U.S. patent application number 10/005510 was filed with the patent office on 2002-10-31 for methods and compositions for the control of coccidiosis.
Invention is credited to Allington, Tony, Bull, Lance, Green, Jackie, Pfannenstiel, Mary Ann, Schasteen, Charles S., Uraizee, Farooq.
Application Number | 20020160022 10/005510 |
Document ID | / |
Family ID | 22932479 |
Filed Date | 2002-10-31 |
United States Patent
Application |
20020160022 |
Kind Code |
A1 |
Schasteen, Charles S. ; et
al. |
October 31, 2002 |
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.
Inventors: |
Schasteen, Charles S.; (St.
Louis, MO) ; Green, Jackie; (O'Fallon, MO) ;
Uraizee, Farooq; (Valley Park, MO) ; Bull, Lance;
(St. John, MO) ; Pfannenstiel, Mary Ann; (Lincoln,
NE) ; Allington, Tony; (Valparaiso, NE) |
Correspondence
Address: |
SENNIGER POWERS LEAVITT AND ROEDEL
ONE METROPOLITAN SQUARE
16TH FLOOR
ST LOUIS
MO
63102
US
|
Family ID: |
22932479 |
Appl. No.: |
10/005510 |
Filed: |
November 8, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60246847 |
Nov 8, 2000 |
|
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Current U.S.
Class: |
424/269.1 |
Current CPC
Class: |
A61K 2039/52 20130101;
C12N 1/10 20130101; A61K 2039/55594 20130101; A61K 39/012
20130101 |
Class at
Publication: |
424/269.1 |
International
Class: |
A61K 039/005 |
Claims
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.times.10.sup.-3 .mu.g 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.times.10.sup.-3 .mu.g
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.times.10.sup.-3 .mu.g 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.times.10.sup.-5 .mu.g 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.times. 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 .mu.g/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-.alpha.,
interferon-.gamma., 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.degree. C. and about
30.degree. C.
44. A method as set forth in claim 43 wherein said sieving is
carried out at a temperature between about 22.degree. C. and about
28.degree. C.
45. A method as set forth in claim 44 wherein said sieving is
carried out at about 25.degree. 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.degree. C.
74. A method as set forth in claim 73 wherein temperature is
maintained from about 15.degree. C. to about 40.degree. C.
75. A method as set forth in claim 74 wherein temperature is
maintained from about 20.degree. C. to about 30.degree. C.
76. A method as set forth in claim 75 wherein temperature is
maintained at about 28.degree. 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.times. 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.degree. 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.degree. 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.degree. 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.degree. 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.times. PBS; and about 30 .mu.g/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.degree. 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.degree. C. or about 5.degree. 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.degree. 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.degree. 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.degree. 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.degree. 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.times.10.sup.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.
Description
BACKGROUND
[0001] Coccidiosis is a disease of various animals in which the
intestinal mucosa is invaded and damaged by a protozoa of the
subclass Coccidia. The economic effects of coccidiosis can be
especially severe in the poultry industry where intensive housing
of birds favors the spread of the disease. Infection by coccidial
protozoa is, for the most part, species specific. Numerous species,
however, can infect a single host. For example, there are seven
species of coccidial protozoa which infect chickens, six of which
are considered to be moderately to severely pathogenic.
[0002] The life cycle of the coccidial parasite is complex. For
example, protozoa of the genera Eimeria, Isospora, Cystoisospora,
or Cryptosporidium typically only require a single host to complete
their life cycle, although Cystoisospora may utilize an
intermediate host. Under natural conditions, the life cycle begins
with the ingestion of sporulated oocysts from the environment. When
sporulated oocysts are ingested by a susceptible animal, the wall
of the sporulated oocyst is broken in order to release the
sporocysts inside. In poultry, the release of the sporocyst is the
result of mechanical disruption of the sporulated oocyst in the
gizzard. Within the sporocysts, are the sporozoites which are the
infective stage of the organism. In poultry, the breakdown of the
sporocyst coat and release of the sporozoites is accomplished
biochemically through the action of chymotrypsin and bile salts in
the small intestine. Once released, the sporozoites invade the
intestinal mucosa or epithelial cells in other locations. The site
of infection is characteristic of the species involved. For
example, in the genus Eimeria, E. tenella is localized in the ceca;
E. necatrix is found in the anterior and middle portions of the
small intestine; E. acervulina and E. praecox occur in the upper
half of the small intestine; E. brunetti occurs in the lower small
intestine, rectum, ceca, and cloaca; E. mitis is found in the lower
small intestine, while E. maxima can be found in any of these
physiological locations.
[0003] Once inside the host animals' cells, sporozoites develop
into multinucleate meronts, also called schizonts. Each nucleus of
the meront develops into an infective body called a merozoite which
enters new cells and repeats the process. After a variable number
of asexual generations, merozoites develop into either
microgametocytes or macrogametes. Microgametocytes develop into
many microgametes which, in turn, fertilize the macrogametes. A
resistant coat then forms around the resulting zygotes. The
encysted zygotes are called oocysts and are shed unsporulated in
the feces. Infected birds may shed oocysts in the feces for days or
weeks. Under proper conditions of temperature and moisture, the
oocysts become infective through the process of sporulation.
Susceptible birds then ingest the sporulated oocysts through normal
pecking activities or ground/litter foraging and the cycle repeats
itself. Ingestion of viable, sporulated oocysts is the only natural
means of infection.
[0004] Infection with coccidial protozoa results in immunity so
that the incidence of the disease decreases over time as members of
the flock become immune. This self-limiting nature of coccidial
infections is widely known in chickens and other poultry. The
immunity conferred, however, is species specific such that
introduction of another species of coccidial protozoa will result
in a new disease outbreak.
[0005] The oocyst wall of coccidial protozoa provides a highly
effective barrier for oocyst survival. Oocysts may survive for many
weeks outside the host. In the laboratory, intact oocysts are
resistant to extremes in pH, detergents, proteolytic, glycolytic,
and lipolytic enzymes, mechanical disruption, and chemicals such as
sodium hypochlorite and dichromate.
[0006] Two methods are currently used to control coccidiosis in
poultry. The first involves control by chemotherapy. Numerous drugs
are available for the control of coccidiosis in poultry. Because of
the number of species which cause the disease, very few drugs are
efficacious against all species, although a single drug may be
efficacious against several species. In modem broiler chicken
production, for example, administration of drugs to control
coccidiosis is routine. The expense for preventative medication
against coccidiosis represents a significant cost of
production.
[0007] Two programs of drug administration are commonly used in the
domestic poultry industry. The simplest is the continuous use of a
single drug from day one following hatching until slaughter. The
second program is to use shuttle or dual drug program which
involves the use of two different drugs, one administered in the
"starter" ration and a second drug administered in the "grower"
ration. This second method is often preferred as a method to
minimize development of drug resistant strains of Coccidia. Using
either method, drugs used are typically rotated two to three times
per year in order to minimize the development of resistant
strains.
[0008] The development of drug resistance by Coccidia is a serious
limitation on the effectiveness of chemotherapy to control the
disease. Surveys in the United States, South America and Europe
have revealed widespread drug resistance in Coccidia. Since drug
resistance is a genetic phenomenon, once established, drug
resistance can remain in the population for many years until
reduced by natural selection pressure and genetic drift.
[0009] The use of drugs in animals used for food production is also
coming under increasing scrutiny by the public. Consumers are
increasingly concerned with the possibility of drug residues in
food. This creates pressure in the poultry industry to reduce the
use of drugs to control coccidiosis.
[0010] Vaccination of birds against coccidiosis is an alternative
to chemotherapy. An advantage of vaccination is that it can greatly
reduce or eliminate the need to administer anti-coccidial drugs,
thus reducing drug costs to poultry producers, preventing the
development of drug-resistant strains, and lessening consumer
concerns about drug residues.
[0011] Numerous methods have been developed to immunize poultry
against coccidial protozoa. The successful methods have all been
based on the administration of live protozoa, either fully virulent
strains or attenuated strains. The most common route of
administration is oral, although other routes have been used.
Edgar, U.S. Pat. No. 3,147,186, teaches vaccination of chickens by
oral administration either directly into the mouth or via the feed
or water of viable E. tenella sporulated oocysts. Davis et al.,
U.S. Pat. No. 4,544,548, teaches a method of vaccination by
continuous administration of low numbers of sporulated oocysts,
with or without simultaneous administration of anti-coccidial
drugs.
[0012] Oral administration of attenuated strains of sporocysts has
also been utilized to confer immunity against coccidiosis. Shirley,
U.S. Pat. No. 4,438,097; McDonald, U.S. Pat. No. 5,055,292; and
Schmatz et al., PCT publication No. WO 94/16725. An alternative to
attenuation is disclosed in Jenkins et al., Avian Dis., 37(1):74-82
(1993), which teaches the oral administration of sporozoites that
have been treated with gamma radiation to prevent merogonic
development.
[0013] Parenteral routes of vaccination have included subcutaneous
or intraperitoneal injection of excysted sporozoites, Bhogal, U.S.
Pat. No. 4,808,404; Bhogal et al., U.S. Pat. No. 5,068,104, and
intra ovo injection of either oocysts or sporocysts, Evans et al.,
PCT publication No. WO 96/40233; Watkins et al., Poul. Sci.,
74(10):1597-602 (1995). Sharma, J. Parasitol., 50(4):509-517
(1964), reported unsuccessful immunization trials involving
intravenous, intraperitoneal, intramuscular, or subcutaneous
injection of either viable oocysts or a mixture of oocysts,
sporocysts and sporozoites. Thaxton, U.S. Pat. No. 5,311,841,
teaches a method of vaccination against Coccidia by administration
of oocysts or sporozoites to newly hatched chicks by yolk sac
injection.
[0014] Regardless of the route of administration, procedures for
the production of coccidiosis vaccines are quite similar. Briefly,
coccidial protozoa are produced by infecting host animals with a
single species of coccidial protozoa. These "seed stocks" are often
clonal in nature, that is, derived from a single organism in order
to insure the presence of only the species of interest. Seed stocks
may be wild type, that is, isolated from the field, or they may be
precocious or attenuated strains. The protozoa are then allowed to
undergo replication in the host, after which, protozoa are
collected from the animals, usually from the excreta. The use of
attenuated strains typically results in fewer shed oocysts from the
host animal. The protozoa are then separated from the excreta by
well known techniques such as salt floatation and centrifugation.
At the time of collection, the protozoa are at the non-infective
oocyst stage of the life cycle. In order to become infective, and
therefore useful for vaccines, the oocysts must be induced to
undergo sporulation. In members of the genus Eimeria, sporulation
typically involves the incubating the oocysts in a 1% to 4% aqueous
solution of potassium dichromate at 19.degree. C. to 37.degree. C.
with constant aeration. Data on oxygen consumption are conflicting,
with Smith and Wilson (J. Parasitol. 30:295-302, 1944) reporting
increased oxygen consumption for E. tenella and Wilson and
Fairbairn (J. Protozool. 8:410-416, 1961) reporting no change in
oxygen consumption for E. acervulina. Sporulation is usually
complete within 12 to 24 hours depending on the temperature used.
Monitoring of the sporulation process is accomplished by
microscopic examination of the protozoa. Storage compositions found
in the prior art typically include an aqueous solution of potassium
dichromate. The sporulated oocysts are usually stored in 1 to 4%
aqueous solution of potassium dichromate to prevent bacterial
growth, however, other storage media have been used.
[0015] Current vaccines available for the prevention of coccidiosis
typically contain a 2.5% weight to volume solution and contain
approximately 1,600 oocyts per dose (400 sporulated oocysts
representing four different species). The current commercially
available vaccines contain from about 1.6.times.10.sup.-2 .mu.g of
potassium dichromate per oocyst to about 0.16 .mu.g of potassium
dichromate per oocyst.
[0016] Although widely used for sporulation and storage, potassium
dichromate has several properties which make its elimination from
biologicals highly desirable. Potassium dichromate is a strong
oxidizer and has been reported to affect the respiratory system,
liver, kidneys, eyes, skin and blood. It is a known carcinogen and
upon disposal is regarded as a hazardous waste. Because of its high
toxicity, compounds containing potassium dichromate are
particularly unsuitable for parenteral administration. Thus, it
would be highly advantageous to eliminate potassium dichromate from
the production and storage of materials to be administered to
animals, especially food animals.
SUMMARY
[0017] Coccidiosis is a disease of animals which has a significant
economic impact, especially in the poultry industry. In many
poultry operations, birds are vaccinated against coccidiosis using
vaccines containing sporulated oocysts. Present methods for the
sporulation and storage of coccidial oocysts use the highly toxic
chemical potassium dichromate. The present invention provides a
method for the sporulation, sterilization and storage of coccidial
oocysts, without the use of potassium dichromate. The present
invention also provides for vaccine compositions that can be
administered to animals, particularly from the class Aves, and more
particularly poultry, said vaccines being characterized as
substantially free of potassium dichromate, both in terms of their
production and storage. Due to its high toxicity, the elimination
of potassium dichromate is particularly desirable in the production
and storage of compositions to be administered to animals, and in
particular to food producing animals. As the vaccine compositions
of the present invention are sterile, the vaccine compositions can
be administered to animals, through various routes, including, but
not limited to orally, e.g, by addition to food or water;
topically, e.g., spraying; parenteral routes, e.g. subcutaneous,
intramuscular or intraperitoneal injection; per os or via
intra-yolk sac injection.
[0018] As used herein, the term substantially free of alkali metal
dichromate indicates that no alkali metal dichromate is added to
the composition during production, including the sporulation and
storage of said composition. Furthermore, as used herein, the term
substantially free of potassium dichromate indicates that no
potassium dichromate is added to the composition during production,
including the sporulation and storage of said composition.
[0019] The present invention provides a method for isolating
oocysts, concentrating oocysts, sporulating oocysts, isolating
sporulated oocysts, sterilizing sporulated oocysts and storage of
sporulated oocysts. More particularly, the present invention
provides a method for sporulating oocysts, isolating sporulated
oocysts, sterilizing sporulated oocysts and storage of sporulated
oocysts.
