U.S. patent application number 11/979177 was filed with the patent office on 2008-05-08 for neospora caninum isolate.
This patent application is currently assigned to University of Technology. Invention is credited to Catherine Margaret Douglas Miller, John Timothy Ellis, Helen Elizabeth Quinn.
Application Number | 20080107602 11/979177 |
Document ID | / |
Family ID | 3823114 |
Filed Date | 2008-05-08 |
United States Patent
Application |
20080107602 |
Kind Code |
A1 |
Ellis; John Timothy ; et
al. |
May 8, 2008 |
Neospora caninum isolate
Abstract
The present invention relates to a novel Neospora caninum
isolate from Nowra and extracts thereof. The strain is useful in
the development of diagnostic assays for the detection of parasites
in animals. The present invention also relates to pharmaceutical
compositions, using live or killed organisms or extracts thereof,
for the treatment and prevention of parasitic infections in
animals.
Inventors: |
Ellis; John Timothy;
(Hornsby, AU) ; Douglas Miller; Catherine Margaret;
(Roseville, AU) ; Quinn; Helen Elizabeth; (Lane
Cove, AU) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Assignee: |
University of Technology
Sydney
AU
|
Family ID: |
3823114 |
Appl. No.: |
11/979177 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10332387 |
Apr 4, 2003 |
|
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PCT/AU01/00908 |
Jul 27, 2001 |
|
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11979177 |
Oct 31, 2007 |
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Current U.S.
Class: |
424/9.1 ;
424/269.1; 435/258.1; 435/325; 435/7.1 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61K 39/00 20130101; C12R 1/90 20130101; C12N 15/11 20130101; C07K
16/20 20130101; A61K 2039/505 20130101; C07K 14/44 20130101; Y02A
50/492 20180101; A61P 33/02 20180101 |
Class at
Publication: |
424/009.1 ;
435/258.1; 435/325; 424/269.1; 435/007.1 |
International
Class: |
A61K 39/002 20060101
A61K039/002; C12N 1/10 20060101 C12N001/10; G01N 33/53 20060101
G01N033/53; A61P 33/02 20060101 A61P033/02; C12N 5/10 20060101
C12N005/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 28, 2000 |
AU |
PQ 9056 |
Claims
1-16. (canceled)
17. A parasitic protozoan isolate having the characteristics of the
isolate deposited as AGAL Accession No. NM01/22338.
18. The host cell infected with an isolate as claimed in claim
17.
19. The host cell as claimed in claim 18 which is derived from the
sample deposited as AGAL Accession No. NM01/22339.
20. The vaccine composition comprising an isolate as claimed in
claim 17, wherein the isolate is in the form of killed parasites or
live attenuated parasites.
21. The method of diagnosing a parasitic infection or disease in an
animal, the method comprising detecting the presence of an isolate
according to claim 17 in the animal, or in a clinical specimen from
the animal.
22. The method as claimed in claim 21 wherein the clinical specimen
is selected from the group consisting of a biopsy, a stool
specimen, a blood sample and foetal tissue.
23. The method for the treatment or prevention of infection or
disease in an animal, the method comprising administering to the
animal a vaccine composition as claimed in claim 20.
24. The method as claimed in claim 21 wherein the infection or
disease is caused by the presence of Neospora in the animal.
25. The method as claimed in claim 23 wherein the infection or
disease is caused by the presence of Neospora in the animal.
26. The method as claimed in claim 21, wherein the animal is a
human or livestock animal.
27. The method as claimed in claim 26, wherein the livestock animal
is a cow.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a novel parasitic protozoan
isolate and extracts thereof. The strain is useful in the
development of diagnostic assays for the detection of parasites in
animals. The present invention also relates to pharmaceutical
compositions, using live organisms or extracts thereof, for the
treatment and prevention of parasitic infections in animals.
BACKGROUND OF THE INVENTION
[0002] The Apicomplexa is a diverse phylum of protozoa containing
some of the most pathogenic organisms known to man. Representatives
include taxa of the well known genera such as Plasmodium, Babesia
and Toxoplasma. In 1984 a research group in Norway reported a
previously unknown cyst-forming protozoan which caused encephalitis
and myositis in dogs (Bjerkas et al. 1984). These clinical signs
were consistent with those caused by Toxoplasma gondii, a widely
distributed member of the Apicomplexa. However, unlike T. gondii,
the organism was not pathogenic to outbred mice, nor were T. gondii
specific antibodies present in the sera of the dogs studied. This
prompted further investigation which demonstrated conclusively the
organism was ultrastructurally and antigenically distinct from T.
gondii (Dubey et al. 1988). Neospora caninum was thus described as
a new member of the Sarcocystidae. Soon after, N. caninum was
implicated as a cause of abortion in cattle (Thilstead & Dubey,
1989).
[0003] An increasing number of N. caninum isolates have now been
obtained, using techniques involving predominantly the direct
inoculation of infected tissues onto tissue culture cells (Dubey et
al. 1988, Conrad et al. 1993), although bovine isolates have proven
difficult to obtain. The primary reason for this has been the
detrimental effects of autolysis on the viability of N. caninum in
aborted bovine fetal tissues. The isolation of a parasite
population from a live animal is therefore preferred.
[0004] Recent studies on the biological properties of bovine and
canine isolates have suggested they belong to a single species
called N. caninum (Holmdahl et al. 1997), despite the record that
wide differences exist between isolates in their biological
properties. These include differences in antigenicity,
ultrastructure, pathogenicity and genetic heterogeneity (Conrad et
al. 1993, Marsh et al. 1995, Lindsay et al. 1995 and Atkinson et
al. 1999). Thus not all isolates of N. caninum may possess the same
properties, and indeed at least one isolate of N. caninum was
mistakenly identified as Hammondia heydorni previously (Schares et
al. 2001). Indeed, others have speculated on whether N. caninum and
H. heydorni are the same or different species (Mehlhorn and
Heydorn, 2000). Hammondia heydorni is also a cyst-forming coccidian
that has a life cycle which is very similar to that of N. caninum.
