Escolar Documentos
Profissional Documentos
Cultura Documentos
t Deceased.
Advances in
PARASITOLOGY
Edited by
J.R. BAKER
Royal Society of Tropical Medicine and Hygiene,
London, England
R. MULLER
International Institute of Parasitology,
St Albans, England
and
D. ROLLINSON
The Natural History Museum,
London, England
VOLUME 39
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Vlll PREFACE
Following this discussion, there is a full treatment by Graham Coombs and his
colleagues at the University of Glasgow, UK, of the biochemistry of the coccidia in
general, including the tissue cyst-forming species of Toxoplasma and Sarcocystis
and the monoxenous genera Eimeria and Cryptosporidium. The chapter considers
the metabolic processes concerned with energy production, proteins and amino
acids, polyamines, purines, pyrimidines, nucleic acids and lipids. There are also
sections on cultivation in vitro, parasite-host cell interactions, and anticoccidial
drugs. The authors conclude by emphasizing that much still remains to be eluci-
dated about the biochemistry of these perhaps rather neglected but increasingly
important parasites.
Advances in our understanding of molecular biology have provided opportu-
nities for the genetic manipulation of parasites. This is a new and challenging area
of research which is currently attracting a great deal of attention from parasitol-
ogists. John Kelly from the London School of Hygiene and Tropical Medicine, who
has been directly involved with many recent research developments, provides an
authoritative overview of progress in genetic transformation of parasitic protozoa.
In this context transformation is considered as the stable acquisition by a cell of a
new gene(s) by uptake of foreign DNA. The first successful stable transformation
of a parasitic protozoan was achieved with Leishmania, and in this chapter parti-
cular attention is given to work with the trypanosomatids Leishmania, Trypano-
soma brucei and Trypanosoma cruzi. Rapid progress has led to research on gene
targeting, functional analysis of genes and analysis of gene expression. One area of
particular interest has been the application of genetic manipulation to elucidate
mechanisms of drug resistance. Other sections deal with the development of
transfection techniques for Toxoplasma and Plasmodium and the intestinal proto-
zoa Entamoeba histolytica and Giardia lamblia.
The final chapter in this volume is a detailed and important contribution by
Patricia Coulson from the University of York, UK, concerning radiation-attenuated
vaccines against schistosomes in animal models. A vaccine against schistosomiasis
is not yet available and control of the disease depends upon many factors such as
health education, snail control and chemotherapy. The need for a vaccine is well
recognized and, despite considerable progress in recent years, much still needs to be
done to understand the immunological response of the human host and the ability of
the parasite to survive within the host. The irradiated vaccine model, which is
unlikely to be suitable for use in humans, can provide a useful example for the
development of a recombinant antigen vaccine. This chapter provides a compre-
hensive account of work associated with radiation-attenuated vaccines, the induc-
tion of immunity and immune effector mechanisms, and concludes with ideas
concerning future research directions.
JOHN BAKER
RALPH MULLER
DAVID ROLLINSON
Clinical Trials of Malaria Vaccines:
Progress and Prospects
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1. The need for a vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2. Aims of a vaccine and immunological constraints .................... 5
1.3. Phases in the evaluation of a malaria vaccine ....................... 7
1.4. Assessing vaccine efficacy: trial end points ......................... 9
1.5. Synthetic peptides for vaccine inclusion require carriers
and adjuvants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.6. New generation nucleic acid (DNA) vaccines ....................... 13
1.7. Strategies for the development of a malaria vaccine . . . . . . . . . . . . . . . . .15
2. Pre-erythrocytic (Infection-blocking) Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.1. The circumsporozoite (or CS) protein and T-cell epitopes . . . . . . . . . . . . . .18
2.2. Cytotoxic T lymphocytes (CTL) for vaccine design . . . . . . . . . . . . . . . . . . . 23
2.3. SSP-PITRAP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.4. Liver stage antigen 1 (LSA-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3. Asexual Blood-stage Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3.1. Role of antibodies? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.2. The problem of parasite antigenic diversity ........................ 29
3.3. MSA-1 and MSA-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.4. RESA/Pfl55 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.5. Other target antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.6. The first chemically synthesized vaccine against P. falciparurn: SPf66 .... 37
3.7. Immune responses to SPf66 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
1. INTRODUCTION
The traveller telephoned to ask where he could obtain the malaria vaccine
that he had read about in the national press. Hopefully a reality in the near
future, but this was April 1993 and a report on the successful outcome of
the first human field trials with the SPf66 malaria vaccine in South America
had just been published (Valero et al., 1993). This was certainly a mile-
stone in the history of parasitology and moved the scientific and medical
communities into a mixed wave of admiration, scepticism and criticism.
Not unusual for malaria vaccines, the evolution of which has been long (yet
not so long as Jenner’s smallpox vaccine which had to wait two centuries
before proof of success), convoluted and not without controversy.
Control of malaria represents one of the world’s greatest health chal-
lenges and never before has a malaria vaccine been more urgently required.
Worldwide, the number of cases is rising at a rate of 5% annually. Increas-
ing mosquito resistance to insecticides is a contributor to this effect, as is
parasite multi-drug resistance. Chloroquine-resistant parasites, which first
appeared in South-East Asia and Latin America simultaneously in the early
1960s, now affect almost every country where the disease is endemic. The
current malaria statistics are frightening, with an estimated 300-500 mil-
lion people infected of whom 2.3 million die every year (Sturchler, 1989;
WHO, 1995). The global impact of malaria is estimated as 35 million
DALYs (Disability Adjusted Life-Years; World Bank, 1993), primarily a
result of infection with Plasmodium falciparum.
As the most important parasitic disease in the world, it is disproportio-
nately prevalent in Africa where approximately 90% of the world’s malaria
sufferers are sub-Saharan Africans (WHO, 1995). This has hindered the
drive to develop new and novel antimalarial drugs as the pharmaceutical
industry sees little return in terms of profit in a market confined to the needs
of impoverished populations in the developing world. Similarly, the world
of commerce is unlikely to underwrite the expensive and long-term clinical
trials required for the development of a malaria vaccine. Fortunately,
however, enlightened governments and charities have been forthcoming
with funds.
CLINICAL TRIALS OF MALARIA VACCINES 3
Vaccines have probably prevented more infectious disease than any other
medical intervention except sanitation, and represent the most cost-effec-
tive approach to control. However, within the discipline of infectious
agents, human parasites stand alone in the absence of any commercially
available vaccine. A human malaria vaccine promises to correct this situa-
tion. The earliest attempt to immunize humans dates back to 1936 (Boyd
and Kitchen, 1936). Following this, vaccination attempts made to improve
immunogenicity by including an adjuvant were successful in that they
achieved partial protection of monkeys immunized with killed blood stage
parasites (Freund et al., 1948). A long-standing difficulty in the develop-
ment of a malaria vaccine has been the lack of a small animal model for
studying P. falciparum, the only appropriate model being P. falciparum in
Aotus monkeys. This together with the increasing conservation of non-
human primates and escalating costs of monkeys for experimentation, has
resulted in some delay in the development of a malaria vaccine.
During 1975, the United Nations, the World Bank and the World Health
Organization jointly advertised funding to cover the development of a
malaria vaccine and this led to a concerted effort and competition between
research groups. Sadly, despite promises of a ‘vaccine around the corner’
and a ‘vaccine within the next 5 years’, it was not forthcoming. Not until
1987 that is, when news of the first Patarroyo SPf66 human vaccine trials in
4 C.A. FACER AND M. TANNER
South America were published and the next crucial step was made (Patar-
royo et aZ., 1987, 1988).
Yet 9 years on in 1996, and at an estimated total cost of all malaria
vaccine development of around 700 million dollars, SF‘f66 continues to
undergo efficacy trials. While it is not the vaccine that will reach routine
application, it is the one that, through attracting controversy, is stimulating
vaccine development. However, at present the most realistic of optimists
will not provide a date when they envisage that a commercially produced
malaria vaccine will be available.
For whom is a malaria vaccine being designed? Malaria vaccines can be
envisaged for protection of non-immune travellers and military personnel
who are transiently exposed to risk as compared to infants and children
living in malarious areas. In order to prevent the death of some 1.2 million
children every year in Africa alone, a malaria vaccine would need to be
‘therapeutic’ in that its aim would best be to prevent life-threatening
disease (in individuals with pre-existing blood-stage infections) whilst
allowing the development of natural immunity through repeated re-
infection. This, of course, requires intimate knowledge of what consti-
tutes ‘malarial disease’. In endemic regions, since the majority of the
morbidity and mortality occurs in young children (Greenwood et aZ.
(1987) estimate that one quarter of African children aged 1 - 4 years die
from malaria), they should be the primary targets for vaccination which
should occur as soon as possible after birth.
The practical importance as well as intellectual appeal of a malaria
vaccine is without question. A vaccine would side step the problems of
multi-drug resistance, it would be more convenient than regular prophy-
laxis and would have the advantage of causing few, if any, side effects.
Last, but not least, the vaccine might synergize with existing drugs and
other tools of control such as bednets (Tanner et al., 1995). In any case, for
most endemic areas, a vaccine is not believed to be the only tool but should
become part of integrated control strategies.
There are attendant intellectual concerns as with any other vaccine:
Thus, there are many basic and important questions that require addressing
right from the planning of the first clinical trials.
y NINFECTED
I+
No ymptoms
I f
UNCOMPLICATED MALARIA
Fever, headache, shivers, rigors, vomiting,
diarrhoea and cough
E MALARIA
it lies beyond the bounds of this review other than to highlight its
importance.
The concept of therapeutic vaccines has stimulated considerable interest
and debate within and outside the subject of malaria. It is regarded with
caution by many researchers who believe it impossible for a vaccine to
improve on the natural immune response, a dogma that has been challenged
for more than a century. Yet the immune system can be supercharged by a
vaccine even after infection as shown recently, for example, in people with
herpes, leprosy or leishmaniasis. The final choice of how many and which
parasite epitopes should be included in a vaccine is a formidable challenge.
Serum from malaria-immune adults identifies reacting malaria antigens,
making the assumption that the best antigens for vaccine inclusion are
those that stimulate good humoral responses. However, this approach
might not identify the best immunogens for a T-cell (helper and cytotoxic)
mediated immunity (Table 1). In addition, the immunodominant epitopes
might not be the ones eliciting the most effective immune responses.
CLINICAL TRIALS OF MALARIA VACCINES 7
Include covalently linked T-cell (helper and cytotoxic and B-cell epitopes. Pro-
blem of antigenic polymorphism (sexual reproduction leads to maintenance of and
increase in heterozygosity). Definition of ‘common, cross-reacting epitopes’ .
Be assessed for the epidemiological implications of its coverage; i.e. for its
effectiveness in different epidemological settings.
In any vaccine trial it is essential to have clear-cut end points against which
the success of the vaccine is assessed. Possible end points include asymp-
tomatic infection through to incidence of either mild disease or severe
disease and death (malaria-specific or all causes; see Figure 1). For these
to become acceptable as outcome variables to allow comparison between
trials, it is important that there are agreed approaches that are feasible and
repeatable, standardized definitions of what is meant by mild and severe
disease and criteria established for the identification of possible malaria
deaths. Recent discussions have clearly outlined these difficulties (Alonso
et al., 1995).
The ultimate objective of a malaria vaccine in sub-Saharan Africa is
reduction in mortality and life-threatening malaria. However, as both are
relatively rare events, very large numbers of individuals must be included
in prospective trials in order to detect as effect on these end points. There-
fore most trials must evaluate the efficacy of candidate vaccines against
uncomplicated malaria. Is fever alone a good indicator of uncomplicated
malaria? There are many causes of fever in children in the tropics of which
malaria is one. The relationship between parasite density and the risk of
fever suggests that setting specific parasite density cut-offs should be used
to distinguish malaria from other causes of fever with high specificity at the
individual level (Alonso et al., 1995).
do not contain any infectious material and hence are devoid of biohazard
risk. A further bonus is that they can be rapidly synthesized in unlimited
quantities. On the other hand, one concern surrounding the use of synthetic
peptides for vaccines is that if included in linear form they may fail to
induce antibodies reacting with the cognate protein, a finding recently
reported for a synthetic sporozoite peptide (see Section 2). This is because
linear peptides may not adopt the conformation of the corresponding region
in the native molecule (Fries et al., 1992a). Various strategies have been
used to induce ‘native-like’ conformation in peptide immunogens, for
example cyclization of peptides whereby the two ends of the peptide are
joined by amino groups. The resulting cyclized peptide now lacks the
‘terminal epitopes’ that frequently elicit the formation of useless antibody
(Etlinger and Trzeciak, 1993). However, cyclization does not always
improve the level of conformational mimicry between peptide and intact
protein and should not be construed as a panacea for increasing reactive
antigenicity of exposed loops of protein.
To be efficacious synthetic vaccines must include epitopes capable of
inducing T-cell effector and T-cell helper activity. The lack of this essen-
tial requirement may be, in part, responsible for the limited efficacy of the
early sporozoite subunit vaccines in clinical trials which focused primarily
on B-cell epitopes (Millet et al., 1992). The malaria vaccines in production
today, in order to bypass this problem, contain an adjuvant and a carrier.
Both potentiate immune responses; the adjuvant by stimulating a higher
and more sustained antibody response (by providing a depot of antigen
with subsequent slow release, macrophage activation and activation of T
helper cells), the carrier by circumventing major histocompatibility com-
plex (MHC) restriction in T-cell epitopes contained within the vaccine.
Recognition of carrier epitopes by specific T helper cells will ultimately
induce B lymphocytes to make antibodies against those B-cell epitopes that
are covalently coupled to the carrier. Adjuvants currently licensed for
human use are few. Some, such as alum, are unfortunately poor stimulators
of cellular effector mechanisms, whilst potent stimulators such as Freund’s
complete adjuvant, are too reactogenic.
Novel adjuvants are being investigated (Hui, 1994). For example, lipo-
somes, concentric lipid layers enclosing the malaria antigens and mono-
phosphoryl lipid, proved to be safe and effective in inducing high titres of
anti-sporozoite antibodies in human volunteers (Fries et al. 1992a).
ISCOMS (immunostimulating complexes), large spherical structures where
immunogens are presented as multimers in a matrix of the adjuvant Quil-A,
have been shown to enhance the immunogenicity in rabbits of a malaria
fusion protein comprising the C-terminal repeat subunit (EENV) of the P.
falciparum blood-stage antigen Pf155 or RESA (ring erythrocyte-asso-
ciated antigen; Sjolander et af., 1993). Other advantages of ISCOMS are
12 C.A. FACER AND M. TANNER
groove, competition between T-cell epitopes for ‘best fit’ results. This
could be a concern where vaccines comprise multiple epitopes.
