Arno N. Vermeulen.
Intervet International PO Box 31 5830 AA Bosmeer The Netherlands arno.vermeulen@intervet.com
Introduction
The family of Coccidian parasites comprising genera
such as Eimeria, Toxoplasma, Neospora and Sarcocystis is more or less
particular, because to date effective vaccines have been developed against each
of its members. Having vaccines available might suggest that problems are solved
and other diseases can be tackled; however, this is an oversimplification of the
complex nature of immunity and disease control in different animal species and
against different members of each genus.
Present vaccines may be improved with respect to better control of parasitemia
and easier and more cost-effective production and distribution.
A major disadvantage of an immunological approach is that for each species to be
controlled a separate and specific vaccine needs to be developed. This may seem
obvious, but it is a drawback when compared to the development of a drug.
Most anti-coccidial drugs developed in the second half of the 20th century are
active against multiple species and even genera and can act in different, albeit
not all, hosts (examples are ionophores and anti-folates).
The occurrence of resistance makes it still necessary to develop new drugs,
preferentially targeting common pathways of Coccidia or even Apicomplexa, of
which some are even absent in vertebrate hosts. Coombs (1) reviews such targets
exemplified as the functional apicoplast, the shikimate pathway, mannitol cycle
and the polyamine metabolism. Recently developed organotypic cultures of neural
tissue may be of benefit in the selection of active compounds (2). It is to be
foreseen that although more and better vaccines become available, several animal
species suffering from the parasites mentioned above, will remain dependent on
drug therapy because vaccines can’t handle the many different species involved
(10 species of Eimeria are attacking young lambs) (3).
Reduction of the use of drugs in consumption animals however also drives the
generation of biological solutions (vaccines).
Eimeria vaccines
Eimeriosis, often designated as coccidiosis, is the
disease caused by Eimeria parasites resulting in severe mucosal damage, weight
loss and sometimes even death. The disease is widespread and many species occur
in poultry, livestock and small animals such as rabbits. Especially the
intensification in poultry production (broiler farms) could only be achieved by
the concurrent development and application of effective drugs (4).
Since chickens readily develop immunity from natural infection, vaccines were
developed based on virulent strains of the most frequently occurring species E.
tenella, E. acervulina and E. maxima. These vaccines were shown to be effective
when administered at low dose, early in life.
Attenuation of strains could be achieved by adaptation to development in
fertilized eggs. Passage of strains through eggs rendered them less pathogenic,
although for instance E.acervulina could not be adapted to the egg (5). Livacox
® contains an embryo-adapted E. tenella line. Precociousness is a
characteristic of most Eimeria referring to a naturally occurring population of
parasites that complete their lifecycle from sporozoite to oocyst 20-30 hrs
faster than their fellow parasites from the same parent. This is a selectable
trait and sometimes this is accompanied with a decrease in proliferative
capacity and pathogenicity. Paracox® was the first line of vaccines that
utilized this feature for live vaccines with a better safety profile (6). Recent
publications report on newly developed precocious vaccines in different parts of
the world (4,7,8). Kawazoe et al. (7) reported that as a result of the selection
for precociousness one strain appeared less pathogenic, whereas the other did
not differ from the parent line in that respect. This emphasizes the fact that
the trait precociousness is not unambiguously linked to the aspect of low
pathogenicity.
Strains selected for naturally occurring low pathogenicity were included in
NobilisCox ATM® (9). Whereas most vaccines allegedly contain sensitive strains,
the latter comprises strains with a defined tolerance for specific drugs.
Thereby it allows the concomitant use of certain ionophores until immunity is
fully developed. The strains are, on the other hand, fully susceptible to
chemical drugs such as diclazuril and toltrazuril, which allows removal if
preferred. Li et al. (8) recently selected ionophore-tolerant, precocious
strains for similar purpose, and Kawazoe et al. (7) demonstrated that a certain
degree of ionophore tolerance is a natural feature of different Eimeria strains,
even if they did not have contact with these drugs before.
Antigenic variability becomes more and more evident, especially when live E.
maxima vaccine strains are applied (10). Only Paracox® and NobilisCox ATM®
have included antigenically different strains. The two strains in the latter
vaccine appear to act synergistically (4).
Vaccine failures were often attributed to highly virulent strains. However, with
improved administration of live oocysts, chickens develop immunity more readily
as a consequence of early cycling of the parasites.
Vaccine spray on day-old birds is nowadays widely considered as the best method
to convey the infection to the chicks. In ovo application of live coccidial
vaccines (such as Inovocox®, Embrex) may also turn out to be effective. It at
least ensures that all birds receive the same dose. Now that more and more
vaccines of similar kind enter the scene, objective criteria need to be set for
these products as presently there are no studies regarding safety and efficacy
requirements.
