| |
| United States Patent
|
5,891,707 |
| Swabby , et al. |
April 6, 1999 |
System for producing Enterocytozoon bieneusi
live organisms
Abstract
Described is a system for efficiently producing live
Enterocytozoon bieneusi (E. bieneusi) organisms using laboratory
animals, preferably Mongolian gerbils. Some important steps are:
providing at least one breeding pair; administering to each of such
breeding pair a sufficient antibiotic in sufficient dosage to
destroy essentially all parasites which might be passed to
offspring, especially Trichomonas; permitting such breeding pair to
produce and rear offspring as production animals; preventing
breeding by the production animals by separating males from females
in separate cages; immune-suppressing each production animal
sufficiently to permit propagation of live E. bieneusi organisms;
infecting each production animal with love E. bieneusi organisms
administered in an amount sufficient for such organisms to propagate
but insufficient to kill the production animal; and, during a
production period following such infecting, collecting the feces of
each production animal. The collected fecal material is then
prepared for use.
| Inventors: |
Swabby; Kenna Diane
(Phoenix, AZ), Cahill; Allen R. (Phoenix, AZ) |
| Appl. No.:
|
08/876,443 |
| Filed: |
June 16, 1997 |
| Current U.S.
Class: |
435/243 ;
435/260; 435/261; 435/947 |
| Current
International Class: |
C12N
1/10 (20060101); C12N 1/02 (20060101); C12N 1/00 (20060101);
C12N 1/04 (20060101); C12N 001/00 (); C12N 001/02 (); C12N
001/04 (); C12N 001/10 () |
| Field of
Search: |
435/243,260,261,947 |
References Cited
[Referenced By]
U.S. Patent
Documents
Other References
Visvesvara et al. J. Eukaryotic Microbiol. vol. 42(5), pp.
506-510, Abstract enclosed, 1995. .
Tzipori et al. J. Infect. Dis. vol. 175 (4), pp. 1016-1020,
Abstract enclosed, 1997..
|
Primary Examiner: Lankford, Jr.; Leon B.
Assistant Examiner: Tate; Christopher R.
Attorney, Agent or Firm:
Stoneman; Martin L.
Claims
What is claimed is:
1. A system for the efficient production of live Enterocytozoon
bieneusi (E. bieneusi) organisms comprising the steps of:
a. providing at least one breeding pair of mammals;
b. administering to each of said breeding pair a sufficient
antibiotic in sufficient dosage to destroy essentially all parasites
which might be passed to offspring and inhibit in said offspring
said efficient production of E. bieneusi organisms;
c. permitting said breeding pair to produce and rear said offspring
as production animals;
d. preventing breeding by said production animals;
e. immune-suppressing each said production animal sufficiently to
permit propagation of E. bieneusi organisms;
f. infecting each said production animal with E. bieneusi organisms
administered in an amount sufficient for said organisms to propagate
but insufficient to kill said production animal; and
g. during a production period following said infecting, collecting
the feces of each said production animal, said feces containing the
live E. bieneusi organisms.
2. A system according to claim 1 further comprising the step of:
a. after said collecting of said feces, screening said feces to
remove any large particles to provide filtered fecal material.
3. The product which results from practicing a system according to
claim 2.
4. A system according to claim 1 further comprising the step of:
a. during said production period, feeding each said production
animal a diet sufficient to provide a non-sticky said feces to a
level consistent with efficient said collecting of said feces.
5. A system according to claim 1 further comprising the step of:
a. after said administering to each of said breeding pair said
sufficient antibiotic in said sufficient dosage to destroy
essentially all said parasites which might be passed to said
offspring and inhibit in said offspring said efficient production of
live E. bieneusi organisms, establishing sufficient gut flora in
each of said breeding pair for adequate digestive function without
interfering with said destruction of said parasites.
6. A system according to claim 1 further comprising the step of:
a. during said production period, feeding each said production
animal a supplement sufficient to prevent dehydration and minimize
intestinal chemical imbalances.
7. A system according to claim 1 wherein said mammals comprise
gerbils.
8. A system according to claim 7 wherein said collecting step
comprises the step of:
a. during said production period, maintaining each said production
animal supported upon a support surface structured and arranged to
permit said feces and other waste material to drop through said
support surface.
9. A system according to claim 8 wherein said collecting step
further comprises the step of:
a. catching said dropping feces and said dropping waste material in
a container holding sufficient water for keeping said feces moist
until said collection.
10. A system according to claim 9 wherein said collecting step
further comprises the step of:
a. collection of all contents of said container, thereby providing a
fecal material comprising said feces, said waste material, and said
water.
11. A system according to claim 10 further comprising the step of:
a. screening said fecal material to remove any large particles,
thereby providing filtered fecal material containing live E.
bieneusi organisms.
12. The product which results from practicing a system according to
claim 11.
13. A system according to claim 11 further comprising the step of:
a. separating live E. bieneusi organisms from said filtered fecal
material.
14. A system according to claim 13 wherein said separating step
comprises the steps of:
a. adding to said filtered fecal material a heavy liquid having a
specific gravity of at least about 1.15 to provide centrifugable
material;
b. centrifuging said centrifugable material to provide a first layer
essentially containing live E. bieneusi organisms between a second
layer of essentially said heavy liquid and a third layer of
essentially said water; and
c. separation of said live E. bieneusi organisms from said
centrifugable material.
15. A system according to claim 14 wherein said separation step
comprises the steps of:
a. removing essentially all the first-layer material in said first
layer from said centrifugable material; and
b. filtering said first-layer material.
16. A system according to claim 15 wherein said filtering step
comprises the steps of:
a. a first filtering through a small enough screen to catch any
particles larger than a said live E. bieneusi organism;
b. a second filtering through about an E.-bieneusi-size screen to
remove most of said live E. bieneusi organisms;
c. placing said removed live E. bieneusi organisms into a clean
container with a small amount of purified water;
d. a third filtering through a substantially-less-than-E. bieneusi
size screen to remove any remaining said E. bieneusi live organisms;
and
e. placing said remaining live E. bieneusi organisms into said clean
container to provide purified live E. bieneusi organisms.
