Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
RECOMBINANT COCCIDIA AND ITS USE IN A VACCINE
The present invention is directed to a
recombinant coccidia in which the DNA of other strains or
species of coccidia has been introduced into a relatively
non-pathogenic strain of coccidia. The invention is also
directed to a coccidiosis vaccine utilizing the recombinant
coccidia to produce immunity to multiple species of
coccidia in a host through inoculation of the host by the
recombinant species.
BACK ,RO ND O mBE rpT~rFrlmTn~
Coccidiosis is an intestinal disease caused as a
result of infection by coccidia, an obligate intercellular
protozoa. As infections are transmitted by environmentally
resistant oocysts released in the feces of infected animals
and because livestock are often kept together in large
numbers, coccidiosis has become an important disease of
livestock throughout the world. In poultry, coccidiosis is
the most frequently recorded disease which according to
some estimates is responsible for losses i.n excess of $1
billion per year. Without adequate measures to control
coccidiosis, the poultry industry could not continue to
exist.
In poultr
y, coccidiosis is a result of infection
by coccidia of the Genera Eimer.ia. There are at present
~~9~'~'~~
2
seven known species of Eimeria which infect chickens;
E.acervulina, E.brunetti, E.max_ima, E.mitis, E.neoatrix,
E.praecox and E.tenella. E.tenella and E.necatrix are the
most pathogenic followed by E.brunetti and E.maxima. In
turkeys, there are also at present seven known species with
E, adenoides considered the most pathogenic and E.
gallopavonis and E. meleagrimitis also considered important
in terms of clinical disease. Eimerians are host specific
in that those species which infect one species of host do
not generally infect other hosts. Not only are the
Eimerians host specific but they parisitize specific sites
:rithin the host and specific cell types within a tissue or
organ. In chicken, species of Eimeria parasitize and
develop in different regions of the gut with E.acervulina
occupying the most proximal region and E.tenella and
E.brunetti the most distal regions. In addition, different
stages of a single species can be specific to different
regions of the intestine and different cell types within
that region. In E.necatrix infections, for example, two
generations of schizogony occur within crypt epithelial
cells of the ileum but gametogony occurs in surface
epithelial cells of the ceca.
Species of Eimeria generally develop in
epithelial cells of endodermal origin, with the majority
parasitizing epithelial cells of the intestinal mucosa.
Coccidiosis infection generally results in villus
flattening or atrophy, crypt hyperplasia and decreases in
villus/crypt ratios accompanied by varying degrees of
malaise, diarrhea, maladsoption, and reduction in growth
rate. Severe infections frequently lead to death.
As early as 1929 (Tyzzer, 1929), ~it was shown
that immunity developed during a coccidial infection such
that birds which recovered from such infections were
generally immune to further infection. Studies have
described the presence of numerous antigens in Eimeria
species with different antigenic profiles associated with
2~~°'"~'~
3
the different stages of the development of the Eimeria
(Wisher 1986; Clarke et al, 1987; Jenkins et al, 1988;
hillehoj et al, 1988; Brothers et al, 1988, Profous-
Juchelba et al, 1988, Castle et al, 1991). The life stage
of Eimeria parasites is intricate consisting of several
differing generations of asexual replication followed by
the development of the sexual stacJes. These different life
cycle stages differ in immunogenicity and antigenic
composition (McDougall et al (1986)). Recent work has
indicated that the gametogony stages are not as immunogenic
as developing asexual forms of the parasite and more recent
studies have indicated that of all the stages, the
sporozoite, particularly in E.tenella, may be the major
target for the development of the immune response in the
host. The nature of the immune response in the host is
generally a combination of antibody mediated and cell-
mediated responses with T-cell dependent immunity being the
prevalent mechanism of immunity to infection (For review
see Rose chapter in Long (1990)).
Coccidiosis control is most commonly
accomplished through the use of anti-coccidial drugs. The
anti-coccidial drugs are mixed into the feed at low levels
to be given continuously to the growing chickens for
continuous prevention of coccidiosis. Amongst the anti-
coccidial drugs most commonly used are the polyether
ionophores with monensin being the most commonly used
ionophore. Monensin is usually administered in the range
of 60 to 90 rpm and is continually administered to the
animal from birth up to slaughter although some producers
continue the practice of weaning off the monensin for 5 to
7 days prior to slaughter. Other commonly utilized anti-
coccidial drugs include salinomycin, lasalocid, narasin,
maduramaicin, and semduramicin, nicarbazin. By far the
most significant use of anti-coccidial drugs is in broiler
chickens which is estimated to cost in excess of $350
million yearly.
4
The intensive use of the anti-coccidial drugs
has lead to the emergence of coccidials with reduced
sensitivity or drug resistance. When a coccidium becomes
resistant to a drug there is usually a collateral
resistance to other drugs of the same chemical class but
not to unrelated drugs. Drug resistance has been found to
be a genetic trait and tends to remain in a population for
many years. Coccidia have more difficulty in becoming
resistant to some drugs than in others. For example,
resistance to halofuginone, clopidol, quinolones and
robenidine has been found within a year of the use while
resistance to monensin and other ionophores were slower to
develop but in recent years the incidence of coccidial
resistance to the ionophores has increased significantly
(See McDougall chapter in Long,1990).