Thus the true identity of the species N. caninum is currently being
debated, and its relationship to H. heydorni is unclear.
[0005] In cattle, abortion due to N. caninum infections usually
occur in mid- to late gestation, although not all infected foetuses
are aborted. Many congenitally infected calves are born healthy and
persistently infected, although some infected calves are diseased
at birth and die in the neonatal period with lesions similar to
those of aborted calves.
[0006] The development and use of serological tests for the
diagnosis of neosporosis in livestock, along with the
identification of animals infected or exposed to N. caninum, has
been reviewed previously in great detail (Bjorkman et al. 1999;
Atkinson et al. 2000a). Significantly, however, there is no
effective vaccine against transplacental transmission or foetal
loss which occurs as a result of neosporosis and attempts to
formulate a vaccine have met with limited success.
[0007] Liddel et al. (1999) injected female BALB/c mice with a
crude N. caninum tachyzoite lysate preparation co-administered with
ImmuMAXSR.TM. adjuvant. These mice were subsequently mated, and
pregnant dams were challenged with N. caninum tachyzoites at 10-12
days of gestation. Results showed a single injection offered
complete protection against transplacental transmission of the
parasite to the pups. All pups in this experimental group were free
from parasitic infection. No results have yet been reported on the
efficacy of this vaccine formulation in the bovine.
[0008] Baszler et al. (2000) examined the possibility of
vaccination of BALB/c mice with soluble N. caninum antigen
formulated in either nonionic surfactant vesicles or Freunds
Complete Adjuvant. This approach resulted in exacerbation of
encephalitis and neurological disease in these mice. These
observations were characterised by increased antigen specific IL-4
secretion and increased IgG1:IgG2a ratios in vivo.
[0009] Adrianarivo et al. (1999) tested four different adjuvants
with a killed whole N. caninum tachyzoite preparation for
immunogenicity. The results indicated that the immune responses, as
determined by IFAT titres, were significantly higher in
experimentally infected cattle compared to immunised cattle.
[0010] Adrianarivo et al. (2000) studied the effect of a killed N.
caninum tachyzoite preparation in pregnant cattle using a
POLYGEN.TM. adjuvant. Heifers were injected at day 35 and day 65 of
gestation and four weeks later were challenged with intravenous or
intramuscular injection of tachyzoites. Post immunisation, heifers
developed both humoral and cell mediated immune responses
characterised by an increase in production of IgG1 and IFN-.gamma.
respectively. Following a challenge with N. caninum tachyzoites,
however, significant cell mediated immune response did not occur.
All foetuses in the study, both from control and experimental
cattle, developed lesions characteristic of N. caninum infection.
Failure to prevent foetal infection by this formulation in pregnant
cattle was concluded.
[0011] Unlike the development of killed or genetically engineered
vaccines against parasites, vaccines based on live populations of
parasites are available, for example against Toxoplasma-induced
abortion in sheep (Buxton & Innes, 1995) and Eimeria parasites
of poultry (Shirley & Bedrnik, 1997). A live vaccine is not,
however, available against N. caninum.
[0012] The literature on live vaccines against N. caninum is
limited. Atkinson et al. (1999) showed that infection of naive mice
by the Nc-SweB1 isolate of N. caninum partially protected them
against a severe infection by Nc-Liverpool. Lindsay et al. (1999)
generated temperature sensitive mutants of N. caninum and
demonstrated that they could prevent clinical signs associated with
neosporosis in mice.
SUMMARY OF INVENTION
[0013] The present inventors have isolated a novel protozoan
parasite from the central nervous system of a dairy calf. The
parasite was identified based on a number of criteria as an isolate
of Neospora caninum and was called the "Nc-Nowra" strain. This
isolate is naturally attenuated in its ability to cause neosporosis
in a laboratory animal, and thus is an ideal isolate to serve as a
basis for a vaccine against this disease.
[0014] A sample of the Nc-Nowra isolate was deposited under the
provisions of the Budapest Treaty on 21 Jun. 2001 with the
Australian Government Analytical Laboratories (AGAL) and accorded
AGAL Accession No NM01/22338. Further, a sample of Vero cells was
deposited under the provisions of the Budapest Treaty on 21 Jun.
2001 with the Australian Government Analytical Laboratories (AGAL)
and accorded AGAL Accession No NM01/22339.
[0015] Accordingly, in a first aspect the present invention
provides a parasitic protozoan isolate having the characteristics
of the isolate deposited as AGAL Accession No. NM01/22338.
[0016] In a preferred embodiment of the first aspect, the isolate
is that deposited as AGAL Accession No. NM01/22338.
[0017] In a second aspect, the present invention provides an
antibody raised against an isolate according to the first aspect.
Preferably, the antibody is a monoclonal antibody.
[0018] In a third aspect the present invention provides a host cell
infected with an isolate of the first aspect. Preferably, the host
cell is derived from the sample deposited as AGAL Accession No
NM01/22339.
[0019] In a fourth aspect, the invention provides a vaccine
composition comprising an isolate of the first aspect, wherein the
isolate is in the form of a killed parasite population or live
attenuated parasites.
[0020] The present invention also provides a vaccine composition
comprising an extract of an isolate according to the first aspect.
Preferably, the extract is selected from the group consisting of
live attenuated, killed and fixed parasites, a cell lysate, an
antigenic polypeptide and a polynucleotide encoding an antigenic
polypeptide.
[0021] In a fifth aspect, the present invention provides a method
for the treatment or prevention of infection or disease in an
animal, the method comprising administering to the animal a vaccine
composition according to the fourth aspect.