Being realistic, we estimate that the vaccination of forty billion broilers each
year will be very difficult if using live oocysts produced by chickens. Thus,
there is a great need for mass production and mass application of effective
vaccines. Subunit vaccines are generally not suited for such purpose, although a
maternal vaccine Coxabic® was licensed. This vaccine is based on E. maxima
gametocyte-derived antigens gam56 and gam82, which should protect the offspring
by the action of maternal antibodies present in the yolk. An Emax250kD antigen
was recently cloned, and predominantly recognized by maternal sera infected with
E. maxima (11). It is not clear what the relation between these approaches is,
but they could work in concert.
Recombinant vaccines should be the only long term solution for the problem in
the future, especially since more drugs will be banned and requirements for live
vaccines will become stricter.
It was shown that target antigens are involved in essential parasite functions.
Surface-expressed and apical complex-associated proteins are prime targets for
antibody attack (12). Effective protection using invasion-related proteins has
not been demonstrated so far. In our lab, we have extensively tested EtMic1 and
EtMic5; protein or DNA-based antigens EtMic2 and EtMic4 have been tested and
showed some protective effect (13,14). Presentation is of essential importance
and may influence the level of the effect observed.
The discovery of an array of 23 different genes (variants), encoding a SAG
family of GPI-anchored proteins (no homology to Toxoplasma this time.) expressed
on the surface of E. tenella merozoites, cannot be currently be evaluated in
relation to vaccine development, but may underline the complexity of our quest
to find a vaccine based on only few components covering the major species of
Eimeria in chickens (15).
Immunogenic soluble/cytoplasmic proteins, such as LDH and enzymes from the
anti-oxidant pool (SOD and 1Cys-peroxidoxin), have been shown to be promising
candidates, since they are recognized by T-cells, CD4+, and CD8+, producing
IFN-g. Partial protection was elicited in vaccinated chickens using Salmonella
typhimurium expressed genes (16).
As said above, the presentation of all candidate antigens to the host immune
system is decisive for inducing good levels of protection.
Lillehoj and coworkers have concentrated their work on this issue as reviewed in
(13). A series of cytokine genes were reported to enhance the effect of the
E.acervulina 3-1E or EtMic2 vaccination, although there was apparently no
consensus cytokine that promoted protective effects for both antigens.
DNA application is more frequently reported. Song et al (17) and Min et al. (18)
used pcDNA3-1E plasmid and co-injected it with cytokine genes in day-old chicks;
however, protection was minimal (<30% oocyst reduction). Wu et al. (19) found
Et1A and TA4 genes effective due to improved weight gain and >60% reduction
in oocyst output.
DNA plasmid deposition was applied using Salmonella typhimurium bacteria (14),
achieving a 50% oocyst reduction after challenge with a pcDNA5401 plasmid.
Scientifically, these approaches will stimulate our thinking about immune
mechanisms involved, the role of cytokines in maturation of an adaptive
response, and antigen processing. Practical cost-effective solutions for a mass
application of Eimeria vaccines will most probably not come from these
approaches. Viral vector vaccines are, in my opinion, the best solution, but
still a number of obstacles have to be overcome. Herpes and Pox viruses are able
to harbor the insert sizes needed to express multiple genes, which are necessary
to control the multiple species involved. Fowl pox and Herpes virus of turkeys
(HVT) are possible candidates (4,20). We have designed cassettes containing
three Eimeria genes, each under the control of its own promoter in HVT, whereby
each of the genes was expressed efficiently.
This indicates that we have the tools to construct vector vaccines for multiple
antigen expression. Proof of principle to find the effective combination needs
further study.
Toxoplasma gondii vaccines
In contrast to the vast experience with vaccination
against Eimeria in chickens, only limited data are published on the induction of
protection against Toxoplasma-related disease in mammals.
Toxoplasma infections occur in nearly all warm-blooded animals, including man.
Apart from felids that can act as definitive host (sexual cycle producing
oocysts) and intermediate host, the remaining hosts are only intermediate hosts
acquiring the infection by ingestion of oocysts or tissue cysts contained in
food (muscle, brain or organs of chronically infected animals). The tachyzoites
are the pathogenic stage of the parasite causing high fever, strong inflammatory
reactions, and inducing abortion or birth of the offspring with neurological
symptoms by the passage from the pregnant host to the fetus. Due to the innate
immunity response of the host, the tachyzoite stage converts into bradyzoites
embedded in tissue cysts, after which a life long immunity is established.
Immunedepression may cause recrudescence, but in man this is only seen in HIV
patients or in patients under post-transplantation treatments, and this seems to
be caused by vast impairment of macrophage function in the brain (21). There is
vertical transmission of the parasite, but in sheep, for instance, solid
immunity is induced after the primary infection and subsequent pregnancies do
not induce recrudescence (22). Some conflicting papers (23) detected high
percentage of familial abortion in sheep, but no association to Toxoplasma was
evidenced.