To attempt to prevent the development of drug
resistance, alternate use of drugs has been proposed using
programs such as shuttle and rotation programs. A shuttle
program is practiced within the growout of a flock where
one drug is used for the first three to four weeks and then
another drug is used fox the remainder of the flock's life.
Generally, in this program, a strong drug is used initially
in the starter feed followed by one of the ionophores such
as monensin in the grower feed. A rotation program simply
alternates the use of two or more drugs at various
intervals between flocks such as over three to six month
periods)
The development of drug resistance has limited
the effectiveness of anti-coccidials and it is likely that
this development will intensify over the next few years.
To overcome this, considerable effort has been spent aimed
at the development of vaccines which will provide~long term
immunity to the animal without the necessity of utilizing
anti-coccidial drugs.
5
Owing to the intricacy of the life stages of the
coccidia and the numerous antigens present with the
different antigenic profiles associated with the different
stages of development, the production of suitable vaccines
has not proceeded as well as had been hoped. while there
have been isolated -reports of immunization against a
specific strain of coccidia through the use of isolated
antigen (hurray et al, 1986), no one antigen or series of
antigens has been developed which will provide protection
against all species of coccidia which infect the animal.
Such protection has only been accomplished through the use
of live vaccines which contain oocysts from all relevant
species of coccidia. Examples of vaccines presently
commercially available include: COCCIVAC and IMMUCOX, both
live, virulent vaccines containing oocysts from the seven
species of Eimeria that parasitize domestic fowl, and
PARACOX, a live, attenuated vaccine.
The vaccines are generally administered either
in one large dose within the first few days of life or
through the use of smaller doses given singularly or at
separate times over the first few weeks of life.
However, the use of the vaccines at present have
some potential problems. The use of the live, virulent
vaccines such as COCCIVAC and IMMUCOX, if not properly
administered could result in some infection of the flock
with coccidiosis. Alternatively, if not enough of any of
the presently available vaccine is administered then the
flock will not attain the necessary immunity to become
resistant to coccidia infection. Additionally to promote
complete protection the vaccines require the combination of
numerous species and strains of Eimeria which may present
problems in the proper formulation of the vaccine.
~~~~r,~e~
6
The present invention provides for a recombinant
coccidia comprising a relatively non-pathogenic strain of
coccidia to which has been introduced DNA from one or more
other strains or species of coccidia.
In an aspect of the invention, there is provided
a recombinant coccidiosis vaccine comprising a relatively
non-pathogenic strain of coccidia to which has been
introduced sufficient DNA of one or more other strains or
species of coccidia t o produce immunity to the multiple
species in a host through inoculation of the host by the
recombinant species.
A preferred embodiment of the present invention
is illustrated in the attached drawings in which:
Figure lA is a photograph of ethidium bromide
stained electrophoretically separated DNA from the parent
strains and the recombinant strain in agarose gel before
transfer of the DNA to nylon membrane, and
Figure 1B is a photograph of X-ray film of the
distribution of radioactive Yeast Artificial Chromosome
(YAC) probe sequence bound to the DNA of Figure 1A after
transfer to a nylon filter.
The present invention is based upon the
principle that genomic DNA of related species of coccidia
is similar enough in genomic organization and gene
expression to permit productive recombination to take place
if DNA from one species were to be introduced into cells of
another species at the right time.
A method for isolating genomic DNA from coccidia
was worked out based on isolation methods for filamentous
fungal DNA. The method basically involves the extraction
of the DNA by use of a French press followed by phenol and
ethanol extractions. The isolated DNA is then transferred
to an recipient host coccidium by suitable means to permit
the expression of the donor DNA in the recipieint host
coccidium. As coccidia DNA is present in the nucleus in
discrete chromosomes, the suitable means for transfer will
allow the donor DNA to either integrate into the host
chromosomes or will allow the donor DNA to replicate as
discrete additional chromosomal material. To permit the
replication of the donor DNA as discrete chromosomes,
chromosome functional sequences such as suitable telomeric
sequences may have to be added. Such suitable telomeric
sequences may be provided by packaging the DNA in a
suitable vector acting as an artificial chromosome which
contains such sequences for transfer of the DNA. Several
such vectors have been developed for yeast which
fortuitously have telomeric sequences derived from
protozoan sources such as Tetrahymena. The use of such
vectors has the added benefit that, not only do they permit
the transfer of the DNA between species of coccidia, but
also enable the cloning of individual genes of the coccidia
in both prokaryotes and yeast as the vector acts as a
plasmid in eukaryotes such as E. coli and as an artificial
chromosome in yeast, in particular S, cerevisiae. Because
the transferred DNA carries marker sequences from the
vector, it is also possible to follow the transformation of
the coccidia by detecting such sequences in genomic DNA of
oocysts recovered after passage by use of conventional DNA-
DNA hybridization methods or a sequence amplification
method (DNA polymerase chain reaction, or PCR).
