, formalin at 0.010% or lower concentrations did not have an adverse effect on oocyst viability. Formalin added at 0.025% affected the viability of only E. acervulina oocysts following long term storage.

[0122] A separate study demonstrated that use of higher concentrations of formalin resulted in increased toxicity towards the oocysts as compared to use of 0.025% or lower, thereby indicating the preferability of concentrations of 0.025% or lower for maintaining oocyst viability. Exemplary results from these studies are shown below in Table 4:

TABLE-US-00004 TABLE 4 Effect of Higher Concentrations of Formalin on Oocyst Viability Eimeria Time Stored % Decrease in species at 2-7 C. Formalin Viability* maxima 2 weeks 0.675% 49% acervulina 4 months 0.1% 37% *Compared to same lot of oocysts stored with 0% Formalin

[0123] In the vaccine preparations, the presence of a final concentration of either 0.0028% or 0.0071% formalin did not affect oocyst viability.

Example4: Effect of Additional Disinfectants on Oocyst Viability

[0124] In this example, additional disinfectant compounds were tested for their ability to inhibit microbial growth in Eimeria oocyst samples as compared to formalin. E. maxima oocysts were spiked with B. subtilis and P. chrysogenum, and the minimal inhibitory concentrations of several disinfectants were determined. The disinfectants were then added to E. maxima oocysts and the oocysts were stored at 2-7 C. for 6 months. The oocysts were assayed for viability and the decrease in viability due to the disinfectant was calculated by comparison to the same lot of oocysts stored in 0.5 PBS only. Exemplary results are summarized below in Table 5.

TABLE-US-00005 TABLE 5 Effect of disinfectants at minimal inhibitory concentration on oocyst viability following 6 months of storage at 2-7 C. Minimal Inhibitory Decrease Disinfectant Concentration in Viability* Citral 0.0125% 24% Thymol 0.0063% 15% *Compared to same lot of oocysts stored with 0% disinfectant

[0125] The results demonstrated that the other disinfectant compounds tested affected oocyst viability to a significantly greater extent than formalin, thereby demonstrating the advantageous properties of formalin in inhibiting microbial bioburden while not significantly impacting oocyst viability.

[0126] In light of the detailed description of the invention and the examples presented above, it can be appreciated that the several aspects of the invention are achieved.

[0127] It is to be understood that the present invention has been described in detail by way of illustration and example in order to acquaint others skilled in the art with the invention, its principles, and its practical application. Particular formulations and processes of the present invention are not limited to the descriptions of the specific embodiments presented, but rather the descriptions and examples should be viewed in terms of the claims that follow and their equivalents. While some of the examples and descriptions above include some conclusions about the way the invention may function, the inventors do not intend to be bound by those conclusions and functions, but put them forth only as possible explanations.

[0128] It is to be further understood that the specific embodiments of the present invention as set forth are not intended as being exhaustive or limiting of the invention, and that many alternatives, modifications, and variations will be apparent to those of ordinary skill in the art in light of the foregoing examples and detailed description. Accordingly, this invention is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and scope of the following claims.