17. A system according to claim 16 further comprising the step of:
a. adding sufficient bacteria suppressant to said purified live E.
bieneusi organisms to suppress any bacteria.
18. A system according to claim 17 further comprising the step of:
a. sealing and refrigerating said clean container, for storage until
use.
19. A system for the efficient production of E. bieneusi organisms
comprising the steps of:
a. providing at least one breeding pair of Mongolian gerbils;
b. administering to each of said breeding pair a sufficient
antibiotic in sufficient dosage to destroy essentially all of
Trichomonas protozoa in each of said breeding pair;
c. permitting said breeding pair to produce and rear offspring as
production animals;
d. preventing breeding by said production animals;
e. immune-suppressing each said production animal sufficiently to
permit propagation of live E. bieneusi organisms;
f. infecting each said production animal with live E. bieneusi
organisms administered in an amount sufficient for said organisms to
propagate but insufficient to kill said production animal; and
g. during a production period following said infecting, collecting
the feces of each said production animal, said feces containing the
live E. bieneusi organism.
20. A system according to claim 19 wherein:
a. said immune-suppressing step comprises injection under the skin
with methylprednisolone acetate in a dosage of about 4 mg. for about
a 100 gram gerbil, with the same dosage repeated about three days
later; and
b. said sufficient antibiotic in sufficient dosage comprises
metronidazole hydrochloride administered once daily for five days
orally at a daily dosage of about 30 mg. per pound of animal weight,
repeated after about six months.
21. A system according to claim 19 further comprising the step of:
a. during said production period, feeding each said production
animal a diet of high-protein dog food, with an addition of a few
black sunflower seeds.
22. A system according to claim 19 further comprising the step of:
a. after said administering to each of said breeding pair said
sufficient antibiotic in said sufficient dosage to destroy
essentially all said Trichomonas protozoa, administering gut flora
and a weak antibiotic to each of said breeding pair sufficiently to
establish sufficient gut flora for adequate digestive function
without interfering with said destruction of said Trichomonas
protozoa.
23. A system according to claim 22 wherein said step of
administering gut flora and a weak antibiotic comprises placing in
the drinking water of each of said breeding pair about 5 mg. of
tetracycline hydrochloride per each cc. of drinking water for about
5 days.
24. A system according to claim 19 further comprising the step of:
a. during said production period, feeding each said production
animal a supplement sufficient to prevent dehydration and minimize
intestinal chemical imbalances;
b. wherein said supplement comprises a pet drink comprising
fructose.
25. A system according to claim 19 wherein said collecting step
comprises the step of:
a. during said production period, maintaining each said production
animal supported upon a support surface structured and arranged to
permit said feces and other waste material to drop through said
support surface.
26. A system according to claim 25 wherein said support surface
comprises:
a. open mesh material sufficient for said feces to fall through, yet
providing ample support and footing for said gerbils;
b. wherein said open mesh material comprises a 1/4-inch wire cloth.
27. A system according to claim 25 wherein said collecting step
further comprises the step of:
a. catching said dropping feces and said dropping waste material in
a container holding sufficient water for keeping said feces moist
until said collection;
b. wherein said container comprises a shallow pan suitable for
holding a pond about 1/4-inch deep of distilled water.
28. A system according to claim 27 wherein said collecting step
further comprises the step of:
a. collection of all contents of said container, thereby providing a
fecal material comprising said feces, said waste material, and said
water.
29. A system according to claim 28 further comprising the step of:
a. screening said fecal material to remove any large particles,
thereby providing filtered fecal material containing live E.
bieneusi organisms.
30. A system according to claim 29 wherein said screening comprises,
using a small amount of additional distilled water, pouring and
mushing through a strainer screen, of about 250 mesh size, placed
atop a beaker.
31. A system according to claim 29 further comprising the step of:
a. separating live E. bieneusi organisms from said filtered fecal
material;
b. wherein said separating step comprises the steps of
i. adding to said filtered fecal material a heavy liquid having a
specific gravity of at least about 1.15 to provide centrifugable
material;
ii. centrifuging said centrifugable material to provide a first
layer essentially containing live E. bieneusi organisms between a
second layer of essentially said heavy liquid and a third layer of
essentially said water; and
iii. separation of said live E. bieneusi organisms from said
centrifugable material.
32. A system according to claim 31 wherein:
a. said adding is done in a glass tube, after filling said glass
tube about half full of said filtered fecal material;
b. said heavy liquid comprises zinc sulfate;
c. said heavy liquid is injected into a bottom portion of said glass
tube with a long needle syringe; and
d. said centrifuging comprises
i. placing said glass tube in said centrifuge; and
ii. spinning down said glass tube for about 15 minutes at about
2,000 RPM;
iii. whereby waste sediment from said filtered fecal material is at
said bottom portion, said zinc sulfate is directly above said waste
sediment and below said live E. bieneusi organisms, and water is
above said live E. bieneusi organisms.
33. A system according to claim 31 wherein said separation step
comprises the steps of:
a. removing essentially all the first-layer material in said first
layer from said centrifugable material; and
b. filtering said first-layer material.
34. A system according to claim 33 wherein said filtering step
comprises the steps of:
a. a first filtering through an about-3-micron membrane pre-filter
above a container with a vacuum source;
b. a second filtering through about an E.-bieneusi-size screen of
about 1 micron to remove most of said live E. bieneusi organisms;
c. placing said removed live E. bieneusi organisms into a clean
container with a small amount of purified water;
d. a third filtering through a less-than-E.-bieneusi size screen of
about 0.4 micron to remove any remaining said live E. bieneusi
organisms from said purified water; and
e. placing said remaining live E. bieneusi organisms into said clean
container to provide purified live E. bieneusi organisms.