The packaged DNA is physically transferred into
the recipient host organism by typical methods such as
electroporation, microinjection or by in vitro incubation
fi
.. 8
of 'the DNA at high ionic concentration with the free living
stages of the organism. Preferably, the transfer of the
DNA is accomplished by elect roporation or microinjection,
most preferably by electroporation.
Electroporation is a simple and rapid method of
introducing foreign DNA into a cell line. Cells are
subjected to a short electric discharge, apparently
producing reversible pores in 'the cell membrane. DNA is
then capable of passing into the cell where it may be
incorporated into the genetic library.
In order to increase the selectivity of the
recombinant coccidia, one or more selectable markers are
preferably used. For example, the donor organism for the
DNA preferrably encodes for a selectable marker, such as,
for example, drug resistance while the recipient organism
is devoid of the marker such as, for example, being
sensitive to the drug. After production of the recombinant
coccidium, the recombinant coccidium is passaged in a
suitable host and the selectable marker is utilized to
permit replication of only the recombinant organism. For
example, if drug resistance i.s utilized as the selectable
marker, the host animal is administered the drug to which
the donor organism is resistant during passage of the
recombinant organism. In this way only those recombinant
organisms which carry the drug resistance marker from the
donor DNA will replicate and reproduce.
The present invention is particularly suitable
for use with coccidia of the genera Eimeria which infect
poultry hosts. The DNA from donor organisms of one or more
particularly pathogenic species is introduced into
recipient host organisms of a less pathogenic species to
produce a recombinant organism which will exhibit the less
pathogenic infectivity of the host recipient organism while
expressing the antigenic character of the more pathogenic
donor. species. In this way, the recombinant organism will
~
w, g ~~~~~~r~c~3
enable the development of a safer vaccine to immunize the
animal against the potentially lethal species of Eimeria.
For example, the DNA from E.tenella and/or E.necatrix which
are the most pathogenic species of Eimeria to infect the
chicken is incorporated into a less pathogenic species such
as E.acervulina. Similarly, for the turkey the DNA from
for example E. adenoides could be incorporated into a less
pathogenic species of Eimeria which infects turkeys. For
the purpose of the detailed examples following, the chicken
model utilizing E. acervulina as the recipient host
organism and E. tenella as the donor of the DNA was
utilized. It will be appreciated by those of skill in the
art that the methodology utilized in the examples is
applicable to other systems, namely other species of '
Eimeria which infect chicken, other species of Eimeria
which infect other hosts, and other species of coccidia in
other hosts.
E. acervulina, the most prolific species of
chicken coccidia, is only mildly pathogenic, causes
maladsorption in the duodenum and is rarely, if ever,
fatal. It evokes protective immunity with greater ease
than E. tenella and has a shorter prepatent period of 5
days versus 7 days fox E, tenella. This gives an apparent
advantage in that, with respect to the incorporated genes
of E. tenella while being able to express antigenic
determinants, the genes would not have sufficient time to
encode for a complete E. tenella life cycle.
In terms of molecular biology, haploid E.
acervulina has a DNA content comparable to that of E.
tenella (7 .25 X 10-15 g/nucleus or about 69, 000 kb)
(Cornelissen et a1 1984). This amount of DNA is about 17
times that of E. coli (4,000 kb), an organism used by
Clarke et al (1987), Danforth and Augustine (1986) and
Murray and Galuska (1987) to produce antigens.
Incorporating the E. tenella genes into E. acervulina not
only results in a biological producer of. the desired
to
antigens, but also results in a .natural vector for use in a
suitable vaccine. This approach circumvents the problems
inherent in methods used by others where monoclonal
antibodies and complementary DNAs have to be synthesized,
purified and then used to produce antigens in unrelated
organisms, before assembly into a subunit vaccine. This
vaccine, if and when available, would still need to be
injected or introduced into individual chickens.
By suitable techniques, the whole or part of the
DNA from the nucleus of unsporulated E. tenella is isolated
and inserted into unsporulated oocysts or sporozoites of E.
acervulina. Recombinant E. acervulina oocysts are then
sporulated and the sporulated oocysts and/or sporozoites
enriched by feeding them to coccidia-free days old chicks
and the feces collected after a suitable patency period.
The presence of E. tenella DNA in the collected oocysts is
confirmed and the progeny utilized to immunize birds which
are then challenged with E. tenella as well as E.
acervulina.