[0022] In a sixth aspect, the present invention provides a method
for the treatment or prevention of infection or disease in an
animal, the method comprising administering to the animal an
antibody according to the second aspect.
[0023] In a seventh aspect, the present invention provides a method
of diagnosing a parasitic infection or disease in an animal, the
method comprising identifying the presence of the isolate according
to the first aspect of the invention.
[0024] In a preferred embodiment of the fifth, sixth or seventh
aspects, the parasitic disease is neosporosis. In a further
preferred embodiment the parasitic infection is due to the presence
of Neospora caninum. In one embodiment the Neospora caninum is the
isolate of the first aspect.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1. Scatterplot showing sera ELISA readings against N.
caninum for individual cows of the study herd.
[0026] FIG. 2. Bar chart showing IgG responses (as ELISA
absorbances) to N. caninum antigen in mice infected with or without
Nc-Nowra before pregnancy. Groups given Nc-Liverpool at day 8 of
pregnancy are indicated. Only mice receiving Nc-Nowra before
pregnancy gave high IgG titres.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The parasitic isolate of the present invention may be used
to develop diagnostic tools or aids for the detection or diagnosis
of parasitic disease in animals. Such diagnostic tools or aids
include antigenic polypeptides derived from the isolate, antibodies
raised against the isolate and molecular probes or primers derived
from the genome of the isolate.
[0028] Standard protein purification techniques can be used to
isolate antigenic polypeptides from the novel parasitic strain of
the present invention. Such techniques include selective
precipitation with such substances as ammonium sulphate, column
chromatography, immunopurification methods, and the like. See, for
instance, R. Scopes, Protein Purification: Principles and Practice,
Springer-Verlag: New York (1982). Proteins and portions thereof
isolated from the strain can be sequenced according to standard
techniques as described for instance in Sambrook et al., Molecular
Cloning, A Laboratory Manual, Cold Spring Harbour Publish., Cold
Spring Harbour, N.Y. 2nd Ed (1989).
[0029] Antibodies raised against the strain may be polyclonal or
monoclonal antibodies.
[0030] If polyclonal antibodies are desired, a selected mammal
(e.g., mouse, rabbit, goat, horse, etc.) may be immunised with an
immunogen preferably a purified protein mixed with an adjuvant.
Serum from the immunised animal is collected and treated according
to known procedures. Further fractionation of the antisera to
enrich for antibodies reactive to the isolated proteins of the
invention can be done if desired.
[0031] Monoclonal antibodies may be obtained by various techniques
by one skilled in the art Briefly, spleen cells in an animal
immunised with a desired immunogen are immortalised, commonly by
fusion with a myeloma cell (See, Koller & Milstein, Eur. J.
Immunol. 6:511-519 (1976)). Alternative methods of immortalization
include transformation with Epstein Barr Virus, oncogenes, or
retroviruses, or other methods well known in the art. Colonies
arising from single immortalised cells are screened for production
of antibodies of the desired specificity and affinity for the
desired antigen.
[0032] Antibodies, both monoclonal and polyclonal, which are
directed against epitopes are particularly useful in diagnosis, and
those which are neutralising are useful in passive immunotherapy.
Monoclonal antibodies, in particular, may be used to raise
anti-idiotype antibodies. Anti-idiotype antibodies are
immunoglobulins which carry an "internal image" of the antigen of
the agent against which protection is desired.
[0033] Techniques for raising anti-idiotype antibodies are known in
the art. These anti-idiotype antibodies may also be useful in
therapy.
[0034] For the purposes of this invention, the term "antibody",
unless specified to the contrary, includes fragments of whole
antibodies which retain their binding activity for a target
antigen. Such fragments include Fv, F(ab') and F(ab').sub.2
fragments, as well as single chain antibodies (scFv).
[0035] In one embodiment, the antibodies of the present invention
bind specifically to the Neospora isolate of the present
invention.
[0036] Antibodies of the invention may be bound to a solid support
and/or packaged into kits in a suitable container along with
suitable reagents, controls, instructions and the like.
[0037] Molecular probes or primers for detecting the isolate of the
present invention may be generated by recombinant or synthetic
means. Preferably, the polynucleotide is at least 16 nucleotides in
length, more preferably at least 20, 25, 30 or 40 nucleotides in
length. The polynucleotide may be used to produce a primer, e.g. a
PCR primer, a primer for an alternative amplification reaction, or
a probe e.g. conjugated by conventional means to a radioactive or
non-radioactive label.
[0038] Diagnosis may be achieved by detecting the presence of the
parasite or by assaying for antibodies to the parasite in the
animal. As there is now concern that humans are susceptible to
these type of parasite infections, the present invention includes
human applications.
[0039] The parasite may be detected by culturing the parasite from
a clinical specimen and identifying the parasite by microscopy, or
may be identified by the use of antibodies to the parasite or by
detecting a portion of the genome of the parasite by molecular
biological techniques. The clinical specimen may be a biopsy, stool
specimen, blood sample, foetal tissue or the like. It will be
appreciated that the discovery of the parasite and its association
with parasitic disease will allow its detection by any of the known
methods of the art. It will also be appreciated that molecular
detection methods like polymerase chain reaction (PCR) can also be
used to identify the presence of the parasite in a clinical
specimen.
[0040] Immunological and immunoassay procedures in general, are
described in Basic And Clinical Immunology 7th Ed. (D. Stites and
A. Terr ed.) 1991. According to the present invention immunoassays
may by performed in any of several configurations, which are
reviewed extensively in Enzyme Immunoassay, E. T. Maggio, ed., CRC
Press, Boca Raton, Fla. (1980); Practice and Theory of Enzyme
Immunoassays," P. Tijssen, Laboratory Techniques In Biochemistry
And Molecular Biology, Elsevier Science Publishers B. V. Amsterdam
(1985). For example, a person skilled in the art would understand
that the proteins and antibodies according to the present invention
can be used in ELISA, immunoblot analysis and agglutination
assays.