When a primary infection is acquired during pregnancy, the embryo is seriously
at risk due to a very efficient infection of placenta and fetal tissue.
In humans it is generally believed that most ocular toxoplasmosis is caused by
congenital infection, but recent studies have shown that ocular lesions are seen
mostly in postnatally acquired infection (24,25).
In animals, the main health problems are abortions in sheep and goats (26). In
UK and Spain, the prevalence of Toxoplasma in sheep is high and it is
responsible for 25% of abortions (26,28). This is probably true in most
sheep-producing countries. Cattle seem to be less efficiently infected by
Toxoplasma gondii (27), and cats usually do not develop clinical symptoms.
Prevalence in pigs is very variable, and very much dependent on management and
facility sanitation. In Argentina, indoor reared pigs only had 4%
seroprevalence, whereas outdoor reared sows were 100% positive (29).
It is generally accepted that the main source for human infection is eating
infected pork or lamb. Since indoor farming started to be practiced in Europe,
the prevalence in pigs and humans has dramatically decreased, with only a
population of young women at risk, since most are seronegative up to
child-bearing age (30).
A risk factor for acquiring Toxoplasma infection also seems to be pregnancy
itself. Dramatic changes in the CD4/CD8 ratio and reduced functionality of
macrophages and NK cells especially in the third trimester of gestation render a
state of high susceptibility for infections such as T. gondii (31). In a recent
multicenter study in Europe 62% of women seroconverted during pregnancy, with
18.5% of the children acquired congenital toxoplasmosis (32).
Vaccine research for farm animals has focused on the control of abortions in
sheep and goats, and the only commercial vaccines available are live vaccines
comprising the S48 strain of T. gondii (Ovilis®Toxovax, Intervet) (Toxovax,
Agvax New Zealand) (33). This strain is peculiar, as it does no longer produce
tissue cysts ,and it is regarded as a deficient strain, that can be safely used
as a live vaccine for livestock. The product is registered for use in sheep, and
reduces the risk of abortion due to T. gondii infection (33). It is applied
either intramuscularly or subcutaneously no later than three weeks prior to
mating. In a challenge study it was shown that the duration of immunity was of
at least 18 months (59). This could indicate that the parasite deposited
antigenic materials at remote sites, which are able to sustain the immunity. The
S48 strain was attenuated by serial passages for over 30 yrs. Mutagenesis has
also been used and has resulted in temperature-sensitive variants, which optimal
proliferation temperature is 28-32 C. These strains (TS4 mutant) (34) could be
used as a vaccines, but have not been developed commercially.
A bradyzoite-based vaccine was developed using a mutant T263, deficient in
sexual replication. This product could be used as a vaccine to reduce oocyst
production in cats. It needed storage in liquid nitrogen and was applied orally
to cats by a straw.
Vaccines for humans probably will not be based on live attenuated parasites.
Recombinant approaches could result in defined products of sustainable quality.
In this respect, progress can be made along the lines of immunology and the
functional role of proteins in the life cycle of the parasite.
The completion of the Toxoplasma genome, the availability of extensive EST
databases, and further detailed studies of stage conversion mechanisms have
elucidated different host-parasite interactions that could be of value for the
future development of vaccines based on individual proteins, genes or
combinations of these.
Some success is reported from latest studies using DNA-encoding granule-dense
antigens in conjunction with SAG1 DNA-plasmids (35). Almost 90% reduction of
mortality after a lethal challenge was achieved in mice, and a significant
reduction of tissue cyst establishment was produced. Similarly, the use of SAG1
DNA-plasmids reduced cysts numbers, but no effect was detected on vertical
transmission (36). The latter indicates that even few tissue cysts can be
responsible for the generation of fetus infection.
Although such studies are performed in animal models, this is certainly
encouraging for future development of vaccines for women during their
reproductive phase of live. However, the progress mentioned in molecular systems
driving stage conversion, such as the role of Hsp90, especially in the
conversion of tissue cysts to tachyzoites, may also provide alternative ways to
fight the disease. Also, drugs that could interfere in this process could be
developed. The main challenge will be how to cross the blood-brain barrier (37).
Neospora caninum vaccines
Neospora caninum has been recognized as the most
commonly diagnosed cause of abortion in cattle, after its discovery in 1984
(38). Its life cycle is very similar to that of T. gondii, with asexual
multiplication through tachyzoites and bradyzoites in different mammals –
though mainly ruminants – and sexual multiplication in canids, such as the
dog. It was demonstrated that coyotes are part of a sylvatic cycle (39).