A carrier epitope taken from the same protein as the ‘protective’ epitope
would be a convenient design strategy. Algorithms can ‘screen’ areas of
native protein adjacent to the candidate vaccine sequence and predict
probable T-cell epitopes. However, the inability to predict the MHC range
to which a given epitope will bind is a problem to be faced.
One solution to overcoming antigen recognition would be to find a
‘promiscuous’, malaria T-cell epitope, that is a molecule capable of react-
ing with T cells irrespective of the MHC background. In fact such an
epitope exists in the carboxy terminus of the circumsporozoite (CS) protein
of P.falciparum. This region (amino acid residues 378-398), when added
to T-cell clones from non-exposed Caucasians of varying HLA-DR status,
induced in all of them a MHC-restricted proliferation (Singaglia et al.,
1988). This particular peptide, however, was not recognized by Gambians
(Good et al., 1988a, b) and in only 13% of Australians who had lived in
Papua New Guinea (Zevering et al., 1990). The search for other universal
epitopes on malaria antigens must continue since this strategy does repre-
sent a promising way forward. Their use would mean that a malaria vaccine
would protect many populations irrespective of their MHC haplotype. The
one drawback is that while universal T-cell epitopes are potentially active
with any B-cell epitope conjugated to them, strong adjuvants are some-
times required for the induction of a long-lasting high titred antibody
response (Del Giudice, 1992).
The possibility of a malaria vaccine designed to induce generation of
cytotoxic T lymphocyte (CTL) effectors is attracting attention as advances
in molecular immunogenetics allows analysis of motifs on human HLA
antigens which bind malaria peptides (see Section 2). However, this pre-
sents a daunting approach since, at present, very few peptide, subunit or
recombinant vaccines have been shown to induce CTL against any foreign
antigen in humans. Improved delivery systems are currently under inves-
tigation to rectify this situation including the intramuscular injection of
parasite DNA, shown to be most effective in inducing CTL activity in
animal models (Hoffman, et al., 1995).
plasmid DNA) are injected intramuscularly into the recipient, the muscle
cells, having successfully incorporated the foreign gene, begin producing
(for prolonged periods of time) the target protein, presumably in the exact
or similar conformation as found in the native form.
Delivery of foreign genes to mammalian somatic cells has still to be
perfected. In addition to delivery using plasmid DNA as described above,
others have used DNA cornplexed with specific protein carriers, attached to
cationically charged molecules such as liposomes or calcium salts, or
incorporated into live attenuated carriers such as vaccinia virus.
DNA vaccines have distinct advantages although, by their very nature,
attendant potential disadvantages (Table 3) when compared with recombi-
nant or subunit vaccines. One clear beneficial effect is that DNA vaccina-
tion stimulates all arms of the immune response including cytotoxic T
cells. The parasite protein, because it originates from inside the host cell,
is processed by the MHC class I pathway. Class I molecules carry peptide
fragments of the parasite protein to the cell surface where they stimulate
CD8+ cytotoxic T lymphocytes. By contrast, standard vaccine antigens are
taken up by phagocytosis and processed through the MHC class I1 system
which primarily stimulates antibody responses. Nevertheless, experience to
date with DNA vaccines indicates that, despite this theory, some DNA
vaccines (e.g. the Schistosomu mansoni Sm23 DNA vaccine) fail to elicit a
CTL response stimulating only THO and TH1 responses (D. Ham, personal
communication).
Disadvantages:
introduced (foreign) DNA may become incorporated into host chromosomes and
subsequent potential for a transformation event (DNA triplex formation)
introduced DNA may become incorporated into germ line cells
0 the DNA may stimulate anti-DNA antibodies
unexpected and untoward consequences of the persistent expression of a foreign
antigen therefore difficult to proceed to clinical trials
CLINICAL TRIALS OF MALARIA VACCINES 15
There are four main strategies in the development of a malaria vaccine: (i)
blocking the sporozoite from invading or developing within hepatocytes
(anti-infection);(ii) blocking merozoite invasion of red cells and inhibiting
development of schizonts (anti-disease or asexual stage); (iii) blocking the
adverse pathology-inducing effects of cytokines and parasite sequestration
16 C.A. FACER AND M. TANNER
Several other pre-E stage antigens have been identified: this list is not fully comprehensive. Pf,Plasmodium falciparum; aa, amino
acids; Abs, antibodies; CTL, cytotoxic T lymphocytes; SZ, sporozoite.
18 C.A. FACER AND M. TANNER
bites, they do not develop a sterile immunity. One explanation for this is
the presumed presence of low levels of protective effector cytotoxic T
lymphocytes that could be enhanced by an appropriate CTL-generating
vaccine (Hill et al., 1992).
The several disadvantages associated with attenuated vaccines (see
Table 2), in particular, practical difficulties in producing en m a n e suffi-
cient numbers of sporozoites or infected mosquitoes for irradiation, has
led to the search for immunogenic epitopes on the sporozoite that could
be produced either by recombinant DNA technology or by chemical
synthesis. In addition, the identification of genes encoding proteins shared
by the liver stages should allow incorporation of those genes into the new
DNA vaccines (see Section 5).
A conservative estimate of the number of different proteins synthesized
by asexual stage parasites is > 2000, the genes for only a small proportion
of these having been cloned. What among these proteins are the important
vaccine candidates? (for summary, see Table 4 and Figure 6). Most have
been chosen using either a rational or an empirical approach. In the former,
one selects specific antigens that will have certain predetermined properties
and that will elicit a specific response (e.g. the choice of antigens which
elicit antibodies that react with the surface of merozoites and prevent
merozoite re-invasion of red cells). In order that all variants of natural
parasites will be susceptible to the elicited antibodies, it is essential that the
immunogen is antigenically conserved. In the alternative empirical
approach to vaccine development, antigens are selected solely on their
ability to protect an animal in a vaccine trial without the necessity of
characterizing the properties of that malaria antigen in other assays. The
SPf66 vaccine is an example of this approach (see Section 3).
The early studies of experimental rodent malaria revealed quite clearly that
MHC class I restricted CTL could provide protection against sporozoite
challenge (Romero et af., 1989; Rodriguez et af., 1991) and lyse-infected
hepatocytes in vitro (Hoffman et al, 1989; Weiss et u f . , 1990).
It was also possible to reverse immunity to sporozoite challenge in mice
by experimental depletion of CD8+ cells coupled with neutralization of
interferon gamma (IFNy) (Schofield et a f . , 1987). MHC restriction in CTL
epitopes occurs since, in two congenic strains of mice (expressing between
them five different MHC class I molecules) vaccinated with attenuated
sporozoites, only a single CS epitope (polymorphic) was recognized
(Kumar et u f . , 1989). These original animal experiments provided consid-
erable impetus to attempt to identify CTL against P. fulciparum in humans
and a current review outlines various approaches to the design of a CTL-
inducing malaria vaccine (Lalvani et af., 1994).
The various lines of evidence for protective CTL responses, potential
epitopes and information pertaining to the generation of CTL in vivo, are
summarized in Table 5 . There are now three lines of evidence implicating
the involvement of CTL in protection against P. falciparum in humans.
First, HLA class I restricted CTL responses have been identified in Africans
repeatedly exposed to P. fufciparum sporozoites (Hill et uf., 1991, 1992,
Lalvani et al., 1994, 1996). Although only low levels of precursor CTL
were present in the circulation, these cells could provide some useful
24 C.A. FACER A N D M. TANNER
and is about to enter phase I1 trials (see Section 5 ) . Another group has
adopted an HLA approach to the design of a CTL vaccine, the aim of which
is to identify the most appropriate malaria CTL-stimulating epitopes in
view of the inevitable human HLA class I restriction of CTL responses
(Lalvani et al., 1994; Aidoo et al., 1995).
Using the reverse immunogenetic method, whereby short P. fakiparum
peptides (8-10 amino acids) are synthesized to match the known charac-
teristics of peptides binding in the groove of the particular HLA class I
molecules, 12 CTL epitopes have been identified in naturally exposed
Gambians, in four P. fakiparum pre-erythrocytic antigens: CS protein,
SSP-2, LSA-1 and STARP (Aidoo et al., 1995). Fortunately, the extent
of polymorphism detected in these epitopes appears limited. The HLA
types used for epitope identification, HLA-B53, HLA-B35, HLA-B8,
HLA-B17, HLA-B7 and HLA-A2 (both HLA-A2.1 and HLA-A2.2) were
chosen because of their high frequencies (70-75%) in Caucasians and
Gambians.
The next step is to develop a sufficiently sensitive in vitro efficacy assay
that will measure the CTL-induced killing of P. fakiparum within hepa-
tocytes. Primary pre-erythrocytic stage peptide-specific and MHC-
restricted CTL clones are now being generated from malaria-naive
individuals for analysis of cytotoxicity against freshly isolated and
malaria-parasitized human hepatocytes in which sporozoite invasion and
multiplication occurs (multiplication does not occur in the commonly used
hepatoma cell line) allowing the surface expression of malaria antigens on
these cells.
Finally, Hill and colleagues (1992) plan to incorporate P. fakiparum
CTL-stimulating epitopes into yeast-derived virus-like particles (Ty-VLR)
and to use this construct as a vaccine. Preliminary experiments have shown
that Ty-VLR in mice induce impressive levels of CTL to various foreign
epitopes. With this vaccine approach however, there remains the potential
problem of having to include in the vaccine P. fakiparum CTL epitopes for
all the HLA types within a given population.
2.3. SSP-PITRAP
liver stage protein (Charoenvit et al., 1987; Knusmith et al., 1991) which is
also expressed in the asexual erythrocytic stages (Robson et al, 1990). It
joins the CS protein (P. falciparum, P. yoelii and P. berghei) and the P .
falciparum liver stage antigen 1 (LSA-1) as the third plasmodia1 protein
described that has been cloned and sequenced and to which CD8+ CTL
have been generated.
SSP-2 in P. falciparum (see Figure 3b) shares a RGD(Arg-Gly-Asp)-
binding motif with the CS protein implying perhaps a function in hepato-
cyte recognition, attachment and invasion (Cowan et al., 1992). SSP-2 has
potential as a component of a human malaria vaccine (Knusmith et al.,
1991) since it has, at least in the experimental mouse models, the ability to
generate protective CD8+ CTL (Wizel et al., 1994). The CTL appear to
recognize SSP-2 peptides with class I HLA molecules on the surface of
infected hepatocytes.
One constraint for inclusion into a vaccine, as with most malaria anti-
gens, is the high degree of polymorphism within this protein (Robson et al.,
1990), although two regions, peptides A6 and BH-1 (see Figure 3b) which
generated CTL, appear conserved (Wizel et al., 1994). In a trial with a
small number of exposed Gambians of HLA-B35 and B53 status, 23
synthetic SSP-2 peptides failed to induce CTL responses in vitro, although
peptide BH-1 was not included (Hill et al., 1992).
These studies have now been extended to include a larger number of
Gambian donors and several CD8+ CTL clones have been established
which are specific for peptides on SSP-2 and LSA-1 (see below) despite
the difficulty in producing such clones because of low precursor frequency
in the peripheral circulation and cytokine requirements in culture (A.V.S.
Hill, personal communication). Two conserved peptides (tr42 and tr43)
located in region A6 of SSP-2 are recognized by a Gambian HLA type,
HLA-B8 (Aidoo et al., 1995).
TRAP, in addition to stimulating a CTL response, also stimulates the
production of anti-TRAP antibodies in malaria-exposed individuals whose
epitope specificity and possible association with protection is currently
being assessed in an age-related study in Mali and the Gambia (Scarselli
et al., 1993; K. Robson, personal communication).
Vaccines targeting the asexual blood stages are not expected to prevent
blood-stage infection but rather to control parasite growth. Natural immu-
nity would develop while the vaccine prevented life-threatening disease. It
is an approach that is suited to areas of high malaria transmission where the
priority target is young children, a high-risk group for malaria morbidity
and mortality.
The largest human trials of an asexual blood stage vaccine have been
achieved with SPf66 (see Section 3.6). The vaccine includes several
antigens, the exact parasite location and function of which remain
obscure.
CLINICAL TRIALS OF MALARIA VACCINES 29
(a) Merozoite
MSA-1 (MSP-1, g ~ 1 9 5 ) 195 Merozoite surface Red cell invasion. Many vaccine studies. Holder ( 1993)
Protection against homologous challenge in
Aotus. Gene incorporated into multi-stage
DNA vaccine (NWAC-F'f7)
MSA-2 (MSP-2) 45 Merozoite surface Function unknown. Phase I trial underway Epping et ul. (1988)
MSA-3 (MSP-3) 48 Merozoite surface Function unknown. Conserved repeats. Oeuvray et al. (1994)
Induces protective cytophilic antibodies
RESA 155 Dense granules Stabilization of rbc spectrin. Found in culture Perkins (1992)
supernatants. Weak protection in Aotus
vaccine trial
EBA-175 175 Micronemestapical Red cell invasion. EBA peptide 4 binds to Sim (1995)
end sialic acid on glycophorin A
AMA- 1 83 Rhoptry organelle Red cell invasion? Found in culture superna- Thomas et al. (1 990)
tant. Gene incorporated into multi-stage
DNA vaccine (NYVAC-Pf7)
QF3 (RAP-112) 80142 Rhoptry organelle Red cell invasion? Impressive protection in Crowther et al. (1990)
Saimiri monkeys
SPf66 (a peptide polymer) 5000 Merozoite proteins Immunogenic in human
Plus (NANPh vaccine trials. Protection not
universal
(b) Infected erythrocyte membrane surface
PEMP- 1 250-400 PRBC surface Ligand for cytoadherence to endothelial cells Howard and
Extensive antigenic variation Gilladoga (1989)
Gene cloned (var genes). Important role in Baruch et al. (1995)
cerebral malaria
PfHRP-2 65-75 PRBC surface Histidine-rich Rock et al. (1987)
Protein very stable (identified in Egyptian
mummies)
Rosettin 22-28 PRBC surface Binds to unifected rbc which rosette around Helmby el al. (1993)
PRBC. Gene not cloned. May be involved
in cerebral malaria
Ag332 2000 PRBC surface Monoclonal blocks binding PRBC to Mattei et al. (1992)
endothelial cells. Parts of gene cloned
EGF motifs
t
Repeats(b1ock 2)
f
19kDa
3.4. RESA/Pfl55
The merozoite rhoptry antigens (QF3 complex, RAP-1/2 and apical mem-
brane antigen-1 [AMA-11, Crowther et al., 1990) and a microneme protein,
erythrocyte binding antigen-175 (EBA-175; Sim, 1995), are discharged on
CLINICAL TRIALS OF MALARIA VACCINES 37
Much attention has been focused on the novel synthetic polymeric blood-
stage vaccine, SPf66, designed and developed by Dr Manuel Patarroyo in
Bogota, Colombia, following reports that the vaccine induced significant
protection against blood-stage challenge in Aotus monkeys (Patarroyo et
al., 1987) and in a small group of army volunteers (Patarroyo et al., 1988).