[0020] Among the several aspects of the invention, is provided a
vaccine for the prevention and/or control of coccidiosis comprising
viable sporulated oocysts of at least one species of protozoa known
to cause coccidiosis, wherein the composition is sterile and
characterized as substantially free of potassium dichromate.
[0021] Another aspect provides a vaccine for the prevention and/or
control of coccidiosis comprising an aqueous diluent and viable
sporulated oocysts of at least one species of protozoa known to
cause coccidiosis wherein the vaccine is sterile and characterized
as substantially free of potassium dichromate.
[0022] Another aspect of the invention provides a method of
isolating and concentrating oocysts to prepare the oocysts for
sporulation comprising a novel combination of isolation, cleansing,
and concentration steps. The methods of the instant invention may
be used individually or in combination with one another.
[0023] The method of isolating and concentrating oocysts comprises
sieving a manure slurry known to contain oocysts. The method
further comprises concentrating the oocysts by centrifugal-based
separation. The method further comprises a flotation step to
further cleanse and isolate the oocysts. The method further
comprises centrifugal-based separation, followed by a flotation
step, followed by another application of centrifugal-based
separation.
[0024] A further aspect provides a method for inducing the
sporulation of oocysts comprising incubating viable oocysts of at
least one species of protozoa known to cause coccidiosis in an
aqueous medium wherein dissolved oxygen concentration is maintained
from about 30% to about 80% of saturation. Temperature is
controlled from a temperature which prevents substantial freezing
up to about 43.degree. C. An oxidizing agent, other than potassium
dichromate, is added at a sporulation inducing concentration to
form a sporulation medium; and the sporulation medium is incubated
to form sporulated oocysts.
[0025] Another aspect provides a method for inducing the
sporulation of oocysts comprising incubating viable oocysts of at
least one species of protozoa known to cause coccidiosis in an
aqueous medium wherein dissolved oxygen concentration is maintained
at at least about 50% of saturation. Temperature is controlled from
a temperature which prevents substantial freezing up to 43.degree.
C. An oxidizing agent, other than an alkali metal dichromate,
soluble dichromate moieties, dichromate ions, or potassium
dichromate, to form a sporulation medium; and wherein the oxidizing
agent is at a sporulation inducing concentration. The sporulation
medium is then incubated to form sporulated oocysts; the sporulated
oocysts are then separated from the sporulation medium; the
sporulated oocysts are then sterilized with a chemical
disinfectant; the chemical disinfectant is removed by tangential
flow filtration. The sporulated oocysts may then be stored in a
diluent substantially free of potassium dichromate as it has been
discovered that an oxidizing agent, other than dissolved oxygen
available in sterile water, is not necessary to preserve viability
for useful periods of time.
[0026] However, the use of an oxidizing agent may increase
longevity of the vaccine, and thus, may be used as part of the
instant invention.
[0027] Still a further aspect provides, a method for monitoring
sporulation of oocysts comprising, incubating viable oocysts in a
medium under sporulation inducing conditions, and monitoring the
medium during the incubation for a change in pH, or a change in the
combination of dissolved oxygen and pH, the change being
characteristic of sporulation.
[0028] Yet another aspect provides, a kit for the prevention and/or
control of coccidiosis comprising a vaccine comprising sterile,
viable, sporulated oocysts of at least one species of protozoa
known to cause coccidiosis, the vaccine being characterized as
substantially free of potassium dichromate; and instructions for
administering the composition to an animal. In another embodiment,
the kit further comprises a diluent, which may be sterile, and
instructions for mixing the oocysts with the diluent to form a
mixture and for administering the mixture to an animal.
[0029] Another aspect provides, a composition for the storage of
sporulated oocysts comprising an aqueous diluent and a bactericide,
the composition being sterile and being characterized as
substantially free of potassium dichromate. In yet another
embodiment the aqueous diluent comprises water. In a further
embodiment the aqueous diluent comprises domestic water. In a
further embodiment the aqueous diluent comprises from about
0.1.times. to about 1.times. phosphate buffered saline (PBS) and
from about 0 to about 30 .mu.g/ml gentamicin.
[0030] A further aspect provides, a method for storing sporulated
oocysts comprising obtaining sterile, sporulated oocysts of at
least one species of protozoa known to cause coccidiosis and
placing the sporulated oocysts in a composition comprising an
aqueous diluent and a bactericide, the composition being sterile
and being characterized as substantially free of added potassium
dichromate.
[0031] Yet another aspect provides, a method for storing sporulated
oocysts comprising obtaining sterile, sporulated oocysts of at
least one species of protozoa known to cause coccidiosis; placing
the sporulated oocysts in a sterile composition comprising
0.5.times. PBS and about 30.mu.g/ml gentamicin, the composition
being characterized as substantially free of potassium dichromate;
and storing the composition containing the sporulated oocysts at
less than about 10.degree. C., preferably between about 1.degree.
C. to about 8.degree. C., more preferably from about 3.degree. C.
to about 6.degree. C., and most preferably, about 4.degree. C.
[0032] In yet another aspect, the invention provides a combination
of species of oocysts from the genus Eimeria comprising a minimum
immunizing dose of oocysts.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, appended claims and accompanying figures
where:
[0034] FIG. 1 shows a graph of percent saturation of dissolved
oxygen (A) and pH (B) versus time of the sporulation medium during
a successful sporulation when percent saturation of dissolved
oxygen is not controlled.
[0035] FIG. 2 shows a graph of percent saturation of dissolved
oxygen (A) and pH (B) versus time of the sporulation medium during
a successful sporulation in which the rise in pH was preceded by a
drop in pH when percent saturation of dissolved oxygen and pH is
not controlled.
[0036] FIG. 3 shows the percent viable oocysts recovered (PVOR)
versus storage time for oocytes stored under the conditions
indicated. All storage medium contained P. acnes and oocysts were
sterilized by 5% NaOCl.
[0037] FIG. 4 shows the percent viable oocysts recovered (PVOR)
versus storage time for oocytes stored under the conditions
indicated. All storage medium contained P. acnes and oocysts were
sterilized by 2% NaOCl.
[0038] FIG. 5 shows a flow diagram of the process used to produce
oral coccidiosis vaccine. The flow chart is divided into four
suites: (1) Challenge Suite; (2) Purification Suite; (3)
Sporulation Suite; and (4) Storage Suite. Steps 1 through 34 are
described for illustrative purposes as follows:
[0039] 1. Feed given to the birds
[0040] 2. Water given to the birds
[0041] 3A. Manure is harvested after birds begin shedding
oocysts
[0042] 3B. Manure is discarded prior to when birds shed oocysts
[0043] 4. Water is added to a slurry tank containing the manure
[0044] 5. The slurry is sieved
[0045] 6. Solid waste from sieving is discarded
[0046] 7. Filtrate collection transferred to and then separated by
centrifugal-based separation
[0047] 8. Liquid waste is discarded while moist solid is
retained
[0048] 9. Moist solid is transferred to mix tank
[0049] 10. High fructose corn syrup is added to tank
[0050] 11. Water is added (if needed) to adjust specific
gravity
[0051] 12. Suspension is transferred for separation
[0052] 13. Solid wastes are discarded
[0053] 14. Liquid phase is transferred to mix tank
[0054] 15. Water is added to mix tank
[0055] 16. Diluted suspension is transferred to and then
centrifuged
[0056] 17. Liquid waste is discarded
[0057] 18. Solid transferred to blend vessel
[0058] 19. Water is added to blend vessel
[0059] 20. Diluted suspension is deposited in holding container
then later transferred to sporulation vessel
[0060] 21. An oxidizing agent is added to the sporulation
medium
[0061] 22. Sporulated oocysts transferred from sporulation vessel
to separation device
[0062] 23. Water is added to sporulated oocysts suspension
[0063] 24. Waste water from separation is removed and discarded
[0064] 25. Sporulated oocysts, now separated from sporulation
medium, are transferred to sterile filtration unit
[0065] 26. Addition of disinfecting agent
[0066] 27. Addition of sterile water
[0067] 28. Waste water and disinfecting agent are discarded
[0068] 29. Transfer to holding vessel
[0069] 30. Addition of buffer
[0070] 31. Addition of bactericide
[0071] 32. Transfer to blending vessel where buffer and
post-challenge performance improvement composition are blended
[0072] 33. Vialing
[0073] 34. To kit or to market
DETAILED DESCRIPTION
[0074] The following detailed description is provided to aid those
skilled in the art in practicing the present invention. Even so,
this detailed description should not be construed to unduly limit
the present invention as modifications and variations in the
embodiments discussed herein can be made by those of ordinary skill
in the art without departing from the spirit or scope of the
present inventive discovery.
[0075] All publications, patents, patent applications, databases
and other references cited in this application are herein
incorporated by reference in their entirety as if each individual
publication, patent, patent application, database or other
reference were specifically and individually indicated to be
incorporated by reference.
[0076] As used herein, the term "domestic water" refers to
available (potable) tap water.
[0077] The present invention provides for the substantial
elimination of added potassium dichromate in the production of
vaccines used for the prevention of coccidiosis in animals, and in
particularly poultry. More specifically, the composition of the
invention is a novel vaccine ready for administration. The
composition of the invention herein, in a preferred embodiment, is
substantially free of alkali metal dichromates, soluble
dichromates, dichromate ions, and potassium dichromate when the
methods disclosed herein are practiced. Any alkali metal
dichromates, soluble dichromates, dichromate ions, and potassium
dichromate present in the compositions described herein or present
in the compositions made by the methods described herein would be
residual and, in a preferred embodiment, would not be present due
to affirmative addition. In addition, the present invention
provides methods for the sporulation of coccidial protozoa, the
production of sterile preparations containing viable sporulated
oocysts that are substantially free of potassium dichromate, and
compositions and methods for storing sporulated oocysts.
[0078] Furthermore, the invention provides a method for the
monitoring of the sporulation process that does not require
microscopic examination of the oocysts. Furthermore, the invention
provides for kits containing a composition used for the prevention
of coccidiosis in animals, and in particularly poultry
characterized as substantially free of potassium dichromate.
[0079] Oocysts used in the practice of the present invention can be
obtained from a variety of sources. For example, oocysts can be
obtained by the inoculation of host animals with coccidial protozoa
of a single species or with a mixture of species. The coccidial
protozoa used can be clonal in nature, that is derived from a
single progenitor, or polyclonal. The oocysts of the present
composition are derived from wild type oocysts. Inoculation can be
by any means which will allow for the replication of the protozoa
in the host animal. The most common route of inoculation is per os,
but other suitable routes may be used. If administered per os, the
protozoa are preferably at the sporulated oocyst stage.
Administration can be by gavage or through the feed and/or water.
Inoculation can also be accomplished by exposing host animals to
environments contaminated with coccidial protozoa. Alternatively,
oocysts can be obtained from animals with naturally occurring
infections.
[0080] The present invention provides a method for the sporulation
of oocysts comprising first orally inoculating host animals with
Eimeria species. Manure from the innoculated animals is then
collected from the inoculated host animals. Oocysts are separated
from the manure using a combination of isolation techniques,
including sieving, centrifugation and density flotation. The
isolated oocysts are then sporulated under certain defined
conditions controlling factors such as temperature, percent
saturation of dissolved oxygen, pH, and agitation in a sporulation
medium. The sporulated ooeysts are then separated from the
sporulation medium and disinfected. After, the sporulated oocysts
have been separated from the sporulation medium and disinfected
they are combined with a diluent or a diluent and a buffer to form
a vaccine. A post-challenge performance improvement composition may
also then be added. Such a composition ameliorates a decrease in
post-challenge performance. The amelioration may be seen in factors
such as bursal growth and appearance. For illustrative purposes
only, a preferred method is described graphically in the form of a
flow sheet in FIG. 5.
[0081] Furthermore, the invention provides for a method of
monitoring sporulation without the need for microscopic
observations. Microscopic observation requires sampling and
technician labor. The elimination of microscopic observation
provides a more efficient method for monitoring sporulation. The
method of the invention for monitoring sporulation comprises
monitoring oxygen consumption, as evidenced by a change in
dissolved oxygen content, and monitoring pH. It has been discovered
that sporulation results in an increase in oxygen consumption, as
evidenced by a decrease in dissolved oxygen in a sporulation
medium, and that sporulation results in an increase in pH. See
FIGS. 1 and 2. Sporulation rates may be monitored by monitoring the
volume and flow rate of an oxygen containing gas added to control
percent saturation of dissolved oxygen and also may be monitored by
monitoring the acid or base added needed to maintain a desired pH.
Thus, the decrease in dissolved oxygen is a differential decrease
sufficient to trigger the addition, by a controlled device, to
supply more oxygen. In addition, the decrease in pH is also a
differential decrease that is sufficient to trigger the addition of
an acid or a base.
[0082] Methods of Isolation, Concentration, and Purification
[0083] In general, a number of different methods of preparing
oocysts for sporulation are known in the art. Any one or
combination of such methods may be used prior to sporulation.
However, a preferred method is set out below. A number of well
known processes are set forth to assist one skilled in the art to
practice the invention in its different embodiments.
[0084] To begin, once host animals begin shedding the organism, the
protozoa can be collected. Most commonly, protozoa are collected
from the feces, but they can also be collected from intestinal
contents and/or scrapings as well as contaminated bedding (see FIG.
5A, "Challenge Suite"). Once collected, the oocysts are preferably
isolated from the extraneous fecal material as decreasing the fecal
content in an oocyst suspension increases the number of oocysts
that will sporulate (Smith and Ruff, Poultry Sci. 54:2083, 1975).
As will be described below, a preferred method for isolating
oocysts is by sieving (see FIG. 5A, step 5). However, several
methods for isolating protozoa are known in the art and may be used
in practicing the present invention. A summary of these isolation
methods are summarized herein followed by a description of the
preferred method. Several methods described herein process the
collected manure to a point wherein sporulation may then begin.
Others require further processing, such as further isolation or
cleansing. This further processing may be accomplished by utilizing
the methods and techniques described herein or a combination
thereof.