35. A system according to claim 34 further comprising the step of:
a. adding sufficient bacteria suppressant to said purified live E.
bieneusi organisms to suppress any bacteria;
b. wherein about 50 mg. of gentamycin sulfate is added to each 1 cc.
of said live E. bieneusi organisms.
36. A system according to claim 35 further comprising the step of:
a. sealing and refrigerating said clean container, for storage until
use.
37. A system for the efficient production of live E. bieneusi
organisms comprising the steps of:
a. providing at least one breeding pair of Mongolian gerbils;
b. administering to each of said breeding pair a sufficient
antibiotic in sufficient dosage to destroy essentially all of
Trichomonas protozoa in each of said breeding pair,
i. wherein said sufficient antibiotic in sufficient dosage comprises
metrodinazole hydrochloride administered once daily for five days
orally at a daily dosage of about 30 mg. per pound of animal weight,
repeated after about six months;
c. permitting said breeding pair to produce and rear offspring as
production animals;
d. preventing breeding by said production animals by separately
caging each sex before breeding capability occurs;
e. immune-suppressing each said production animal sufficiently to
permit propagation of live E. bieneusi organisms
i. wherein said immune-suppressing step comprises injection under
the skin with methylprednisolone acetate in a dosage of about 4 mg.
for about a 100 gram gerbil, with the same dosage repeated about
three days later;
f. infecting each said production animal with live E. bieneusi
organisms administered in an amount sufficient for said organisms to
propagate but insufficient to kill said production animal
i. wherein said amount comprises about 10,000 live organisms per 50
grams of animal weight administered about the same time as said
second injection of said immune-suppressing step;
g. during a production period following said infecting, collecting
the feces of each said production animal, said feces containing the
live E. bieneusi organisms.
38. A system according to claim 37 further comprising the steps of:
a. after said administering to each of said breeding pair said
sufficient antibiotic in said sufficient dosage to destroy
essentially all said Trichomonas protozoa, administering gut flora
and a weak antibiotic to each of said breeding pair sufficiently to
establish sufficient gut flora for adequate digestive function
without interfering with said destruction of said Trichomonas
protozoa
i. wherein said step of administering gut flora and a weak
antibiotic comprises placing in the drinking water of each of said
breeding pair about 5 mg. of tetracycline hydrochloride per cc. of
drinking water for about 5 days;
b. during said production period, feeding each said production
animal a diet of high-protein dog food, with an addition of a few
black sunflower seeds;
c. during said production period, feeding each said production
animal a supplement sufficient to prevent dehydration and minimize
intestinal chemical imbalances;
d. wherein said collecting step comprises the step of
i. during said production period, maintaining each said production
animal supported upon a support surface structured and arranged to
permit said feces and other waste material to drop through said
support surface,
ii. wherein said support surface comprises open mesh material
sufficient for said feces to fall through, yet providing ample
support and footing for said gerbils,
iii. wherein said open mesh material comprises a 1/4-inch wire
cloth;
e. wherein said collecting step further comprises the step of
i. catching said dropping feces and said dropping waste material in
a container holding sufficient water for keeping said feces moist
until said collection,
ii. wherein said container comprises a shallow pan suitable for
holding a pond about 1/4-inch deep of distilled water;
f. wherein said collecting step further comprises the step of
collection of all contents of said container, thereby providing a
fecal material comprising said feces, said waste material, and said
water;
g. screening said fecal material to remove any large particles,
thereby providing filtered fecal material containing live E.
bieneusi organisms,
i. wherein said screening comprises, using a small amount of
additional distilled water, pouring and mushing through a strainer
screen, of about 250 mesh size, placed atop a beaker.
39. A system according to claim 38 further comprising the steps of:
a. separating live E. bieneusi organisms from said filtered fecal
material;
b. wherein said separating step comprises the steps of
i. adding to said filtered and fecal material a heavy liquid having
a specific gravity of at least about 1.15 to provide centrifugable
material,
ii. centrifuging said centrifugable material to provide a first
layer essentially containing live E. bieneusi organisms between a
second layer of essentially said heavy liquid and a third layer of
essentially said water, and
iii. separation of said live E. bieneusi organisms from said
centrifugable material
iv. wherein
(1) said adding is done in a glass tube, after filling said glass
tube about half full of said filtered fecal material,
(2) said heavy liquid comprises zinc sulfate,
(3) said heavy liquid is injected into a bottom portion of said
glass tube with a long needle syringe,
(4) said centrifuging comprises
(a) placing said glass tube in said centrifuge, and
(b) spinning down said glass tube for about 15 minutes at about
2,000 RPM,
(c) whereby waste sediment from said filtered fecal material is at
said bottom portion, said zinc sulfate is directly above said waste
sediment and below said live E. bieneusi organisms, and water is
above said live E. bieneusi organisms,
(5) said separation step comprises the steps of
(a) removing essentially all the first-layer material in said first
layer from said centrifugable material; and
(b) filtering said first-layer material
(c) wherein said filtering step comprises the steps of
(i) a first filtering through an about-3-micron membrane pre-filter
above a container with a vacuum source;
(ii) a second filtering through about an E.-bieneusi-size screen of
about 1 micron to remove most of said live E. bieneusi organisms;
(iii) placing said removed live E. bieneusi organisms into a clean
container with a small amount of purified water;
(iv) a third filtering through a less-than-E.-bieneusi size screen
of about 0.4 micron to remove any remaining said live E. bieneusi
organisms from said purified water, and
(v) placing said remaining live E. bieneusi organisms into said
clean container to provide purified live E. bieneusi organisms;
c. adding sufficient bacteria suppressant to said purified live E.
bieneusi organisms to suppress any bacteria
i. wherein about 50 mg. of gentamycin sulfate is added to each 1 cc.
of said live E. bieneusi organisms;
d. sealing and refrigerating said clean container, for storage until
use.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a system for producing Enterocytozoon
bieneusi (E. bieneusi) live organisms. More particularly, this
invention concerns efficient production methods for Enterocytozoon
bieneusi live organisms and the products resulting therefrom.