In the latter phase I trial, infection was not prevented but the three volun-
teers had mild infections with a steady decrease in parasite counts (all
parasitaemias were c 0.5%) and total recovery 21 days following
challenge.
Following these encouraging results, extensive phase I11 trials in Latin
America, most of them in Colombia, were performed with the first report of
the vaccine’s success in a randomized double-blind placebo-controlled trial
in 1993 (Valero et al., 1993). This represented a crucial milestone in
malaria vaccine development and provided the first evidence that immu-
nization with this synthetic peptide polymer has the potential of reducing
the risk of clinical malaria in populations under natural exposure to P .
falciparum. Its other attractions were a comparatively low estimated cost
(around 25 US cents for the three doses), stability and ease of administration.
What peptides are included in the construct, how were they chosen and
what is the vaccine’s efficacy under different degrees of malaria trans-
mission?
Patarroyo and his colleagues first identified, with the aid of natural
malaria immune sera, several proteins (and then several constituent pep-
tides) of P. falciparum which provided protection against experimental
infection in monkeys. Three of the most promising peptides, representing
sequences from three P. falciparum blood-stage antigens, formed the basis
of the vaccine. The peptides 35.1 and 55.1 were based on partial sequences
from two, as yet unidentified, P. falciparum molecules. The third peptide,
83.1, corresponds to the highly conserved region 45-53 of MSA- 1. The
construct also includes two NANP sequences from the B-cell immunodo-
minant repeat of the CS protein which apparently are added to create hair-
pin bends in the sequence rather than for their immunogenicity. The
sequence, shown in Figure 5, is artificial notably because, in order to
38 C.A. FACER AND M. TANNER
Spf 55.1
Asp-GIu-Leu-GIu-Ala-GIu-Thr-GIn-Asn-Val-Tyr-Ala-Ala
Tyr-Ser-Leu-Phe-Glnl-t Met-Val-Leu
Tyr-GIy-Gly-Pro-Ala-Asn-Lys-Lys-Asn-Ala-GIy
-
Cys-Gly- (SPf 55.1) - Pro-Asn-Ala-Asn-Pro (SPf 83.1) Pro-Asn- -
Ala- Asn-Pro- ( SPf 35.1) -Cys
Figure 5 The synthetic SPf66 vaccine construct and the amino acid sequences of
the peptides contained within it. The peptide numbering corresponds to the mole-
cular weight of the malaria protein from which the sequence is derived. Peptide
83.1 is an 11 amino acid synthetic peptide corresponding to residues 43-53 of
MSA-1 (a conserved epitope in the majority of P. falciparum isolates) and contains
the red cell binding motif Lys-Glu-Lys (KEK, shown boxed). One Cys residue is
added at end of the molecule to permit polymerization via formation of disulphide
bridges between pairs of Cys residues on oxidation (Patarroyo et al., 1987).
" Vaccine efficacy based on overall incidence rates of first clinical episodes of
malaria (P. falciparum) in vaccine and placebo groups.
DB/RCT (double-blind, randomized, placebo-controlled trial conditions).
' Efficacy 22 months after last vaccine dose.
The acid test for the vaccine has been its performance in African and
other countries where the transmission of malaria is several orders of
magnitude greater than in South America. Three such trials recently com-
pleted are a Swiss/Spanish sponsored and led independent phase I11 trial in
Tanzania, another in the Gambia initiated by the British Medical Research
Council, and one run by the US Walter Reed Army Institute of Research
(WRAIR) in Thailand (Ballou et al., 1995). All batches of SPf66 used were
produced under good manufacturing practice (GMP) conditions and given
Food Drug Administration Approval (FDA) approval or European Com-
mission licences, for the Thailand and African human trials, respectively.
One requirement specified in the GMP conditions is that each batch of
vaccine produced must be biochemically analysed for evaluation of the
content of peptide monomers and polymers. The potential for the hybrid
peptide to cyclize during or after production is also being considered an
important issue since such peptides may be superior to linear forms in
CLINICAL TRIALS OF MALARIA VACCINES 41
Despite the Gambian and Thai trial results, the WHO is supporting
further trials in Tanzania. The second series of Tanzanian trials designed
to examine the effect of vaccinating children at a very young age, which
might provide better results, started in February 1996 and addresses two
essential questions. First, does the reduction in the risk of clinical malaria
as shown in the first trials translate into similar reductions against all
episodes of clinical malaria, particularly life-threatening malaria morbid-
ity? Second, as more than half of all malaria-related morbidity and mor-
tality in Kilombero takes place before the age of 1 year, can SPf66
vaccination be delivered following the existing EPI (extended programme
of immunization) schedules and schemes?
In a first step, a randomized, double-blind, placebo-controlled study of
the safety, immunogenicity, potential interactions with EPI vaccines and
efficacy against clinical episodes of malaria (phase YIII trial) is being
carried out among 1000 infants with the three doses of SPf66 (or placebo)
being administered at age 1, 2 and 7 months (Alonso and Tanner, 1995).
The study will have sufficient power to detect a 25% reduction in the
incidence of malaria episodes. Providing the results confirm the safety
and minimum estimated efficacy of 25%, then a further phase I11 trial (to
be completed in 1999) will be conducted among 6000 children under 2
years of age in the same region. This second series of trials is a con-
sequence of the initial trial in Tanzania (Alonso et d . , 1994) and will
conclude the debate on the real potential of SPf66 to reduce the risk of
clinical malaria in highly endemic areas.
P. v i v a
Duffy K E W 2 C No
Pv200 LKKL 4 C No
KEK 1 C No
‘Fever is probably caused by a little toxin which escapes from each parasite
. . .’ (Ross,1911). A hallmark of all malaria infections is fever which
occurs when schizonts rupture, an event recognized as early as 1889 by
Golgi (see Kean et al., 1978). There is now compelling evidence that much
of the pathophysiology of malaria, including fever, is mediated by proin-
flammatory cytokines (TNFa, IL- 1 and IL-6) released from macrophages,
monocytes and T cells activated by malaria ‘endotoxins’ originating from
ruptured schizonts (Figure 6).
TNF is a prime candidate for fever induction and this is supported by
experiments showing that injection of TNF alone can elicit many of the
clinical symptoms of acute malaria, such as hyperexia, hypotension, pul-
monary oedema and diffuse intravascular coagulation (Beutler and Cerami,
1988). Another feature of malaria caused by P. falciparum in humans and
linked to high TNF production, is cerebral malaria (CM, see below), a
relatively rare yet frequently fatal event occurring in about 1% of
infections (Marsh, 1992).
Any vaccine intervention which could reduce the morbidity (‘anti-toxic’
vaccine) and mortality (vaccine to prevent or reverse CM) would be a
useful adjunct to existing vaccines. Disease-modifying vaccines would
alleviate symptoms without imposing a lethal pressure on the parasite.
The target antigens may not be critical for parasite survival and would
ideally have the advantage of not being countered by parasite immune
evasion mechanisms. By reducing childhood mortality, disease-modifying
vaccines would extend life until naturally acquired anti-parasite immunity
develops at a later stage.
It has been recognized for many years that children living within malaria-
endemic regions develop what is known as ‘anti-toxic’ immunity, manifested
as a progressive reduction in disease severity, following repeated malarial
attacks and occurring several years before parasitaemias begin to fall.
Thus, it is common to find 5-10 year old African children who are
asymptomatic yet have levels of parasitaemia that would be associated
with fever and disease symptoms in a non-exposed malaria-naive indivi-
dual. As fever is density dependent, that is fever only occurs when para-
sitaemia reaches a certain level known as the fever threshold (Kitchen,
1949), it is obvious that these children must have acquired an altered
Figure 6 Erythrocytic cycle of P. falciparum showing expression and/or release of antigens proposed for inclusion into anti-
parasite and anti-disease vaccines.
48 C.A. FACER AND M. TANNER
-
CIDR domain of
300 400aa : some Hydrophobic
-
homology with EBA 175
transmembrane
segment
I
t
1-
Conserved 1 - 3 variable domains
I
II
head structure Conserved
acid terminal
segment
(anchor to rbc
cytoskeleton?)
tion of the respective endothelial ligands with an anti- ‘toxic’ vaccine might
modify the degree or site of sequestration.
6. THE FUTURE
There has been a remarkable increase over the past decade in our under-
standing and knowledge of the malaria parasite and the disease that it
causes. Never before has one been in the position to appreciate what a
sophisticated and complex organism we are attempting to defeat through
vaccine development. Nevertheless, we believe that pre-erythrocytic and
asexual blood stage vaccines are now a reality and, given their further
development in order to overcome the remarkable plasticity and diversity
of this parasite, they should assure a high efficacy. However, this will
require the continued funding by the relevant agencies and the determina-
tion and innovation of researchers given this support to produce a vaccine
that will, finally, be universally licensed to effectively control malaria at a
time when it has never been needed more.
ACKNOWLEDGEMENTS
We thank Dr Gerd Pluschke and Dr Tom Smith for reading the manuscript
and for their helpful suggestions.
CLINICAL TRIALS OF MALARIA VACCINES 57
REFERENCES
Aidoo, M., Lalvani, A., Allsopp, C.E.M., Plebanski, M. Meisner, S.J., Krausa, P.,
Browning, M., Moms-Jones, S., Gotch, F., Fidock D.A., Takiguchi, M.,
Robson, K.J.H., Greenwood, B.M., Druilhe, P., Whittle, H.C. and Hill,
A.V.S. (1995). Identification of conserved antigenic components for a cytotoxic
T-lymphocyte inducing vaccine against malaria. Lancet 345, 1003-1008.
Aley, S.B., Shenvood, D.A. and Howard, R.J. (1984). Knob-positive and knob-
negative Plasmodium falciparum differ in expression of strain-specific malaria
antigen on the surface of infected erythrocytes. Journal of Experimental
Medicine 160, 1585-1590.
Allan, R.J., Rowe, A. and Kwiatkowski, D. (1993). Plasmodium fakiparum
varies in its ability to induce tumour necrosis factor. Infection and Immunity
61, 4772-4776.
Alonso, P.L., Smith, T., Armstrong Schellenberg, J.R.M., Masanja, H., Mwankusye,
S., Urassa, H., Bastos de Azevedo, I., Chongela, J., Kobero, S., Menendez, C.,
Hurt, N., Thomas, M.C., Lyimo, E., Weiss, N.A., Hayes, R., Kitua, A.Y., Lopez,
M.C., Kilama, W.L., Teuscher, T. and Tanner, M. (1994). Efficacy of the SPf66
vaccine against P.falciparum malaria in African children. Lancet 344,1175-1 18 1.
Alonso, P.L. and Tanner, M. (1995a). The Ifakara SPf66 malaria vaccine pro-
gramme: a research programme for the further evaluation and development of
the SPf66 malaria vaccine towards applicability in malaria control strategies and
programmes. Study Protocol, Hospital Clinic i Provincial, Swiss Tropical
Institute and Ifakara Centre, d d .
Alonso, P.L., Molyneux, M.E. and Smith, T. (1995b). Design and methodology
of field-based intervention trials of malaria vaccines. Parasitology Today 11,
197-200.
Alonso, P.L., Smith, T., Armstrong-Schellenberg, J., Kitua A.Y., Masanja, H.,
Hayes, R. et al. (1996). Duration of protection and age dependence of the effects
of the SPf66 malaria vaccine in African children exposed to intense transmission
of Plasmodium falciparum. Journal of Infectious Diseases 174, 367-372.
Anders, R. (1991). Antigenic diversity in Plasmodium falciparum. Acta Leidensia
60, 57-67.
Anders, R.F. and Brown, G.V. (1990). Vaccines against asexual blood stages of P.
falciparum. In: Progress in Allergy 41 (K. Ishizaka, P. Kallos, P. Lachmann and
B.H. Waksman, eds), pp. 491-512.
Amot, D. (1989). Malaria and the major histocompatibility complex. Parasitology
Today 5 , 138-144.
Babiker, H.A., Ranford-Cartwright, L.C., Cume, D., Charlwood, J.D., Billingsley
P., Teuscher, P. and Walliker, D. (1994). Random mating in a natural population
of the malaria parasite Plasmodium falciparum. Parasitology 109, 41 3-423.
Babiker, H.A., Charlwood, J.D., Smith, T., Walliker, D. (1995). Gene flow and
cross-mating in P. falciparum in households in a Tanzanian village. Parasitology
111,433442.
Ballou, W.R., Hoffman, S.L., Shenvood, J.A., Hollingdale, M.R., Neva, F.A.,
Hockeyer, W.T., Gordon, D.M., Schneider, I., Wirtz, R.A. et al. (1987). Safety
and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine.
Lancet i, 1277-1281.
Ballou, W.R., Blood, J., Chongsuphajaissidhi, T., Gordon, D.M., Heppner, D.G.,
Kyle, D.E. et al. (1995). Field trials of an asexual blood stage malaria vaccine:
58 C.A. FACER AND M.TANNER
studies of the synthetic polymer SPf66 in Thailand and the analytic plan for a
phase IIb efficacy study. Parasitology 110 (Suppl.), S25-S36.
Baruch, D.I., Pasloske, B.L., Singh, H.B., Bi, X., Ma, X.C., Feldman, M., Taraschi,
T.F. and Howard, R.J. (1995). Cloning of the P. falciparum gene encoding
PfEMPI, a malaria variant antigen and adherence receptor on the surface of
parasitized human erythrocytes. Cell 82, 77-87.
Bate, C.A.W., Taverne, J., Roman, E., Moreno, C. and Playfair, J.H.L. (1991). TNF
induction by malaria exoantigens depends on phospholipid. Immunology 75,
129- 135.
Bate, C.A.W. and Kwiatowski, D. (1994). Inhibitory immunoglobulin M antibodies
to tumour necrosis factor-inducing toxins in patients with malaria. Infection and
Immunity, 62, 3086-309 1.
Bate, C.A.W. Taverne, J., Kwiatkowski, D. and Playfair, J.H.L. (1993). Phospho-
lipids coupled to a carrier induce IgG antibody that blocks tumour necrosis factor
induction by toxic malaria antigens. Immunology 79, 138-145.
Bathurst, I.C., Gibson, H.L., Kansopon, J., Hahm, B.K., Green, K.M., Chang, S.P.