A method for isolating genomic DNA from oocysts
of Eimeria species was developed utilizing a French press
and phenol and ethanol extractions. Such DNA appears to be
very "crude"; it is sheared (<100 kbp in size), and it has
variable amounts of contaminants (nuclease activity,
polysaccharides and membraneous material) associated with
it. This crude DNA responds to exogenously added, purified
restriction endonucleases but does not yield very many
discrete fragments like yeast DNA which is similar in
genomic complexity. Not much evidence for repeat DNA
sequence was seen using usual methods. This suggests that
the DNA is of low quality but that it was still useable in
testing DNA transfer methods.
An existing vector containing protozoan
teleomeric sequences and which functions as an artificial
chromosome in yeast (YAC) was selected for packaging the
11
donor DNA for transfer of the donor DNA into the recipient
organism. A recent vector pYAC-RC (provided by Douglas
Marchuk and Francis Collins, used in Cystic Fibrosis gene
mapping), was utilized.
Since YAC vectors use telomeric sequences from a
protozoan, such sequences are likely to also work in
Emeria. Upon removal of a segment of the p-YAC-RC vector
DNA by cutting with BamH1 nuclease (p-YAC-RC is a circular
DNA plasmid), DNA sequences that function as telomeres
(ends of chromosomes) are exposed. The remaining large DNA
fragment is further cut with SmaI nuclease to generate two
"arms". E. tenella DNA cut with SmaI nuclease is combined
with the vector fragments and ligated to create
"chromosomal-like°' fragments with one or more of the
"arms". On introducing these hybrid DNAs into
E. acervulina oocysts, such sequences replicate alone or
with host chromosomal DNA via integration during subsequent
stages of chicken infection. Because the transferred DNA
carries YAC sequence it is possible to detect/identify such
sequences in genomic DNA of oocysts recovered after passage
through a chicken by use of conventional DNA-DNA
hybridization methods or a sequence amplification method
(DNA polymerise chain reaction or PCR).
Construction of vectors containing the donor DNA
employs standard ligation techniques. Isolated DNA
fragments are cleaved, tailored and religated in the form
desired to form the vectors required.
Cleavage or digestion'is performed by treating
with restriction enzyme (or enzymes) in suitable buffer.
In general, about lE~g of DNA is used with about 1 unit of
enzymes and about 20~.L of buffer solution. Appropriate
buffers and substrate amounts for particular restriction
enzymes are generally specified by the manufacturer of the
enzyme. Owing to the size of the DNA from the coccidia
incubation times of about 4 hours to 24 hours at 37°C are
12 ~~~V~~~
preferable. After incubation protein is removed by
extraction with phenol and chloroform and nucleic acids
recovered from the aqueous fraction by precipitation with
ethanol.
Ligation refers to the process of forming
phosphodiester bonds between two double stranded nucleic
acid fragments. For ligation, approximately equal molar
amounts of the desired components suitably end tailored to
provide correct matching are treated with about 10 units T4
DNA ligese per 0.5~,g of DNA. When cleaved vectors are
used, it may by useful to prevent religation of the cleaved
vector by pre-treatment o.f vector DNA with alkaline
phosphatase.
Southern analysis is a method by which the
presence of DNA sequences in a digest of DNA containing
compositions is confirmed by hybridization to a known
labelled oligonucleotide or DNA fragment. For the purposes
herein unless otherwise provided, southern analysis shall
mean separation of digest on a 0.8o agarose gel and
transfer to nitrocellulose by the method of E. Southern,
(1975) J. Mol Biol x$:503-517 and hybridization as
described by T. Maniacus et al (1978) Cell ~ 687-701
Transformation means introducing DNA into an
organism so that the DNA is replicable either as an extra
chromosomal elemental or a chromosomal integrant.
The following example is given to illustrate the
invention, but the invention is not to be limited thereto.
TSOT_,ATTOj~ OF FTAj RTA 00 YSTS AND SPC~RIyOTTF~
Unsporulated E. tenella and E. acervulina
oocysts were isolated from fecal droppings of infected
chickens by flotation in saturated NaCl. Tf desired, the
~..~
13
oocysts are allowed to sporulate by incubation in t he salt
solution for 18 t o 20 hours at 30°C. The oocysts were
surface-sterilized and decocted by washing in distilled
water followed by treatment with 6a sodium hypochlorite far
10 min at 4°C. The decocted oocysts were washed thoroughly
by centrifugation at 125 g for 1 min periods followed by
resuspension. The washings were repeated at least 5 times
in 0.9o NaCl and twice in homogenizing buffer containing
0.25 M sucrose and 0.1 M EDTA in 0.01 M Tris-HC1 buffer pH
7.0 with added penicillin (100 units/ml) and streptomycin
(50 ~.g/ml). The washed oocysts were centrifuged at 1,500 g
for 10 min and the pellets resuspended and counted.
Sporocysts were released by fracturing the
oocyst walls using unsiliconized glass beads. Oocysts were
concentrated to approximately 8x106 oocysts/ml. 10.58 of
450-500 glass beads per 2.5m1 of oocyst suspension is
placed in a one-ounce universal bottle and vortexed on high
for 15 seconds. The beads were rinsed in sterile water and
gravity filtered through a glass bead funnel in order to
remove most of the wall debris and unbroken oocysts.