[0085] A review of several methods for the isolation of oocysts can
be found in Ryler et al.(Parasitology 73:311-326, 1976). In one
method, described in U.S. Pat. No. 3,147,186, oocysts are only
crudely isolated following the addition of the oxidizing agent
potassium dichromate. In this method, the moist droppings of host
animals are directly mixed with an aqueous solution containing
between one and four percent potassium chromate, preferably 2.5%
or, less preferably, water, so that a suspension of thin
consistency is obtained. The method indicates that a concentration
of at least about one to four percent potassium chromate solution
is necessary to obtain adequate oocyst sporulation. Larger
insoluble debris, such as feathers and partially digested or
undigested feed, is removed. Removal can be done conventionally by
filtering the suspension through a mesh screen. The suspension is
then allowed to stand for about five minutes to allow heavier
coarser particles of debris that passed through the screen to
settle to the bottom of the holding container. The supernatant
liquid containing the oocysts is then removed. The sporulated
oocysts are viable for up to about 18 hours.
[0086] Another method for separating oocysts from droppings
comprises flotation using solutions of sufficient specific gravity,
typically having a specific gravity of about 1.2, so that oocysts
float to the top of the suspension. Generally these solutions are
made up of water to which a sugar (e.g. sucrose), ZnSO.sub.4, or
NaCl has been added to increase the specific gravity to the desired
value. Useful solutions include solutions comprising 58% (w/v)
sucrose, 37% (w/v) ZnSO.sub.4 7H.sub.2O and saturated NaCl
solutions, which all have a specific gravity from about 1.09 to
about 1.2. Other solutions which have a comparable specific gravity
and are not harmful to the oocysts can also be used.
[0087] In the flotation method of isolation, a preliminary step of
filtering diluted collected manure through, e.g., gauze, a sieve or
cheesecloth to remove large particles of undesired fecal matter may
be included. After mixing harvested oocysts with the flotation
solution, the oocyst slurry may be centrifuged and the oocyst
removed from the surface layer of the supernatant. The
centrifugation step may be repeated several times to further purify
the oocysts by resuspending the captured supernatant in a flotation
medium having a specific gravity similar to that used in previous
centrifugation steps and centrifuged again. This step may be
repeated until the desired level of purity is reached.
[0088] Another method for isolation of oocysts available in the art
comprises gradient centrifugation. The gradient used can be
discontinuous or continuous. An example of a typical gradient for
coccidial oocysts is 0-50% sucrose. In this method the material
containing the oocysts is placed on top of the gradient and the
oocyst containing material is then centrifuged along with the
gradient. Following centrifugation, the layer containing the
oocysts is recovered. The process may be repeated in order to
increase the purity of the resulting oocyst preparation. As with
flotation, this method is preferably preceded by filtration of the
collected manure.
[0089] Additional methods of oocyst isolation include, the use of
glass bead columns (Ryler et al., Parasitology, 73:311-326, 1976)
and the bicarbonate ether method (Smith and Ruff, Poultry Sci.
54:2081-2086, 1975). In the glass column method, the aqueous
suspension of fecal matter is added to a mixture of glass beads and
a detergent, for example 5% Tween 80. The mixture is then applied
to a column of glass beads and the oocysts are allowed to flow
through while much of the undesired fecal matter is retained in the
column. The effluent may then be concentrated by
centrifugation.
[0090] In the bicarbonate ether method, the feces from infected
chickens is strained, through cheese cloth for example, and the
liquid fraction is captured while the solid fraction is discarded.
The liquid fraction is then concentrated by centrifugation. The
solid fraction is recovered and the supernatant is discarded. The
recovered solid fraction is then resuspended in a solution of 1%
sodium bicarbonate. To the resuspended solid fraction, now in
suspension, is then added ether in a volume approximately equal to
the volume of 1% solution of sodium bicarbonate. The mixture is
then centrifuged. The debris plug and supernatant is discarded
while the sediment is washed by resuspension in water. This
suspension is then centrifuged and the supernatant discarded. The
sediment is then recovered for use. (Smith and Ruff, Poultry Sci.
54:2081-2086, 1975).
[0091] The various methods for isolating, concentrating, and
purifying oocysts describe above may be used in combination with
one another or in combination with the preferred embodiments of the
instant invention. Regardless of the methods used, the greater the
isolation, concentration, and purification the greater percent
sporulation during the sporulation suite (see FIG. 5, "Sporulation
Suite"). Therefore, it has been discovered that the following
methods of isolation, concentration, purification, sporulation,
sterilization, and storage provide a novel and improved method for
the production of sporulated oocysts.
[0092] One aspect of the method of the instant invention comprises
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.
[0093] In another aspect the method for isolating oocysts comprises
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.
[0094] In yet another aspect, the method for isolating oocysts
comprises 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.
[0095] A further aspect of the methods provided herein describes a
method for sporulating 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; and introducing an oxidizing agent
into said medium at a rate sufficient to maintaining the dissolved
oxygen content of the medium at at least 30% of saturation; said
medium containing less than about 0.8% by weight alkali metal
dichromate during incubation of said oocysts.
[0096] Another aspect of the methods provided herein describes a
method for sporulating 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; and introducing an oxidizing agent
into said medium at a rate sufficient to maintaining the dissolved
oxygen content of the medium at between about 30% and about 80% of
saturation; said medium containing less than about 0.8% by weight
alkali metal dichromate during incubation of said oocysts.
[0097] In another aspect of the methods provided herein is
described a method for separating sporulated oocysts from a
sporulation medium; sterilizing sporulated sporocysts by contacting
said sporulated oocysts with a chemical disinfectant; and storing
said sporulated oocysts in a sterile diluent, wherein said diluent
contains less than about 0.8% by weight alkali metal
dichromate.
[0098] In another aspect of the methods described herein is
described 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; and introducing an
oxidizing agent having a standard reduction potential of at least
about 0.5 V 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; said medium containing less than about
0.8% by weight alkali metal dichromate during incubation of said
oocysts.
[0099] In yet another aspect of the instant invention, there is
described a method for monitoring sporulation 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.
[0100] In another aspect of the instant invention, there is
provided a composition for the storage of sporulated oocysts
comprising 0.5.times. PBS; and about 30 .mu.g/ml gentamicin,
wherein said composition is characterized as substantially free of
alkali metal dichromate, and further 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.degree. C.
[0101] In a further aspect of the instant invention, a method of
storing sporulated oocysts is provided that comprises contacting
sporulated oocysts with the storage composition described
above.
[0102] In yet but another aspect, the instant invention provides
for a kit 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.
[0103] The preferred methods for isolation, concentration,
flotation, sporulation, monitoring, separation, and sterilization,
are now described.
[0104] Oocyst Isolation
[0105] The initial isolation of oocysts from the gross fecal matter
is desired to remove large debris and fecal matter from a manure
slurry. Thus, the step begins with a manure slurry highly
contaminated with gross particles and results in a aqueous manure
slurry substantially free of gross particles. Although isolation
may be achieved by one of the above methods known in the art,
isolation in the present invention is accomplished by filtering. In
a preferred embodiment, the filtration is by sieving.
[0106] In one embodiment, the initial isolation is achieved by
collecting manure from host animals, mixing the manure with
domestic water, and then sieving. In one embodiment, the process
begins with collected manure, e.g., a batch of several hundred
pounds, made into a aqueous slurry and processed to concentrate
oocysts as a suspension in a relatively small volume of aqueous
medium, e.g., a several hundred bound batch of manure may
ultimately yield about two liters of oocysts in an aqueous
suspension (see FIG. 5A, "Challenge Suite", steps 1-3B). In this
embodiment of the invention, sieving is by means of shaker screens,
such as multiple tier shaker screens.
[0107] In one embodiment, the manure from the chickens is placed
into a mixing vessel either by hand, e.g. using a shovel, or by
using a mechanical dumper. Domestic water is added at a minimum
ratio of about 1 gallon per each six birds' manure (see FIG. 5A,
steps 3A and 4). Alternatively, the collected manure is mixed with
domestic water at a ratio from about 2 to about 6 pounds of
collected manure per gallon of water. As manure quantity is
approximate and dilution is realized by using an approximation of
the manure quantity, the dilution range is therefore also
approximate. The slurry is then mixed until homogeneous. Feathers
and other large, floating debris may be skimmed off the surface
with an appropriate tool, e.g., a wire screen.
[0108] Typically, a sample is taken from the homogenous slurry
prior to the screening/isolation process to assess oocyst count.
Such count is done, for example, by microscopic visual examination.
The homogenous slurry is then pumped onto a two-tier vibratory
shaker screen. The top screen can be from about 150-mesh to about
350-mesh while the bottom screen can be from about 25-mesh to about
75-mesh. In a preferred embodiment, the top deck is equipped with a
50-mesh screen, the "top screen," while the lower deck has a
250-mesh screen, the "bottom screen." Larger unwanted fecal solids
are separated at the top deck 50-mesh screen while smaller unwanted
fecal solids are separated from the slurry at the lower deck, 250
mesh screen. A preferred flow rate onto the top deck screen is
approximately 1 liter per minute per 12.1 m.sup.2. The optimal flow
rate of the pumping varies with the solids content and the
condition of the screen. Larger or smaller screens may be used
depending on the scale of the operation.
[0109] The oocysts are contained in the liquid fraction of the
screening/isolation process. If the solid material coming off
either the top or the bottom screen is too wet, recovery is
unacceptably low as isolation of the oocysts from the homogenous
slurry is not occurring. On the other hand, if too much water is
removed, the solids stick to the screen and do not clear
themselves. Eventually, depending on solid matter content and flow
rates, so much material can accumulate that it may be necessary to
remove it. The screens can be lubricated with water, allowing the
screens to clear themselves. The liquid fraction is collected and
sent on for concentration while the solid fraction is discarded
(see FIG. 5A, step 6).
[0110] The solids coming off both of the screens are checked for
oocysts and then discarded. If less than 5% of the oocysts loaded
onto the screens are found in the solids, then the solids are
discarded. If more than 5% of the oocysts loaded onto the screens
are found in the solids, the solids are resuspended in an aqueous
slurry and recycled through the sieves. The liquid that passes
through both screens, the filtrate, is the fraction that contains
the oocysts. This filtrate is collected into a receiving vessel
(see FIG. 5A, step 7) and then sent to a centrifuge, preferably a
bottle centrifuge or, alternatively, a decanter centrifuge.
[0111] In this non-limiting embodiment, the sieving method can be
carried out in temperatures ranging from a low temperature that
substantially avoids freezing to a high temperature that
substantially avoids damage to the oocysts, preferably at room
temperature. Lower temperatures, about 4.degree. C., are preferred
when sieving procedures take over more than three hours to protect
the viability of the oocysts. In addition, the sieving process can
be carried out at any rate throughput allows as long as the screens
do not accumulate excessive solid matter and at a rate rapid enough
to prevent the manure from drying. As with other steps of the
invention, equipment should be clean prior to use.
[0112] Concentrating the Filtrate
[0113] The liquid oocyst-containing fraction recovered, from any
one of the preceding methods used to initially isolate oocysts, is
then concentrated. Isolated oocysts are concentrated to increase
sporulation rates and output. Concentration is realized by various
means, including substantial separation from the aqueous slurry,
centrifugal-based separation or centrifugal-based separation
followed by filtration. Concentrating the oocysts is accomplished
by utilizing one or more of such techniques in combination with
others. As used herein, centrifugal-based separation includes
processing in either a mechanical rotated centrifugation or a
static hydrocyclone. In one embodiment, centrifugation is by
decanter centrifugation. Other methods are also known in the art
and disclosed herein. Centrifuge scale and capacity varies by batch
size. For larger batch size, the use of a decanter centrifugation
or use of a hydrocyclone would be preferred. For smaller batch
size, bottle centrifugation is preferred.
[0114] In one method, a combination of first sieving the collected
manure, as described in the preferred sieving process above, is
followed by continuous centrifugation and then filtration. In this
method preliminary purification is achieved by sieving a homogenous
slurry of collected manure through sieves having progressively
smaller openings. Further purification is achieved by continuous
centrifugation of the liquid fraction captured from the sieving
process using a suspension wherein the suspension has a specific
gravity preferably between about 1.01 to about 1.08 g/l. As a
further purification step, the solid material recovered from the
centrifugation, which contains the oocysts, is re-slurried and
filtered using a membrane of a pore size that retains the oocysts,
but allows the passage of smaller material, including bacteria.
This process eliminates the flotation step, as described below.
[0115] In an example of a further alternative procedure, the
material passing through the sieves can be further purified by
being collected and pumped into a continuous flow centrifuge
maintained at about 40-50.degree. F., discarding the centrate, and
collecting the solid material containing the oocysts (such as seen
in FIG. 5A, step 8).
[0116] In a preferred embodiment, the filtrate recovered from the
multi-screen screening/isolation process described above is then
concentrated using a centrifuge. In one embodiment, the filtrate
recovered from the multi-screen screening process described above
is then concentrated using a bottle centrifuge. In a preferred
embodiment, the filtrate recovered from the isolation/screening
process is then concentrated using a bottle centrifuge wherein the
filtrate is poured into centrifuge bottles and centrifuged at
1200.times. g for about ten minutes. The solid fraction formed
contains the oocysts. The supernatant is then poured off (see FIG.
5A, step 8) and, if the solid fraction volume allows, more filtrate
is poured on top of the solid fraction. At some point, the solid
fraction will need to be removed as more oocysts collect. The
oocysts may be loosened or removed with a spoon or spatula.
Residual material that is still in contact with the centrifuge
bottles may be rinsed out using a minimal amount of domestic water.
The oocysts then require suspension in sufficient water to bring
the solids content to less than about 70% of the total volume of
the suspension, preferably less than about 60% of the total volume,
and most preferably less than about 50% of the total volume, and
should bring the suspension to substantial homogeneity. During this
suspension process mixing of the aqueous suspension should be
sufficient to keep the solids suspended, but the mixing should not
create foaming. This suspension may then be further processed by
the flotation step, described herein below.