2. Description of the Prior Art
Typically, in the testing of possible AIDS victims, testing kits
attempt to help in assessing whether the patient is infected with E.
bieneusi live organisms, an infection of the type rarely found in
persons with normal immune systems. Companies making such testing
kits ideally would like to provide a large supply of IFA (Indirect
Fluorescent Antibody), and/or DFA (Direct Fluorescent Antibody),
and/or ELISA (Enzyme-Linked Immuno-Sorbent Assay), and/or C-ELISA
(modified ELISA), as well as live E. bieneusi live organisms for use
as controls, for use in such testing kits. But it has been found to
be difficult to grow such live organisms except in AIDS patients,
making the supply of such live organisms, both for direct use and
for making such sera, very expensive and difficult to obtain in
large quantities.
OBJECTS OF THE INVENTION
A primary object of the present invention is to fulfill the
above-mentioned need by the provision of an efficient system for
producing such E. bieneusi live organisms using other animals (and
not humans). In addition, it is a primary object of this invention
to provide such a system for such and other hard-to-grow organisms,
in connection with its resulting novel products, in a useful form
for test kit manufacturers. Other objects of this invention will
become apparent with reference to the following invention
descriptions.
SUMMARY OF THE INVENTION
According to a preferred embodiment of the present invention, this
invention provides a system for the efficient production of E.
bieneusi live organisms comprising the steps of: providing at least
one breeding pair of mammals; administering to each of such breeding
pair a sufficient antibiotic in sufficient dosage to destroy
essentially all parasites which might be passed to offspring and
inhibit in such offspring such efficient production of E. bieneusi
live organisms; permitting such breeding pair to produce and rear
such offspring as production animals; preventing breeding by such
production animals; immune-suppressing each such production animal
sufficiently to permit propagation of E. bieneusi live organisms;
infecting each such production animal with E. bieneusi live
organisms administered in an amount sufficient for such organisms to
propagate but insufficient to kill such production animal; and
during a production period following such infecting, collecting the
feces of each such production animal. It also provides such a system
further comprising the step of, after such collecting of such feces,
screening such feces to remove any large particles to provide
filtered fecal material. And it provides the product which results
from practicing such a system.
Furthermore, this invention provides such a system further
comprising the step of, during such production period, feeding each
such production animal a diet sufficient to provide a non-sticky
such feces to a level consistent with efficient such collecting of
such feces; and, further comprising the step of, after such
administering to each of such breeding pair such sufficient
antibiotic in such sufficient dosage to destroy essentially all such
parasites which might be passed to such offspring and inhibit in
such offspring such efficient production of E. bieneusi live
organisms, establishing sufficient gut flora in each of such
breeding pair for adequate digestive function without interfering
with such destruction of such parasites. And it provides such a
system further comprising the step of, during such production
period, feeding each such production animal a supplement sufficient
to prevent dehydration and minimize intestinal chemical imbalances;
and, further, wherein such mammals comprise gerbils. It also
provides such a system wherein such collecting step comprises the
step of, during such production period, maintaining each such
production animal supported upon a support surface structured and
arranged to permit such feces and other waste material to drop
through such support surface; and, further, the step of catching
such dropping feces and such dropping waste material in a container
holding sufficient water for keeping such feces moist until such
collection; and, further, the step of collection of all contents of
such container, thereby providing a fecal material comprising such
feces, such waste material, and such water. Also, it provides such a
system further comprising the step of screening such fecal material
to remove any large particles, thereby providing filtered fecal
material containing E. bieneusi live organisms. And it provides the
product which results from practicing such a system.
Moreover, the present invention provides such a system further
comprising the step of separating E. bieneusi live organisms from
such filtered fecal material; and, further, wherein such separating
step comprises the steps of adding to such filtered fecal material a
heavy liquid having a specific gravity of at least about 1.15 to
provide centrifugable material, centrifuging such centrifugable
material to provide a first layer of essentially E. bieneusi live
organisms between a second layer of essentially such heavy liquid
and a third layer of essentially such water, and separation of such
E. bieneusi live organisms from such centrifugable material; and,
further, wherein such separation step comprises the steps of
removing essentially all the first-layer material in such first
layer from such centrifugable material, and filtering such
first-layer material. It further provides such a system wherein such
filtering step comprises the steps of a first filtering through a
small enough screen to catch any particles larger than a such E.
bieneusi live organism, a second filtering through about an E.-bieneusi-size
screen to remove most of such E. bieneusi live organisms, placing
such removed E. bieneusi live organisms into a clean container with
a small amount of purified water, a third filtering through a
substantially-less-than-E.-bieneusi size screen to remove any
remaining such E. bieneusi live organisms, and placing such
remaining E. bieneusi live organisms into such clean container to
provide purified E. bieneusi live organisms. And it provides such a
system further comprising the step of adding sufficient bacteria
suppressant to such purified E. bieneusi live organisms to suppress
any bacteria; and, further comprising the step of sealing and
refrigerating such clean container, for storage until use.
Additionally, in accordance with a preferred embodiment thereof, the
present invention provides a system for the efficient production of
E. bieneusi live organisms comprising the steps of: providing at
least one breeding pair of Mongolian gerbils; administering to each
of such breeding pair a sufficient antibiotic in sufficient dosage
to destroy essentially all of Trichomonas protozoa in each of such
breeding pair; permitting such breeding pair to produce and rear
offspring as production animals; preventing breeding by such
production animals; immune-suppressing each such production animal
sufficiently to permit propagation of E. bieneusi live organisms;
infecting each such production animal with E. bieneusi live
organisms administered in an amount sufficient for such organisms to
propagate but insufficient to kill such production animal; and,
during a production period following such infecting, collecting the
feces of each such production animal. Also, it provides such a
system wherein such immune-suppressing step comprises injection
under the skin with methylprednisolone acetate in a dosage of about
4 mg. for about a 100 gram gerbil, with the same dosage repeated
about three days later, and such sufficient antibiotic in sufficient
dosage comprises metrodinazole hydrochloride administered once daily
for five days orally at a daily dosage of about 30 mg. per pound of
animal weight, repeated after about six months.