Glass bead funnels were prepared by filling 150m1 sintered
glass Buchner funnels with 200~L unsiliconized glass beads
up to a height of 5cm and rinsing once with filtered water
before loading with the smashed oocyst solution.
Sporocysts were rinsed through the glass beads until
oocysts are visible in the filtrate.
After filtering the sporocysts, they were
centrifuged and resuspended at 10x106 oocyst/10m1 of
medium-199. (Gibco Instant Tissue Culture Medium pH
adjusted to 7:5) Sporocysts were then incubated at 41°C
for 1/2 hour in Difco Bacto Bile Salts at a concentration
of 50mg/l0ml of solution followed by Trypsin at lOmg/ml or
solution and incubated at 41C for the additional one hour
to release sporozoites. E. acervulina sporozoites are
fragile relative to other stages in the Eimerian life cycle
and should be kept on ice immediately after excystration
CA 02098773 1998-12-10
14
and should be subjected to minimal centrifugation to retain
viability.
Glass beads were rinsed once with Ringer's
solution before sporozoite/bile/trypsin suspension were
loaded onto the glass bead funnels and sporozoites passed
through without the aid of suction. If significant residue
blocks the filter, the surface beads were stirred using a
wooden stirring rod. Sporozoites were then rinsed through
until the effluent contained increased percentages (greater
than lo) of sporulated oocysts. The sporozoites were
purified by passing them through a second, clean glass bead
filter using the same method described above. Sporozoites
were then collected by centrifugation at medium speed for 3
minutes and resuspended in Ringer's solution. Recovery of
sporzoites was usually 70% or better with purities greater
than 98.50.
Glass beads were cleaned by soaking them in
nitric acid followed by extensive rinsing in tap water.
Nitric acid is removed from the beads by stirring them in
distilled water until the pH of the distilled water added
remained constant.
E. tenella oocysts were collected and washed in
JavexTM as set out above. They were then washed 3 times in
20mM Tris buffer (pH adjusted to 7.5) and lOmM EDTA
solution before smashing in the French press. Cells were
concentrated and resuspended in approximately lOml of
Tris/EDTA solution and passed once through the French press
at 10-12 tonnes. Sodium dodecal sulfate (SDS) was added to
a final concentration of to in order to denature protein.
Caution must be taken not to exceed to SDS since the DNA
may precipitate and centrifugation should be done at room
temperature.
r'~. 15 ~~(.~~~~~~~
Proteinase K was added to a concentration of
100~.g/ml and the pH was adjusted to 8 using a concentrated
Tris base. Digestion of protein was allowed to proceed
overnight at 37°C. DNA was extracted with an equivolume of
phenol and inverted gently (taking care not to shear DNA)
for 5 minutes doing all extraction in the fumehood and the
centrifuged at room temperature 10-15 minutes at 5000rpm.
If the top layer was not clear, the top layer was removed,
reextracted with, phenol and centrifuged.
The top layer was pipetted off and extracted
with equivolume of 50:50 phenol: half chloroform, inverted
gently for 5 minutes and centrifuged for 10-15 minutes at
5000 rpm. The top layer was removed, extracted with
equivolume of chloroform, inverted gently for 3-5 minutes
and centrifuged for 10-15 minutes at 5000 rpm. The
supernatant was removed and the molarity increased to 0.3M
by addition of 1/10 volume of 3NI sodium acetate. (pH of
5.2) Two volumes of absolute ethanol or 1 volume
isopropanol was added quickly in order to precipitate DNA.
The precipitated DNA was centrifuge at 3000 rpm
to form a pellet. The ethanol was carefully poured off so
as not to drain off the soft pellet. DNA was solubilized
in a minimum TE solution ensuring that all DNA has
solubilized as some may remain as a clear gel. ire-boiled
RNase was added up to 50~.g/ml and before incubation at 37C
for 1 hour a small aliquot was measured and its absorption
level at 260nm recorded.
In order to remove RNase proteinase K was added
up to 200~.1.g/ml and incubated at 37C for 1 hour. To purify
DNA phenol, phenol/chloroform(1:1), and chloroform were
added respectively at an equivolume. After each successive
addition the extracts were inverted gently for 5 minutes,
centrifuge 10-1.5 minutes at 5000 rpm and the bottom layer
discarded.
16 ~~~U~~~~:3
After the last centrifugation was complete, the
top layer was removed and the solution was placed into a
dialysis bag and dialyzed in a sterile 1L beaker of TE
solution overnight in order to reduce nucleotides in the
solution. The TE solution was replaced and allowed to sit
for an additional 1 to 2 hours. A spectometer reading at
260nm of the TE solution should approach 0 as fewer
nucleotides are being removed from the dialysed DNA
solution.