[0041] In brief, immunoassays to measure antibodies or antigens are
either competitive or noncompetitive binding assays. In competitive
binding assays, the sample analyte (e.g., anti-N. caninum
antibodies) competes with a labelled analyte (e.g., anti-N. caninum
monoclonal antibody) for specific binding sites on a capture agent
(e.g., isolated N. caninum protein) bound to a solid surface. The
concentration of labelled analyte bound to the capture agent is
inversely proportional to the amount of free analyte present in the
sample.
[0042] Noncompetitive assays are typically sandwich assays, in
which the sample analyte is bound between two analyte-specific
binding reagents. One of the binding agents is used as a capture
agent and is bound to a solid surface. The second binding agent is
labelled and is used to measure or detect the resultant complex by
visual or instrument means.
[0043] Alternatively, the immunoassay is carried out in liquid
phase and a variety of separation methods are employed to separate
the bound labelled component from the unbound labelled components.
These methods are known to those skilled in the art and include,
but are not limited to, immunoprecipitation, column chromatography,
adsorption, addition of magnetisable particles coated with a
binding agent and other similar procedures.
[0044] In another alternate procedure, an immunoassay is carried
out in liquid phase without a separation procedure. Typically, in
these procedures, the assayed protein or other analyte competes
with a compound for binding to an antibody attached to a label. The
binding of the analyte to an antibody, but not the binding of the
compound to the antibody, causes a change in the signal emitted by
the label, so that analyte binding is measured without separating
the bound from the unbound labelled component.
[0045] Western blot (immunoblot) analysis is also used to detect
the presence of antibodies to the parasitic isolate in a biological
sample. This technique is a reliable method for confirming the
presence of antibodies against a particular protein in the
sample.
[0046] Pharmaceutical compositions prepared using extracts of the
isolate of the present invention may be used for the treatment
and/or prevention of parasitic infections, preferably N. caninum
infections.
[0047] Preferably, the extract is selected from the group
consisting of live attenuated parasites, killed and fixed
parasites, cell lysates, antigenic polypeptides and polynucleotides
encoding antigenic polypeptides.
[0048] Vaccines of the invention may comprise a crude extract of
the novel parasitic isolate. Killed and/or chemically fixed
parasites or cells can also be used. Vaccines may also comprise
partially or completely purified polypeptide preparations derived
from the parasitic isolate. The polypeptide may be an antigen
produced by recombinant DNA technology.
[0049] In addition, polynucleotide sequences derived from the novel
parasitic isolate may be cloned into viruses that transfect host
cells in animals. Live attenuated viruses, such as vaccinia or
adenovirus, are convenient alternatives to vaccines because they
are inexpensive to produce and are easily transported and
administered. It will be appreciated that polynucleotides derived
from the parasitic isolate of the present invention may also be
incorporated into non-viral vaccine vectors.
[0050] The preparation of vaccines which contain an immunogenic
polypeptide(s) as active ingredient(s), is known to one skilled in
the art. Typically, such vaccines are prepared as injectables,
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. The preparation may also be emulsified, or the protein
encapsulated in liposomes. The active immunogenic ingredients are
often mixed with excipients which are pharmaceutically acceptable
and compatible with the active ingredient. Suitable excipients are,
for example, water, saline, dextrose, glycerol, ethanol, or the
like and combinations thereof.
[0051] In addition, if desired, the vaccine may contain minor
amounts of auxiliary substances such as wetting or emulsifying
agents, pH buffering agents, and/or adjuvants which enhance the
effectiveness of the vaccine.
[0052] Further examples of adjuvants and other agents include
aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate
(alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil
emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial
endotoxin, lipid X, Corynebacterium parvum (Propionobacterium
acnes), Bordetella pertussis, polyribonucleotides, sodium alginate,
lanolin, lysolecithin, vitamin A, saponin, liposomes, levamisole,
DEAE-dextran, blocked copolymers or other synthetic adjuvants. Such
adjuvants are available commercially from various sources, for
example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco
Laboratories, Detroit, Mich.).
[0053] Typically, adjuvants such as Quil A, Amphigen
(oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of
Amphigen and Alhydrogel are used. Other immunostimulatory molecules
such as cytokines (for example, .gamma.-interferon) may also be
used.
[0054] The proportion of immunogen and adjuvant can be varied over
a broad range so long as both are present in effective amounts. For
example, aluminum hydroxide can be present in an amount of about
0.5% of the vaccine mixture (Al.sub.2O.sub.3 basis). Conveniently,
the vaccines are formulated to contain a final concentration of
immunogen in the range of from 0.2 to 200 .mu.g/ml, preferably 5 to
50 .mu.g/ml, most preferably 15 .mu.g/ml.
[0055] After formulation, the vaccine may be incorporated into a
sterile container which is then sealed and stored at a low
temperature, for example 4.degree. C., or it may be freeze-dried.
Lyophilisation permits long-term storage in a stabilised form.
[0056] The vaccines are conventionally administered parenterally,
by injection, for example, either subcutaneously or
intramuscularly. Additional formulations which are suitable for
other modes of administration include suppositories and, in some
cases, oral formulations. For suppositories, traditional binders
and carriers may include, for example, polyalkylene glycols or
triglycerides; such suppositories may be formed from mixtures
containing the active ingredient in the range of 0.5% to 10%,
preferably 1% to 2%. Oral formulations include such normally
employed excipients as, for example, pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, and the like. These compositions
take the form of solutions, suspensions, tablets, pills, capsules,
sustained release formulations or powders and contain 10% to 95% of
active ingredient, preferably 25% to 70%. Where the vaccine
composition is lyophilised, the lyophilised material may be
reconstituted prior to administration, e.g. as a suspension.