[0117] In an alternative preferred embodiment, the filtrate from
the multi-screen screening process described above, is pumped into
a decanter solid bowl continuous centrifuge at a rate of
approximately 3 to 4 liters/min. The decanter is set with a bowl
speed of at least about 4000 RPM and a conveyor speed of at least
about 2500 RPM and no more than about 3600 RPM. A receiving vessel
is placed to catch the solids as they are expelled from the solid
discharge of the decanter centrifuge. Under these conditions,
solids are discharged as a runny paste. The liquid coming out of
the liquid discharge of the decanter centrifuge is checked for
oocysts and discarded if the liquid contains less than about 2% of
oocysts initially loaded into the decanter centrifuge. If the
liquid waste contains greater than or about 2% of oocysts initially
loaded into the decanter centrifuge, the liquid should be re-mixed
with the solids and run again until the liquid waste contains less
than 2% of oocysts initially loaded into the decanter
centrifuge.
[0118] In a preferred embodiment, once all the filtrate is pumped
into the decanter centrifuge, the centrifuge is allowed to run for
a period of time sufficient to move the residual solids out of the
decanter. In one embodiment, this period of time is more than about
two but less than about five minutes. Use of various sized
centrifuges will vary the period of time and may be adjusted by one
skilled in the art. In a preferred embodiment, the speed of the
bowl is then lowered to about 1000 RPM and the speed of the
conveyor lowered to about 500 RPM. The length of time and the bowl
speed also varies according to batch size and can be properly
adjusted by one skilled in the art. Domestic water may be sprayed
into the access port to wash solids off the inner surfaces of the
body of the decanter. The solids are then moved to an appropriate
volume centrifuge bottle for the flotation step, described below.
It is important to clean the equipment after each run and may be
accomplished by the use domestic water sprayed from a hose in order
to obtain greater yield.
[0119] In yet another embodiment, a hydrocyclone is used to
concentrate the filtrate obtained from sieving. It has been
discovered that a hydrocyclone, traditionally used in the
petrochemical and environmental science fields is useful for
concentrating oocysts. Hydrocyclones use the principle of
centrifugal separation to remove or classify solid particles from a
fluid, based on size, shape, and density. The use of a
hydrocyclone, not known to be used for living organisms, was
previously believed to fatally damage the oocysts due to intense
sheer forces. The instant invention provides a method of utilizing
a hydrocyclone to concentrate oocysts. In one embodiment, the
hydrocylone used is a Dorr-Oliver DOXIE Type 5 Hydrocyclone
(available from GL&V/Dorr-Oliver, Millford, Conn.).
[0120] In a preferred embodiment involving the use of a
hydrocyclone, a reservoir containing the filtrate obtained from
sieving is connected to a pump. The pump delivers the filtrate to
the hydrocyclone at a pressure of between about 120 psi and about
130 psi and at a feed rate from about 1 to about 3 gallons per
minute, preferably about 2 gallons per minute. A preferred
hydrocyclone has one inlet and two outlets. Each outlet is equipped
with a needle valve to regulate the flow through each orifice. By
regulating the flow between the upper and lower outlets, it is
possible to remove a significant amount of liquid through the upper
outlet while retaining most of the denser materials, including the
oocysts, in a concentrated suspension through the lower outlet. In
a preferred embodiment, a 2 to 1 ratio between the flow of material
collected from upper outlet and lower outlet. Such 2 to 1 ration
produces an optimal recovery of oocysts. The recovered concentrated
material, that is, the material collected from the lower outlet,
may be recycled through the hydrocyclone for greater concentration
if further volume reduction is desired. The suspension collected
from the upper outlet is discarded. For large volumes of filtrate,
it may be advantageous to operate hydrocyclones in parallel or
utilize larger scale equipment to increase throughput.
[0121] Floating the Oocysts
[0122] To further isolate the oocysts collected from the
concentration methods described above from unwanted solids, such as
fecal matter, grit, etc., the oocysts are floated to the top of a
solution using density variations. In an alternative embodiment,
the oocysts may be added to a sucrose solution and centrifuged. In
yet another alternative embodiment, the oocysts may be added to a
sucrose solution and then filtered. In a preferred embodiment, the
oocysts are floated to the top of a solution comprising domestic
water and high fructose corn syrup and having sufficient density to
allow the oocysts to float to the top of the suspension while the
heavier unwanted solids migrate to the bottom of a holding vessel
or vessels. In a preferred embodiment, the oocysts are isolated
from the dense solutions using centrifugation. The oocysts are then
recovered from the liquid phase in this step of the invention.
[0123] In one embodiment, the solid material containing the oocysts
recovered from centrifugation is transferred to a mix tank and to a
concentrated sucrose or high fructose corn syrup (HFCS) of volume
equal to that of the oocysts is added. A water of a volume equal to
that of the oocyst/HFCS solution is added for a total final volume
of about four times the volume of the initial solids (FIG. 5A,
steps 9-11). The final mixture is then pumped into a continuous
centrifuge at a rate to allow the oocysts to remain in the centrate
and solids are discarded (FIG. 5B, steps 12-13). If desired,
further concentration of the oocysts and dilution of and
substantial removal of the residual sugar solution is accomplished
by addition of domestic water and continuous flow centrifugation at
a feed rate which allows separation of the phase containing the
oocysts from the sugar solution phase. In an alternative
embodiment, the oocysts/HFCS solution can be centrifuged in a
bottle centrifuge. In this case, the supernatant is discarded and
the oocysts in the resulting solid fraction are resuspended in
water. In yet another alternative embodiment, the residual sugar
can be removed by filtration using filters with a pore size which
excludes the oocysts. When filtration is used, tangential flow is
preferred. Tangential flow filtration is characterized in that an
influent stream is separated into two effluent streams, known as
permeate and retentate. The permeate is that fraction which has
passed through the "semi-permeable" membrane (or filter pad). The
retentate is that stream which has been enriched with the solutes
of suspended solids which have not passed through the membrane (or
filter pads). Water can be continually added to the retentate
vessel at the same rate at which the sucrose-rich permeate is
leaving in order to avoid over concentration of the solids. Once
sufficiently filtered, the retentate, containing the isolated
oocysts, can then be stored in any suitable medium and temperature
until sporulation. In one embodiment, the isolated oocysts are
placed in sterile water and stored at about 2-8.degree. C.
[0124] In a preferred embodiment, the decanter centrifuge method of
concentration, described above, is used to concentration the
filtrate retained from sieving and the volume of solids obtained
from a decanter centrifuge is measured by volumetric measurement.
Such measurement may be taken by centrifuging about 50 ml of the
concentrated filtrate for about 10 minutes at 1,500.times. g
(r.sub.average) in a centrifuge with 50 ml conical tube adapters.
Any centrifuge that produces the preferred forces on the filtrate
may be used. The percent solids is calculated by multiplying the
volume of the solid by 2. Other well known methods may also be used
to calculate solids and can be determined by one skilled in the
art. The solids content is then adjusted to less than 60% solids,
with domestic water, if necessary. More preferably, the percent
solids is brought to below about 50% solids by the addition of
domestic water, and most preferably the percent solids is brought
to below about 40% solids by the addition of domestic water.
[0125] Then a HFCS solution, in a percent volume from about 30% to
about 40% of the solid collected from the
concentration/centrifugation step, is added. This typically brings
the density of the liquid phase up to the point where the oocysts
float. The density of the liquid is brought up to at least 1.09
g/ml and can be brought up to an amount higher than 1.09 g/ml. The
density of the liquid is preferably between 1.09 g/ml and about
1.20 g/ml, more preferably to be between 1.09 g/ml and about 1.14
g/ml, and most preferably to be between about 1.09 g/ml and about
1.10 g/ml. If the density of the liquid is less than 1.09 g/ml,
remix the oocyst-containing liquid with additional HFCS solution
until the density is at least 1.09 g/ml. This dense liquid is then
poured into vessels proper for centrifuging, the vessels are
balanced with respect one another in their placement in the
centrifuge, and then centrifuged.
[0126] In a preferred embodiment, the dense liquid is centrifuged
at a temperature from about 4.degree. C. to about 10.degree. C. The
density of the liquid phase is then measured following the first
centrifuge run using methods well known in the art. If the density
of the liquid is less than 1.09 g/ml, one should re-mix the liquid
phase and the solid phase and add more high fructose corn syrup
solution to obtain a density of 1.09 g/ml or greater. These steps
can be repeated if necessary in order to obtain the highest yield
of oocysts.
[0127] In a preferred embodiment, to the resuspended oocysts from
the concentration step described above, is added a volume of HFCS
equal to about 30% to about 40% of the volume of the solid
fraction. HFCS is added until the density of the liquid phase is
brought up to the point where the oocysts float, a density of about
1.09 to about 1.14 g/ml. The entire suspension of oocysts in the
HFCS/domestic water suspension is then separated from the HFCS (see
FIG. 5B, step 12).
[0128] In one embodiment, the HFCS/domestic water suspension is
poured into centrifuge bottles, balanced with respect one another
in their placement in the centrifuge, and centrifuged for about 15
minutes at 3750.times. g (r.sub.max) and at a temperature from
about 4.degree. C. to about 10.degree. C. The buoyant oocysts float
to the top of the suspension while heavier unwanted solids settle
to the bottom of the bottles. The solids contained in the
supernatant should contain no more than about 40% solids. If the
percent solids found in the supernatant, measured according to the
volumetric method described above, is higher than about 40%, then
the density is too high and the entire suspension needs to be
diluted with domestic water and re-centrifuged.
[0129] In using the bottle centrifuge method of centrifuging, a
large number of the oocysts will remain in contact with the bottle
near the top of the supernatant. This oocyst-containing material
may be freed and returned to the supernatant, for e.g., by swirling
the bottles or by using a tool, such as a spatula. This will not
disturb the solid phase. The bottles may be swirled by hand at room
temperature to remove the crust of oocysts on the bottle. In larger
batch sizes the vessel used for centrifuging can be cleaned by
those methods familiar to one skilled in the art in order to clean
the vessel and recover a higher percentage of oocysts.
[0130] The supernatant is then poured off into a vessel. If using
the bottle centrifuge, rotating the bottles while pouring helps
rinse the oocysts off the sides. The solid fraction can then be
discarded. The same centrifuge bottles can then be refilled and the
process repeated until all of the dense liquid has been
centrifuged. The oocysts are now ready to go to the second
concentration step which removes residual sucrose.
[0131] Concentrating the Oocysts after Flotation
[0132] The liquid fraction from the floatation step described above
is then diluted with domestic water and the separated from the
HFCS. In this process, the oocysts are concentrated prior to the
sporulation step. The concentrated oocysts are then diluted yet
again and held prior to sporulation.
[0133] In one embodiment the liquid phase recovered from the
flotation centrifugation step is first diluted with domestic water
till the oocysts sink and then centrifuged to capture the oocysts
in the solid phase (FIG. 5B, steps 15-18) to remove a substantial
amount of HFCS. In another embodiment, the liquid phase recovered
from the flotation centrifugation step is first diluted with
domestic water till the oocysts sink and the suspension is
processed with a hydrocyclone. In using the hydrocyclone, the upper
fraction is recovered. Subsequent separation of the HFCS, the
concentrated oocyst containing suspension is again diluted with
domestic water and transferred to a holding vessel prior to
sporulation (FIG. 5B, steps 19-20).
[0134] In a preferred embodiment, the volume of the liquid fraction
recovered from the flotation step is measured and a sample is taken
to assess oocyst count. Sufficient domestic water is added to lower
the density of the supernatant to less than about 1.04 g/ml. This
allows the oocysts to sink. The density is measured following the
addition of the domestic water using techniques well know in the
arts. If the density is not less than about 1.04 g/ml and/or the
oocysts have not sunk, additional domestic water is added until
such density is reached and/or the oocysts sink. The oocyst
suspension is then poured into centrifuge bottles and centrifuged
for about 10 minutes at 1200.times. g from about 4.degree. C. to
about 10.degree. C. The supernatant is tested for oocyst presence
by counting using a microscope and hemocytometer and the
supernatant is discarded if an acceptable amount of oocysts are
counted in the supernatant. An acceptable amount of oocysts in the
supernatant is from about 1% to about 5%, preferably about 2%, of
the total oocysts loaded at the beginning of flotation step. More
of the mixture from the flotation step is then poured on top of the
solid fraction generated by centrifugation. While not necessarily
being resuspended, the solid fraction is loosened somewhat,
particularly by inverting the bottle a few times. The resuspended
solid fraction suspension is then centrifuged as before, for about
10 minutes at 1200.times. g from about 4.degree. C. to about
10.degree. C., and the process is repeated until the flotation step
mixture has all been centrifuged.
[0135] When using the bottle centrifugation method, at this point,
there should be several bottles, each with a solid fraction in the
bottom. Note, however, with larger batch size the vessel or vessels
vary with equipment that is of appropriate volume and recovery
methods may be determined by one skilled in the art. The solid
fractions in the centrifugation vessels are then resuspended by
shaking them with a minimal amount of domestic water. The solid
fractions are rinsed into one or two of the bottles and the bottles
filled and balanced with water if necessary. These bottles are
centrifuged one last time as before, for about 10 minutes at
1200.times. g from about 4.degree. C. to about 10.degree. C. The
supernatant is then discarded. Any loose solid fractions that comes
out with the supernatant can be ignored. The solid fraction is then
resuspended in a minimal amount of domestic water and stored in a
single bottle from about 2.degree. C. to about 5.degree. C. pending
sporulation while freezing should be avoided.
[0136] In an alternative embodiment, the HFCS in the liquid phase
recovered from the flotation step can be remove by filtration using
filters with a pore size which excludes the oocysts. When
filtration is used, tangential flow is preferred. Tangential flow
filtration (TFF) is characterized in that an influent stream is
separated into two effluent streams, known as permeate and
retentate. The permeate is that fraction which has passed through
the "semi-permeable" membrane (or filter pad). The retentate is
that stream which has been enriched with the suspended solids which
have not passed through the membrane (or filter pads). Once
sufficiently filtered, the retentate, containing the isolated
oocysts, can then be stored in any suitable medium and temperature
until sporulation. In one embodiment, the isolated oocysts are
placed in sterile water and stored at about 2-8.degree. C. Note
that the tangential flow filtration is an alternative embodiment to
concentrating the oocysts after flotation and TFF at this step
should not be confused with TFF used during sterilization.