( 1993). An experimental vaccine cocktail for Plasmodium falciparum malaria.
Vaccine, 11, 449456.
Beck, H.P., Felger, I., Huber, W., Steiger, S., Smith, T., Weiss, N., Alonso, P. and
Tanner, M. (1997). Analysis of multiple P. falciparum infections in Tanzanian
children during the Phase 111 trial of the malaria vaccine, SPf66. Journal of
Infectious Diseases (in press).
Berendt, A.R., Simmons, D., Tansey, J., Newbold, C.I. and Marsh, K. (1989).
Intercellular adhesion molecule 1 (ICAM- 1) is an endothelial cytoadherence
receptor for Plasmodium falciparum. Nature 341, 57-59.
Berendt, A.R., Ferguson, D.J.P., Gardner, J., Turner, G., Rowe, A., McCormick,
C., Roberts, D., Craig, A., Pinches, R., Elford, B.C. and Newbold, C.I. (1994).
Molecular mechanisms of sequestration in malaria. Parasitology 108 (Suppl.),
S 19-S28.
Beutler, B. and Cerami, A. (1988). Tumour necrosis factor, cachexia shock
and inflammation: a common mediator. Annual Review of Biochemistry 57,
505-5 18.
Blackman, M.J., Scott-Finnizan, T.J., Shai, S., and Holder, A. (1994). Antibodies
inhibit the protease-mediated processing of a malaria merozoite surface protein.
Journal of Experimental Medicine 180, 389-393.
Borst, P., Bitter, W., McCulloch, R., van Leewen, F., and Rudenko, G. (1995).
Antigenetic variation in malaria. Cell 82, 1-4.
Boyd, M.F., and Kitchen, S.F. (1936). Is the acquired homologous immunity to
Plasmodium v i v a equally effective against sporozoites and trophozoites?
American Journal of Tropical Medicine. 10, 317-322.
Chappel, J.A. and Holder, A.A (1993). Monoclonal antibodies that inhibit P. falci-
parum invasion in vitro recognise the first growth factor-like domain of merozoite
surface protein-1. Molecular and Biochemical Parasitology 60, 303-3 12.
Charoenvit, Y., Leef, M.F., Yuan, L.F., Sedegah, M. and Beaudoin, R.L. (1987).
Characterization of Plasmodium yoelii monoclonal antibodies directed against
stage-specific sporozoite antigens. Infection and Immunity 55, 604-608.
Clark, J.T., Donachie, S., Anand, R., Wilson, C.F., Heidrich, H.G. and McBride,
J.S. (1989). A 46-53 kilodalton glycoprotein from the surface of Plasmodium
falciparum merozoites. Molecular and Biochemical Parasitology 32, 15.
Clyde, D.F., McCarthy, V.C., Miller, R.M., and Woodward, W.E. (1975). Immu-
CLINICAL TRIALS OF MALARIA VACCINES 59
J.U., Cryz, S.J. and Sadoff, J.C. (1992b). Safety immunogenicity and efficacy of
a Plasmodium falciparum vaccine comprising a circumsporozoite protein repeat
region conjugated to Pseudomonas aeruginosa toxin A. Infection and Immunity
60, 1834-1839.
Genton, B., Al-Yaman, F., Anders, R., Brown, G., Saul, A., Stuerchler, D. et al.
(1997). Asexual blood stage vaccine (p.190, MSP-2, RESA) trials against P.
falciparum malaria: relevance for travellers and long term residents. Proceedings
5th International Conference on Travel Medicine, Geneva.
Good, M.F. (1995). Harnessing cytotoxic T lymphocytes for vaccine design.
Lancet 345, 999-1000.
Good, M.F., Berzofsky, J.A. and Miller, L.H. (1988a). The T cell response to the
malaria circumsporozoite protein: an immunological approach to malaria
vaccine development. Annual Review of Immunology 6, 663-688.
Good, M.F., Pombo, D., Quakyi, I.A., Riley, E.M., Houghton, R.A., Menon, R.,
Alling, D.W., Berzofsky, J.A. and Miller, L.H. (1988b). Human T cell recogni-
tion of the circumsporozoite protein of Plasmodium falciparum. Immunodomi-
nant T cell domains map to the polymorphic regions of the molecule.
Proceedings of the National Academy of Sciences USA 85, 1199-1203.
Greenwood, B.M., Bradley, A.K., Greenwood, A.M., Byass, P., Jammen, K., Marsh,
K., Tulloch, S., Oldfield, F.S.J. and Hayes, R. (1987). Mortality and morbidity
from malaria among children in a rural area of The Gambia, West Africa. Trans-
actions of the Royal Society of Tropical Medicine and Hygiene 81, 478486.
Gupta, S. and Day, K.P. (1994). A strain theory of malaria transmission.
Parasitology Today 10, 476-48 1.
Heath, A.W. and Playfair, J.H.L. (1992). Cytokines as immunological adjuvants.
Vaccine 10, 427431.
Hedstrom, R.C., Sedegah, M. and Hoffman, S.L. (1994). Prospects and strategies
for development of DNA vaccines against malaria. Research in Immunology
145, 476-483.
Helmby, H., Cavalier, L., Peterson, U., Wahlgren, M. (1993). Rosetting P.
falciparum-infected erythrocytes express unique strain-specific antigens on their
surface. Infection and Immunity 61, 284-288.
Herrera, M.A., Rosero, F., Herrera, S., Caspers, P., Rotmann, D., Sinigaglia, T. and
Certa, U. (1992). Protection against malaria in Aotus monkeys immunized with a
recombinant blood stage antigen fused to a universal T cell epitope: correlation
of serum gamma interferon levels with protection. Infection and Immunity 60,
154-158.
Herrington, D.A., Clyde, D.F., Losonsky, G., Cortesia, M., Murphy, J.R., Davis, J.,
Baqar, S., Felix, A.M., Heimer, E.P. et al. (1987). Safety and immunogenicity in
man of a synthetic peptide malaria vaccine against Plasmodium falciparum
sporozoites. Nature 328, 257-259.
Herrington, D.A., Losonsky, G.A., Smith, G., Volvovitz, F., Cochran, M., Jackson,
K., Hoffman, S.L., Gordon, D.M., Levine, M.M. and Edelman, R. (1992). Safety
and immunogenicity in volunteers of a recombinant Plasmodium falciparum
circumsporozoite protein malaria vaccine produced in Lepidopteran cells.
Vaccine 10, 841-845.
Hill, A.V.S., Allsopp, C.E.M., Kwiatkowski, D., Anstey, N.M., Twumasi, P.,
Rowe, P.A., Bennett, S., Brewster, D., Mcmichael, A.J. and Greenwood, B.M.
( 1991). Common West African HLA antigens are associated with protection
from severe malaria. Nature 352, 595-600.
Hill, A.V.S., Elvin, J., Willis, A.C., Aidoo, M., Allsopp, C.E.M., Gotch, F.M., Gao,
X.M., Takiguchi, M., Greenwood, B.M., Townsend, A.E.M., Mcmichael, A.J.
and Whittle, H.C. (1992). Molecular analysis of the association of HLA B53 and
resistance to severe malaria. Nature 360, 434439.
62 C.A. FACER AND M. TANNER
Valero, M.V., Amador, L.R., Aponte J.J., Narrez, A., Galindo, C. Silva, Y. et al.
(1996). Evaluation of SPf66 malaria vaccine during a 22 month follow up field
trial in the Pacific coast of Colombia. Vaccine 14, 1466-1470.
Vreden, S.G., Verhave, J.P., Oettinger, T., Sauerwein, R.W. and Menwissen, J.H.
(1991). Phase I clinical trial of a recombinant malaria vaccine consisting of the
circumsporozoite repeat region of P. falciparum coupled to hepatitis B surface
antigen. American Journal of Tropical Medicine and Hygiene 45, 533-538.
Wahlgren, M., Carlson, J., Udomsangpetch, R. and Perlmann, P. (1989). Why do
Plasmodium falciparum-infected erythrocytes form spontaneous rosettes?
Parasitology Today 5 , 183-186.
Waine, G.J. and McManus, D.P. (1995). Nucleic acids: vaccines of the future.
Parasitology Today 11, 113-1 16.
Wang, G., Seidman, M.M. and Glazer, P.M. (1996). Mutagenesis in mammalian
cells induced by triple helix formation and transcription-coupled repair. Science
271, 802-805.
Weber, L. and Hockmeyer, W.T (1985). Structure of the circumsporozoite protein
gene in eighteen strains of Plasmodium falciparum. Molecular and Biochemical
Parasitology 15, 305-3 16.
Weiss, W.R., Mellouk, S., Houghten, R.A., Sedegah, M., Kumar, S . , Good, M.F.,
Berzofsky, J.A., Miller, L.H. and Hoffman, S.L. (1990). Cytotoxic T cells
recognize a peptide on malaria-infected hepatocytes. Journal of Experimental
Medicine 171, 763-773.
WHO (1995). Control of Tropical Diseases (CTD). Malaria Control. Geneva:
World Health Organization.
Williams, T.N., Maitland, K., Bennett, S . , Ganczakowski, M., Peto, T.E.A.,
Newbold, C.I., Bowden, D.K., Weatherall, D.J. and Clegg, J.B. (1996). High
incidence of malaria in a-thalassaemic children. Nature 383, 522-525.
Wizel, B., Rogers, W.O., Houghten, R.A., Lanar, D.E., Tine, J.A. and Hoffman,
S.L. (1994). Induction of murine cytotoxic T lymphocytes against Plasmodium
falciparum sporozoite surface protein 2. European Journal of Immunology 24,
1487-1495.
World Bank, World Development Report (1993). Investing in Health. Oxford:
Oxford University Press.
Yang, C., Shi, Y.-P., Udhayakumar, V., Alpers, M., Povoa, M.M., Hawley, W.A.,
Collins, W.E. and Lal, A.A. (1995). Sequence variations in the non-repetitive
regions of the liver stage specific antigen (LSA-1) of Plasmodium falciparum
from field isolates. Molecular and Biochemical Parasitology 71, 29 1-294.
Zavala, F., Tam, J.P., Ban-, P.J., Romero, P.J., Ley, V., Nussenzweig, R.S. and
Nussenzweig, V. ( 1987). Synthetic peptide vaccine confers protection against
murine malaria. Journal of Experimental Medicine 166, 1591-1596.
Zevering, Y., Houghton, R.A., Frazer, I.H. and Good, M.F. (1990). Major popula-
tion differences in T cell response to a malaria vaccine candidate. International
Immunology 2, 945-955.
Zevering, Y., Khamboonruang, C. and Good, M.F. (1994). Natural amino acid
polymorphisms of the circumsporozoite protein of Plasmodium falciparum abro-
gate specific human CD4+ T cell responsiveness. European Journal of
Immunology 24, 1418-1425.
Zhu, J. and Hollingdale, M. (1991). Structure of Plasmodium falciparum liver stage
antigen 1 . Molecular and Biochemical Parasitology 48, 223-226.
Phylogeny of the Tissue
Cyst-forming Coccidia
1
Institut fur Parasifologie. Tierarztliche Hochschule Hannover.
Bunteweg 17. 30559 Hannover. Germany and
2Molecular Parasitology Unit. Faculty of Science.
University of Technology. Sydney. GPO Box 123. Broadway.
New South Wales 2007. Australia
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
2. Taxonomy. Nomenclature and Currently Used Classifications . . . . . . . . . . . . . . 71
2.1. The family Sarcocystidae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
2.2. The problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3. Traditional Characters Used for the Classification of Tissue
Cyst-forming Coccidia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.1. Morphology and ultrastructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
3.2. Life cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
3.3. Inference of phylogenetic relationships from phenotypic characters . . . . . 94
3.4. Conflicting hypotheses based on phenotypic characters . . . . . . . . . . . . . . .98
4. The Molecular Answer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.1. Characteristics of SSU rRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
4.2. Nucleotide sequence determination . . . . . . . ...................... 102
4.3. Sequence alignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4.4. Outgroup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.5. Tree-building methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.6. Comparison of different phylogenetic trees inferred from SSU rRNA
sequencedata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5. Phylogenetic Relationships and Genetic Relatedness of Tissue Cyst-forming
Coccidia Inferred from SSU rRNA Sequence Comparisons . . . . . . . . . . . . . . . 112
5.1. Phylogenetic relationships of tissue cyst-forming coccidia
to homoxenous coccidia and other apicomplexan protozoa . . . . . . . . . . . 113
5.2. Phylogenetic relationships of tissue cyst-forming coccidia to each other . 114
5.3. Genetic divergence among tissue cyst-forming coccidia . . . . . . . . . . . . . .117
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
1. INTRODUCTION
many years and are still uncertain today. Although the tissue cyst or oocyst
stages of several tissue cyst-forming coccidia have been known for more
than a century (Miescher, 1843; Kuhn, 1865; Stiles, 1891), it was not until
only two decades ago that the complete heteroxenous life cycles of some of
these parasites were elucidated (Hutchison et al., 1969; Rommel et al.,
1972; Frenkel and Dubey, 1975; Rommel and Krampitz, 1975; Wallace
and Frenkel, 1975). Thus far, the classification of tissue cyst-forming
coccidia has been based on phenotypic characters, such as morphology
and life cycle. However, these characters provide only limited phylogenetic
information, because morphological features are often subject to change
during the developmental cycle of the parasite and the complete life cycles
are not known for all species (reviewed by Rommel, 1989; Dubey, 1993;
Tenter, 1995). Therefore, we still know very little about the phylogeny and
genetic relatedness of different species of tissue cyst-forming coccidia,
both to each other and to members of other taxa in the phylum Apicom-
plexa (see Tadros and Laarman, 1982; Vivier, 1982; Kreier and Baker,
1987; Barta, 1989; Frenkel, 1989; Cox, 1991, 1994; Tenter, 1995).
This review gives an overview of currently used classifications of tissue
cyst-forming coccidia and of recent phylogenetic studies based on mole-
cular data that have provided new insights into the phylogeny and genetic
relatedness of some of these parasites. In particular, the genetic relation-
ships of species placed into the two oldest genera of tissue cyst-forming
coccidia (i.e. Sarcocystis Lankester, 1882 and Toxoplasma Nicolle and
Manceaux, 1909) are discussed.
Table I Continued
Genus Besnoitia Henry, 1913
Meronts in fibroblasts and other cells; host cell nucleus within meront wall; tissue
cyst wall thick; definitive hosts mammals; intermediate hosts mammals or reptiles;
about seven named species; type species Besnoitia besnoiti (Marotel, 1913) Henry,
1913.