Reconstitution is preferably effected in buffer.
[0057] Capsules, tablets and pills for oral administration to a
patient may be provided with an enteric coating comprising, for
example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose
acetate phthalate or hydroxypropylmethyl cellulose.
[0058] Vaccine compositions of the present invention may be
administered to animals susceptible to or otherwise at risk of
infection to elicit an immune response against the parasite and
thus enhance the animals own immune response capabilities. Such an
amount is defined to be an "immunogenically effective amount". In
this use, the precise amount depends on the judgement of the
prescribing veterinarian or doctor and would include consideration
of the patient's state of health and weight, the mode of
administration, the nature of the formulation, and the like.
[0059] A variety of vaccination regimes may be effective in
immunising cattle and other animals. Preferably, female cattle are
vaccinated just prior to or at the time of breeding so as to
prevent abortion and reduce the possibility of congenital
infections. A second immunisation may be given at other time
periods before and during gestation. Calves and adult males may
also be vaccinated, if desired. Animals that have previously been
exposed to N. caninum or have received colostral antibodies from
the mother may require booster injections. The booster injection is
preferably timed to coincide with times of maximal challenge and/or
risk of abortion.
[0060] In order that the present invention may be more clearly
understood preferred forms will be described with reference to the
following examples and drawings.
EXAMPLE 1
Isolation of Bovine Neospora sp. in New South Wales
[0061] The isolation and characterisation of a Neospora parasite
from a clinically normal neonatal Australian Holstein-Friesian calf
is described. The calf was born from a dam maintained in a
commercial dairy herd of Holstein-Friesian cattle located on the
south coast of New South Wales (NSW) Australia, near Nowra. This
herd has been endemically infected with neosporosis for a number of
years and has been studied extensively (Atkinson et al. 2000b). The
dam was first identified as being serologically positive with an
IFAT titre of 1280 for N. caninum in March 1995, having previously
aborted in January 1994 but not since. The calf, which was
seropositive to N. caninum antigen by western blotting, remained
healthy and showed no clinical signs of N. caninum infection prior
to it's euthanasia (NSW Agriculture) with intravenous potentiated
pentabarbitone (Euthetal) via jugular puncture with a 21 gauge
needle and 20 ml syringe, dosed to effect. On cessation of life,
the brain and spinal cord (including thoracic segments T10 to T15)
were removed following dissection of the tissues of the skull and
cervical region and exposure of the central nervous system (CNS)
using a saw and bone cutters. With the exception of a 3 cm diameter
segment of left frontal cerebral cortex which was fixed in 10%
formalin for histological processing, the CNS tissues were placed
immediately in antibiotic saline solution (0.9% NaCl containing
penicillin and streptomycin) and homogenised. Trypsin was then
added to 0.05% and the homogenate incubated for 30 min at
37.degree. C. The homogenate was centrifuged for 10 min at 1200 g
and resuspended in antibiotic saline. The pellet was washed twice
in antibiotic saline with centrifugation.
[0062] For tissue culture, the pellet was resuspended in 8 ml of
tissue culture medium and placed using aseptic technique into a
flask containing a monolayer of vero cells (Barber et al. 1995).
Unfortunately the cultures became contaminated and were
discarded.
[0063] Homogenates in antibiotic saline were injected
intraperitoneally into interferon-gamma receptor knockout
(IFN-.gamma.RKO, 129/Sv) mice with a 23 gauge needle. Mice were
injected with homogenate from either infected brain (6 mice) or
spinal cord extract (6 mice) and then monitored daily for clinical
signs of disease. Clinical signs suggestive of neosporosis
including ruffled coat, huddled lethargic appearance and weight
loss (Atkinson et al. 1999), were observed in both groups of mice
(brain and spinal cord injected) within 23 days post-inoculation
with homogenate. Mice were euthanased with carbon dioxide and their
brains removed and processed as above for infection of tissue
culture cells with bovine brain tissue, however using smaller
volumes. Brain homogenates were also passaged back into naive
IFN-.gamma.RKO knock-out mice, which developed clinical signs of
neosporosis by 23 days post infection. A third passage in mice
resulted in clinical signs being observed from 10 days post
infection.
[0064] Histopathological examinations, performed by NSW
Agriculture, of multiple sections of cerebrum from the clinically
normal calf identified very mild multifocal non-suppurative
encephalitis characterised by rare foci of gliosis and perivascular
cuffing. No parasitic elements were observed, however, the lesions
were considered consistent with very mild intrauterine cerebral
infection with N. caninum (Boulton et al. 1995). Histological
examination of cerebrum from 5 mice inoculated with calf brain
homogenates identified mild multifocal non-suppurative encephalitis
in all 5, with 2 apicomplexan structures resembling N. caninum
tachyzoites identified in the cerebral choriod tissue section of a
single mouse. Immunohistochemistry performed on this section
identified positive immunoperoxidase staining with anti-Neospora
antisera.
[0065] Vero cells infected with brain homogenate from one of these
mice resulted in the proliferation of tachyzoites 14 days post
infection. The in vitro growth of these parasites has now been
successfully achieved for 15 months. The isolate was identified as
a member of the genus Neospora for the reasons outlined below and
was named Nc-Nowra, after the geographical location of the study
herd.
EXAMPLE 2
Characterisation of Isolated Organisms
[0066] Electron microscopy. Tachyzoites were recovered from in
vitro culture and examined by transmission electron microscopy.