Although extensively studied, there is no indication that human infections occur
on any significant scale. Very low seroprevalence is detected in man and no
relation to any pregnancy problems could be found (38).
Cattle do mount an effective immune response protecting the fetus from aborting,
although repeated abortions may occur in 5% of the animals. Previously infected
animals have a greater chance of abortion than seronegative animals due to
recrudescence of existing infection during pregnancy, whereas exogenous
infections from dog-spread oocysts are relatively rare (40). The efficiency of
transplacental transmission is over 90%, which makes this disease hard to
control, since no drugs are available against the acute phase, and certainly not
the tissue cyst stage.
The negative effects of N. caninum infection are not only abortion, but also
embryo mortality, reduction in milk production, higher culling rate, birth of
calves with congenital abnormalities, and decreased growth rate (60). The
consequences of N. caninum infection depend on the time of gestation in which
the parasitemia occurs (44). Infection during late gestation seldom results in
abortion, but in the birth of congenitally infected calves or calves with
congenital abnormalities.
Control measures are focused on reducing the chance of infection during
gestation and on preventing the vertical spread of infection by not breeding
infected cows. Intensive systems of screening of serum or milk tank antibody
levels have been developed to identify farms at risk, but these systems require
guidance and are very costly.
Vaccination therefore seems to provide the best tool for long lasting control of
neosporosis.
As with the use of live vaccines against Toxoplasma-induced abortions, it was
also shown that experimental infection of naïve animals prior to pregnancy can
reduce or even prevent abortion if challenged at 10 weeks of gestation (41), and
can even reduce vertical transmission if animals were challenged around 130 days
of gestation (42). However, no effect was seen on the recrudescing infection
during mid-gestation in already infected animals (41).
This indicates that vaccination with live tachyzoites is feasible in
seronegative animals. However, since no attenuated strain of Neospora caninum
that would not induce a chronic infection is available, no such vaccine was
developed. Some naturally low pathogenicity strains may be used for this
purpose, as reported for the Nowra strain in Australia (43). Moreover, the main
target is to develop a vaccine that prevents vertical transmission of the
parasite in previously infected animals and not only in naïve animals.
Since the disease is associated to immunological changes that occur during
gestation, the main focus of research has been on the immunological responses of
both the cow and the fetus during the whole gestational period in relation to
the effects needed to control an active or reactivated infection of N. caninum.
Innes et al (44) reviewed the state of the art in this subject, and concluded
that the reduction of maternal T-cell mediated immunity or the tilting of the
balance towards the Th-2 type of response reduces the ability of the mother to
generate IFN-gamma, TNF-alpha and other typical responses of the
anti-inflammatory repertoire, which clears the way for the parasite to
proliferate more or less uncontrolled and pass to the foetus.
The timing of this passage during gestation determines the outcome of infection.
Early infection leads to high innate IFN-g response, resulting in abortion or
mummification. Infection during mid-gestation is accompanied by a reduction in
maternal response and the outcome is mainly abortion. When the infection occurs
late in gestation, the immune system of the fetus is already able to cope with
the infection and a healthy but infected calf is born (44).
Congenital acquired infection leads to life-long infection as a postnatal
infection would, but the immune status of the animal may differ according to the
timing of infection acquisition. There may be a point during which tolerance
converts into responsiveness.
In Bovine Viral Diarrhoea (BVD), disease-tolerant calves are known to carry BVD
virus when infected before gestational day 120; these calves do not produce
antibodies, but shed virus during their entire life (45).
The immunological studies have been mainly carried out in rodent models for the
availability of different cytokines, antibodies, and knockout strains. These
models confirm the consensus hypothesis that the key cytokines during pregnancy
are IL-4 and IL-10, which modulate the response towards a Th-2 bias (46).
Cattle were vaccinated with killed tachyzoites, and this was found to be
effective in reducing the chance of abortion. The commercial vaccine resulting
from these studies (Bovilis® Neoguard or in USA called NeoGuard, Intervet)
consists of killed tachyzoites with Havlogen adjuvant in an oil-in-water
emulsion given subcutaneously to 1-3 months pregnant cattle (47). Romero et al.
(48) applied this vaccine in farms in Costa-Rica and found a 50% reduction in
the risk of abortion in vaccinated animals (total n=876). Most abortions
occurred during 5-6 months of gestation, which is consistent with earlier
observations. Heuer et al. (47) used the same vaccine in New Zealand, and
demonstrated similar efficacy. Interestingly, Heuer was able to detect a
vaccination effect in seropositive cattle as well as seronegative cattle,
although sample size was too small to be significant. Killed tachyzoite
preparations were also efficacious in reducing transplacental transmission in
pregnant mouse studies (49), whereas no data are reported that this is also true
for cattle.