[0137] In an alternative embodiment, the volume of the liquid
fraction recovered from the flotation step is measured and a sample
is taken to assess oocyst count. Sufficient domestic water is added
to lower the density of the supernatant to less than about 1.04
g/ml. This allows the oocysts to sink. The density is measured
following the addition of the domestic water using techniques well
know in the arts. If the density is not less than about 1.04 g/ml
and/or the oocysts have not sunk, additional domestic water is
added until such density is reached and/or the oocysts sink. The
oocysts suspension is then processed through a hydrocyclone at a
flow rate of about 2 gallons per minute and at a pressure between
about 120 psig and about 130 psig.
[0138] Batch size and scale will lead one skilled in the art to
utilize various centrifugation processes based on batch size while
one skilled in the art will also adjust the correct centrifugation
speeds based on the batch size.
[0139] Sporulation
[0140] Sporulation is performed to transform the cleaned and
concentrated oocysts into their next life form, the sporulated
oocyst (see FIG. 5B, "Sporulation Suite"). Sporulation may be
performed in any suitable container, however, a fermentation vessel
is preferred in order to best control temperature, dissolved
oxygen, pH, and mixing in addition to monitoring these parameters
of the sporulation medium. The capacity of the sporulation vessel
varies with batch size and can be adequately selected by one
skilled in the art. A preferred fermentor is the New Brunswick
BioFlow 3000 (available from New Brunswick Scientific Company,
Edison, N.J.).
[0141] Sporulation is achieved by subjecting the oocysts to an
oxidative challenge. In this step, the oocysts are contacted with
an oxidizing agent which is effective to promote sporulation but
does not result in the death of the oocysts. As described below,
the oxidizing agent comprises a principal oxidant other than a
source of dichromate. Preferably, the sporulation medium is
substantially devoid of potassium dichromate, an alkali metal
dichromate, dichromate ions or other dichromate salt. In a
preferred embodiment, the sporulation medium contains less than
about 0.8% by weight of alkali metal dichromate, or less than about
0.6% by weight of alkali metal dichromate, or less than about 0.4%
by weight of alkali metal dichromate, or less than 0.2% by weight
of alkali metal dichromate, more preferably 0.1% by weight of
alkali metal dichromate, and most preferably the sporulation medium
is substantially free of an alkali metal dichromate. In another
preferred embodiment, the sporulation medium contains less than
about 3.0% by weight of dichromate ions. In yet another preferred
embodiment, the sporulation medium contains less than about 1.5% by
weight hexavalent chromium.
[0142] In one embodiment of the invention, oocysts concentrated by
the methods and processes of the instant invention and described
above are collected over a time period sufficient to create a batch
size suitable for a fermentation vessel of desired volume. The
collected concentrated oocysts are then deposited in to a
sporulation vessel, an oxidizing agent is added and sporulation is
allowed to occur (see FIG. 5B, step 21). Domestic cold water is
used to rinse the container or containers holding the oocysts prior
to their contact with the fermentation vessel. The sporulated
oocysts and the rinse is then transferred to a separation device
(FIG. 5B, step 22). The instant invention also provides for
sporulation by depositing concentrated oocysts in a fermentation
vessel, subjecting the oocysts to an oxidative challenge by
contacting the oocysts with an oxidizing agent, such as oxygen or
sodium hypochlorite, in an aqueous medium, wherein the percent
saturation of dissolved oxygen in the medium is maintained at
preferred levels, pH is controlled between preferred levels by the
alternative addition of an acid or a base, the suspension is mixed
to near homogeneity, and temperature is between preferred
temperatures over a preferred period of time. In a further aspect
of the instant invention an anti-foaming agent is added during the
sporulation process. Preferably solids do not exceed more than
about 50%. Preferably solids are less than about 35%. More
preferably still, solids are less than about 25%. Sufficient
domestic water is added to the sporulation vessel to achieve this
ratio between solid and liquid phase. The liquid phase in the
fermentation vessel is termed the sporulation medium.
[0143] In a preferred embodiment the oxidizing agent used is
oxygen. Oxygen may be added in the form of air or as pure
oxygen.
[0144] In an alternative embodiment, sufficient 5.25% sodium
hypochlorite is added to the fermentor to achieve the following
initial concentrations upon dilution of active chlorine
concentrations for the individual subspecies. The values are
approximate and indicate preferred maximum concentrations that do
not inhibit sporulation:
1 TABLE 1 Spp. of Eimeria weight % E. Acervulina 0.01 E. Maxima
0.05 B. Tenella 0.05
[0145] During sporulation, the percent saturation of dissolved
oxygen content in the aqueous medium is maintained at at least 30%
of saturation, preferably at at least 40% of saturation, and more
preferably at least 50% of saturation. Percent saturation of
dissolve oxygen is controlled, by supplying air or molecular
oxygen, to achieve consistent and higher yields of sporulated
oocysts.
[0146] In a preferred embodiment of the invention, percent
saturation of dissolved oxygen is maintained by bubbling air
through the mixture at a rate sufficient to meet the above ranges.
Pure oxygen may also be bubbled through the mixture to maintain the
requisite percent dissolved oxygen. Care should be taken so that
the flow of oxygen is not so rapid as to cause foaming. If desired,
an anti-foaming agent may be added, such as Antifoam A (available
from Sigma-Aldrich, St. Louis, Missouri). Oxygen is added by any
means practicable. Oxygen may be added by adding both air when
lesser flow rates are needed. e.g., when oxygen consumption is
relatively low to peak sporulation, to maintained the preferred
percent dissolved oxygen saturation while molecular oxygen may be
added when the need is greater, e.g., when oxygen consumption is
greatest. Oxygen is preferably added at a flow rate of from about
0.1 to about 2.0 liters of gas per liter of material and more
preferably from about 0.3 to about 0.5 liters of gas per liter of
material. The flow rate may be kept constant despite a greater need
to maintain preferred percent saturation of dissolved oxygen as the
gas added may consist of air when less oxygen is needed and may
consist of molecular oxygen when more oxygen is needed. The
preferred fermentor automatically converts from the addition of air
to molecular oxygen as needed while controlling a nearly constant
flow rate.
[0147] The pH level is preferably maintained from about 7.0 to
about 7.7, more preferably from 7.2 to about 7.5, and more
preferably still the pH is maintained about 7.4. The pH level of
the sporulation medium is controlled by adding an acid or a base.
In a preferred embodiment, either sodium hydroxide (5N) or sulfuric
acid (5N) is alternatively added to the sporulation medium as
needed to maintain the pH near 7.4. When using a fermentation
vessel, the acid and/or the base may be added by using a
fermentation vessel's automatically controlled peristaltic pumps on
the fermentor.
[0148] The temperature of the sporulation medium is controlled
throughout sporulation. Oocysts are placed in a sporulation vessel
at a temperature from a temperature that substantially avoids
freezing to about 43.degree. C.; preferably between about
15.degree. C. to about 38.degree. C.; and more preferably between
about 20.degree. C. to 30.degree. C. and more preferably still at
about 28.degree. C..+-.1.degree. C. It will be apparent to those of
ordinary skill in the art that the rate of sporulation is
temperature dependent, so that the time required for sporulation
will generally be less at higher temperatures.
[0149] Throughout the sporulation process, the sporulation medium
is mixed. Any suitable method of mixing can be used to mix the
sporulation medium to about a homogenous state. The exact method of
mixing varies depending on the container used. For example, when
bottles or flasks are used, mixing can be achieved by the use of
shakers, or magnetic or mechanical stirrers. When vats or
fermentors are used, a mechanical stirrer, such as a paddle stirrer
is preferred.
[0150] Although sporulation is substantially complete within 12 to
18 hours, removal of the sporulated oocysts prior to about 72 hours
decreases viability. Therefore, sporulated oocysts are preferably
kept under the above sporulation conditions for a preferred time
period to provide a more stable population of sporulated oocysts.
The oocysts are preferably maintained in the above conditions for
approximately 72 to 120 hours, more preferably for 72 to 110 hours,
and more preferably still for 72 to 96 hours, to allow sporulation
to occur.
[0151] Sporulation start point, end point and rate maybe monitored
by monitoring: (1) the rate at which oxygen must be added to the
sporulation medium to control percent saturated dissolved oxygen;
and/or (2) by monitoring the amount of acid or based required to be
added to control the pH of the sporulation medium. It has been
discovered that sporulation results in an increase in oxygen
consumption, as evidenced by a decrease in dissolved oxygen in the
sporulation medium, and an increase in pH, that is, if percent
saturation of dissolved oxygen and pH are not controlled. When no
additional oxygen is added to the sporulation medium, sporulation
is indicated by a drop in dissolved oxygen to less than 60% of
saturation, more preferably less than 40%, and more preferably
still less than 20%. The change in dissolved oxygen can also be
measure in terms of percent change. Thus, sporulation can also be
indicated by a decrease of at least 10% (i.e., from 50% to 40%),
preferably at least 20%, more preferably at least 30%, and more
preferably still at least 40% in dissolved oxygen content as
expressed in percent of saturation (see FIG. 1A). When pH is not
controlled by the alternative addition of an acid or a base,
increase in pH of at least about 0.25 pH units, more preferably at
least about 0.5 pH units, is indicative that sporulation is
occurring (see FIG. 2).
[0152] The change in dissolved oxygen and pH do not occur
independently. An increase in the oxygen consumption indicates the
start point of sporulation. Note, however, that background oxygen
consumption will be seen as the sporulation medium is not sterile
at this point and so various bacteria will be consuming oxygen as
well as the oocysts. However, the increase in oxygen consumption
will be significant over the background oxygen consumption so that
the sporulation start point, end point, and rate, including peak,
are readily ascertainable. A decrease in oxygen consumption
indicates a drop in sporulation rate. Once oxygen consumption
becomes low and consistent, sporulation is substantially complete,
usually after about 18 hours. However, as mentioned above, the
sporulated oocysts should be maintained under the sporulation
conditions for at least an additional 36 to 48 hours to increase
yield. Monitoring of sporulation will assist the practitioner in
reaching higher yields of viable sporulated oocysts. Optionally,
sporulation can be confirmed by microscopic examination of the
oocysts. However, the method of present invention obviates the need
for sampling and microscopic examination.
[0153] Sterilization
[0154] Following sporulation, the sporulated oocysts, are removed
from the sporulation vessel, and washed free of the sporulation
medium and concentrated by any suitable method, preferably
filtration. The entire sterilization process is generally conducted
in two phases: (1) contaminants are first removed non-aseptically
(see FIG. 5C, steps 23-25); followed by (2) disinfection of
sporulated oocysts medium carried out under sterile conditions (see
FIG. 5C, steps 26-28). The purpose of this process is to collect
sporulated oocysts and filter out contaminants. A further purpose
is to concentrate oocysts, preferably by filtration. However,
centrifugation may also be used to concentrate the sporulated
oocysts. A further purpose is to sterilize the suspension with a
disinfectant, preferably sodium hypochlorite (leaving the
sporulated oocysts intact), then to remove the disinfectant from
and then concentrate the sporulated oocysts. Then, to the
sporulated oocysts, is added an appropriate quantities of buffer
and antibiotic, preferably PBS and gentamicin (FIG. 5C, steps
30-31). This sporulated oocysts-containing suspension is then
transferred into suitable storage containers for bulk storage prior
to final packaging for distribution to consumers.
[0155] In one embodiment, separation of the sporulated oocysts from
the sporulation medium may be achieved by centrifugal-based
separation, such as by bottle centrifuge, decanter centrifuge, or
by hydrocyclone. The volume of the batch size will be determinative
of the mode of centrifugal-based separation and can be determined
by one skilled in the art. The solid fraction from any one of the
centrifugal-based separation methods is recovered. If more than
about 5% of the oocysts loaded into the centrifugal-based
separation unit are in the refuse fraction, a liquid fraction in
this embodiment, said fraction is mixed with the solid fraction and
recycled through the centrifugal-based separation unit. The
recovered solids are then diluted to a volume appropriate for
sterilization, preferably by filtration, more preferably by
tangential flow filtration.
[0156] In a preferred embodiment, once sporulation is complete, the
resultant aqueous suspension of sporulated oocysts is transferred
from the fermentation vessel into a receiving vessel of appropriate
volume. The transfer of the oocysts from the fermentation vessel is
preferably accomplished by using air forced through the
fermentation vessel, e.g., pressurizing the headspace, thereby
forcing the sporulated oocysts into the awaiting container. A
sample of the sporulation medium from the container is then taken
to assess sporulated oocyst count and sporulation ratio. Any
material still in contact with the fermentation vessel may be
removed by using a rinse, e.g., a sufficient amount of domestic
water, and the rinse may then be combined with the suspension of
sporulated oocyst already transferred from the fermentation
container (FIG. 5B-C, step 22). Again, a sample is then taken to
assess sporulated oocyst count and sporulation ratio in an effort
to ascertain yield. The manner and method in which the sporulated
oocysts are harvested from the fermentation vessel varies with the
batch size and type of fermentation vessel and may be properly
determined by one skilled in the art.
[0157] After removal of the suspension of sporulated oocysts from
the fermentation vessel, the oocysts are allowed to settle from
sporulation medium over a period of several hours, e.g., 8 to 20
hours, while the medium and the oocysts are held at a temperature
from a temperature that prevents freezing to about 10.degree. C.,
more preferably from about 2.degree. C. to about 6.degree. C., and
most preferably about 4.degree. C. Sporulated oocysts settle to the
bottom of the storage container while contaminants remain suspended
or dissolved in the aqueous layer. In a preferred embodiment, the
supernatant is decanted, poured or pumped off using a small
peristaltic pump or other method suitable to the volume being
removed. Domestic water is then added at a sufficient volume to
resuspend the sporulated oocysts. Settling can be carried out in
the receiver or in a separate settling vessel.
[0158] The sporulated oocysts, now resuspended after collection
from settling, are then separated from the sporulation medium. In a
preferred embodiment, separation is by filtration. However, any
appropriate means may be used to separate the sporulated oocysts
from the sporulation media. In a more preferred embodiment, the
filtration process is by means of tangential flow filtration.
Tangential flow filtration ("TFF") is used in this procedure to
separate sporulated oocysts from other material that may be present
in the suspension, e.g., grit, other microorganisms, etc. In
addition to the filter membrane, two essential parts of the TFF
system are a retentate vessel, which holds the sporulated oocysts,
and a low shear pump that circulates the retentate through the
membranes and back into the retentate vessel. The oocysts are
retained in the retentate while the permeate is discarded.