Tachyzoites were fixed in 2.5% glutaraldehyde, 2% paraformaldehyde
in phosphate buffer pH 7.2 for 2 hrs; washed three times with 0.1M
cacodylate buffer and post-fixed in 1% aqueous osmium tetroxide.
After excess fixative was removed by washing with water, parasites
were dehydrated in ethanol, transferred to anhydrous acetone and
infiltrated and embedded in Epon resin. The blocks were cut and
ultrathin sections examined with a JEM 1010 transmission electron
microscope. Tachyzoites were identified as N. caninum by virtue of
their size, shape and internal structures including the presence of
characteristic intracellular organelles such as electron dense
rhoptries (Speer et al. 1999).
[0067] Western blotting. Tachyzoite lysates of Nc-Nowra,
Nc-Liverpool and the ME49 strain of T. gondii were analysed by
western blotting using sera obtained from mice acutely infected
with Nc-Liverpool (Atkinson et al. 1999). The antigen profile
detected for Nc-Nowra was similar, but not identical to, that
obtained from Nc-Liverpool. The antigen profile possessed no
similarity to that shown by T. gondii.
[0068] PCR and DNA sequencing. PCR amplification of Nc-Nowra
genomic DNA was performed using DNA prepared by standard
techniques, from in-vitro cultured tachyzoites. The primers Tim3
and Tim11 were used and the amplification products were analysed by
agarose gel electrophoresis (Payne and Ellis, 1996). PCR
specifically amplified an internal transcribed spacer (ITS1)
sequence, which was similar in size to that obtained from
Nc-Liverpool DNA. The PCR product was purified using a Qiagen
purification kit and the DNA sequenced by cycle sequencing using
primers Tim3 and Tim11. A consensus sequence derived for the ITS1
of Nc-Nowra was 99% similar to that previously reported for N.
caninum (AF029702 and AF038860 for example).
EXAMPLE 3
Nc-Nowra Tachyzoites Used in a Serological Test such as an
ELISA
[0069] A dairy herd, located in the Southern Highlands of NSW,
contained 177 Friesians with a history of abortion, with 37
abortions being recorded over 18 months spanning 1999/2000. There
were two dogs and numerous foxes on this property, and N. caninum
was previously diagnosed on the property by post mortem examination
of aborted foetuses by NSW Agriculture. The herd is regularly and
fully vaccinated against leptosporidiosis. The dairy herd was bleed
for routine investigation of the cause of the abortions and these
sera were screened for antibodies to Nc-Nowra using an indirect
ELISA protocol developed at UTS and described below.
[0070] Nc-Nowra tachyzoites were recovered from in vitro culture
and reduced to protein extracts by resuspension in lysis buffer (20
mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 2 mM DTT,
1 mM benzimidine HCl) and disruption by sonication at 50 W/20 KHz
for 10-20 secs. The resulting preparation was dialysed against PBS
overnight at 4.degree. C. with one change of buffer. Protein
concentration was determined using the Bradford dye-binding assay
(Biorad). Nunc 96 well plates were coated with antigen diluted to 1
.mu.g/well in carbonate buffer at 4.degree. C. overnight. Plates
were washed three times with PBS/0.03% Tween and the test bovine
serum diluted in ELISA buffer 2 (0.3% Tween, 0.05% bovine
haemoglobin) was added to wells. The sera used were from dairy
cattle which were prone to abortion. Plates were again incubated
overnight at 4.degree. C. and then washed three times with
PBS/0.03% Tween. Anti-bovine IgG-alkaline phosphatase conjugate was
diluted 1:6,000 in buffer 2 and plates were incubated at 37.degree.
C. for 3 hrs. Plates were washed with PBS/0.03% Tween and 1 mg/ml
of p-nitrophenylphosphate in carbonate buffer was added. Plates
were incubated at 37.degree. C. for 30 min and absorbance at 405 nm
read in a Biorad ELISA plate reader. A positive serum was
identified as being 2 standard deviations greater than the mean
negative O.D. result (from 20 sera).
[0071] Results. Nc-Nowra crude lysate was used in an ELISA to
screen a dairy herd suspected of containing N. caninum positive
cows. A graph showing a summary of the O.D. readings obtained from
the herd ELISA is in FIG. 1. Four (4) of 177 (2.3%) cows in the
herd were seropositive to N. caninum using this assay. There was a
high correlation between ELISA absorbances obtained using the
Nc-Nowra ELISA and the results from a commercial N. caninum ELISA
kit, which detected 16 of 177 (9%) cows as seropositive within the
herd. The commercial kit is also based on a crude lysate antigen,
however the NC-1 N. caninum isolate is used. This was isolated in
the U.S.A. and has different biological properties to the Nc-Nowra
isolate.
EXAMPLE 4
A Cell Extract of Nc-Nowra Used to Induce an Immune Response in an
Animal
[0072] Thirty (30) QS mice were divided equally into 10 groups,
each of which received one of the following treatments, 4 weeks
apart: TABLE-US-00001 GROUP TREATMENT 1. None 2. Freund's
incomplete adjuvant (FIA) alone 3. FIA + Nc-Nowra
[0073] Two (2) weeks after the second injection, mice were killed
by CO.sub.2 asphyxiation, bled and their spleens removed for a cell
proliferation assay.
[0074] An Nc-Nowra extract was made in the following way and used
for either injection into animals or for ELISA. Nc-Nowra
tachyzoites were recovered from in vitro culture and reduced to
protein extracts by resuspension in lysis buffer (20 mM Tris pH
7.5, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, 2 mM DTT, 1 mM
benzimidine HCl) and disruption by sonication at 50 W/20 KHz for
10-20 secs. The resulting preparation was dialysed against PBS
overnight at 4.degree. C. with one change of buffer. Protein
concentration was determined using the Bradford dye-binding assay
(Biorad).