Rodent studies showed that recombinant antigens can be effective either as
proteinaceous vaccine for SRS2 (50), for combinations of SAG1, SRS2 and DG1 and
DG2 (51) or as plasmid-DNA for SAG1 and SRS2 (52), or presented by vaccinia
virus for SRS2 (53). The extrapolation of such studies to the problems in cattle
needs to be further studied.
In conclusion, N. caninum is an important cause of abortion and congenital
infection of cattle. Although a killed vaccine is available, which reduces the
chance of abortion, prevention of transplacental infection is the final
challenge. Such vaccine could derive from defined antigens, which should be
tested in the final host. A vaccine for dogs has limitations, since other canids
or stray dogs can also transmit the infection, and are sources which are hard to
control.
Sarcocystis neurona vaccine for horses
Sarcocystis neurona is the causative agent of a
neurological disease in horses known as EPM (equine protozoal
myeloencephalitis), observed especially in the Americas. The definitive host for
this coccidian parasite is the opossum. Horses acquire the disease from
sporocysts spread by roaming opossums. Although until recently parasites were
detected only in horses that were severely immunocompromised (54), Rossano et al
(55) described the culture of viable merozoites from the blood of an
immunocompetent horse artificially infected with sporocysts obtained from
opossum. The incidence of clinical EPM in the USA is rather low (<0.15%), but
over 40% of horses are seropositive in areas where the opossum is prevalent
(56). It is suggested that stress factors induced by transportation or heat can
elicit the clinical phase of the disease, but that has not been proven yet.
Cutler et al. (57) showed that dexamethasone-treated horses developed
neurological symptoms when infected, but the parasite could not be detected as
the unambiguous cause of the diseased state.
Notwithstanding the low chance of clinical disease, a vaccine has been
conditionally launched in USA consisting of killed merogonic stages.
The efficacy is not documented since no challenge model is established. Field
serology seems to be interfered by vaccination titers (58).
So, in conclusion, coccidial parasites are highly immunogenic and, due to their
role in causing disease in animals and man, have been target for development of
vaccines based on either live or killed parasites. Due to their diversity in
species and hosts, vaccination not always the first method of choice, and new
drugs are needed. However, there has been progress in the development of the
current solutions into more sustainable products for the future. The increasing
knowledge of molecular processes by unraveling of the genomic organization of
the first three genera will pave the path for the development of more defined
therapeutics and vaccines.
ACKNOWLEDGEMENTS
I would like to thank Drs. T. Schetters, R. Koopman,
and J. Munoz-Bielsa for their helpful comments on this manuscript. I thank the
organizers of the 9th International Coccidiosis Conference, Foz do Iguaçu,
Brazil, for inviting me to present this review paper.
Reference List 1. Coombs,G.H. & Muller,S. (2002) Recent advances
in the search for new anti-coccidial drugs. International Journal for Parasitology
32, 497-508.
2. Vonlaufen,N., Gianinazzi,C., Muller,N., Simon,F., Bjorkman,C., Jungi,T.W.,
Leib,S.L., & Hemphill,A. (2002) Infection of organotypic slice cultures
from rat central nervous tissue with Neospora caninum: an alternative approach
to study host-parasite interactions. International Journal for Parasitology
32, 533-542
3. Gauly,M., Krauthahn,C., Bauer,C., & Erhardt,G. (2001) Pattern of Eimeria
oocyst output and repeatability in naturally infected suckling Rhon lambs. J.Vet.Med.B
Infect.Dis.Vet.Public Health 48, 665-673.
4. Vermeulen,A.N. (2004) Avian coccidiosis: a disturbed host-parasite relationship
to be restored. Symp.Soc.Exp.Biol. 211-241.
5. Gore,T.C., Long,P.L., Kogut,M., & Johnson,J. (1983) Attenuation of Eimeria
necatrix and E. tenella of U.S. origin by serial embryo passage. Avian Dis.
27, 569-576.
6. Williams,R.B. (2002) Anticoccidial vaccines for broiler chickens: pathways
to success. Avian Pathol. 31, 317-353.
7. Kawazoe,U., Bordin,E.L., de Lima,C.A., & Dias,L.A.V. (2005) Characterisation
and histopathological observations of a selected Brazilian precocious line of
Eimeria acervulina. Veterinary Parasitology 131, 5-14.
8. Li,G.Q., Kanu,S., Xiang,F.Y., Xiao,S.M., Zhang,L., Chen,H.W., & Ye,H.J.
(2004) Isolation and selection of ionophore-tolerant Eimeria precocious lines:
E. tenella, E. maxima and E. acervulina. Veterinary Parasitology 119, 261-276.
9. Vermeulen,A.N., Schaap,D.C., & Schetters,T. (2001) Control of coccidiosis
in chickens by vaccination. Veterinary Parasitology 100, 13-20.