Even further, this invention provides such a system further
comprising the step of, during such production period, feeding each
such production animal a diet of high-protein dog food, with an
addition of a few black sunflower seeds. It also provides such a
system further comprising the step of, after such administering to
each of such breeding pair such sufficient antibiotic in such
sufficient dosage to destroy essentially all such Trichomonas
protozoa, administering gut flora and a weak antibiotic to each of
such breeding pair sufficiently to establish sufficient gut flora
for adequate digestive function without interfering with such
destruction of such Trichomonas protozoa; and, further, wherein such
step of administering gut flora and a weak antibiotic comprises
placing in the drinking water of each of such breeding pair about 5
mg. of tetracycline hydrochloride per cc. of drinking water for
about 5 days. Further, it provides such a system further comprising
the step of, during such production period, feeding each such
production animal a supplement sufficient to prevent dehydration and
minimize intestinal chemical imbalances, wherein such supplement
comprises a pet drink comprising fructose. And it provides such a
system wherein such collecting step comprises the step of, during
such production period, maintaining each such production animal
supported upon a support surface structured and arranged to permit
such feces and other waste material to drop through such support
surface; and, further, wherein such support surface comprises open
mesh material sufficient for such feces to fall through, yet
providing ample support and footing for such gerbils, wherein such
open mesh material comprises a 1/4-inch wire cloth.
Even additionally, this invention provides such a system wherein
such collecting step further comprises the step of catching such
dropping feces and such dropping waste material in a container
holding sufficient water for keeping such feces moist until such
collection, wherein such container comprises a shallow pan suitable
for holding a pond about 1/4-inch deep of distilled water; and,
further, wherein such collecting step further comprises the step of
collection of all contents of such container, thereby providing a
fecal material comprising such feces, such waste material, and such
water; and, further comprising the step of screening such fecal
material to remove any large particles, thereby providing filtered
fecal material containing E. bieneusi live organisms; and, further,
wherein such screening comprises, using a small amount of additional
distilled water, pouring and mushing through a strainer screen, of
about 250 mesh size, placed atop a beaker.
Yet further, this invention provides such a system further
comprising the step of separating E. bieneusi live organisms from
such filtered fecal material, wherein such separating step comprises
the steps of: adding to such filtered and fecal material a heavy
liquid having a specific gravity of at least about 1.15 to provide
centrifugable material; centrifuging such centrifugable material to
provide a first layer of essentially E. bieneusi live organisms
between a second layer of essentially such heavy liquid and a third
layer of essentially such water; and separation of such E. bieneusi
live organisms from such centrifugable material; and, further,
wherein such adding is done in a glass tube, after filling such
glass tube about half full of such filtered fecal material such
heavy liquid comprises zinc sulfate, such heavy liquid is injected
into a bottom portion of such glass tube with a long needle syringe,
and such centrifuging comprises placing such glass tube in such
centrifuge, and spinning down such glass tube for about 15 minutes
at about 2,000 RPM, whereby waste sediment from such filtered fecal
material is at such bottom portion, such zinc sulfate is directly
above such waste sediment and below such E. bieneusi live organisms,
and water is above such E. bieneusi live organisms.
Yet additionally, it provides such a system wherein such separation
step comprises the steps of removing essentially all the first-layer
material in such first layer from such centrifugable material, and
filtering such first-layer material; and, further wherein such
filtering step comprises the steps of a first filtering through an
about-3-micron membrane pre-filter above a container with a vacuum
source, a second filtering through about an E.-bieneusi-size screen
of about 1 micron to remove most of such E. bieneusi live organisms,
placing such removed E. bieneusi live organisms into a clean
container with a small amount of purified water, a third filtering
through a less-than-E.-bieneusi size screen of about 0.4 micron to
remove any remaining such E. bieneusi live organisms from such
purified water, and placing such remaining E. bieneusi live
organisms into such clean container to provide purified E. bieneusi
live organisms. And it provides such a system further comprising the
step of adding sufficient bacteria suppressant to such purified E.
bieneusi live organisms to suppress any bacteria, wherein about 50
mg. of gentamycin sulfate is added to each 1 cc. of said E. bieneusi
live organisms; and, further comprising the step of sealing and
refrigerating such clean container, for storage until use.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of a first generation breeding pair of
Mongolian gerbils used in the preferred embodiment of the system of
the present invention for producing E. bieneusi live organisms.
FIG. 2 is a perspective view of a first generation adult gerbil,
being treated to remove Trichomonas protozoa.
FIG. 3 is a perspective view of a first generation adult gerbil,
being treated with a weak antibiotic to help establish a proper
amount and balance of gut flora.
FIG. 4 is a perspective view of a first generation adult gerbil,
raising second generation gerbils.
FIG. 5 is a perspective view of second generation offspring gerbils,
separated, in preparation for producing E. bieneusi live organisms.
FIG. 6 is a perspective view of a second generation gerbil receiving
an immune suppressant in preparation for producing E. bieneusi live
organisms.
FIG. 7 is a perspective view of a second generation gerbil being
infected with E. bieneusi live organisms.
FIG. 8 is a perspective view of a second generation gerbil feeding
upon a specialized diet.
FIG. 9 is a perspective cut-away view, detailing a specialized
production cage, for collecting droppings from infected second
generation gerbils.
FIG. 10 is a perspective view of a gerbil being euthanized.
FIG. 11 is a perspective view of the retrieval from the production
cage of the collecting water, droppings, and E. bieneusi live
organisms.
FIG. 12 is a perspective view of the initial separation of the E.
bieneusi live organisms from the large waste material.