[0075] Sera, made by standard procedures, was assayed by ELISA
using the following method. Nunc 96 well plates were coated with
Nc-nowra antigen extract diluted to 1 .mu.g/well in carbonate
buffer at 4.degree. C. overnight. Plates were washed three times
with PBS/0.03% Tween and mouse serum diluted in ELISA buffer 2
(0.3% Tween, 0.05% bovine haemoglobin) was added to wells. Serum
used was from mice in the treatment groups as described above.
Plates were again incubated overnight at 4.degree. C. and then
washed three times with PBS/0.03% Tween. Anti-mouse IgG-alkaline
phosphatase conjugate was diluted 1:6,000 in buffer 2 and plates
were incubated at 37.degree. C. for 3 hrs. Plates were washed with
PBS/0.03% Tween and 1 mg/ml of p-nitrophenylphosphate in carbonate
buffer was added. Plates were incubated at 37.degree. C. for 30 min
and absorbance at 405 nm read in a Biorad ELISA plate reader.
[0076] Cell proliferation assays were performed on mouse spleen
cells as follows. Spleens were individually placed in wash medium
(DMEM containing penicillin and streptomycin, DMEM/PS), and forced
through a 70 .mu.m nylon disposable sieve to yield a single cell
suspension. The cells were concentrated by centrifugation and
resuspended in 2 mls red cell lysis buffer and left at room temp.
for 1 min. The lysis buffer was diluted out with more DMEM/PS,
centrifuged and the pellets resuspended in fresh DMEM/PS for
counting. 1.times.10.sup.4 cells per well of each suspension were
added in triplicate to wells of a microtitre plate. Stimulating
antigen at a concentration of 10 .mu.g/well for Con A and Nc-Nowra
lysate was added to the cells. A blank (containing no cells) was
included. The cells were incubated at 37.degree. C. in 5% CO.sub.2
for 4 days. BrdU was then added and the cells incubated for a
further 4 hours. The cells were pelleted to the bottom of the
microtitre plate by centrifugation, the supernatants collected for
cytokine assay, and the cells dried and fixed. BrdU incorporation
was then detected using a commercial immunoassay kit (Roche)
encompassing an anti-BrdU-POD conjugate.
[0077] Results. An increase in absorbance was observed in the serum
samples taken at each time point indicating an IgG response was
raised in mice against a crude antigen extract of Nc-Nowra. This
response was significantly greater than that seen in uninjected
mice or mice injected with adjuvant alone. The time course showed a
significant IgG response at and after 6 weeks post injection in the
mice receiving Nc-Nowra extract.
[0078] A crude extract of Nc-Nowra, when used as a stimulating
antigen, also stimulated cell proliferation in spleen cells from
mice, showing a cell mediated immune response was induced.
EXAMPLE 5
Nc-Nowra is Only Mildly Pathogenic in the Mouse
[0079] A single experiment was performed to investigate the
pathogenicity of Nc-Nowra in the BALB/C mouse. This model has been
used previously to investigate the pathogenicity of N. caninum
(Nc-Liverpool and Nc-SweB1) in vivo (Atkinson et al. 1999). Groups
of female mice were injected intra-peritoneally with either saline,
10.sup.6 (Nc-Liverpool or Nc-Nowra) or 10.sup.4 (Nc-Nowra)
tachyzoites grown in-vitro. Mice were weighed daily and euthanased
when clinical signs dictated (to alleviate unnecessary suffering)
or at the end of the experiment at day 31 post-infection. The
brains of all mice in the experiment were removed, fixed in 10%
formalin and examined pathologically.
[0080] Results. All mice injected with saline survived until the
end of the experiment and gained weight steadily over this time
period. Mice given Nc-Liverpool rapidly became ruffled and
lethargic and euthanasia of affected mice began at day 20 post
infection. By day 31, only 4 out of 9 mice remained in this group.
Mice given Nc-Nowra were not as rapidly affected with 7 out of 17
mice surviving until day 31 post infection.
[0081] Histological sections from 3 of the mice injected with
saline failed to identify significant histological lesions (no
further mice were examined in this group). Lesions of varying
severity and characterised as multifocal non-suppurative to
necrotising encephalitis were identified in 5 of 8 mice receiving
10.sup.4 Nc-Nowra, all of 9 mice receiving 10.sup.6 Nc-Nowra and
all of 9 mice receiving 10.sup.6 Nc-Liverpool. Lesions were graded
in severity as per previous studies (Atkinson et al. 1999) with 4
of the mice receiving 10.sup.4 Nc-Nowra, 4 of the mice receiving
10.sup.6 Nc-Nowra and all 9 mice of the 10.sup.6 Nc-Liverpool mice,
judged to have moderate to severe lesions. These findings show
that, like Nc-SweB1, which was also isolated from the bovine
(Stenlund et al. 1997), Nc-Nowra is less pathogenic in the BALB/C
mouse, compared to Nc-Liverpool.
[0082] Five (5) BALB/C mice injected i.p. with Nc-Nowra infected
brain from a knock-out mouse were maintained in a healthy condition
for 7 months, and showed no clinical signs of neosporosis. These
mice seroconverted to N. caninum antigen (by western blotting).
After euthanasia, analysis of brain pathology showed mild brain
lesions consistent with N. caninum infection. No parasitic stages
were seen during the pathologic analyses.