Genus Hammondia Frenkel and Dubey, 19775
Meronts in numerous cell types; oocysts not infectious to definitive host; definitive
hosts felids or canids; intermediate hosts mammals; about three named species; type
species Hammondia hammondi Frenkel and Dubey, 1975.
Genus Neospora Dubey, Carpenter, Speer, Topper and Uggla, 1988
Oocysts and intestinal stages unknown; merozoites divide by endodyogeny in
numerous tissues, especially brain and spinal cord, usually without formation of
parasitophorous vacuole; tissue cysts thick-walled, found generally in the central
nervous system; in canids; one named species; type species Neospora caninum
Dubey, Carpenter, Speer, Topper and Uggla, 1988.
I Nodevelopment
I on theground
I I
I I
cysts
FRENKELlA
SARCOCYSTIS
I
I
intermediate hosts
W
Endomites
TOXOPLASMA BESNOlTlA
I
/ Development in
\
and gamogony 1 on the
9 final host and
in the final host 1 ground
I exogenousstages ? \
U I unknown
I I
HAMMONDIA NEOSPORA
Figure I Life cycles of different genera of tissue cyst-forming coccidia. A. The genus Sarcocystis. B. The genus Frenkelia. C. The
genus Toxoplasma. D. The genus Besnoitia. E. The genus Hammondia. F. The genus Neospora. Illustrations are arranged as follows:
development in the intermediate host is shown below the full line, development in the definitive host is shown on the left of the
dashed line, exogenous life cycle stages are shown on the right of the dashed line (modified with permission from Rommel, 1989).
Table 3 Taxa used for the classification of tissue cyst-forming coccidia since the elucidation of their life cycles in the 1970s.
Number of lower
Taxon taxa" References
Family
Eimeriidae Minchin, 1903 5 8 subfamilies Tadros and Laarman (1976), Overdulve (1978), Kreier
5 28 genera and Baker (1987), Mehlhorn and Ruthmann (1992)
Isosporidae Minchin, 1903 3 subfamilies EuzCby (1987)
7 genera
Sarcocystidae Poche, 1913 5 3 subfamilies Levine (1973, 1988), Frenkel (1974), Hiepe and
2-6 genera Jungmann (1983), Frenkel et al. (1987), Current et al.
(1990), Eckert et al. (1992)
Toxoplasmatidae Biocca, 1956 2 4 genera Frenkel (1974), Eckert et al. (1992)
Subfamily
Sarcocystinae Poche, 1913 2-3 genera Levine (1973, 1988), Hiepe and Jungmann (1983),
Euzkby (1987), Frenkel et al. (1987), Current et al.
(1990)
Toxoplasmatinae Biocca, 1957 2-4 genera Levine (1973, 1988), Hiepe and Jungmann (1983),
Euzkby (1987), Frenkel et al. (1987), Current et al.
( 1990)
Isosporinae Wenyon, 1926 2 genera EuzCby (1987)
Cyclosporinae Wenyon, 1926 6 genera Kreier and Baker (1987)
Besnoitiinae Garnham, 1966 1 genus Levine (1973)
Eimeriinae Wenyon, 1926 > 2 genera Tadros and Laannan (1976)
Endorimosporinae Tadros and Laarman, 1976 1 genus Tadros and Laarman (1976)
Cystoisosporinae Frenkel, Heydorn, Mehlhorn and 1 genus Hiepe and Jungmann (1983), Frenkel et al. (1987)
Rommel, 1979
Genusb
Isospora Schneider, 1881' > 240 species Overdulve (1978), Levine (1988), Current et al. (1990),
Dubey (1993)
Sarcocystis Lankester, 1882 > 120 species Levine (1986, 1988), Tenter (1995)
Toxoplasma Nicolle and Manceaux, 1909d 1-9 species Dubey and Beattie (1988), Levine (1988), Current et al.
(1990)
Besnoitia Henry, 1913 6 7 species Levine (1988), Rommel (1989), Dubey (1993)
Frenkelia Biocca, 1968 2 species Levine (1988), Rommel (1989), Dubey (1993)
* Hammondia Frenkel and Dubey, 197Sd 3 4 species Rommel (1989), Dubey (1993)
* Arthrocystis Levine, Beamer and Simon, 1970 1 species Levine and Baker (1987), Levine (1988)
* Endorimospora Tadros and Laarman, 1976' - Tadros and Laarman (1982)
* Levineia Dubey, 1977f - Dubey (1977a, b)
* Cystoisospora Frenkel, 1977' 5 4 species EuzCby (1980), Hiepe and Jungmann (1983), Greene and
Prestwood (1984), Rommel (1989)
Neospora Dubey, Carpenter, Speer, Topper and Uggla, 1 species Dubey (1993), Dubey and Lindsay (1993)
1988
" For references see Levine and Tadros (1980), Levine (1986, 1988).
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 87
dimensions of the tissue cyst, the size or shape of the so-called ‘spores’
within the tissue cyst, or the structure of the tissue cyst wall. By the end of
the century, names had been assigned to nine species of Sarcocystis (see
LabbC, 1899). The list of named Sarcocystis species had increased to 35 by
1926, to 44 by 1932, and to 54 by 1975 (Wenyon, 1926; Babudieri, 1932;
Kalyakin and Zasukhin, 1975). However, only after the elucidation of the
complete heteroxenous life cycles of some of these species in 1972 (see
Section 3.2) did it become clear that what had been known as Miescher’s
tubules for more than a century were in fact last generation meronts (or
tissue cysts) of obligately heteroxenous coccidian parasites in their inter-
mediate hosts, and that the ‘spores’ were last generation merozoites (or
cystozoites); that is, the infectious stage for the definitive host. Since 1972,
the validity of several Sarcocystis species has been evaluated by transmis-
sion studies, and some of the earlier names assigned to species in the genus
Sarcocystis are now obsolete. After an extensive review of the literature,
Levine and Tadros (1980) gave a list of 93 different species of Sarcocystis
with their synonyms and hosts. However, new species of Sarcocystis are
continually being described, even today. Although both the intermediate
and definitive hosts are not known for all species, the last review of the
taxonomy of species in the genus Sarcocystis regarded 122 as valid
(Levine, 1986), and since then at least another 20 species have been named.
Thus, the genus Sarcocystis is now by far the largest genus of tissue cyst-
forming coccidia and is one of only eight genera in the phylum Apicom-
plexa that comprise more than 100 species (see Levine, 1988).
In 1908, asexual stages of another tissue cyst-forming protozoan were
found in the spleen, liver and blood of gondis, a species of North African
rodent, by Nicolle and Manceaux (1908). The authors first thought that this
parasite resembled a species of Leishmania Ross, 1903 and thus assigned to
it the specific name Leishmania gondii. However, a more detailed study of
this parasite showed that it lacked a kinetoplast, and therefore the authors
concluded that the parasite was not related to Leishmania and, in the
following year, proposed the generic name Toxoplasma for it (Nicolle
and Manceaux, 1909). Thus, the second genus of tissue cyst-forming
coccidia was established and Toxoplasma gondii (Nicolle and Manceaux,
1908) Nicolle and Manceaux, 1909 became the type species of the genus
Toxoplasma. However, the tissue cyst stage of T. gondii remained unknown
until the late 1950s (reviewed by Jacobs et al., 1960a, b), and thus no
relationship was recognized between the genus Toxoplasma and the genus
Sarcocystis.
As with Sarcocystis, several species of Toxoplasma were named during
the first half of this century, mainly based on differences in the host
species infected (Wenyon, 1926; GrassC, 1953). However, whereas sev-
eral transmission studies carried out over the last three decades confirmed
88 A.M. TENTER AND A.M. JOHNSON
F
Light microscopy
Discovery of Miescher's tubules 1
(today tissue cyst of Sarcocysris muris)
Description of the tissue cyst of 2
Synchyrrium miescherianum
I882 (today the type species Sarcocysris miescherianu)
Introduction of the genus Surcocysfrs 3
Placement of the Sarcosporidia 3,4
into the class Sporozoea
1882 Discovery of the asexual stages of Toxoplasmu gondii 11
1
-'i
in gondis
Description of the type species Toxoplusma gondir 12
(only asexual stages) 1909
1-
Introduction of the'genus Toxoplasmu 12
I
CLASSIFICATION OF EVENT CLASSIFICATION OF REFERENCE
SARCOCYSTIS TOXOPLASM
I
Subclass Sarcosporidia Genus incertae sedis in the order 15, 17
j
in the class Sporozoea Coccidiida, subclass Coccidiomorpha,
1 class Swrozoea
Electron microscopy
1956 1956
Discovery of ultrastructural similarities between 18-2 1
1957 Sarcocystis and Toxoplasma 1957
T
Family Sarcocystidae in the order Family Toxoplasmatidae in the order 22-24
Toxoplasmida, class Toxoplasmea, Toxoplasmida, class Toxoplasmea,
I
subphylum Sporozoa subphylum Sporozoa
-It 1970
Discovery of ultrastructural similarities between
Sarcocystis, Toxoplasma, and Eimeria
Cell culture
Cultivation of the sexual stages of Sarcocystis
25-28
21,37
CLASSIFICATION OF EVENT CLASSIFICATION OF REFERENCE
SARCOCYSTIS TOXOPLASMA
1 1
1977 Elucidation of the life cycle of Besnoitiu 1971 46,48
Molecular biology
1988 1988
Inference of the phylogenetic relationship between 53, 56,51,
1996 Sarcocystis and Toxoplasma based on SSU rRNA 1996 59-64
The final elucidation of the life cycle of Toxoplasma began with the
discovery that faecal material recovered from a cat previously fed mouse
carcasses and brains infected with Toxoplasma tissue cysts induced Toxo-
facultatived
Type of development
in definitive host gamogony, gamogony, endopolygeny, endopolygeny, endopolygeny, ?
sporogony sporogony gamogony gamogony g~ogonY
in environment none none sporogony sporogony sporogony ?
in intermediate host endopolygeny, endopolygeny, endodyogeny endodyogeny endodyogeny endodyogeny
endodyogeny endodyogeny
Natural route of transmission'
definitive to intermediate host via free via free via sporulated via sporulated via sporulated ?
sporocysts sporocysts oocysts oocysts oocysts
intermediate to definitive host via tissue cysts via tissue cysts via tissue cysts via tissue cysts via tissue cysts ?
definitive to definitive host none none via sporulated none none ?
oocysts or
endozoites
intermediate to intermediate host none or via none or via via tissue cysts via tissue cystsd none via endozoites (or
endozoitesd endozoitesd or endozoites tissue cysts?)
Extraintestinal stages in definitive absent absent present present or absentdpresent or absentd ?
host
Morphology and location of developmental stages
Zygote location in lamina propria in lamina propria in epithelium in epithelium in epithelium ?
Oocyst disporous, disporous, disporous, disporous, disporous, ?
tetrazoic tetrazoic tetrazoic tetrazoic tetrazoic
Tissue cyst
location striated muscles, central nervous many tissues connective tissue striated muscles, central nervous
neural tissue system brain system
shape variabled lobulated or subspherical subspherical subspherical subspherical
subspherical
tissue cyst wall variabled, within thin, within thin, within thick, surrounds thin, within thick, within
host cell host cell host cell host cell host cell host cell
septa present present absent absent absent absent
stages within tissue cyst metrocytes, metrocytes, cy stozoites cystozoites cystozoites cystozoites
cystozoites cystozoites
cystozoite banana-shaped, banana-shaped, crescent-shaped, crescent-shaped, crescent-shaped, crescent-shaped,
broad broad slender slender slender slender
Location of host cell nucleus outside tissue outside tissue outside tissue within tissue outside tissue outside tissue
cyst wall cyst wall cyst wall cyst wall cyst wall cyst wall
Over the past decade, the introduction of new molecular biological meth-
ods to the study of phylogenetic relationships has had great impact on
previously held beliefs on the taxonomy, systematics and phylogeny of
almost all life forms (Woese, 1987; Sogin, 1991; Knoll, 1992; Patterson
and Sogin, 1992; Olsen and Woese, 1993; Wainright et ul., 1993; Schlegel,
100 A.M. TENTER AND A.M. JOHNSON
used to generate SSU rRNA sequence data for tissue cyst-forming coccidia
and to infer their phylogenetic relationships, we briefly discuss some of the
factors that may have influenced the topology of the phylogenetic trees
constructed in these studies. More detailed reviews of various aspects of the
reconstruction of organismal phylogenies from nucleotide sequence data
have recently been published by Felsenstein (1988), Hillis and Moritz
(1990), Hillis and Dixon (1991), Beanland and Howe (1992), Miyamoto
and Cracaft (1992), Hillis et al. (1993) and Morrison (1996).
SSU rRNA sequence data can be derived either directly from the SSU
rRNA or from its gene. Accordingly, molecular biological methods that
have been employed to generate sequence data for phylogenetic analyses
include gene cloning and sequencing, reverse transcription of RNA and,
more recently, gene amplification by polymerase chain reaction (PCR).
Figure 3 shows phylogenetic trees of species in the genera Sarcocystis,
Toxoplasma and Neospora that have been derived from SSU rRNA
sequence data of these parasites. These trees were obtained using different
methods to generate the nucleotide sequences used for comparison.
The tree shown in Figure 3A was obtained in one of the earlier studies on
tissue cyst-forming coccidia and was constructed from sequence data
generated by reverse transcription of SSU rRNA (Tenter et al., 1992).
This method was developed in the mid 1980s (Qu et al., 1983; Lane et
al., 1985) and has been widely used to generate data derived from phylo-
genetically informative regions of SSU rRNA over the past decade
(reviewed by Johnson and Baverstock, 1989; Barta et al., 1991). Thus, in
the first phylogenetic studies on tissue cyst-forming coccidia this method
was used to obtain SSU rRNA sequences of the parasites (Johnson et al.,
1987b, 1988; Tenter et al., 1992). The advantages of the technique are that
it is quick and inexpensive, and therefore allows the determination of SSU
rRNA sequences of a much larger number of taxa in a shorter period of
time than had been possible with earlier methods. The disadvantages of the
technique are that it generates information on only part of the SSU rRNA
and that the sequence data obtained are only 95-99 % accurate (Lane et al.,
1985; Johnson and Baverstock, 1989; Baverstock and Johnson, 1990). In
addition, the technique is based on RNA as starting material and therefore
requires a reasonable number (usually >lo8) of parasite cells for RNA
extraction.
By the late 1980s, a method became available that employs PCR ampli-
fication and subsequent sequencing of the SSU rRNA gene for the genera-
tion of SSU rRNA sequence data (Medlin et al., 1988). This method has
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 103
A S. tenella
S. capracanis
S. arieticanis
S. cruzi
901 Tgondii
85 E. maxima
-
I
Cryptosporidium sp.