FIG. 13 is a cross-section elevation view of a tube containing the
screened collecting water, E. bieneusi live organisms, and remaining
small waste material.
FIG. 14 is a cross-section elevation view of a tube containing the
screened collecting water, E. bieneusi live organisms, remaining
small waste material, and zinc sulfate.
FIG. 15 is a perspective view illustrating a centrifuge separating
the E. bieneusi live organisms from the remaining waste material.
FIG. 16 is a cross-section elevation view of a tube containing E.
bieneusi live organisms separated from the remaining waste material.
FIG. 17 is a cross-section elevation view of the E. bieneusi live
organisms being removed from the tube containing the remaining waste
material.
FIG. 18 is a perspective view of the final purification filtering of
the E. bieneusi live organisms.
FIG. 19 is a perspective view of the final treatment of the E.
bieneusi organisms.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT AND THE BEST MODE OF
PRACTICE
The method for the production of E. bieneusi live organisms begins
with the preparatory steps of preparing from birth suitable host
animals for said production. In FIG. 1 is illustrated a breeding
pair of Mongolian gerbils (i.e., a female 31 and a male 32), a
preferred type of gerbil, which are to be prepared for the purpose
of producing suitable offspring for the production of E. bieneusi
live organisms. The breeding pair 31 and 32 of Mongolian gerbils of
FIG. 1, which are referred to as first generation, are not suitable
for E. bieneusi production, and will be used for the sole purpose of
producing and rearing second generation production gerbils.
Mongolian gerbils are the preferred mammal for use in the system of
the present invention; but other mammals may be used as
less-preferred alternates. Such alternates may include, but are not
limited to, goats, sheep, non-human primates, calves, cats, mice,
etc.
Preparing the first generation of gerbils for producing suitable
second generation offspring begins with treatment of each of the
breeding pair 31 and 32 of the first generation to remove
Trichomonas protozoa as shown in FIG. 2. Mongolian gerbils, as
received from the supplier, are carriers of such protozoa.
Trichomonas (a type of protozoa) acts as an inhibitor which
interferes with the growing of the E. bieneusi live organisms. After
the first generation of gerbils have had the Trichomonas
successfully destroyed, their offspring, the second generation, will
be free of this protozoa, and will not require like treatment. Thus,
according to this invention, the step is provided of administering
to each of the breeding pair a sufficient antibiotic in sufficient
dosage to destroy essentially all of Trichomonas protozoa in each of
the breeding pair, wherein a preferred such sufficient antibiotic in
such sufficient dosage is metrodinazole hydrochloride (a product
example is "Flagyl", a trademark of Searle Pharmaceutical, which now
manufactures and sells such product) administered once daily for
five days orally at a daily dosage of about 30 mg. per pound of
animal weight, repeated after about six months. FIG. 2 illustrates
one of the breeding pair 31 and 32 (the female 31 is illustrated,
but the male, as described, gets the same treatment) about to be
administered such antibiotic orally by means of measuring syringe
33, including feeding or dispensing tube 34. Such metrodinazole
hydrochloride is not 100% effective, and unfortunately, Trichomonas
will often regrow, and so it is preferred to repeat such dosage
again six months later.
An unfortunate side effect of the metrodinazole hydrochloride
treatment is the destruction of gut flora in the breeding pair, the
digestive bacteria for proper digestive functioning. The types of
gut flora will also be out of balance in that more of the anaerobic
flora will be destroyed than the aerobic flora. Therefore gut flora
must be re-established, and in proper balance, to allow proper
digestive functioning. Thus, in the method of this invention, the
step is provided of administering gut flora and a weak antibiotic to
each of the breeding pair sufficiently to establish sufficient gut
flora for adequate digestive function without interfering with the
destruction of such Trichomonas protozoa. It is preferred that this
step embody the giving to each of the breeding pair 31 and 32, in
their drinking water, tetracycline hydrochloride (a product example
is "Panmycin", a trademark of Upjohn Pharmaceutical, which now
manufactures and sells such product) administered in a dosage of
about 5 mg. per cc. of drinking water for 5 days. It is preferred
that this step also embody the giving to each of the breeding pair
31 and 32, in such drinking water, a small amount of fecal material
from healthy such gerbils, thus promoting the growth of proper gut
flora, especially providing some replacement anaerobic flora for
faster flora balancing. As illustrated in FIG. 3, such tetracycline
hydrochloride (and such fecal material) is administered to such an
example gerbil 31 by way of its drinking water dispenser 35. Such
tetracycline hydrochloride administration begins immediately after
such metrodinazole hydrochloride treatment, or concurrently if
desired. As with the "Flagyl" (trademark) treatment, "Panmycin" (a
trademark) need not be administered to the second generation.
After the first generation breeding pair 31 and 32 have been
prepared as previously described, they are allowed to produce and
rear their offspring 41 as suggested by the illustration of FIG. 4.
These offspring 41 are referred to herein as second generation or as
production animals 41, suitable for E. bieneusi live organism
production. Thus, the present invention provides the step of
permitting such breeding pair to produce and rear second generation
offspring as production animals. Many times the first generation
female gerbil 31 is received from the supplier already pregnant. If
the delivery of her offspring takes place at least two days after
the mother has been on her "Flagyl" (a trademark) treatment, the
offspring may be used as second generation E. bieneusi producers,
i.e., as production animals 41. Earlier births may contain
Trichomonas and would render the young not suitable as second
generation E. bieneusi producers. In this event, they could still be
considered and used as first generation gerbils requiring the above
described treatments.
Once the second generation gerbils, (the immediate offspring of the
treated first generation gerbils), the production animals 41, reach
an age of from approximately 30 to 60 days, they may be used to
produce the desired E. bieneusi live organisms. Being free of the
Trichomonas protozoa, they are suitable for introduction of the
infecting E. bieneusi live organisms. In preparation for infection
and growth of E. bieneusi, they are separated by sex (females 41a
from males 41b) to prevent breeding, and placed into appropriate
cages as 43 illustrated by FIG. 5. Thus, this invention provides a
step of preventing breeding by the production animals by separately
caging each sex before breeding capability occurs. Specific
preferred cage construction features are later illustrated and
described with reference to FIG. 9.