[0159] The pore size of the filter membrane should be small enough
so that sporulated oocysts cannot enter the pores, but large enough
to allow bacteria to pass through. In one embodiment, the filter
has a pore size of approximately 10-microns. In yet another
embodiment the filter has a pore size of approximately 5-microns. A
preferred filtration unit is a Consep membrane unit manufactured by
North Carolina SRT (available from North Carolina SRT, Inc., 221
James Jackson Ave., Cary, N.C. 27513). However, other filtration
units may be used, such as those produced by Millipore (available
from Millipore Corporation, 80 Ashby Road, Bedford, Mass. 01730). A
preferred filter is the Spectra/Mesh polyester filters (Spectrum
Laboratories, Inc., Rancho Dominguez, Calif.; cat no: 146524).
Tangential filtration units such as an OPTISEP CL, OPTISEP, or
CONSEP may be used, also available from North Carolina SRT.
Throughput can be increased by utilizing a larger scale filtration
unit. One skilled in the art will recognize that the type of
filtration unit needed depends on the volume of the sporulated
oocyst suspension. In one embodiment, an OPTISEP CL unit is used to
run about a 1L sporulated oocyst suspension. In another embodiment,
a CONSEP unit is used to run about a 10L sporulated oocyst
suspension.
[0160] When a filtration process is applied to a sporulated
oocyst-containing medium the permeate is discarded. Water is added
as permeate is removed if the filtration process is conducted at a
constant volume. Filtration can be accomplished by gravity flow or
by the use of a pump, for example, a peristaltic pump. In a
preferred embodiment, the mixture is pumped tangentially over the
filter. If a pump is used, the rate of pumping varies with such
well known factors as the surface area of the filter, the path
length, the total area of the flow channel, and the pore size.
Optimum pumping rates can be determined by one of ordinary skill in
the art without undue experimentation as such flow rates are a
function of surface area of the filtration unit and solids
content.
[0161] In a further embodiment, the inlet and the outlet tubing for
the tangential flow unit are placed into a vessel containing the
sporulated oocysts while the permeate tubing is placed in a
separate vessel. The pump, for example, a diaphragm pump, is then
started to begin filtration. A preferred flow rate is about 1 LPM
per 160 cm.sup.2. The pump rate may also be expressed in terms of
lineal velocity. Lineal velocities may be between 20 and 50
centimeters per second. A preferred lineal velocity when using a
CONSEP filtration unit is 28 centimeters per second. The pump may
be kept running to maintain the flow rate throughout the process.
The permeate is sampled and, using a glass slide, observed for
sporulated oocysts. The optical density of the permeate sample is
also measured using a spectrophotometer at 600 nm (OD.sub.600).
Circulation of retentate over the filter medium is continued if the
concentration of oocysts in the permeate has not increased to or
exceed a maximum tolerable level. An acceptable concentration is
from about not more than 5% of the total sporulated oocysts loaded
into the filtration unit. If the sporulated oocysts concentration
measures to or above the maximum acceptable level, filtration is
stopped. The permeate is recycled and mixed with the retentate and
filtration is resumed. Filtration is stopped when the measured
OD.sub.600 is about less than about 0.5 at a lineal velocity of
about 28 centimeters per second. However, filtration may be stopped
when the measured OD.sub.600 is about 0.6, again at a lineal
velocity of about 28 centimeters per second. Once the desired OD is
reached, oocysts from the membranes and the tubing are transferred
to the retentate vessel and any oocysts remaining in the membranes
and tubing are then flushed with water into the retentate
vessel.
[0162] The retentate, containing the concentrated oocysts, is then
placed in a vessel under refrigeration at about 4.degree. C. from
about 15 to about 24 hours. This allows the sporulated oocysts to
settle in bottom solid phase and a liquid phase will normally form
above the solid phase. The liquid phase is then substantially
removed after refrigeration to reduce unwanted volume, e.g., by
decanting, pumping, or siphoning. In an alternative embodiment, the
retentate is processed immediately after filtration. However, it is
preferred to let the retentate rest as described above.
[0163] Once the sporulated oocysts have been concentrated by
filtration they can be sterilized by means of a chemical
disinfectant or sterilizing agent other than an alkali metal
dichromate, soluble dichromate moieties, dichromate ions, or
potassium dichromate. Sterilization processes are conducted in
sterile environments. In a preferred embodiment, sterilization is
accomplished within the filtration device used to concentrate the
sporulated oocysts. In an alternative embodiment the retentate
containing the sporulated oocysts can be washed from the filter and
sterilization is accomplished in a vessel separate from the
filtration device. Any filtration unit used to sterilize the
sporulated oocysts should be sterilized prior to the addition of
the unsterilized sporulated oocysts. In one embodiment, the
filtration unit is sterilized by autoclaving. In an alternative
embodiment, the filtration unit is sterilized by passing steam at
approximately 250.degree. C. through the system for at least about
30 minutes at approximately 20 psi. In yet another alternative
embodiment, the unit is chemically sterilized by treating the
system with 5% sodium hypochlorite for at least about 10 minutes
wherein the sodium hypochlorite contains at least about 5%
available chlorine by weight.
[0164] The agent used for sterilizing the sporulated oocysts
preferably is one which kills bacteria and viruses, but does not
kill the sporulated oocysts. Preferably, the disinfectant used
kills the infectious bursal disease (IBDV), chick anemia (CAV)
viruses, and related bacteria. As IBDV is known to be a robust
virus, a sterilization agent that kills IBDV will kill other, less
robust microorganisms as well. An agent that eliminates IBDV is
considered to substantially eliminate microorganisms.
[0165] In a preferred embodiment, the disinfectant used is sodium
hypochlorite. The concentration of disinfectant used varies with
the agent chosen to accomplish sterilization. In more preferred
embodiment, sodium hypochlorite is used at a concentration
preferably in the range from about 1% to about 10%, and more
preferably in the range of about 2% to about 5% wherein the percent
represents the percent of available chlorine by weight. The time
during which the sporulated oocysts are exposed to the disinfectant
varies depending upon factors such as the concentration of the
disinfectant and the volume of the batch of sporulated oocysts. In
one embodiment, the sporulated oocysts are treated with
approximately 5% sodium hypochlorite, wherein the percent
represents the percent of available chlorine by weight, from about
2 to about 20 minutes, more preferably from about 5 to about 18
minutes, and most preferably for about 10 minutes.
[0166] Once the filtration unit is sterilized, the vessel holding
the sporulated oocyst suspension is then removed from
refrigeration. The clear upper layer is removed by pumping,
pouring, or suctioning off the supernatant, leaving the bottom
sporulated oocyst fraction. The latter fraction is then transferred
to a retentate vessel of adequate volume. The previous vessel is
then rinsed with domestic water. The domestic water rinse is then
added to holding vessel and stirred by adequate means. In one
embodiment, stirring is accomplished by means of a magnetic stir
bar while the retentate vessel is sitting on a magnetic stirrer. In
another embodiment, stirring is accomplished by means appropriate
for the volume of the retentate vessel, such as with a paddle.
[0167] Next, a volume of about 10% aqueous sodium hypochlorite
solution that is approximately equal to the volume of suspension in
the retentate vessel is added to the sporulated oocyst suspension.
The sporulated oocyst suspension containing sodium hypochlorite
should have a solids content of preferably less than about 30%,
more preferably to less than about 25%, and even more preferably to
less than about 15%, and most preferably to less than 7.5%. A
solids content of less than about 7.5% is preferred as the reduced
solids content produces a higher assurance of sterility. When
solids are brought to about 15%, there is approximately 5% sodium
hypochlorite, wherein the percent represents the percent of
available chlorine by weight, in the suspension. In an alternative
embodiment, the solids concentration is adjusted to about 15% prior
to sodium hypochlorite addition. After this addition, the solids
are 7.5% and the sodium hypochlorite is at 5%. With either
embodiment, the suspension is mixed thoroughly by adequate means
and, in one embodiment, allowed to stand for preferably 5 minutes,
more preferably 8 minutes, and most preferably about 10 minutes.
Standing time will vary depending on batch size and volume and may
be adjusted as needed. However, standing may be avoided and
filtration may occur immediately after dilution.
[0168] The retentate vessel containing the sporulated oocysts
suspension is then connected to the filtration unit. Autoclaved or
otherwise sterile water is used as a water source during the
sterilization process. The retentate pump is then activated while
the permeate line is pinched or clamped closed. Once any air
bubbles have been substantially eliminated from the membranes and
tubing the permeate line may be opened and directed to a collection
vessel outside a sterile environment. Permeate is then analyzed to
verify that there is no significant loss of oocysts via the
permeate.
[0169] In an alternative to using filtration to remove the sodium
hypochlorite from the oocyst suspension, centrifugation may be
utilized.
[0170] While the hypochlorite-rich permeate leaves the filtration
unit, sterile water is added to replenish the volume. Filtration is
continued and water is added as needed to control the percent
solids during filtration. A sample is then taken from the permeate
to determine total chlorine level. Chlorine level can be detected
using CHEMetrics Vacuette Kit, available from CHEMetrics, Inc.,
Route 28, Calverton, Va., 20138. The total level of chlorine should
is reduced to less than about 1 ppm by. When the permeate contains
less than about 1 ppm of the chlorine the retentate, which contains
the oocysts, also contains less than about 1 ppm. Once the desired
level of chlorine is reached, filtration may be continued without
adding more water to reduce the overall volume of the sporulated
oocysts suspension.
[0171] When the desired retentate volume is reached, the pump feed
tubing is removed from the retentate vessel and placed into a
vessel containing sterile water and filtration is continued. Once
substantially all of the suspension has been flushed out of the
tubing and the filter housing, the pump is shut off and filtration
is complete.
[0172] Next, if desired, a buffer, such as PBS, and a bactericide
is added to the sterilized sporulated oocyst suspension. In a
preferred embodiment, 1.times. PBS containing 60 .mu.g/ml
gentamicin is added in a 1:1 ratio to the disinfected sporulated
oocyst suspension to result in a suspension of sporulated oocysts
in 0.5.times. PBS with 30 .mu.g/ml gentamicin. The suspension is
then stored under refrigeration, preferably around 4.degree. C. for
future use. In an alternative embodiment, 1.times. PBS is added to
the sporulated oocyst suspension while no bactericide is added to
the sporulated oocyst suspension. Average yields for all three
subspecies, E. tenella, E. acervulina, and E. maxima, is about 70%
for disinfection and final filtration.
[0173] For the purposes of the present invention, sporulated
oocysts and compositions containing sporulated oocysts are
considered sterile if samples of liquids containing the oocysts do
not have detectable amounts of live bacteria, IBD virus or CAV
virus. Detection of live bacteria can be accomplished by any method
known in the art. For example, bacteria can be detected by
incubation on bacterial agar plates at 35-37.degree. C. for 18 to
24 hours. One preferred method of testing is set forth in 9 C.F.R.
113.27(1999), hereby incorporated in its entirety by reference.
Briefly, to test for bacterial contamination, a sample of the
preparation of the present invention can be innoculated into
soybean casein digest medium and incubated at 30 to 35.degree. C.
for 14 days. To test for fungal contamination, a sample of the
preparation of the present invention can be innoculated into
soybean casein digest medium and incubated at 20 to 25.degree. C.
for 14 days. After the incubation period, the vessels can be
examined macroscopically for microbial growth. If growth cannot be
determined reliably by visual examination, the judgment can be
confirmed by microscopic examination.
[0174] Detection of IBDV virus or CAV virus can be by any method
known in the art. A non-limiting example of IBDV and CAV detection
is by the methods set forth in 9 C.F.R. 113.47 (1999), herein
incorporated in its entirety by reference. Briefly, to test for
CAV, MSB-1 cells from the Maine Biological Laboratories,
Waterville, Me. are used as the indicator cell line for Chicken
Anemia Virus. MSB-1 cells are a lymphoblastoid cell line from a
Marek's disease lymphoma that show cytopathic effect when infected
with Chicken Anemia Virus. Cells are maintained in Opti-MEM.RTM.
(Life Technologies, Gaithersburg, Md.) or other suitable media at
41.degree. C. for at least 24 days prior to testing. Cells are
subcultured 10-12 times during the maintenance period with all but
the last subculture resulting in a monolayer of at least 75
cm.sup.2. The last subculture is at least 6 cm.sup.2.
[0175] Three groups of MSB-1 monolayers are used for each test, a
negative control group, a positive control group, and a test group.
At the start of the 24 day maintenance period, the positive control
group is inoculated with 10.sup.5.75 TCID.sub.50/ml of Chicken
Anemia Virus, Del Ros strain originally obtained from the Center
for Veterinary Biologics Laboratory (Ames, Iowa) and the test group
inoculated with the test preparation. The negative control group is
inoculated with a preparation known to be free of Chicken Anemia
Virus. The cells are then maintained for at least 24 days as
described above.
[0176] Two days after the last subculture, the three groups of
monolayers are fixed and treated with a specific chicken polyclonal
Chicken Anemia Virus antibody (Hy-Vac, Adel, Iowa). The monolayers
are then washed and treated with a fluorescein labeled goat anti
chicken IgG (H&L, Jackson Immunoresearch, West Grove, Pa.) and
examined for specific fluorescence. If the positive control shows
specific fluorescence and there is no difference in fluorescence
between the test and negative control groups, the preparation is
considered free of Chicken Anemia Virus.
[0177] Alternatively, the presence of Chicken Anemia Virus can be
detected by the polymerase chain reaction (PCR). Three days after
the last subculture, DNA is extracted from the three groups of
monolayers using well established procedures. See, for example,
Ausubel et al., Short Protocols in Molecular Biology, 2.sup.nd Ed.,
John Wiley & Sons, 1992; Sambrook et al., Molecular Cloning, A
Laboratory Manual, 2.sup.nd Ed., Cold Spring Harbor Laboratory
Press, 1989; Davis, et al., Basic Methods in Molecular Biology,
Elsevier, 1986. Briefly, cells are lysed by two cycles of freezing
to -80.degree. C. and thawing at 37.degree. C. Cellular debris is
removed by centrifugation at about 3,500.times. g for 20 minutes.