B S. tenella
S.arieticanis
S. gigantea
T. gondii
S.muris
E. tenella
C. parvum
B. bovis
Th. annulata
Perkinsus sp.
Cr cohnii
104 A.M. TENTER AND A.M. JOHNSON
100 7 S. tenella
C
S. arieticanis
S. gigantea
S. muris
I: gondii
97
I
E. tenella
C. pawum
B. bovis
Th. annulata
I ’ Perkinsus sp.
Cr cohnii
S. tenella
- S. arieticanis
-
93 S. firsiformis
S. gigantea
-
100
S. muris
-
98 100 N. caninum
-
71
T. gondii
E. tenella
-63 79 B. bovis
- Th. annulata
C. pawum
Perkinsus sp.
Cr. cohnii
several advantages over the earlier method (Sogin, 1989; Baverstock and
Johnson, 1990). First, it uses DNA, which is more stable than RNA, as
starting material. Only a very small amount (about 50 ng) of DNA is
needed for PCR amplification, and thus the technique is ideal for those
parasites of which only a low number of cells can be obtained. Second, the
fidelity of thermostable DNA polymerases is greater than that of reverse
transcriptase and therefore the sequences obtained are more accurate (prob-
ably 99.7-99.9%) than those obtained by reverse transcription (Medlin et
al., 1988; Saiki et al., 1988; Ellis et al., 1994b). In addition, the entire SSU
rRNA gene sequence can be obtained in both directions. The increased
accuracy of the sequences generated by this method enables the phyloge-
netic comparison of more closely related organisms. Consequently, this
method has now become the method of choice for the generation of SSU
rRNA gene sequences of tissue cyst-forming coccidia (Gajadhar et al.,
1991; Ellis et al., 1994a; Fenger et al., 1994; Holmdahl et al., 1994; Jeffries
et al., 1997). The trees shown in Figure 3B, C, D, which were constructed
in more recent studies on tissue cyst-forming coccidia, were also derived
from sequence data generated by the latter method (Ellis and Morrison,
1995; Ellis et al., 1995).
Figure 3 Continued
(redrawn with permission from Tenter et al., 1992). B-D. Nucleotide sequences
were generated by PCR amplification and sequencing of the SSU rRNA gene. B.
The data set used to construct the tree consisted of the entire SSU rRNA gene
sequences apart from the 5' and 3' ends which were truncated, i.e. 1868 nucleotide
positions (redrawn with permission from Ellis et al., 1995). C. The data set used to
construct the tree consisted of 1567 nucleotide positions of conserved and semi-
conserved SSU rRNA gene regions (redrawn with permission from Ellis et al.,
1995). D. The tree was constructed from a sequence alignment based on the
secondary structure of SSU rRNA. The data set consisted of 1301 nucleotide
positions located in the helices of the SSU rRNA (redrawn with permission from
Ellis and Morrison, 1995).
SSU rRNA sequences of the following taxa were used in the analyses: Protozoa,
phylum Apicomplexa, order Eucoccidiida LBger and Duboscq, 1910, family Sar-
cocystidae: Sarcocystis tenella, Sarcocystis capracanis, Sarcocystis arieticanis,
Sarcocystis cruzi, Sarcocystis fusifonnis, Sarcocystis gigantea, Sarcocystis
muris, ,Toxoplasma gondii, Neospora caninum; family Eimeriidae: Eimeria
tenella, Eimeria stiedai, Eimeria maxima; family Cryptosporidiidae: Cryptospor-
idium parvum, Cryptosporidium sp.; order Piroplasmida Wenyon, 1926: Babesia
bovis, Theileria annulata; order Perkinsida Levine, 1978: Perkinsus sp.; amoebae:
Acanthamoeba castellanii (outgroup in A); dinoflagellates: Crypthecodinium coh-
nii (outgroup in B-D), Prorocentrum micans; Metazoa: Homo sapiens. For sources
of sequences see Tenter et al. (1992), Ellis and Morrison (1995), and Ellis et al.
(1995).
106 A.M. TENTER AND A.M. JOHNSON
It should be noted that the alignment of the SSU rRNA sequences for
optimal homology is straightforward in regions of relatively conserved
primary and secondary structure of the molecule, but is much more arbi-
trary in the more variable regions. Therefore, for optimal inference of
phylogenetic relationships the latter regions, for which one cannot be
confident of the alignment, should be excluded from the analysis (Hase-
gawa et al., 1985; Baverstock and Johnson, 1990; Beanland and Howe,
1992; Olsen and Woese, 1993; Morrison, 1996). However, there are few
objective criteria from which to determine what regions should be removed
as being phylogenetically uninformative before phylogenetic reconstruc-
tion (Gatesby et al., 1993).
Sequence alignment is probably the most important aspect of the recon-
struction of phylogenetic trees, and is the most problematic (Morrison,
1996). In most of the phylogenetic studies on tissue cyst-forming coccidia
carried out so far, the sequence alignments were based on the primary
structure of SSU rRNA (Johnson et al., 1987b, 1988, 1991; Barta et al.,
1991; Tenter et al., 1992; Gagnon et al., 1993; Ellis er al., 1994a, b, 1995;
Fenger et al., 1994; Holmdahl et al., 1994; Escalante and Ayala, 1995).
While most of these studies used information derived from full-sequence
alignments, with only few sequences of uncertain homology excluded from
the data set used in the analysis (Gajadhar et al., 1991; Gagnon et al., 1993;
Ellis et al., 1994a, 1995; Fenger et al., 1994; Holmdahl et al., 1994;
Escalante and Ayala, 1995; Relman et al., 1996), a few studies concen-
trated on using only information derived from semi-conserved regions of
the molecule; that is, those regions in which the nucleotide sequences were
neither highly variable nor totally conserved among the taxa included in the
analysis and which are believed to contain the phylogenetically informative
nucleotide positions (Johnson et al., 1987b, 1988, 1991; Tenter et al.,
1992). However, the blocks of semi-conserved nucleotide positions were
often chosen by eye, and this limited the number of taxa that could be
aligned.
More recently, several studies have used knowledge of the secondary
structure constraints of SSU rRNA to optimize the alignment (Van de Peer
et al., 1994) and have then used information derived from phylogenetically
informative nucleotide positions located in the helices of the molecule to
infer phylogenetic relationships of tissue cyst-fonning coccidia (Ellis and
Morrison, 1995; Jeffries et al., 1997). This alignment is then edited using
the Dedicated Comparative Sequence Editor of De Rijk and De Wachter
(1993) and it appears to be a significant advance over either full-sequence
alignment based on primary structure or alignment of semi-conserved
nucleotide sequence blocks by eye. Figure 3D shows an example of a
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 107
4.4. Outgroup
1995; Relman et al., 1996; Jeffries et al., 1997). The more advanced
methods used for phylogenetic reconstruction in recent studies include
neighbour joining, maximum parsimony, and maximum likelihood, with
maximum parsimony being the method that has been used most fre-
quently (Ellis er al., 1994a, 1995; Fenger er al., 1994; Ellis and Morrison,
1995; Escalante and Ayala, 1995; Relman et al., 1996; Jeffries et al.,
1997).
All four trees shown in Figure 3 were derived using maximum parsimony
analysis for tree construction (Tenter et al., 1992; Ellis and Morrison, 1995;
Ellis et al., 1995). This method counts the minimum number of character
state changes (i.e. base substitutions and sometimes also insertions and/or
deletions) that are required for each proposed tree to accommodate the
observed sequence data. That tree or trees requiring the fewest changes (i.e.
the most parsimonious tree(s)), is/are preferred over all other trees (Felsen-
stein, 1988; Swofford and Olsen, 1990; Beanland and Howe, 1992; Hillis et
al., 1993; Morrison, 1996). The robustness of the monophyletic groups
(nodes) of the trees was tested using the bootstrap method. This method
creates a new data set by randomly resampling sites of the original data set
with replacement until the resampled data set is of the same size as the
original one. This can be repeated hundreds of times, with the constructed
tree showing how often the nodes were supported by all of the bootstraps
(Felsenstein, 1985, 1988).
Genus Neospora
N. caninum ? mammals high central nervous system 5 100 pm probably world-wide
Genus Isospora
I. felis felids rodents and non-pathogenic mainly lymphoid tissues dormozoited world-wide
some other
mammals
a For further information on these species see Levine (1988), Dubey (1993) and Tenter (1995).
This finding still needs to be confinned by transmission studies, see Fenger et al. (1995).
For risk groups such as pregnant animals or immunocompromised hosts.
Single dormozoites surrounded by a tissue cyst wall.
112 A.M. TENTER AND A.M. JOHNSON
trees shown in Figure 3A and B, the latter tree showed the Sarcocystis
species to be monophyletic, which was supported by high (96100%)
bootstrap values. However, it should be noted that 10 of 37 trees, whose
lengths were within 14 steps of the most parsimonious tree (817 steps long)
found in this analysis, showed paraphyly of the Sarcocystis species, with
the length of the shortest of those trees being 823 steps; that is, only six
steps longer than the most parsimonious tree (Ellis et al., 1995). Thus, there
was no significant number of steps (Felsenstein, 1988) between the most
parsimonious tree, which suggested monophyly of the Sarcocystis species,
and its next best competitor, which suggested paraphyly of the Surcocystis
species.
Therefore, it appears that although all three trees derived from sequence
alignments based on the primary structure of the SSU rRNA were consis-
tent in showing the dog-transmitted pathogenic Sarcocystis species to be
monophyletic, the relationships of the cat-transmitted non-pathogenic Sar-
cocystis species could not be unambiguously resolved by these analyses
(Figure 3A, B, C). By contrast, strong evidence for the monophyly of the
genus Surcocystis was obtained in a recent study on the phylogenetic
relationships of tissue cyst-forming coccidia (Ellis and Morrison, 1995),
in which the sequence alignment was based on the secondary structure of
SSU rRNA (Van der Peer et al., 1994). In this study, monophyly of the
genus Surcocystis was supported by 83% of the bootstrap replicates when
the entire data set containing 2050 positions was used in the analysis and
by 93% of the bootstrap replicates when the data set was restricted to 1301
positions located in the helices of the SSU rRNA molecule (Figure 3D).
Over the past decade, several authors have used SSU rRNA sequence
comparisons to examine the phylogenetic relationships of different species
and genera of tissue cyst-forming coccidia (Johnson et al., 1987b, 1988;
Gajadhar et al., 1991; Tenter et ul., 1992; Gagnon et al., 1993; Ellis et al.,
1994a, b, 1995; Fenger et al., 1994; Holmdahl et al., 1994; Ellis and
Morrison, 1'995; Jeffries et al., 1997). Other authors have included taxa
of tissue cyst-forming coccidia in their studies on different protozoa
currently classified in the phylum Apicomplexa (Johnson et al., 1990b,
1991; Barta et al., 1991; Ellis et ul., 1992; Goggin and Barker, 1993;
Allsopp et al., 1994; Escalante and Ayala, 1995; Relman et ul., 1996). In
addition, comparisons of SSU rRNA sequences have been used to examine
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 113
the extent of genetic divergence among different species and strains in the
genera Sarcocystis, Toxoplasma and Neospora (see Luton et al., 1995;
Marsh et al., 1995; Tenter, 1995). The information gained from these
studies will be an important complement to the information gained from
comparisons of phenotypic characters of tissue cyst-forming coccidia (see
Section 3).
However, when interpreting phylogenetic trees inferred from phenotypic
or molecular data it should be kept in mind that no one method is likely to
generate the ‘true’ phylogenetic tree of the organisms under study. Instead,
the results obtained are a statistical estimation of the probable phylogenies
of the organisms (Felsenstein, 1988; Swofford and Olsen, 1990; Nei, 1992;
Hillis et al., 1993; Morrison, 1996). Thus, rather than aiming at finding the
one tree that shows the ‘most likely’ phylogeny of the organisms, one
should evaluate a range of trees on the assumption that the phylogenetic
relationships that are supported by most or all of those trees are the most
probable phylogenetic relationships of the organisms (see Section 4).
As described above (see Sections 4.6 and 5.1), all four phylogenetic trees
shown in Figure 3 are consistent in showing tissue cyst-forming coccidia
(i.e. members of the genera Sarcocystis, Toxoplasma and Neospora) as a
monophyletic group (Tenter et al., 1992; Ellis and Morrison, 1995; Ellis
et al., 1995). This is in agreement with other phylogenetic analyses of
tissue cyst-forming coccidia based on SSU rRNA sequence comparisons
(Johnson et al., 1988, 1991; Johnson and Baverstock, 1989; Barta et al.,
1991; Gagnon et al., 1993; Ellis et al., 1994a, b; Fenger et al., 1994;
Holmdahl et al., 1994; Escalante and Ayala, 1995; Relman er al., 1996).
In addition, the phylogenetic trees constructed in these studies are con-
sistent in showing the genera Toxoplasmu and Neospora to be monophy-
letic (Ellis et al., 1994a, b; Holmdahl et al., 1994; Ellis and Morrison,
1995; Escalante and Ayala, 1995; Jeffries et al., 1997).
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 115
The SSU rRNA molecule is one of the slowest evolving molecules found
throughout the range of living organisms (Hillis and Dixon, 1991). It has
been suggested that SSU rRNA sequence comparison for inference of
phylogenetic relationships is useful only for organisms that diverged
more than 80-100 million years ago (Baverstock and Johnson, 1990).
This technique may reach its limits if organisms are more closely related,
that is, if they diverged less than 80 million years ago. If phylogenetic
relationships are inferred from SSU rRNA sequences of very closely
related species, the extent of microheterogeneity between repeated SSU
rRNA sequences of one species may exceed the extent of nucleotide
differences among the SSU rRNA sequences of different species (Schlegel,
1991; Cai et al., 1992). Usually, phylogenetic relationships inferred from
SSU rRNA sequences become statistically uncertain if the distances among
the compared sequences are less than one nucleotide change per 100
positions (Sogin et ul., 1989; Patterson and Sogin, 1992). Thus, even if
the outgroup, the alignment of homologous nucleotide positions and the
data set used for analysis have all been optimized for inference of phylo-
genetic trees from SSU rRNA sequences, the technique can still be limited
by the organisms themselves. Therefore, the differences found among the
phylogenetic trees of tissue cyst-forming coccidia inferred from SSU rRNA
sequence comparisons (see Sections 4.6 and 5.2) are consistent with the
hypothesis that tissue cyst-forming coccidia are highly derived protozoa. In
other words, the phylogenetic relationships or genetic relatedness of these
parasites are so close that there are few differences among their SSU rRNA
sequences, so that even small changes in the SSU rRNA regions and
methods used for phylogenetic analysis make large differences to the trees
obtained and the probability of obtaining the wrong tree is high (Nei,
1992).