After dialysis spectrometer readings were taken
at wavelengths of 260nm and 280nm. One OD unit at 260nm is
equivalue to 50~g/ml of DNA. A ratio of OD units from
readings at wavelengths 260nm and 280nm indicate the purity
of the sample. Ratios between 1.6 and 1.8 indicate pure
DNA whereas ratios of 2 and upwards indicate significant
residue present.
Three samples of E.tenella DNA (150 ~1g in 250 ~Ll
final volume of digestion buffer) were digested for 0.5, 2,
and 4 hr respectively at 37°C with 10 units of Smal
endonuclease (cuts at sequence: 5'-NNCCC< > GGGNN-3'). 15
~.g of YAC vector (plasmid pYAC-RC) was digested under the
same conditions with 1 unit each of BamH1 and SmaI. After
stopping the respective reactions with 0.1o SDS and 25mM
EDTA at 70°C for 5 min, all of the samples were pooled and
extracted twice with 1 volume of, phenol-chloroform. DNA
was precipitated with 2.5 volumes of 95o ethanol (v/v) and
centrifugation (Beckman 12 microfuge). DNA was semi-dried
with N2 gas and dissolved in 250.1 of 1mM Dithothreitol in
H20 at 36°C for 24 h before addition of 5X ligation buffer
(66mM Tris-HC1 pH 7.8, 6mM MgCl2 final cone.), freshly made
ATP (0.5mM final) and 30 ~1 (60 units) of T4 ligase
(Pharmacia). Ligation of E.tenella DNA to plasmid vector
~~~(~~~~~
17
DNA was carried out for 30 hr at 5°C, before further
dilution with 1X ligation buffer without ligase to approx.
500,1 final volume. Ligated DNA was subsequently stored at
4°C until used.
Sporozoites were prepared as described earlier.
The cell-porator unit supplied by Bethesda Research
Laboratories Life Technologies, Inc, in conjunction with a
cell-porator voltage were used containing two electrodes
4cm apart, 1 ml of sporozoite suspension at concentrations
greater than 1x106/ml was loaded into the chamber using a
micro pipette. Although cell fusions may occur at such
high concentrations, a greater percent of transformation is
thought to occur when cell concentrations exceed 1x106/ml.
DNA was added to each cell suspension at a concentration
not less than 20~,g/ml. Chambers were kept an ice while
cells were subjected to the electrical pulses at 400V,
330~1F capacitance, 100kV booster and high resistance. The
sporozoite suspensions were thereafter allowed to remain on
the lab bench for at least 10 minutes in order to enhance
DNA uptake.
Several trials were carried out using different
stages of the Eimerian life cycle, sporocysts and oocysts.
As well some sporozoite and oocyst suspensions containing
acridine orange in addition to the DNA were studied as
acridine orange may increase DNA uptake.
Groups of days old chicks were inoculated per os
with about 500,000 sporozoites each of untreated
E.acervulina, E.acervulina into which had been tranferred
Clostridium sp. DNA, E.acervulina into which had been
transferred E. tenella DNA and E.acervulina into which had
been transferred acridine orange labelled E. tenella DNA.
CA 02098773 1998-12-10
18
Thirteen days after inoculation each group was challenged
blind with 50,000 E. tenella oocysts per bird. Six days
after challenge, each bird was scored blind for lesion size
and the results tabulated.
10
i) Cenomic DNA- Approx. 50 ug of DNA from
E_tenella, E.acerv~lina and the two putative E.acervulina x
E. tenella recombinants isolated as desribed above were
each incubated with SO units of BamHl enzyme (cuts at
sequence: 5'NNG< >GATCCNN-3') at 37°C for 4 hr and 24 hr in
buffer containing lOmM Tris.HCl pH 7.8, 50mM KCl, O.lmM
EDTA, 1mM Dithiothreitol. Reactions were stopped by
addition of sodium dodecylsulphate (0.2o final) and EDTA
(25mM) .
ii) Marker DNA: plasmid pYAC-RC (5 ~.lg) and lambda
bacteriophage, Charon 4A (5~.g) were digested as described
fur genomic DNA (see above) with 1 unit each of BamHl,
HindIII and Eco Rl.
b) F~ectrophores~s, DNA Transfer to Membranes and
DNA samples were loaded into slots (lanes in a l5cm x 20cm,
0.80 (w/v) agarose slab containing Tris-Acetate, EDTA,
ethidium bromide (5ug/ml) buffer and electrophoresed
(submerged in same buffer without ethidium bromide) for
6hrs at 40mA, 18°C. DNA in gel was photographed (Polaroid
type 52 film using UV light.and filter). Gel was further
treated with 100mM HC1 at 20°C for 10 min. and 400mM NaOH
for 30 min to break and denature the DNA strands. After
three brief washes in gel running buffer, DNA in the gel
was electrophoretically transferred to Hybond-NTM (Amersham)
19 ~~~Jr~~~
0.45 ~m nylon membrane (same size as gel). The membrane
was rinsed in a solution containing 1M NaCl and 1mM EDTA
and dried by air (30min) and heat (30°C, 1 hr in vaccuo).