The supernatant is treated with DNase and RNase A to remove
cellular contaminants and the proteins and/or virus precipitated
with polyethylene glycol. The precipitate is treated with
Proteinase K and extracted three times with
phenol/chloroform/isoamyl alcohol. DNA is precipitated with sodium
acetate-ethanol and pelleted by centrifugation at 14,000.times. g
for 15 minutes. The resulting pellet is resuspended in double
distilled water and stored at -20.degree. C.
[0178] Conserved regions of the Chicken Anemia Virus viral genome
are amplified using standard techniques (Innis et al., PCR
Protocols, Academic Press, 1990). Information on the Chicken Anemia
Virus viral genome for designing suitable primers can be found on
databases well known to those in the biomedical arts such as the
databases available through the U.S. National Institutes of Health
website at http://www.ncbi.nlm.nih.gov, all herein incorporated by
reference. PCR products are analyzed by agarose gel electrophoresis
and ethidium bromide staining. If the PCR amplification does not
result in a band corresponding to the band found in the positive
control, the preparation is considered free of Chicken Anemia
Virus.
[0179] One preferred, but non-limiting method for detection of IBDV
in the preparation of the present invention is the same as for
Chicken Anemia Virus with the following changes. A preferred cell
line used for IBDV testing is a primary chick embryo fibroblast
cell line. After addition of a sample of the preparation or
10.sup.8.25 TCID.sub.50 of IBDV originally obtained from American
Type Culture Collection (ATCC VR-2041 strain D78), the monolayers
are maintained in basal medium Eagle (BME) or other suitable media
at 37.degree. C. and 5% CO.sub.2 for at least 14 days. Detection is
preferably accomplished using an IBDV specific polyclonal chicken
antiserum and a fluorescein labeled goat anti-chicken IgG (Jackson
Immunoresearch, West Grove Pa.). If the positive control shows
specific fluorescence and there is no difference in fluorescence
between the test and negative control groups, the preparation is
considered free of IBDV.
[0180] Alternatively, the test for the IBDV contamination can be
accomplished using PCR as described for Chicken Anemia Virus.
Information on the IBDV viral genome for designing suitable primers
can be found on databases well known to those in the biomedical
arts such as the databases available through the U.S. National
Institutes of Health website at http://www.ncbi.nlm.nih.gov, all
herein incorporated by reference. If the PCR amplification does not
result in a band corresponding to the band found in the positive
control, the preparation is considered free of Infectious Bursal
Disease Virus.
[0181] Post-challenge performance improvement compositions may also
be added to the sterilized sporulated oocyst suspension. A
preferred post-challenge performance improvement composition is
Propionibacterium acnes (P. acnes) and can be added to the
sporulated oocyst suspension prior to the time of filling vials for
consumer use. The P. acnes may be obtained from independent
manufactures in 1-liter glass bottles suspended in PBS or water and
having gentamicin at a cell density equivalent to 10.sup.12
cells/ml of the pre-autoclaved P. acnes count. The P. acnes
suspension is preferably included in the final vaccine at a dry
weight dose equivalent to 50 .mu.g per bird.
[0182] Storage
[0183] The instant invention also provides for compositions to
store sterile sporulated oocysts. The sporulated oocysts can be
held in sterile water or other suitable diluent (see FIG. 5C,
"Storage Suite"). In a further embodiment, an oxidizing agent is
added to the storage composition. In one embodiment, the suspension
of sporulated oocysts is transferred to vials that are prepared in
a kit containing the suspension as a ready-to-administer
vaccine.
[0184] Sterile sporulated oocysts are preferably stored at any
temperature between room temperature and a low temperature that
substantially avoids freezing. In a preferred embodiment, the
sporulated oocysts are stored at between about 1.degree. C. and
about 10.degree. C., more preferably between about 2.degree. C. to
about 7.degree. C., and in a most preferred embodiment between
about 4.degree. C. to about 5.degree. C. In an alternative
embodiment, the sporulated oocysts are stored at a temperature
between 20.degree. C. to about 30.degree. C., more preferably
between 22.degree. C. to about 27.degree. C., and most preferably
at about 25.degree. C.
[0185] Although not necessary, a buffering agent may be added to
the diluent in which the sporulated oocysts are stored. Buffers are
utilized in the storage composition as they prolong viability over
the use of sterile water or sterile water containing gentamicin.
Many suitable buffers are known in the art including, but not
limited to, phosphate buffer, bicarbonate buffer, citric acid and
tris buffers. In one preferred embodiment, the diluent comprises
0.5.times. PBS. In a preferred embodiment, a volume of buffer is
used that results in a concentration of sporulated oocysts suitable
for transfer to containers that are ultimately used by the consumer
as a vaccine for the prevention of coccidiosis.
[0186] The diluent may also optionally comprise a bactericide or
other preservative. Any bactericide that is suitable for use in
pharmaceutical compositions, and especially compositions that are
administered to food animals, can be used. Non-limiting examples or
bactericides include potassium perchlorate, sodium hypochlorite,
hydrochlorous acid, sodium hydroxide and antibiotics. Preferred
concentrations of chemical bactericides in final concentration in
the vaccine, include: from about 0.10 wt % to about 0.25% potassium
perchlorate, from about 0.001 wt % to about 0.01 wt % sodium
hypochlorite, from about 1 ppm to about 5 ppm hydrochlorous acid,
and from about 0.5 mM to about 1 mM sodium hydroxide. In another
aspect of the instant invention, any antibiotic which is suitable
for incorporation into compositions to be administered to animals,
and especially food animals can be used.
[0187] In one embodiment, after the sterilization and final
concentration, rather than adding water back to 100% of the volume
prior to sterilization in the filtration unit, the volume is
brought back up to 50% of the volume prior to sterilization and
filtration. In this particular embodiment, the tangential flow
filter and associated tubing are flushed with a sterile PBS buffer
solution to collect any residual sporulated oocysts and the rinse
is added to collected retentate. In a preferred embodiment,
1.times. PBS containing 60 .mu.g/ml gentamicin is added in a 1:1
ratio to the disinfected sporulated oocyst suspension to result in
a suspension of sporulated oocysts in 0.5.times. PBS with 30
.mu.g/ml gentamicin. A sample is then taken for assay purposes.
Thereafter, the material is subdivided into pre-sterilized
containers, sealed, labeled and stored in the cold room pending
filling of containers for commercial distribution, such as vials.
This material constitutes a bulk lot.
[0188] In yet another embodiment, the diluent used for storage
purposes includes a composition that ameliorates a decrease in
post-challenge performance and thickening agents to maintain the
sporulated oocysts in suspension. Suitable thickening agents
include starches, gums, polysaccharides, and mixtures thereof.
Suitable compositions to ameliorate a decrease in post-challenge
performance include, but are not limited to, cytokines, growth
factors, chemokines, mitogens and adjuvants. Such compositions to
improve post-challenge performance are well known to those skilled
in the art and can be found, for example, in Plotkin and Orenstein,
Vaccines, Third Ed., W.B. Saunders, 1999; Roitt et al., Immunology,
Fifth Ed., Mosby, 1998; and Brostoff, et al., Clinical Immunology,
Gower Medical Publishing, 1991. Examples of compositions to improve
post-challenge performance, include, but are not limited to, Alum
(aluminum phosphate or aluminum hydroxide), Freund's adjuvant,
calcium phosphate, beryllium hydroxide, dimethyl dioctadecyl
ammonium bromide, saponins, polyanions, e.g. poly A:U, Quil A,
inulin, lipopolysaccharide endotoxins, liposomes, lysolecithins,
zymosan, propionibacteria, mycobacteria, and cytokines, such as,
interleukin-1, interleukin-2, interleukin-4, interleukin-6,
interleukin-12, interferon-.alpha., interferon-.gamma.,
granulocyte-colony stimulating factor. In one preferred embodiment,
the diluent includes Propionibacterium acnes (P. acnes) at from
between 10 .mu.g and 100 .mu.g per dose (dry weight) and more
preferred at about 50 .mu.g per dose (dry weight). The preferred
concentration is from about 3.0 to about 5.0 milligrams per
milliliter of vaccine, most preferably about 4.2 milligrams per
milliliter.
[0189] In a further embodiment of the present invention, sterile
sporulated oocysts are stored in a composition comprising an
oxidizing agent. The oxidizing agent preferably has a reduction
potential of greater than 0.5 V, more preferably between 0.75 and
3.0 V, most preferably between about 1.0 and 2.0 V in the
sporulation medium. The oxygen present in sterile water may also be
used as the oxidizing agent. When sterile water, or any other
oxidizing agent is used, no additional oxygen or air is
incorporated in to the storage composition. Examples of other
suitable oxidizing agents include, but are not limited to, aqueous
bromine, chlorine dioxide, hydrogen peroxide, potassium
permanganate, potassium perchlorate, sodium hypochlorite, and
hydrochlorous acid which have reduction potentials of about 1.09 V,
1.64 V, 1.78 V, 1.49 V, 1.37 V, 1.49 V, and 1.63 V, respectively.
The requisite amount of oxidizing agent added varies with the agent
used and the species of protozoa and can be determined empirically
by one skilled in the art. For protozoa of the genus Eimeria,
preferred concentrations of the oxidizing agents once added to the
sporulated oocyst suspension include from about 0.1 to about 0.75
wt % for potassium perchlorate, from about 0.5 to about 2.9 wt %
for potassium permanganate, from about 0.001 to about 0.1 wt %
sodium hypochlorite and from about 1 ppm to about 5 ppm for
hydrochlorous acid.
[0190] In one embodiment, the sporulated oocysts in contact with
the diluent retain greater than 60% viability when stored for 13
weeks at 25.degree. C. In a preferred embodiment of the instant
invention, the sporulated oocysts in contact with the diluent
retain greater than 70% viability for at least 26 weeks when stored
at 4.degree. C. Because the resulting vaccine is sterile and lacks
potassium dichromate, the product is suitable for administration by
a variety of routes including, but not limited to, intravenous,
subcutaneous, intramuscular and intraperitoneal injection. Thus,
the sporulated oocyst/diluent composition can be used for
vaccinating animals against coccidiosis.
[0191] The instant invention comprises a composition containing
viable sporulated oocysts of a single species or combination of
species of protozoa known to coccidiosis. The combined species of
sporulated oocysts are present in a number sufficient to comprise
the minimum number of sporulated oocysts required to comprise an
effective dose for immunizing purposes. The number of sporulated
oocysts per dose is further determined by the estimated half-life
of the sporulated oocysts in the storage composition claimed
herein. As the sporulated oocysts age a certain number cease to be
functional. A preferred shelf-life is approximately 12 months. An
example of half-life determinations may be found in FIGS. 4 and 5
and Example 4. Therefore, a minimum amount of a single species or
combination of sporulated oocysts is added to the compositions for
consumption that will result in the minimum immunizing dose
computed as a function of half-life determinations.
[0192] The storage medium of the current invention contains less
than about 0.8% by weight of alkali metal dichromate. In more
preferred embodiments, the instant invention contains less than
about 0.6% by weight of alkali metal dichromate, or less than about
0.4% by weight of alkali metal dichromate, or less than about 0.2%
by weight of alkali metal dichromate, and less than about 0.1% by
weight of alkali metal dichromate.
[0193] Again, as the production of the concentrated vaccine is
without the use of an alkali metal dichromate, the storage
composition will contain less than about 0.3% by weight of
dichromate ion. In another embodiment, the storage composition of
the instant invention will contain less than about 0.15% by weight
of hexavalent chromium.
[0194] Furthermore, a most preferred embodiment of the instant
invention comprises a coccidiosis vaccine for chickens using the
sporulated oocyst/diluent composition of the present invention
containing at least about 1.5.times.10.sup.4 viable wild type
sporulated oocysts per milliliter and is characterized as
substantially free of potassium dichromate.
[0195] In addition, a dye may be added to the vaccine to encourage
consumption. Dyes well known in the art may be used. A preferred
dye is 0.02% FD+C Emerald Green.
[0196] Vaccine Composition
[0197] The present invention also provides a vaccine composition
for the prevention and control of coccidiosis. The vaccine may be
concentrated, requiring dilution before administration, or the
vaccine may be ready for administration. The concentrated
embodiment of the instant invention may be diluted with any
suitable diluent to concentrations suitable for various forms of
administration, including intra-yolk sac administration, per os,
oral gavage, delivery via spray cabinet, or top-fed via spray on to
food, such as OASIS Hatchling Supplement.
[0198] The vaccine composition of the instant invention comprises
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. In more preferred
embodiments, the instant invention contains about 10,000 oocysts
per milliliter and less than about 0.6% by weight of alkali metal
dichromate, or about 10,000 oocysts per milliliter and less than
about 0.4% by weight of alkali metal dichromate, or about 10,000
oocysts per milliliter and less than about 0.2% by weight of alkali
metal dichromate, or about 10,000 oocysts per milliliter and less
than about 0.1% by weight of alkali metal dichromate. In addition,
the vaccine composition of the current invention will contain at
least about 10,000 oocysts per milliliter and less than about 3.0%
by weight of dichromate ion or less than about 0.15% by weight of
hexavalent chromium.
[0199] In a further embodiment, the concentrated vaccine may be
diluted prior to administration, for example, from 10 milliliters
to about a 250 milliliter. In another embodiment, the concentrated
vaccine may be diluted prior to administration from about 10
milliliters to about 2.5 Liters. Such dilute vaccine is sterile and
comprises wild type oocysts known to cause coccidiosis. In a
preferred embodiment, said diluted vaccine composition comprises at
least about 1,000 oocysts per milliliter and less than about 0.002%
by weight of alkali metal dichromate. In a more preferred
embodiment, said diluted vaccine comprises at least about 1,000
oocysts per milliliter and is characterized as substantially free
of alkali metal dichromate.
[0200] In a most preferred embodiment, the instant invention is a
concentrated vaccine ready for dilution and then administration
wherein said concentrated vaccine contains at least about 10,000
sporulated viable wild type oocysts per milliliter and is
characterized as substantially free of alkali metal dichromate.