118 A.M. TENTER AND A.M. JOHNSON
s r e n e ~ aI - 0.005 0.010 0.024 0.030 0.024 0.023 0.024 0.031 0.030 0.035 0.032
s capracanrs I 8 - 0.008 0.022 0.029 0.022 0.020 0.022 0.029 0.028 0.030 0.028
s arierrcanis I 15 I3 0.025 0.027 0.025 0.024 0.025 0.033 0.032 0.037 0.033
s mouler ’ 37 33 39 - 0.031 0.000 0.010 0.000 0.018 0.017 0.022 0.018
s gigantea ’ 45 44 42 48 - 0.031 0.031 0.031 0.042 0.041 0.046 0.043
s fuslformrs ‘ 37 33 39 0 48 - 0.010 0.000 0.018 0.017 0.022 0.018
s murrs ’ 35 31 37 15 48 15 - 0.010 0.021 0.020 0.025 0.023
s neurona ’ 37 33 39 0 48 0 15 - 0.018 0.017 0.022 0.0I8
N canrnum 47 44 50 27 64 27 32 27 - 0.000 0.005 0.010
T gondrr S48 46 43 49 26 63 26 31 26 0 - 0.005 0.010
T gondir SAlLlE 53 46 56 33 70 33 38 33 7 7 - 0.012
I Jars 49 43 51 27 66 27 35 27 16 15 18
ic Ea Eb Ema Emi En Ep El Cb Cm cp cw
I.p i s 0.088 0.091 0.090 0.091 0.087 0.088 0.083 0.107 0.099 0.111 0.110
E. acervulina 135 0.011 0.014 0.012 0.014 0.008 0.010 0.134 0.123 0.135 0.134
E brunerri 139 17 0.014 0.013 0.021 0.014 0.017 0.138 0.127 0.136 0.136
E. maxima 138 22 22 0.018 0.022 0.014 0.018 0.138 0.127 0.134 0.135
E. milis 139 18 20 27 0.019 0.012 0.015 0.141 0.128 0.141 0.139
E. necalrix I33 22 32 34 29 - 0.016 0.004 0.134 0.125 0.134 0.132
E. praecox I35 13 21 22 19 25 - 0.012 0.134 0.123 0.133 0.133
E. tenella 127 16 26 28 23 6 19 0.133 0.122 0.132 0.131
C. baileyr I62 205 21 1 210 214 205 205 203 - 0.034 0.024 0.024
C muris I50 I88 193 I94 I95 190 188 I86 52 - 0.045 0.046
c.parvum I69 206 207 204 214 204 203 202 36 69 - 0.003
C. wrairi I68 204 207 206 212 202 203 200 36 70 5 -
S. tenella I 0.005 0.024 0.030 0.031 0.035 0.095 0.097 0.089 0.085 0.089 0.100
s capracanis I 8 - 0.022 0.029 0.029 0.030 0.094 0.095 0.087 0.083 0.087 0.100
s moulei’ 37 33 - 0.031 0.018 0.022 0.088 0.088 0.081 0.077 0.095 0.110
S. gigantea ’ 45 44 48 - 0.042 0.046 0.103 0.105 0.099 0.096 0.102 0.113
N canrnum 47 44 27 64 - 0.005 0.087 0.088 0.083 0.079 0.092 0.105
T. gondii SAlLlE 53 46 33 70 7 - 0.092 0.092 0.088 0.084 0.097 0.109
E. maxima 145 144 134 157 134 141 - 0.018 0.022 0.018 0.127 0.135
E. miris I48 145 134 160 134 141 27 0.019 0.015 0.128 0.139
- 0.004
~
6. CONCLUSIONS
Figure 5 Continued
Cryptosporidiurn rnuris; Cp, Cryptosporidiurn parvurn; Cw, Cryptosporidiurn
’
wrairi. Pathogenic Sarcocystis species using canids as definitive hosts. * Non-
pathogenic Sarcocystis species using felids as definitive hosts. Pathogenic Sar-
cocystis species with uncertain definitive host (probably opossum). For sources of
sequences see Tenter (1995) and Jeffries et al. (1997).
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 121
ACKNOWLEDGEMENTS
The studies described here were partially funded by grants from the
Australian Research Council and Deutsche Forschungsgemeinschaft. We
thank Kim Luton and Michael Johnson for technical assistance, and Alex
Jeffries, John Ellis, David Morrison, Peter Baverstock and Michel
Rommel for critical discussion, prepublication information and review
of the manuscript.
REFERENCES
Allsopp, M.T.E.P., Cavalier-Smith, T., De Waal, D.T. and Allsopp, B.A. (1994).
Phylogeny and evolution of the piroplasms. Parasitology 108, 147-152.
Ashford, R.W. (1977). The fox, Vulpes vulpes, as a final host for Sarcocystis of
sheep. Annals of Tropical Medicine and Parasitology 71, 29-34.
Babudieri, B. (1932). I sarcosporidi e le sarcosporidiosi. Studio monografico.
Archiv fur Protistenkunde 76, 421-580.
Baker, J.R. (1987). Reply to Frenkel, Mehlhorn and Heydorn. Parasitology Today
3, 252.
Balbiani, G . (1882). Les sporozoaires. Journal de Micrographie (Paris) 6, 80-89.
Barretto, M.P. (1940). Contribuigiio ao estudo dos Sarcosporidia Butschli, 1882,
com a descriqgo de uma nova espCcie: Sarcocystis jacarinae, n. sp., parasita do
‘tiziu’ (Volatinia jacarina L.). Arquivos do Zoologia S6o Paul0 1, 339-368.
Barta, J.R. (1989). Phylogenetic analysis of the class Sporozoea (phylum Apicom-
plexa Levine, 1970): evidence for the independent evolution of heteroxenous life
cycles. Journal of Parasitology 75, 195-206.
Barta, J.R., Jenkins, M.C. and Danforth, H.D. (1991). Evolutionary relationships of
avian Eimeria species among other apicomplexan protozoa: monophyly of the
Apicomplexa is supported. Molecular Biology and Evolution 8, 345-355.
126 A.M. TENTER AND A.M. JOHNSON
Baverstock, P.R. and Johnson, A.M. (1990). Ribosomal RNA nucleotide sequence:
a comparison of newer methods used for its determination, and its use in
phylogenetic analysis. Australian Systematic Botany 3, 101-1 10.
Beanland, T.J. and Howe, C.J. (1992). The inference of evolutionary trees from
molecular data. Comparative Biochemistry and Physiology 102B, 643-659.
Bernard, P.N. and Bauche, J. (1912). Filariose et atherome aortique du buffle et du
boeuf. Bulletin de la Socie'te' de Pathologie Exotique 5, 109-114.
Bettencourt, A., Franqa, C. and Borges, J. (1907). Un cas de piroplasmose bacilli-
forme chez le daim. Arquivos do Instituto Bacterioldgia Camara Pestana 1,341.
Biocca, E. (1956). Schema di classificazione dei protozoi e proposta di una nuova
classe. Atti della Accademia Nazionale dei Lincei, Rendiconti, Classe di Scienze
Fisiche, Matematiche e Naturali, Serie 8, 21,453455.
Biocca, E. (1957). Alcune considerazioni sulla sistematica dei protozoi e sulla
utilith di creare una nuova classe di protozoi. Revista Brasileira de Malariologia
e Doengas Tropicais 8, 91-102.
Biocca, E. (1968). Class Toxoplasmatea: critical review and proposal of the new
name Frenkelia gen. n. for M-organism. Parassitologia 10, 89-98.
Blanchard, R. (1885). Note sur les sarcosporidies et sur un essai de classification de
ces sporozoaires. Bulletin de la Socie'te' Zoologique de France 10, 244276.
Britten, R.J. (1986). Rates of DNA sequence evolution differ between taxonomic
groups. Science 231, 1393-1398.
Butschli, 0. (1882). 111. Sarcosporidia. In: Die Klassen und Ordnungen des Thier-
reichs, Vol. 1, (H.G. Bronn, ed.), pp. 604-616. Leipzig: Akademische Verlags-
gesellschaft Geest & Portig.
Cai, J., Collins, M.D., McDonald, V. and Thompson, D.E. (1992). PCR cloning and
nucleotide sequence determination of the 18s rRNA genes and internal
transcribed spacer 1 of the protozoan parasites Cryptosporidium parvum and
Cryptosporidium muris. Biochimica et Biophysica Acta 1131, 3 17-320.
Cavalier-Smith, T. (1993). Kingdom Protozoa and its 18 phyla. Microbiological
Reviews 57, 953-994.
Cawthorn, R.J. and Speer, A.C. (1990). Sarcocystis: infection and disease of
humans, livestock, wildlife, and other hosts. In: Coccidiosis of Man and Domes-
tic Animals (P.L. Long, ed.), pp. 91-120. Boca Raton: CRC Press.
Ctrn6, Z., Kol6rov6, I. and Sulc, P. (1978). Contribution to the problem of cyst-
producing coccidians. Folia Parasitologica (Praha) 25, 9-16.
Cheissin, E.M. and Poljansky, G.I. (1963). On the taxonomic system of Protozoa.
Acta Protozoologica 1, 327-352.
Collins, G.H., Atkinson, E. and Charleston, W.A.G. (1979). Studies on Sarcocystis
species III: The macrocystic species of sheep. New Zealand Veterinary Journal
27, 204-206.
Corliss, J.O. (1994). An interim utilitarian ('user-friendly') hierarchical classifica-
tion and characterization of the protists. Acta Protozoologica 33, 1-5 1.
Cox, F.E.G. (1981). A new classification of the parasitic protozoa. Protozoological
Abstracts 5, 9-14.
Cox, F.E.G. (1991). Systematics of parasitic protozoa. In: Parasitic Protozoa, 2nd
edn, Vol. 1, (J.P. Kreier and J.R. Baker, eds), pp. 55-80. San Diego: Academic
Press.
Cox, F.E.G. (1994). The evolutionary expansion of the Sporozoa. International
Journal for Parasitology 24, 1301-1316.
Current, W.L., Upton, S.J. and Long, P.L. (1990). Taxonomy and life cycles. In:
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 127
Coccidiosis of Man and Domestic Animals (P.L. Long, ed.), pp. 1-16. Boca
Raton: CRC Press.
DardB, M.L., Riahi, H.,Bouteille, B. and Pestre-Alexandre, M. (1992). Isoenzyme
analysis of Hammondia hammondi and Toxoplasma gondii sporozoites. Journal
of Parasitology 78, 731-734.
De Rijk, P. and De Wachter, R. (1993). DCSE, an interactive tool for sequence
alignment and secondary structure research. Computer Applications in Biologi-
cal Sciences 9, 735-740.
Doflein, F. (1901). Die Protozoen als Parasiten und Krankheitserreger nach
biologischen Gesichtspunkten dargestellt. III. Klasse: Sporozoa, pp. 93-225.
Jena: Gustav Fischer.
Doflein, F. and Reichenow, E. (1953). Lehrbuch der Protozoenkunde. Eine Dar-
stellung der Naturgeschichte der Protozoen mit besonderer Beriicksichtigung
der parasitischen und pathogenen Fonnen, 6th edn, pp. 777-1080. Jena: Gustav
Fi scher.
Dubey, J.P. (1976). A review of Sarcocystis of domestic animals and of other
coccidia of cats and dogs. Journal of the American Veterinary Medical Associa-
tion 169, 1061-1078.
Dubey, J.P. (1977a). Taxonomy of Sarcocystis and other coccidia of cats and dogs.
Journal of the American Veterinary Medical Association 170, 778-782.
Dubey, J.P. (1977b). Toxoplasma, Hammondia, Besnoitia, Sarcocystis, and other
tissue cyst-forming coccidia of man and animals. In: Parasitic Protozoa, Vol. 3,
(J.P. Kreier, ed.), pp. 101-237. New York: Academic Press.
Dubey, J.P. (1990). Neospora caninum: a look at a new Toxoplasma-like parasite
of dogs and other animals. Compendium on Continuing Education for the
Practicing Veterinarian 12, 653-663.
Dubey, J.P. (1992). A review of Neospora caninum and Neospora-like infections in
animals. Journal of Protozoology Research 2, 40-52.
Dubey, J.P. (1993). Toxoplasma, Neospora, Sarcocystis, and other tissue cyst-
forming coccidia of humans and animals. In: Parasitic Protozoa, 2nd edn,
Vol. 6, (J.P. Kreier, ed.), pp. 1-158. San Diego: Academic Press.
Dubey, J.P. and Beattie, C.P. (1988). Toxoplasmosis of Animals and Man. Boca
Raton: CRC Press.
Dubey, J.P. and Fayer, R. (1983). Sarcocystosis. British Veterinary Journal 139,
371-377.
Dubey, J.P. and Lindsay, D.S. (1993). Neosporosis. Parasitology Today 9,452458.
Dubey, J.P., Miller, N.L. and Frenkel, J.K. (1970). The Toxoplasma gondii oocyst
from cat feces. Journal of Experimental Medicine 132, 636-662.
Dubey, J.P., Carpenter, J.L., Speer, C.A., Topper, M.J. and Uggla, A. (1988).
Newly recognized fatal protozoan disease of dogs. Journal of the American
Veterinary Medical Association 192, 1269-1285.
Dubey, J.P., Speer, C.A. and Fayer, R. (1989). Sarcocystosis of Animals and Man.
Boca Raton: CRC Press.
Dubey, J.P., Davis, S.W., Speer, C.A., Bowman, D.D., de Lahunta, A., Granstrom,
D.E., Topper, M.J., Hamir, A.N., Cummings, J.F. and Suter, M.M. (1991).
Sarcocystis neurona n. sp. (Protozoa: Apicomplexa), the etiologic agent of
equine protozoal myeloencephalitis. Journal of Parasitology 77, 212-21 8.
Eckert, J., Rommel, M. and Kutzer, E. (1992). Systematik und Taxonomie. In:
Veterinunnedizinische Parasitologie, 4th edn (J. Eckert, E. Kutzer, M. Rommel,
H.J. Burger and W. Korting, eds), pp. 4-22. Berlin: Paul Parey.
128 A.M. TENTER AND A.M. JOHNSON
Goldman, M., Carver, R.K. and Sulzer, A.J. (1958). Reproduction of Toxoplasma
gondii by internal budding. Journal of Parasitology 44, 161-171.
Gothe, R. and Reichler, I. (1990). Zur Befallshaufigkeit von Kokzidien bei Hun-
defamilien unterschiedlicher Haltung und Rassen in Siiddeutschland. Tierdrz-
tliche Praxis 18, 407413.