The membrane was incubated for 1 hr at 68°C in 200 ml of
hybridization buffer (1M NaCl, 1mM EDTA, 0.1o SDS, 10~.g/ml
sheared and dematured calf thymus DNA (Sigma Chem).
Hybridization was carried out in a plastic bag containing
ml of same buffer stock with 10~ cpm of random primed
pYAC-RC (0.5 ug) template. Membrane was washed four times
10 with 100m1 of hybridization buffer at 1X and four times
with 100m1 of hybridization buffer at 0.5X at 68°C. Filter
was air dried and exposed to Kodak XR X-ray film with
single screen for three days and 5 days.
Differences in samples of DNA were noted during
processing of E. acervulina and E. tenella DNA. After
French pressing, the E. acervulina solution seemed very
gel-like, whereas the viscosity of the E. tenella did not
visibly change. Upon phenol addition, E. acervulina DNA
formed a very tight white precipitate at the interface
whereas E. tenella precipitate breaks up at the interface.
After addition of ethanol, E. tenella turns the DNA
suspension cloudy whereas the E. acervulina suspension
remains clear and DNA is visible as a fluffy cotton ball
type precipitate. E. tenella DNA precipitate after
freezing is a fine powder. In general the DNA samples
prepared from the first generation electroporated oocysts
appeared as the unaltered E. acervulina DNA however, after
precipitation with ethanol some of the precipitate floated
and some separated and sank to the bottom of the tube.
This may be an indication of an alteration in the E.
acervul.ina DNA.
a
Fig. 1A shows a photograph of ethidium bromide stained,
electrophoretically separated DNA in agarose gel before
5 transfer of DNA to nylon membrane. Fig 1B shows a
photograph of X-ray film indicating distribution of
radioactive YAC probe sequence bound to DNA on the .nylon
filter.
10 Lanes a and m, negative control (does not hybridize to YAC
probe as expected) and DNA size markers: lambda
bacteriophage Charon 4A DNA digested with BamHl, HindIII
and EcoRl. Charon 4A digest should yield 11 fragments but
only six large fragments 4.9-11.5 kbp in size (see six bars
15 right side of photo) were observed.
Lane h, BamH1+ HindIII + SmaI digested PYAC-RC (positive
control) should yield at least eight ethidium bromide
staining bands (more with partial digestion as appears to
20 be the case as SmaI does nat cut well). Probe made from
this DNA hybridizes to all fragments (Fig. 1B).
Lanes b-f and lanes i-1 correspond to Emeria sp. DNA
digested with BamH1 (see Experimental for details). Lane
~ DNA from a Clostridium sp. also isolated from chicken
faeces (serves as another negative hybridization control).
E.tenella DNA (lanes b and c), E. acervulina (lanes d, e)
and putative E. acervulina x E.tenella recombinants fixst
trial (lanes i, j) and ~on~, rial (k and 1). Lanes b,
d, g, j and k are DNA samples digested for 4 hrs at 37°C.
Lanes c, e, f, i, and 1 are samples of DNA digested with
same amount of BamHl enzyme at 37°C but for 24hrs.
b) Tn-.rpretations:
Fig. 1A indicates that all DNA samples have
endogenous nucleases (compare 4 hrs vs. 24 hr digests).
Other tests indicated that this nuclease activity is
~7~~i~~~~
inhibited mostly by EDTA, requiring MgCl2 which is present
in the restriction enzyme buffer. There is also evidence
that the "crude" DNA samples contain (see ethidium bromide
staining material particularly at top of gel) cellular
debris rich in polysaccharide and lipid. This material is
entangled with high molecular DNA and is released from
extensively degraded DNA (24hr samples); most of it floated
out of the gel wells during electrophoresis, the rest
escaped during handling of the gel. for photography. These
observation are very typical of crude DNA preps of most
lower organisms.
No YAC probe hybridization to lamda Charon 4A in
spite of very high concentration of this DNA which is a
very good control for probe specificity, suggesting that
hybridization to extra bands in lane j may be real. No
hybridization to Clostridium sp DNA observed also is
another good sign of specificity.
No hybridization to low molecular weight DNA of
Emeria parental DNAs (4hr digests); slightly more to
E.tenella than E.acervulzna DNA. The ethidium bromide
staining patterns (FiglA) of Eimeria parental DNA show
traces of repeat sequences which do not hybridize to YAC
probe. However the E.acervulina recombinant # 1 DNA which
gave the lowest lesion score (1.2) has a pattern of
hybridization a bit like E.tenella DNA but it also has two
positive hybridization bands, one being similar in size to
YAC plasmid DNA used originally in the ligations. In late
digestion (24hr) all of this hybridization material is
likely lost as might be expected by extensive endogenous
nuclease nicking of the DNA.
Recombinant # 2 DNA also has a small amount of
hybridization in the same position but the concentration is
much lower. Since these samples represents pools of
oocysts this could mean that #1 has been amplified more
than #2.