[0201] The vaccine composition of the present invention also
comprises viable wild type sporulated oocysts containing less than
about 5.0.times.10.sup.-3 .mu.g of alkali metal dichromate per
oocyst. In a preferred embodiment the vaccine composition contains
less than about 3.8.times.10.sup.-3 .mu.g of alkali metal
dichromate per oocyst. In a more preferred embodiment the vaccine
composition contains less than about 1.3.times.10.sup.-3 .mu.g of
alkali metal dichromate per oocyst. In a highly preferred
embodiment the vaccine contains less than about 6.3.times.10.sup.-5
.mu.g of alkali metal dichromate per oocyst. In a most preferred
embodiment the vaccine composition is characterized as
substantially free of alkali metal dichromate.
EXAMPLES
[0202] 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.
Example 1
Oocyst Collection and Isolation
[0203] Five hundred fifty, 15 day old broiler chickens were
infected with approximately 7000 viable oocysts per bird of E.
tenella by oral gavage or by ingestion via drinking water or feed.
Excreta were collected over a three day period beginning 6 days
later at 21 days of age. Total excreta collected over the three day
period was 189 kg. Excreta were processed on the day collected. The
excreta collected were put in a dilution tank maintained at
approximately 40-50.degree. F. and diluted with water at 0.687 to
0.630 L/bird. The diluted excreta was pumped through a 30" diameter
vibrating sieve fitted with a 50 mesh (297 micron) top screen and a
250 mesh (61 micron) bottom screen at a rate of approximately 6
LPM. The top two fractions were discarded and the filtrate,
containing the oocysts, was pumped into a chilled (about
40-50.degree. F.) collection tank and continuously agitated. The
filtrate was then pumped at a feed rate of approximately 2.9-3.5
LPM into a Sharples Super-D-Canter centrifuge. The centrifuge
settings were: bowl speed 3990-4004 RPM; auger speed 2306-3990 RPM
and RPM delta 16.84-17.33. RPM delta is a measure of the difference
between bowl and auger speeds. Total run time ranged from 97 to 100
minutes. The centrate was discarded and the solids (cake), which
contained oocysts, were collected into a stainless steel tray,
weighed and stored in a tank at about 40-50.degree. F. The solids
obtained from each of three collection days were combined.
[0204] The volume of solids from the combined three runs was 28 L.
To these solids was added 25.6 L of high fructose corn syrup and
46.4 L of water to give a total volume of 100 L and a specific
gravity of 1.094 g/l. This material was then centrifuged using a
Sharples Super-D-Canter centrifuge at a bowl speed of 5998 RPM, an
auger speed of 3998 RPM and a RPM delta of 20.41. The feed rate was
1.1/min and the total run time was 95 minutes. The centrate,
containing the oocysts, was collected and stored in a tank at
approximately 40-50.degree. F. and the unwanted excreta solids
discarded.
[0205] In order to remove the residual sugar in solution and to
concentrate the oocysts further, the centrate was subjected to an
additional centrifugation. To the 96 L of centrate obtained was
added 114 L of water to give a final volume of 210 L. The
centrifuge settings were bowl speed 6011 RPM and auger speed 4050
RPM. The initial RPM delta was 20.01 but was decreased to 15 and
then 10 during the run to increase centrate flow. The centrate was
discarded and the solid containing the oocysts was retained. The
oocysts were placed in a sterilized container with sterile water at
a preferred concentration of between about 5.times.10.sup.6/ml and
about 50.times.10.sup.6/ml and held at from about 2.degree. C. to
about 8.degree. C. until transferred to the sporulation vessel to
undergo sporulation.
Example 2
Separation by Hydrocyclone
[0206] The manure from 400 host birds inoculated with E. maxima was
collected from a one day period resulting in 45 kg of manure. This
manure was diluted and sieved according to the method of Example 1
to give a filtrate volume of 270 L containing 6% solids and
4.64.times.10.sup.9 oocysts. The filtrate was then introduced to
the hydrocyclone by a high pressure pump at a feed rate of
approximately 2 gallons per minute and at a pressure of 126
psi.
[0207] The first run resulted in the an upper outlet volume
(overflow) of 182 L containing 1.58.times.10.sup.8 oocysts and a
lower outlet volume of 88 L containing 8% solids and
4.02.times.10.sup.9 oocysts. Overflow material was discarded after
each run. A second run resulted in an upper outlet volume of 56 L
containing 1.46.times.10.sup.8 oocysts and a lower outlet volume of
28 L containing a 13% solids and 3.25.times.10.sup.9 oocysts. A
third and final run resulted in an overflow volume of 18 L
containing 1.27.times.10.sup.8 and a final volume for the lower
outlet of 10.5 L containing 27% solids and 3.56.times.10.sup.9
oocysts.
Example 3
Sporulation
[0208] To the oocysts obtained as described in Example 1, was added
enough of a 5.25% sodium hypochlorite solution (CLOROX) to obtain a
final concentration 0.05 wt % sodium hypochlorite. This
oocyst/sodium hypochlorite mixture was added to a 10 liter
fermentor set at 28+1.degree. C. and an agitation rate of 200 RPM.
Oxygen was provided by portable oxygen cylinders and bubbled
through the mixture at a rate sufficient to obtain a percent
saturation of dissolved oxygen value of at least 50% of saturation
of dissolved oxygen. Oxygen flow was adjusted so as not to cause
foaming of the mixture. The oocysts were maintained under these
conditions for about 72 hours. During sporulation, dissolved oxygen
and pH were constantly monitored. It was observed that beginning at
approximately 12 hours into the sporulation process there was a
decrease in the percent saturation of dissolved oxygen (increased
oxygen consumption) followed by an increase in pH and a return of
dissolved oxygen to previous 1.0 levels (FIG. 1). In some, but not
all cases, the increase in pH was preceded by a decrease in pH at
about the same time as the decrease in the percent saturation of
dissolved oxygen (FIG. 2). These changes in dissolved oxygen and pH
were found to be reliable indicators of sporulation. Examination of
oocysts following these change showed a high degree of sporulation.
In contrast when these changes were not observed, the sporulation
rate was dramatically reduced from approximately 90% to
approximately 10%. Although sporulation was complete at
approximately 24 to 36 hours, the incubation was continued for
another 36 to 48 hours to provide a more stable sporulated oocyst
population.
[0209] Oocyst quantities for individual species are approximately
as follows:
2 TABLE 2 Spp. of Eimeria Quantity E. acervulina 22 million/mL E.
maxima 13.6 million/mL E. tenella 13.7 million/mL
[0210] The average oocyst sporulation ratio determined for
individual species was as follows:
3 TABLE 3 Spp. of Eimeria Percent average sporulation ratio E.
acervulina 80% E. maxima 90% E. tenella 90%
[0211] The average oocyst viability determination for individual
species was as follows:
4 TABLE 4 Spp. of Eimeria Average oocyst viability E. acervulina
80% B. maxima 70-80% E. tenella 80%
Example 4
Tangential Flow Filtration/Sterilization
[0212] Following sporulation, the sporulated oocysts were
concentrated by tangential flow filtration. To begin, the integrity
of the filter membrane was visually observed prior to assembling
the CONSEP system. The filter unit was then assembled according to
the appropriate standard operating procedure ("SOP") as provided in
the manufacture's manual. After the system was assembled about 2 to
4 liters of cold domestic water was run through the system to check
for leaks. If leaks were found, the system would be disassembled
and reassembled after checking for the source of the leakage.
Particular attention was paid to inspecting the gaskets to assure
lack of damage to the gaskets and for proper seating.
[0213] The concentrated sporulated oocysts medium was then placed
into the retentate vessel of the filter unit. Domestic cold water
was added to adjust the retentate vessel volume to the desired
operating level and also maintain less than a desired amount of
solids. The water source was then connected to the retentate vessel
through an air-tight fitting. This facilitates operating
diafiltration at a constant volume.
[0214] The permeate flow valve was then closed. The control of the
diaphragm pump was set to give the desired flow and then the pump
was started. The permeate flow valve was then opened after
substantially all of the bubbles were removed from the membranes
and a steady flow was established across the membrane. The permeate
was then directed to a separate collection container. The permeate
sample was collected after about 2 to 5 minutes of operation and
checked for sporulated oocysts. The sporulated oocysts are to be
retained in the system. If sporulated oocysts were found in the
permeate, the filtration would have been stopped and the source of
retentate leakage identified. Retentate leakage often occurs from
gaskets around the membrane or when the integrity of the membrane
is compromised. The source of a leak must be detected and corrected
before proceeding with permeation. Permeate collection, found to be
without sporulated oocysts, was then discarded. If sporulated
oocysts were found, the permeate would have been returned to the
filter unit to recover any sporulated oocysts that may have leaked
through into the permeate.
[0215] The flow rate of the permeate was checked periodically by
measuring the volume of permeate by collecting the permeate in a
graduated cylinder. The retentate tank volume was maintained at a
constant volume. A small sample of the permeate was collected after
every 2 liters of permeate was collected from the permeate line and
the optical density at 600 nm (OD.sub.600) was measured. Once the
OD.sub.600 of the permeate was less than 0.5, the diafiltration was
stopped by closing the permeate value and disconnecting the water
source. The pump's inlet lines were then removed from the retentate
vessel and connected to a clean water source. The membrane were
then flushed with about 500 to about 1000 ml of water to recover
any sporulated oocysts. The retentate vessel was then stored
overnight at about 4.degree. C. in a refrigerator. The retentate
was stored overnight to allow the sporulated oocysts to settle to
the bottom of the retentate vessel. The layer of retentate over the
settled oocysts was then siphoned off.
[0216] The sporulated oocysts were then sterilized. The filter unit
was sterilized by using 5.25% sodium hypochlorite solution to
disinfect the system. After the sodium hypochlorite was added, all
subsequent procedures were conducted in a HEPA filtered laminar
flow hood to maintain asepsis.
[0217] The Optisep filter unit was assembled according to the
manufacture's directions. For E. maxima and E. tenella a 10-micron
Spectra/Mesh filter was used. For E. acervulina a 5-micron or a
10-micron filter was used. With all species the following
procedures were the same.
[0218] The inlet (retentate return) and outlet tubing (permeate)
were placed in a beaker containing approximately 400 ml of about
5.25% sodium hypochlorite. The pump on the filter unit was then
started to flush the system with the 5.25% sodium hypochlorite. The
pump was then stopped and all the valves were closed to let the
system equilibrate with the 5.25% sodium hypochlorite in the
chamber and tubing for about 15 minutes.
[0219] The sporulated oocyst containing vessel was then removed
from refrigeration. The supernatant was pumped out without
disturbing the sporulated oocyst layer. Enough supernatant was left
behind so that the total solids were less than approximately 15% by
volume. The sporulated oocyst suspension was then transferred to a
retentate vessel.
[0220] An equal volume of about 10% sodium hypochlorite was added
to the sporulated oocyst suspension to result in a final
concentration of sodium hypochlorite of approximately 5% and a
solids concentration of less than about 7.5% solids in suspension.
The medium was mixed thoroughly and allowed to stand for about 15
minutes.
[0221] The filtration was then begun. Autoclaved water was used as
the water source for filtration. The retentate pump was activated
and set at 0.6 liters per minute. The permeate line was pinched
closed at this time. Once air bubbles were worked through the
filter membrane and tubing the permeate line was opened and
directed to a collection vessel outside the laminar flow hood. The
retentate flow was then increased to 2 liters per minute. A sample
of the permeate was then taken and sampled for sporulated oocysts.
Finding no sporulated oocysts it was determined that there was no
breach of a membrane or failure of a gasket.
[0222] The volume of the retentate vessel was maintained
substantially constant by the addition of either autoclaved or
sterilized water. The filtration was continued until there was no
chlorine odor emanating from the permeate. This required about 10
volumes of retentate to run through the system. A sample of the
permeate was then analyzed for residual chlorine. The filtration
was run until the permeate sample contained less than about 1 ppm
of chlorine.
[0223] Once the chlorine level was sufficiently reduced, the
retentate volume was then reduced by discontinuing the addition of
autoclaved or sterilized water. The concentrated retentate was then
aseptically transferred to a glass vessel wherein an equal volume
of 1.times. PBS with 60 .mu.g/ml of gentamicin was added to the
disinfected sporulated oocysts suspension. This resulted in a
suspension of sporulated oocysts in 0.5.times. PBS with
approximately 30 .mu.g/ml gentamicin. The solution was then placed
in refrigeration at approximately 4.degree. C. for future use.
Example 5
Storage
[0224] Sterile sporulated oocysts were stored in 0.1% potassium
perchlorate, 0.001% sodium hypochlorite, reverse osmosis/deionized
(RO/DI) water or 0.5.times. PBS containing 30 .mu.g/ml gentamicin
and at either 4.degree. C. or room temperature (25.degree. C.). In
some instances the storage medium also contained Propionibacterium
acnes at a concentration of 10-100 .mu.g/dose (dry weight). As used
herein, a dose is the amount to be administered to an individual
animal at one time. At the times indicated in FIGS. 3 and 4,
samples were aseptically removed from the storage containers and
tested for viability by vital staining.
[0225] The results are shown in FIGS. 4 and 5. The method of
sterilization, either 2% or 5% sodium hypochlorite did not appear
to have a significant effect on viability during storage.
Sporulated oocysts which had not been sterilized, however, showed a
rapid decrease in viability when stored.
[0226] Storage temperature was found to have an effect on ability
of sporulated oocysts to remain viable during storage. Sporulated
oocysts maintained at 4.degree. C. maintained their viability for
at least 26 weeks when stored in any of the medium tested.
Sporulated oocysts stored at room temperature, however, showed a
marked decrease in viability by 20 weeks in storage. When stored at
4.degree. C., all groups of sporulated oocysts maintained at least
70% viability over the 26 week test period. In terms of change in
percent viable oocysts recovered (PVOR), a comparison of PVOR at
the first and last sampling periods shows that in no case did the
decrease in PVOR exceed 10% when stored at 4.degree. C. These
results show that it is possible to maintain sporulated oocysts for
extended periods of time in sterile medium lacking potassium
dichromate without a significant loss in viability.
[0227] 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.
[0228] 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.
[0229] 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.
* * * * *
References