EXAMPLE 6
A Live Infection by Nc-Nowra can Prevent Neosporosis in Mice
Infected with Nc-Liverpool
[0083] Tachyzoites of the Nc-Nowra isolate were harvested from an
in vitro culture of Vero cells and diluted in 0.9% saline to a
concentration of 10.sup.5/ml. Ten BALB/c mice were infected
subcutaneously with 10.sup.4 tachyzoites per mouse. A further
fifteen mice were injected with 0.9% saline only. Three weeks post
infection the ten Nc-Nowra infected mice and ten of the saline
injected mice were infected with 10.sup.6 Nc-Liverpool tachyzoites
recovered from tissue culture. The five remaining saline injected
mice were injected with saline again. The mice were monitored daily
for clinical signs (ruffled and lethargic) and weighed daily from
day 13 p.i. Mice were euthanased after losing 20% of their body
weight. Blood was collected from all mice and brains from a
selection from each group. The blood was allowed to clot, spun and
the serum removed. The brains were fixed in 10% formalin and sent
to NSW Agriculture for pathologic examination.
[0084] Results.
[0085] Group 1: injected with saline and challenged with
Nc-Liverpool (positive control, n=10):
[0086] Clinical signs were observed in the Nc-Liverpool only
infected mice on day 14 p.i. and euthanasia of mice started on day
21. All ten mice in this group died between day 21 and day 25
p.i.
[0087] Group 2: infected with Nc-Nowra and challenged with
Nc-Liverpool (n=10):
[0088] In contrast, only 2 mice in the Nc-Nowra/Nc-Liverpool
infected mice developed clinical signs and these were euthanased on
days 19 and 20. Both of these mice were small and light in weight
for their age at the beginning of the experiment. Such mice
normally succumb quickly to neosporosis and were included as an
internal positive control. All eight other mice in this group
remained healthy throughout the experiment and showed no signs
normally associated with N. caninum infection such as weight loss,
head tilting or motor discoordination. It was concluded that the
Nc-Nowra infection had induced protective immunity against the
clinical signs of neosporosis associated with Nc-Liverpool
infection.
[0089] Group 3: injected with saline and given no parasite
challenge (negative control, n=5)
[0090] All mice in this group remained healthy.
[0091] Brain Pathology
[0092] Five (5) micron sections were cut from brains, stained with
H and E and examined for evidence of brain lesions. Lesions were
graded on a scale of 0 to 5 with 5 being the most severe type of
lesion. The average score for the Nc-Liverpool infected brains
(Group 1) examined was 3.5 while the average score for the
Nc-Nowra/Nc-Liverpool (Group 2) infected brains examined was 1.5.
It was concluded that the prior Nc-Nowra infection had
significantly reduced brain lesion score in this experiment.
EXAMPLE 7
Prior Infection by Nc-Nowra has No Effect on Litter Size in the
Pregnant QS Mouse
[0093] Five (5) week-old, female QS mice were injected
subcutaneously with 10.sup.4 in-vitro derived tachyzoites of
Nc-Nowra. Three weeks later ovulation in all mice was synchronised
by two hormone injections (PMSG and hCG) given 48 hours apart. At
day 8 of pregnancy 1 of the treatment groups was given 10.sup.6
tachyzoites of Nc-Liverpool. All mice were killed at day 14 of
pregnancy and the uterine implantation sites were analysed for
viable foetuses.
[0094] Results: The table below summarises the treatment groups and
the mean litter sizes obtained. No significant difference in mean
litter size was found between the treatment groups, indicating
Nc-Nowra does not cause foetal loss in this model. TABLE-US-00002
Number of mice in Treatment Mean Litter Size (S.E.) the group None
15.2 (1.18) 23 Nc-Nowra 16.9 (1.73) 12 Nc-Nowra/Nc-Liverpool 17.3
(1.45) 19
Sera from all dams killed at day 14 were assayed for IgG to N.
caninum antigen using the ELISA method described in Example 4. Mice
infected with Nc-Nowra showed high levels of IgG to N. caninum,
whereas mice in the other two groups did not (FIG. 2).
EXAMPLE 9
Infection of Pregnant QS Mice with Nc-Nowra
[0095] Nine (9) week-old female QS mice were mated following
synchronisation of ovulation with 2 hormone injections (PMSG and
hCG) 48 hrs apart. At day 8 of gestation mice were given either a
subcutaneous injection of saline (control group), 1.times.10.sup.4
or 1.times.10.sup.6 Nc-Nowra tachyzoites derived from tissue
culture. All mice were euthanased at day 14 of gestation and
uterine implantation sites were analysed for viable and non-viable
foetuses. Statistical analyses was performed using an Analysis of
Variance.
[0096] Total nucleic acid was made from the excised foetal tissue
by standard methods involving lysis in buffer containing SDS, EDTA
and proteinase K, followed by phenol chloroform extraction and
ethanol precipitation. These DNAs were analysed for the presence of
N. caninum DNA by PCR using primers that were designed to be
specific for N. caninum from the sequence of the ITS1 reported
previously (Payne and Elis, 1996). The PCR reaction was heated at
95.degree. C. for 2 min, then subject to 35 cycles of 95.degree. C.
45 sec, 50.degree. C. 45 sec and 72.degree. C. for 2 min. The
reaction was then treated at 72.degree. C. for 5 min to allow
primer extension to be complete.
[0097] Results: The table below summarises the results of this
experiment. Injection of mice with Nc-Nowra at day 8 of gestation
caused no clinical signs of disease and no significant foetal loss
(p=0.3355). TABLE-US-00003 Group Mean % of resorptions/litter
(S.E.) Saline 6.813 .+-. 3.535 1 .times. 10.sup.4 Nc-Nowra 26.75
.+-. 24.43 1 .times. 10.sup.6 Nc-Nowra 7.243 .+-. 3.626
[0098] No N. caninum-specific PCR product was obtained from these
foetal DNAs, showing no evidence for N. caninum infection at day 14
of gestation. The positive control (N. caninum DNA) gave the
required band of approx. 400 bp. This study does not exclude the
possibility that N. caninum may be detected in older foetuses or
pups using a similar experimental design with Nc-Nowra.
[0099] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
[0100] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0101] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed in Australia before the priority date of
each claim of this application.
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