10. Smith,A.L., Hesketh,P., Archer,A., & Shirley,M.W. (2002) Antigenic diversity
in Eimeria maxima and the influence of host genetics and immunization schedule
on cross-protective immunity. Infect.Immun. 70, 2472-2479.
11. Witcombe,D.M., Ferguson,D.J.P., Belli,S.I., Wallach,M.G., & Smith,N.C.
(2004) Eimeria maxima TRAP family protein EmTFP250: subcellular localisation
and induction of immune responses by immunisation with a recombinant C-terminal
derivative. International Journal for Parasitology 34, 861-872.
12. Vermeulen,A.N. (1998) Progress in recombinant vaccine development against
coccidiosis. A review and prospects into the next millennium. Int.J.Parasitol.
28, 1121-1130.
13. Dalloul,R.A. & Lillehoj,H.S. (2005) Recent advances in immunomodulation
and vaccination strategies against coccidiosis. Avian Dis. 49, 1-8.
14. Du,A. & Wang,S. (2005) Efficacy of a DNA vaccine delivered in attenuated
Salmonella typhimurium against Eimeria tenella infection in chickens. International
Journal for Parasitology 35, 777-785.
15. Tabares,E., Ferguson,D., Clark,J., Soon,P.E., Wan,K.L., & Tomley,F.
(2004) Eimeria tenella sporozoites and merozoites differentially express glycosylphosphatidylinositol-anchored
variant surface proteins. Molecular and Biochemical Parasitology 135, 123-132.
16. Kuiper, C. M., Roosmalen-Vos, S. v., Beek-Verhoeven, N. vd., Schaap, T.
C., and Vermeulen, A. N. Eimeria tenella anti-oxidant proteins: differentially
expressed enzymes with immunogenic properties. Proceedings of the VIIIth International
Coccidiosis Conference, Palm Cove, Australia.J.T.Ellis, A.M.Johnson, D.A.Morrison,
N.C.Smith eds. 102-103. 2001.
17. Song,K.D., Lillehoj,H.S., Choi,K.D., Yun,C.H., Parcells,M.S., Huynh,J.T.,
& Han,J.Y. (2000) A DNA vaccine encoding a conserved Eimeria protein induces
protective immunity against live Eimeria acervulina challenge. Vaccine 19, 243-252.
18. Min,W., Lillehoj,H.S., Burnside,J., Weining,K.C., Staeheli,P., & Zhu,J.J.
(2001) Adjuvant effects of IL-1[beta], IL-2, IL-8, IL-15, IFN-[alpha], IFN-[gamma]
TGF-[beta]4 and lymphotactin on DNA vaccination against Eimeria acervulina.
Vaccine 20, 267-274.
19. Wu,S.Q., Wang,M., Liu,Q., Zhu,Y.J., Suo,X., & Jiang,J.S. (2004) Construction
of DNA vaccines and their induced protective immunity against experimental Eimeria
tenella infection. Parasitol.Res. 94, 332-336.
20. Cronenberg,A.M., van Geffen,C.E., Dorrestein,J., Vermeulen,A.N., & Sondermeijer,P.J.
(1999) Vaccination of broilers with HVT expressing an Eimeria acervulina antigen
improves performance after challenge with Eimeria. Acta Virol. 43, 192-197.
21. Gazzinelli,R.T., Eltoum,I., Wynn,T.A., & Sher,A. (1993) Acute cerebral
toxoplasmosis is induced by in vivo neutralization of TNF-alpha and correlates
with the down-regulated expression of inducible nitric oxide synthase and other
markers of macrophage activation. J.Immunol. 151, 3672-3681.
22. Munday,B.L. (1972) Transmission of Toxoplasma infection from chronically
infected ewes to their lambs. Br.Vet.J. 128, lxxi-lxxii.
23. Williams,R.H., Morley,E.K., Hughes,J.M., Duncanson,P., Terry,R.S., Smith,J.E.,
& Hide,G. (2005) High levels of congenital transmission of Toxoplasma gondii
in longitudinal and cross-sectional studies on sheep farms provides evidence
of vertical transmission in ovine hosts. Parasitology 130, 301-307.
24. Holland,G.N. (2003) Ocular toxoplasmosis: a global reassessment. Part I:
epidemiology and course of disease. Am.J.Ophthalmol. 136, 973-988.
25. Silveira,C., Belfort,R., Jr., Muccioli,C., Abreu,M.T., Martins,M.C., Victora,C.,
Nussenblatt,R.B., & Holland,G.N. (2001) A follow-up study of Toxoplasma
gondii infection in southern Brazil. Am.J.Ophthalmol. 131, 351-354.