~:~~~~~~9
22
The positive bands in lane j tend to indicate
the presence of true YAC sequence, but not all of the
sequence. This would be the simplest and most consistent
interpretation of the data and would not be surprising
because the specific E.tenella DNA or E.acervulina DNA
associated with it would could have fused and discarded
either all or part of the YAC arms through sequences
related to the Trpl, Ura3, TEL or His3 gene sequences. The
yeast genes are conserved in lower eukaryotes and so
possibly is the TEL gene.
The observed bands may be related to Eimeria
repeat sequences which would require highly intact DNA to
confirm but the control data do not strongly support this
possibility. The possibility of spurious hybridization is
also unlikely due to predictability of results from the
other controls. Also the same trend was continued when
hybridizations were repeated on a more limited scale by dot
method due to lack of sample.
Groups of days old chicks were inoculated with
samples of each of untreated E. acervulina, E. acervulina
in to which have been transferred Clostridium sp. DNA, E.
acervulina in which have been transferred E. tenella DNA,
and E. acervulina in to which have been transferred
acridine orange labelled E. tenella DNA. Thirteen days
after inoculation the chicks were challenged with 50,000 E.
tenella oocysts and after suitable patency period, birds
were explored for lesion size. Summary results of a number
of tests are given in the following Tables 1 and 2.
23
TABLE 1
Group Ave Lesion Score Redy~!esior~/Total
Control 3.0 0/8
Clostridium DNA 2.7 2./14
E. tenella DNA 2.8 3/13
Labelled
E. tenella DNA trial#1 1.5 6/7
trial#2 1.2
TABr.E 2
(~rnnn 2lzrcrarro T.oeinn Cnnro
Not porated 3.0
Control
Porated with 3.3
Clostridium DNA
Porated with 2.8
E. tenella DNA
Porated with
labelled
E. tenella DNA
trial #1 1.5
trial #2 1.2
Not porated 2.6
Sporozoites
Not porated 3.3
Oocysts
2n
Porated with 2.1
Clostridium DNA
Porated with 2.7
E. tenella DNA
The above results clearly demonstrate the
effectiveness of the transfered DNA of the E, tenella donor
to confer immunity to E. tenella .in birds immunized with
the recombinant E. acervulina. This is evidenced by the
reduced lesion scores in those animals inoculated with the
recombinant organism. The reduct_Lon in lesion scores seen
with the Clostridium sp. was postulated to arise as a
result of possibility of cross contamination between the
birds inoculated with the two recombinant organisms. It is
also clear from the results that, at least under the
conditions selected for the electroporation, the use of the
acridine orange label increases significantly the
efficiency of the transfer of the donor DNA to the
recipient host. The average lesion scores of 1.5 and 1.2
and the degree of protection with 6 out of 7 birds having
significantly reduced lesions are similar to results that
are normally observed upon challenge of birds inoculated
with native E. tenella in traditional immunization
programs.
The above example clearly demonstrates the
ability of the recombinant coccidia in the present
invention to confer immunity in an animal host against
infection by the coccidium which acts as a donor organism. '
While the example has only utilized whole DNA from one
species it would be readily apparent to those of skill in
the art that the invention could be practiced utilizing
more than one species as a donor organism thereby
conferring immunity to multiple specie of coccidia in an
animal host. Also as the understanding of coccidial
antigenic determinants and the immunity evoked in the host
animal by immunization with a coccidia vaccine increases in
W 25 ~~~~~?r~~~
the future the invention may be utilized to take advantage
of this increased knowledge. For example, as significant
antigens and the genes encoding them are identified, such
genes may be isolated and packaged in accordance with the
present invention to permit the expression of the gene and
production of the antigenic determinant in the recombinant
coccidium. Such genes could include genes from many
species of coccidia thereby conferring immunity to a
multitude of species through the use of the recombinant
coccidium of the present invention.
The recombinant coccidia of the present
invention permits the development of novel vaccines which
induce immunity to more pathogenic species of coccidia by
incorporating the DNA from such species into less
pathogenic species. Such a vaccine has a major impact on
the concept of coccidial vaccine manufacturing. The vaccine
will be used with less impunity than present live vaccines
and the industry now has a viable and longer-lasting
alternative to medication. By utilizing a less pathogenic
species of coccidia as the recipient, the potential for
accidentally inducing disease in a flock through
misadministration of the vaccine is greatly reduced.
Vaccine doses can be increased relative to traditional
vaccines to ensure that even if individual chicks receive
reduced doses, there will be sufficient antigen present to
confer immunity. Unlike present live vaccines, the vaccine
of the present invention does not require anticoccidials
either in the feed or the water to assist in control of
coccidiosis.
Although various preferred embodiments of the
present invention have been described herein in detail, it
will be appreciated by 'those skilled in the art, that
variations may be made thereto without departing from the
spirit of the invention or the scope of the appended
claims.
t~B~~~'~~~~
26
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27
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35