Prior to being infected with E. bieneusi live organisms, the
production animals 41 are immune-suppressed. A weakened immune
system will allow the E. bieneusi live organisms to easily
propagate. Thus, this invention provides a step of
immune-suppressing each such production animal, preferably by
injection under the skin with methylprednisolone acetate (a product
example is "Depo-Medrol", a trademark of Upjohn Pharmaceutical,
which now manufactures and sells such product). Preferred dosage is
4 mg. (for a 90-100 gram gerbil, typical). This dosage is preferably
repeated again three days later. FIG. 6 illustrates a production
animal 41 being immune-suppressed by injection under the skin using
hypodermic needle 44.
Once the immune system of the production animals 41 is weakened,
they are infected with E. bieneusi live organisms by inoculating
them orally with a feeding tube 34 as shown in FIG. 7. Infection is
conducted as early as the same day as the second administration of
the immune suppressant as described with reference to FIG. 6. Thus,
this invention provides the step of immune-suppressing each
production animal sufficiently to permit propagation of E. bieneusi
live organisms, preferably by an inoculation dosage of about 10,000
live organisms per 50 grams of animal weight. The initial E.
bieneusi live organisms may be obtained from an AIDS patient (a
donor), and subsequently purified; and thereafter from production
animals as herein described. The dosage size required to promote the
desired infection rate is based on the goal of obtaining a good
practical standard production time. If the dosage is too low, the
infection will not propagate, or the onset time will be slow and the
disease weak. This will also occur if the immune system has not been
suitably weakened. The larger the dosage, the faster and more
intense the disease will be. If the dosage is too high, it will
overtake the gerbil's immune system and kill the gerbil. The
specified dosage rate is as high as is practical to get a repeatable
term so they will propagate themselves. Production typically
continues for approximately 3 weeks after infection.
While the production animal 41 is growing and producing E. bieneusi
live organisms, such live organisms are collected and harvested from
the production animal's feces as described in FIGS. 9, 11, and
following. As will be further described, and will become more
apparent, the physical condition of the production animal's stool is
most important. Therefore the production animal 41 is placed on a
diet to provide an optimum consistency stool. Thus, this invention
provides a step of, during such production period, feeding each such
production animal 41 a diet of high-protein dog food 46, with an
addition of a few black sunflower seeds 47 (as a promoter of
gastro-colonic reflex and thus ingestion and production), as
illustrated in FIG. 8. This results in feces which are not sticky
and from which the E. bieneusi live organisms can be suitably
separated. In contrast, a normal diet, which may consist of corn,
barley, oats, or normal pellet feed, results in droppings which are
sticky and difficult to process. In addition, this invention
provides the step of, during such production period, feeding each
such production animal a supplement sufficient to prevent
dehydration and minimize intestinal chemical imbalances, preferably
by adding to the diet of high-protein dog food a water supplement a
pet drink which contains fructose and other known ingredients given
to counteract intestinal upsets and acid base imbalances and to
prevent dehydration, such water supplement being given much as shown
in FIG. 3. An example such pet drink is that called "Thirsty Dog" (a
trademark).
The cages 43 in which the production animals 41 are placed, in
reference to FIG. 5, are of specialized design and construction,
with features necessitated by the production task at hand and
illustrated in FIG. 9. The floor surface 51 is of open mesh
material, with openings sufficient for the feces to fall through,
yet providing ample support and footing for the production animals
41. For Mongolian gerbils, 1/4" wire cloth fulfills this
requirement. Below this open mesh floor 51 is positioned a removable
shallow tray or pan 52, suitable for containing a shallow
(preferably abut 1/4") pond 53 of distilled water, and positioned to
catch all droppings which fall through the mesh floor 51. With this
cage configuration, the E. bieneusi live organisms, produced, as by
the infected production gerbils, and eliminated by such gerbils
through their feces, are collected within the pan, and kept moist
within the pond of distilled water. They may then be easily
collected by the lab technician. Thus, this invention provides steps
of, during such production period, maintaining each such production
animal supported upon a support surface structured and arranged to
permit such feces and other waste material to drop through such
support surface, wherein such support surface comprises open mesh
material sufficient for such feces to fall through, yet providing
ample support and footing for such gerbils, wherein such open mesh
material comprises a 1/4-inch wire cloth; wherein such collecting
step further comprises the step of catching such dropping feces and
such dropping waste material in a container holding sufficient water
for keeping such feces moist until such collection, wherein such
container comprises a shallow pan suitable for holding a pond about
1/4-inch deep of distilled water.
A suitable production rate of E. bieneusi live organisms by the
production animals continues for approximately 3 weeks then
typically begins tapering off. At this point production may be
restarted again by re-introducing the E. bieneusi live organisms as
previously described in FIG. 7. After this re-infecting, production
of the E. bieneusi live organisms typically resumes in about 2 days.
This method of restarting production by re-infection may be
accomplished numerous times until the production animal is overtaken
by an overgrowth of amoeba leaving no opportunity for further
production. This point may occur after up to several months of
successful production. Therefore when the production lowers to an
unsuitable rate and restarting fails, the production animal 41 is to
be destroyed (see FIG. 10) in a humane and safe manner. Destroying
the production animal is accomplished by being put to sleep with an
injectable 56 and then cremation. The reason for this safety
precaution is that the production animal remains a carrier of the E.
bieneusi live organism, and, if turned loose or escaping, could
infect or contaminate in an undesirable manner.
As shown in FIG. 11, collection of the E. bieneusi live organisms
from the cage 43 is accomplished by simply removing the tray 52 from
the bottom of the cage 43 and emptying all its contents 57 by
pouring into a suitable container 58. The material 57 then collected
includes water, feces and other waste and the E. bieneusi live
organisms which are mainly held in suspension in the water. Thus,
this invention provides the step of collection of all contents of
such pan container, thereby providing a fecal material 57 comprising
such feces, such waste material, and such water.