26. Buxton,D. (1998) Protozoan infections (Toxoplasma gondii, Neospora caninum
and Sarcocystis spp.) in sheep and goats: recent advances. Vet.Res. 29, 289-310.
27. Munday,B.L. (1978) Bovine toxoplasmosis: experimental infections. Int.J.Parasitol.
8, 285-288.
28. Pereira-Bueno,J., Quintanilla-Gozalo,A., Perez-Perez,V., Alvarez-Garcia,G.,
Collantes-Fernandez,E., & Ortega-Mora,L.M. (2004) Evaluation of ovine abortion
associated with Toxoplasma gondii in Spain by different diagnostic techniques.
Veterinary Parasitology 121, 33-43.
29. Venturini,M.C., Bacigalupe,D., Venturini,L., Rambeaud,M., Basso,W., Unzaga,J.M.,
& Perfumo,C.J. (2004) Seroprevalence of Toxoplasma gondii in sows from slaughterhouses
and in pigs from an indoor and an outdoor farm in Argentina. Veterinary Parasitology
124, 161-165.
30. Kortbeek,L.M., De Melker,H.E., Veldhuijzen,I.K., & Conyn-Van Spaendonck,M.A.
(2004) Population-based Toxoplasma seroprevalence study in The Netherlands.
Epidemiol.Infect. 132, 839-845.
31. Avelino,M.M., Campos,D., Jr., Parada,J.B., & Castro,A.M. (2004) Risk
factors for Toxoplasma gondii infection in women of childbearing age. Braz.J.Infect.Dis.
8, 164-174.
32. Gilbert,R. & Gras,L. (2003) Effect of timing and type of treatment on
the risk of mother to child transmission of Toxoplasma gondii. BJOG: An International
Journal of Obstetrics and Gynaecology 110, 112-120.
33. Buxton,D. & Innes,E.A. (1995) A commercial vaccine for ovine toxoplasmosis.
Parasitology 110 Suppl, S11-S16.
34. Dubey,J.P. (1996) Strategies to reduce transmission of Toxoplasma gondii
to animals and humans. Vet.Parasitol. 64, 65-70.
35. Mevelec,M.N., Bout,D., Desolme,B., Marchand,H., Magne,R., Bruneel,O., &
Buzoni-Gatel,D. (2005) Evaluation of protective effect of DNA vaccination with
genes encoding antigens GRA4 and SAG1 associated with GM-CSF plasmid, against
acute, chronical and congenital toxoplasmosis in mice. Vaccine 23, 4489-4499.
36. Couper,K.N., Nielsen,H.V., Petersen,E., Roberts,F., Roberts,C.W., &
Alexander,J. (2003) DNA vaccination with the immunodominant tachyzoite surface
antigen (SAG-1) protects against adult acquired Toxoplasma gondii infection
but does not prevent maternofoetal transmission. Vaccine 21, 2813-2820.
37. Echeverria,P.C., Matrajt,M., Harb,O.S., Zappia,M.P., Costas,M.A., Roos,D.S.,
Dubremetz,J.F., & Angel,S.O. (2005) Toxoplasma gondii Hsp90 is a Potential
Drug Target Whose Expression and Subcellular Localization are Developmentally
Regulated. Journal of Molecular Biology 350, 723-734.
38. Dubey,J.P. & Lindsay,D.S. (1996) A review of Neospora caninum and neosporosis.
Veterinary Parasitology 67, 1-59.
39. Gondim,L.F.P., McAllister,M.M., Pitt,W.C., & Zemlicka,D.E. (2004) Coyotes
(Canis latrans) are definitive hosts of Neospora caninum. International Journal
for Parasitology 34, 159-161.
40. Davison,H.C., Otter,A., & Trees,A.J. (1999) Estimation of vertical and
horizontal transmission parameters of Neospora caninum infections in dairy cattle.
International Journal for Parasitology 29, 1683-1689.
41. Williams,D.J.L., Guy,C.S., Smith,R.F., Guy,F., McGarry,J.W., McKay,J.S.,
& Trees,A.J. (2003) First demonstration of protective immunity against foetopathy
in cattle with latent Neospora caninum infection. International Journal for
Parasitology 33, 1059-1065.
42. Innes,E.A., Wright,S.E., Maley,S., Rae,A., Schock,A., Kirvar,E., Bartley,P.,
Hamilton,C., Carey,I.M., & Buxton,D. (2001) Protection against vertical
transmission in bovine neosporosis. International Journal for Parasitology 31,
1523-1534.
43. Miller,C., Quinn,H., Ryce,C., Reichel,M.P., & Ellis,J.T. (2005) Reduction
in transplacental transmission of Neospora caninum in outbred mice by vaccination.
International Journal for Parasitology 35, 821-828.
44. Innes,E.A., Andrianarivo,A.G., Bjorkman,C., Williams,D.J.L., & Conrad,P.A.
(2002) Immune responses to Neospora caninum and prospects for vaccination. Trends
in Parasitology 18, 497-504.
45. McClurkin,A.W., Littledike,E.T., Cutlip,R.C., Frank,G.H., Coria,M.F., &
Bolin,S.R. (1984) Production of cattle immunotolerant to bovine viral diarrhea
virus. Can.J.Comp Med. 48, 156-161.
46. Quinn,H.E., Miller,C.M.D., & Ellis,J.T. (2004) The cell-mediated immune
response to Neospora caninum during pregnancy in the mouse is associated with
a bias towards production of interleukin-4. International Journal for Parasitology
34, 723-732.
47. (2004) Intervet symposium: Bovine neosporosis. Veterinary Parasitology 125,
137-146.
48. Romero,J.J., Perez,E., & Frankena,K. (2004) Effect of a killed whole
Neospora caninum tachyzoite vaccine on the crude abortion rate of Costa Rican
dairy cows under field conditions. Vet.Parasitol. 123, 149-159.
49. Jenkins,M.C. (2001) Advances and prospects for subunit vaccines against
protozoa of veterinary importance. Veterinary Parasitology 101, 291-310.
50. Pinitkiatisakul,S., Mattsson,J.G., Wikman,M., Friedman,M., Bengtsson,K.L.,
Stahl,S., & Lunden,A. (2005) Immunisation of mice against neosporosis with
recombinant NcSRS2 iscoms. Veterinary Parasitology 129, 25-34.
51. Cho,J.H., Chung,W.S., Song,K.J., Na,B.K., Kang,S.W., Song,C.Y., & Kim,T.S.
(2005) Protective efficacy of vaccination with Neospora caninum multiple recombinant
antigens against experimental Neospora caninum infection. Korean J.Parasitol.
43, 19-25.
52. Cannas,A., Naguleswaran,A., Muller,N., Eperon,S., Gottstein,B., & Hemphill,A.
(2003) Vaccination of mice against experimental Neospora caninum infection using
Nc. Parasitology 126, 303-312.
53. Nishikawa,Y., Xuan,X., Nagasawa,H., Igarashi,I., Fujisaki,K., Otsuka,H.,
& Mikami,T. (2001) Prevention of vertical transmission of Neospora caninum
in BALB/c mice by recombinant vaccinia virus carrying NcSRS2 gene. Vaccine 19,
1710-1716.
54. Long,M.T., Mines,M.T., Knowles,D.P., Tanhauser,S.M., Dame,J.B., Cutler,T.J.,
MacKay,R.J., & Sellon,D.C. (2002) Sarcocystis neurona: parasitemia in a
severe combined immunodeficient (SCID) horse fed sporocysts. Experimental Parasitology
100, 150-154.
55. Rossano,M.G., Schott II,H.C., Murphy,A.J., Kaneene,J.B., Sellon,D.C., Hines,M.T.,
Hochstatter,T., Bell,J.A., & Mansfield,L.S. (2005) Parasitemia in an immunocompetent
horse experimentally challenged with Sarcocystis neurona sporocysts. Veterinary
Parasitology 127, 3-8.
56. Dubey,J.P., Lindsay,D.S., Saville,W.J.A., Reed,S.M., Granstrom,D.E., &
Speer,C.A. (2001) A review of Sarcocystis neurona and equine protozoal myeloencephalitis
(EPM). Veterinary Parasitology 95, 89-131.
57. Cutler,T.J., MacKay,R.J., Ginn,P.E., Gillis,K., Tanhauser,S.M., LeRay,E.V.,
Dame,J.B., & Greiner,E.C. (2001) Immunoconversion against Sarcocystis neurona
in normal and dexamethasone-treated horses challenged with S. neurona sporocysts.
Veterinary Parasitology 95, 197-210.
58. Duarte,P.C., Daft,B.M., Conrad,P.A., Packham,A.E., Saville,W.J., MacKay,R.J.,
Barr,B.C., Wilson,W.D., Ng,T., Reed,S.M., & Gardner,I.A. (2004) Evaluation
and comparison of an indirect fluorescent antibody test for detection of antibodies
to Sarcocystis neurona, using serum and cerebrospinal fluid of naturally and
experimentally infected, and vaccinated horses. J.Parasitol. 90, 379-386.
59. Buxton, D., Thomson K.M., Maley, S., Wright, S., Bos, H.J. (1993) Experimental
challenge of sheep 18 months after vaccination with a live (S48) Toxoplasma
gondii vaccine. The Veterinary Record 133, 310-312.
60. Trees AJ, Davison HC, Innes EA, Wastling JM. (1999)Towards evaluating the
economic impact of bovine neospororsis. Int J Parasitol 29:1195-1200.