The collected fecal material 57 is then poured through a strainer
61, preferably of a 250 mesh size, which is placed atop a convenient
container such as a beaker 62, as shown in FIG. 12. The majority of
the E. bieneusi live organisms are held in suspension in the water
passing through strainer 61, with some in the sediment at the bottom
of the container, and some remaining in the feces caught by the
strainer 61. To capture the E. bieneusi live organisms in the feces
retained in the strainer 61, the feces are mushed through the
strainer 61 with a suitable tool 63 while utilizing a small amount
of additional distilled water 64 to aide in washing them through.
Thus, this invention provides a step of screening such fecal
material 57 to remove any large particles, thereby providing
filtered fecal material 65 containing E. bieneusi live organisms,
wherein such screening comprises, using a small amount of additional
distilled water, pouring and mushing through a strainer screen, of
about 250 mesh size, placed atop a beaker. After the large waste
material has been removed by the screening process, the strained
solution 65 is further cleaned and purified and refined as described
in FIGS. 13 through 18.
FIG. 13 illustrates a glass tube 71 filled approximately half full
with the strained solution, the filtered fecal material 65, a first
step in what follows. Next, as illustrated in FIG. 14, zinc sulfate
(ZnSO4) 72 is injected into the bottom of the glass tube 71 and
under the filtered fecal material 65, with a long needle syringe 73.
As the zinc sulfate 72 has a specific gravity of 1.18, it will float
the filtered fecal material 65, with its E. bieneusi live organisms
and the remaining waste sediment. This step embodies the step in the
present invention of adding to such filtered fecal material a heavy
liquid having a specific gravity of at least about 1.15 to provide
centrifugable material.
As illustrated in FIG. 15, next, the glass tube 71, with its
filtered fecal material 65 and its zinc sulfate 72, is placed into a
centrifuge 74, and spun down for approximately 15 minutes at 2,000
RPM. After such spinning, the contents of the glass tube 71 will
have been separated as illustrated in FIG. 16. The zinc sulfate 72
remains at the bottom of the glass tube 71 and the waste sediment 75
part of filtered fecal material 65 that previously was floated above
the zinc sulfate 72 is now at the bottom of the tube 71. The water
76 part of the filtered fecal material 65 remains at the top of the
tube above the zinc sulfate. The E. bieneusi live organisms 77, with
a specific gravity of 1.1 to 1.15, have now been separated into an
interface area between the water (1.0 specific gravity) and the zinc
sulfate with its specific gravity of 1.18, as shown. This step
embodies in this invention the step of centrifuging such
centrifugable material to provide a first layer of essentially E.
bieneusi live organisms between a second layer of essentially such
heavy liquid and a third layer of essentially such water.
Next, the E. bieneusi live organisms 77, in suspension in the
interface area between the water 76 and the zinc sulfate 72, are
drawn out with a pipette 78, as shown in FIG. 17, embodying the step
in this invention of removing essentially all the first-layer
material in such first layer from such centrifugable material. Next,
the E. bieneusi live organisms 77 are placed upon a 3 micron
membrane pre-filter 80, above a container 81 which is connected to a
vacuum source 82, as shown in FIG. 18.
When using the vacuum source 82 to evacuate the container 81, the E.
bieneusi live organisms 77 in suspension, being approximately
0.75-1.75 micron in size, are pulled into the container 81, and any
associated waste material larger than 3 micron remains on the
pre-filter 80. This step embodies in this invention the step of a
first filtering through a small enough screen to catch any particles
larger than a such E. bieneusi live organism. Next, the E. bieneusi
live organisms 77 in suspension are removed from the container and
in like manner (as FIG. 18) are placed upon a 1 micron membrane
filter (otherwise, like filter 80). Upon evacuation of the new
container, the majority of the E. bieneusi live organisms 77 are
caught on the 1-micron filter, with the suspension water being drawn
into the container. The separated E. bieneusi live organisms 77 are
then removed from the membrane filter, placed into a glass tube 91
and a small amount of distilled water 92 is added. The E. bieneusi
organism 77, not being rigid, but having a flexible shape will pass
through an opening smaller than its size. Therefore, the suspension
water with some remaining E. bieneusi live organisms, is filtered
again (as in FIG. 18) with a 0.4 micron membrane filter (otherwise,
like filter 80), catching the remaining live organisms 77. These
then are added to the glass tube 91. These steps embody herein the
steps of: a second filtering through about an E.-bieneusi-size
screen to remove most of such E. bieneusi live organisms; placing
such removed E. bieneusi live organisms into a clean container with
a small amount of purified water; a third filtering through a
substantially-less-than-E.-bieneusi size screen to remove any
remaining such E. bieneusi live organisms; and placing such
remaining E. bieneusi live organisms into such clean container to
provide purified E. bieneusi live organisms. Lastly, as shown in
FIG. 19, about 50 mg. of gentamycin sulfate (per each cc. of such
purified E. bieneusi live organisms 77), in solution (as in the
product "Gentocin", a trademark), is added to the glass tube 91 as
by drop 93. This addition is to suppress bacteria that may
inadvertently pass through the 1 micron filter. The glass tube 91 is
then sealed and refrigerated until use. These steps embody in this
invention the steps of: adding sufficient bacteria suppressant to
such purified E. bieneusi live organisms to suppress any bacteria;
and sealing and refrigerating such clean container, for storage
until use.
Although applicant has described applicant's preferred embodiments
of this invention, it will be understood that the broadest scope of
this invention includes such modifications as diverse shapes and
sizes and materials. Such scope is limited only by the below claims
as read in connection with the above specification.
Further, many other advantages of applicant's invention will be
apparent to those skilled in the art from the above descriptions and
the below claims.
* * * * *
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