Grass& P.P. (1953). Sous-embranchement des Sporozoaires. In: Traite' de Zoolo-
gie. Anatomie, Syste'mtique, Biologie. Tome 1, Fascicule 2, Protozoaires:
Rhizopodes, Actinopodes, Sporozoaires, Cnidosporidies (P.P. Grass& ed.), pp.
545-1005. Paris: Masson et Cie.
Greene, C.E. and Prestwood, A.K. (1984): Coccidial infections. In: Clinical Micro-
biology and Infectious Diseases of the Dog and Cat (C.E. Greene, ed.). Chap. 57,
pp. 824-858. Philadelphia: W.B. Saunders.
Gunderson, J.H., McCutchan, T.F. and Sogin, M.L. (1986). Sequence of the small
subunit ribosomal RNA gene expressed in the bloodstream stages of Plasmodium
berghei: evolutionary implications. Journal of Protozoology 33, 525-529.
Guo, Z.G. and Johnson, A.M. (1995a). Genetic comparison of Neospora caninum
with Toxoplasma and Sarcocystis by random amplified polymorphic DNA PCR.
Parasitology Research 81, 365-370.
Guo, Z.G. and Johnson, A.M. (1995b). Genetic characterisation of Toxoplasma
gondii strains by random amplified polymorphic DNA polymerase chain
reaction. Parasitology 111, 127-132.
Hasegawa, M., Iida, Y., Yano, T., Takaiwa, F. and Iwabuchi, M. (1985). Phylo-
genetic relationships among eukaryotic kingdoms inferred from ribosomal RNA
sequences. Journal of Molecular Evolution 22, 32-38.
Hasselmann, G. (1923). Parasitoses das carnes de consumo (4" nota prkvia). Brazil
Medico 2, 341.
Henry, A. (1913). Besnoitia besnoiti (Marotel, 1913). Recueil de Me'decine Ve'te'r-
inaire 90, 328.
Henry, D.P. (1932). Zsospora buteonis sp. nov. from the hawk and owl, and notes
on Isospora lacazii (Labb6) in birds. University of California Publications in
Zoology 37, 291-300.
Heydorn, A.O. (1985). Zur Entwicklung von Sarcocystis arieticanis n. sp. Berliner
und Munchener Tierdrztliche Wochenschrift 98, 23 1-241.
Heydorn, A.O. and Rommel, M. (1972a). Beitrage zum Lebenszyklus der Sarko-
sporidien. II. Hund und Katze als aertrager der Sarkosporidien des Rindes.
Berliner und Miinchener Tierdrztliche Wochenschrift 85, 121-123.
Heydom, A.O. and Rommel, M. (1972b). Beitrage zum Lebenszyklus der Sar-
kosporidien. IV. Entwicklungsstadien von S. fusiformis in der Diinndarm-
schleimhaut der Katze. Berliner und Miinchener Tierdrztliche Wochenschrift
85, 333-336.
Heydorn, A.O., Gestrich, R., Mehlhorn, H. and Rommel, M. (1975). Proposal for
a new nomenclature of the sarcosporidia. Zeitschrift fur Parasitenkunde 48,
73-82.
Hiepe, T. and Jungmann, R. (1983). Veterintimedizinische Protozoologie, Stamm
Apicomplexa. In: Lehrbuch der Parasitologie, Vol. 2 (T. Hiepe, ed.), pp. 11-15,
63-210. Stuttgart: Gustav Fischer Verlag.
Hillis, D.M. and Dixon, M.T. (1991). Ribosomal DNA: molecular evolution and
phylogenetic inference. Quarterly Review of Biology 66, 41 1 4 5 3 .
Hillis, D.M. and Moritz, C. (1990). Molecular Systematics. Sunderland: Sinauer.
Hillis, D.M., Allard, M.W. and Miyamoto, M.M. (1993). Analysis of DNA
sequence data: phylogenetic inference. Methods in Enzymology 224, 456-487.
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 131
Holmdahl, O.J.M., Mattsson, J.G., Uggla, A. and Johansson, K.E. (1994). The
phylogeny of Neospora caninum and Toxoplasma gondii based on ribosomal
RNA sequences. FEMS Microbiology Letters 119, 187-192.
Honigberg, B.M., Balamuth, W., Bovee, E.C., Corliss, J.O., Gojdics, M., Hall,
R.P., Kudo, R.R., Levine, N.D., Loeblich, A.R., Weiser, J. and Wenrich, D.H.
(1964). A revised classification of the phylum Protozoa. Journal of Protozoology
11,7-20.
Ho-Yen, D.O. and Joss, A.W.L. (1992). Human Toxoplasmosis. Oxford: Oxford
University Press.
Hutchison, W.M. (1965). Experimental transmission of Toxoplasrna gondii. Nature
206, 961-962.
Hutchison, W.M., Dunachie, J.F., Siim, J.C. and Work, K. (1969). Life cycle of
Toxoplasma gondii. British Medical Journal iv, 806.
Hutchison, W.M., Dunachie, J.F., Siim, J.C. and Work, K. (1970). Coccidian-like
nature of Toxoplasma gondii. British Medical Journal i, 142-144.
Hutchison, W.M., Dunachie, J.F., Work, K. and Siim, J.C. (1971). The life cycle of
the coccidian parasite, Toxoplasma gondii, in the domestic cat. Transactions of
the Royal Society of Tropical Medicine and Hygiene 65, 380-399.
Jackson, M.H. and Hutchison, W.M. (1989). The prevalence and source of Toxo-
plasma infection in the environment. Advances in Parasitology 28, 55-105.
Jacobs, L., Remington, J.S. and Melton, M.L. (1960a). The resistance of the
encysted form of Toxoplasma gondii. Journal of Parasitology 46, 11-21.
Jacobs, L., Remington, J.S. and Melton, M.L. (1960b). A survey of meat samples
from swine, cattle, and sheep for the presence of encysted Toxoplasrna. Journal
of Parasitology 46, 23-28.
Jeffries, A., Schnitzler, B., Tenter, A.M., Heydorn, A.O. and Johnson, A.M. (1997).
Identification of synapomorphic characters in the genus sarcocystis. Journal of
Eukaryotic Microbiology, (in press).
Joachim, A., Jeffries, A., Tenter, A.M. and Johnson, A.M. (1996). A RAPD-PCR
derived marker can differentiate between pathogenic and non-pathogenic Sarco-
cystis species in sheep. Molecular and Cellular Probes 10, 165-172.
Johnson, A.M. (1990). Toxoplasma: biology, pathology, immunology, and treat-
ment. In: Coccidiosis of Man and Domestic Animals (P.L. Long, ed.). Chap. 7,
pp. 121-153. Boca Raton, CRC Press.
Johnson, A.M. and Baverstock, P.R. (1989). Rapid ribosomal RNA sequencing and
the phylogenetic analysis of protists. Parasitology Today 5, 102-105.
Johnson, A.M., Illana, S., Dubey, J.P. and Dame, J.B. (1987a). Toxoplasma gondii
and Hammondia hammondi: DNA comparison using cloned rRNA gene probes.
Experimental Parasitology 63, 272-278.
Johnson, A.M., Murray, P.J., Illana, S. and Baverstock, P.R. (1987b). Rapid
nucleotide sequence analysis of the small subunit ribosomal RNA of
Toxoplasma gondii: evolutionary implications for the Apicomplexa. Molecular
and Biochemical Parasitology 25, 239-246.
Johnson, A.M., Illana, S., Hakendorf, P. and Baverstock, P.R. (1988). Phylogenetic
relationships of the apicomplexan protist Sarcocystis as determined by small
subunit ribosomal RNA comparison. Journal of Parasitology 74, 847-860.
Johnson, A.M., Adoutte, A., Cavalier-Smith, T. and Lynn, D.H. (1990a). Phylo-
geny and evolution of protozoa. Zoological Science, Supplement 7, 179-188.
Johnson, A.M., Fielke, R., Lumb, R. and Baverstock, P.R. (1990b). Phylogenetic
relationships of Cryptosporidium determined by ribosomal RNA sequence
comparison. International Journal for Parasitology 20, 141-147.
132 A.M. TENTER AND A.M. JOHNSON
Johnson, A.M., Fielke, R., Ellis, J., O'Donoghue, P.J. and Baverstock, P.R. (1991).
The phylogenetic relationships of the genus Eimeria based on comparison of
partial sequences of 18s rRNA. Systematic Parasitology 18, 1-8.
Kalyakin, V.N. and Zasukhin, D.N. (1975). Distribution of Sarcocystis (Protozoa:
Sporozoa) in vertebrates. Folia Parasitologica 22, 289-307.
Knoll, A.H. (1992). The early evolution of eukaryotes: a geological perspective.
Science 256, 622-627.
Krampitz, H.E. and Rommel, M. (1977). Experimentelle Untersuchungen uber das
Wirtsspektrum der Frenkelien der Erdmaus. Berliner und Munchener Tierarz-
tliche Wochenschrifr 90, 17-19.
Krampitz, H.E., Rommel, M., Geisel, 0. and Kaiser, E. (1976). Beitrage zum
Lebenszyklus der Frenkelien. II. Die ungeschlechtliche Entwicklung von Fren-
kelia clethrionomyobuteonis in der Rotelmaus. Zeitschriftfur Parasitenkunde 51,
7-14.
Kreier, J.P. (1993). Parasitic Protozoa, 2nd edn, Vols. 4-6. San Diego: Academic
Press.
Kreier, J.P. and Baker, J.R. (1987). Parasitic Protozoa, pp. 1-1 1, 123-158. Boston:
Allen & Unwin.
Krylov, M.V. (1992). The origin of heteroxeny in Sporozoa. Parassitologia 26,
361-368.
Kudo, R.R. (1950). Protozoology, 3rd edn. Class 3 Sporozoa Leuckart, pp.
427-544. Springfield: Charles C. Thomas.
Kuhn, D. and Weiland, G. (1969). Experimentelle Toxoplasma-Infektionen bei der
Katze. I. Wiederholte ijbertragung von Toxoplasma gondii durch Kot von mit
Nematoden infizierten Katzen. Berliner und Munchener Tieriirztliche Wochen-
schrift 82,401-404.
Kuhn, J. (1865). Untersuchungen uber die Trichinenkrankheit der Schweine. Mitt-
eilungen des landwirthschafrlichen Instituts der Universitat Halle, 1-84.
LabbC, A. (1899). Sporozoa. In: Das Tierreich. Eine Zusammenstellung und
Kennzeichnung der rezenten Tieqormen 5. Lieferung (F.E. Schulze and 0.
Butschli, eds), pp. 115-1 19. Berlin: R. Friedlander.
Landau, 1. (1974). Hypothtses sur la phylogCnie des Coccidiomorphes de
vertCbrCs. Zeitschrift fur Parasitenkunde 45, 63-75.
Lane, D.J., Pace, B., Olsen, G.J., Stahl, D.A., Sogin, M.L. and Pace, N.R. (1985).
Rapid determination of 16s ribosomal RNA sequences for phylogenetic
analyses. Proceedings of the National Academy of Sciences of the USA 82,
6955-6959.
Lankester, E.R. (1882). On Drepanidium ranarum, the cell-parasite of the frog's
blood and spleen (Gaule's Wurmschen). Quarterly Journal of Microscopical
Science 22, 53-65.
Lankester, E.R. (1875-1 889). Protozoa. In: The Encyclopaedia Britannica: A
Dictionary of Arts, Sciences, and General Literature, 9th edn, pp. 830-866.
Edinburgh: Black.
LCger, L. (1911). Caryospora simplex, coccidie monosporCe et la classification des
coccidies. Archiv fur Protistenkunde 22, 7 1-85.
LCger, L. and Duboscq, 0. (1910). Selenococcidium intermedium LCg. et Dub. et la
systkmatique des sporozoaires. Archives de Zoologie Expe'rimentale et Ge'ntrale
45, 187-237.
Leuckart, R. (1879-1886). Die Parasiten des Menschen und die von ihnen herr-
hurenden Krankheiten. Vol. 1, pp. 221-334, 959-968. Leipzig: C.F. Winter.
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 133
Swofford, D.L. and Olsen, G.J. (1990). Phylogeny reconstruction. In: Molecular
Systematics (D.M. Hillis and C. Moritz, eds), pp. 41 1-501. Massachusetts:
Sinauer.
Tadros, W. and Laarman, J.J. (1976). Sarcocystis and related coccidian parasites:
a brief general review, together with a discussion on some biological aspects of
their life cycles and a new proposal for their classification. Acta Leidensia 44,
1-137.
Tadros, W. and Laarman, J.J. (1982). Current concepts on the biology, evolution
and taxonomy of tissue cyst-forming eimeriid coccidia. Advances in Parasitol-
ogy 20,293-468.
Tenter, A.M. (1995). Current research on Sarcocystis species of domestic animals.
International Journal for Parasitology 25, 1311-1330.
Tenter, A.M., Baverstock, P.R. and Johnson, A.M. (1992). Phylogenetic relation-
ships of Sarcocystis species from sheep, goats, cattle and mice based on ribo-
somal RNA sequences. International Journal for Parasitology 22, 503-5 13.
Thomford, J.W., Conrad, P.A., Telford, S.R., Mathiesen, D., Bowman, B.H.,
Spielman, A. , Eberhard, M.L., Herwaldt, B.L., Quick, R.E. and Persing, D.H.
(1994). Cultivation and phylogenetic characterization of a newly recognized
human pathogenic protozoan. Journal of Infectious Diseases 169, 1050-1056.
Tyzzer, E.E. (1907). A sporozoan found in the peptic glands of the common mouse.
Proceedings of the Society for Experimental Biology and Medicine 5, 12-13.
Uggla, A. and Buxton, D. (1990). Immune responses against Toxoplasma and
Sarcocystis infections in ruminants: diagnosis and prospects for vaccination.
Revue ScientiJque et Technique, OfJice International des Epizooties 9,44 1-462.
Van de Peer, Y.,Van den Broek, I., De Rijk, P. and De Wachter, R. (1994).
Database on the structure of small subunit ribosomal RNA. Nucleic Acids
Research 22, 3488-3494.
Vivier, E. (1982). Rtflexions et suggestions A propos de la systtmatique des
sporozoaires: crtation d’une classe des Hematozoa. Protistologica 18,449-457.
Wainright, P.O., Hinkle, G., Sogin, M.L. and Stickel, S.K. (1993) Monophyletic
origins of the metazoa: an evolutionary link with fungi. Science 260, 340-342.
Wallace, G.D. and Frenkel, J.K. (1975). Besnoitia species (Protozoa, Sporozoa,
Toxoplasmatidae): recognition of cyclic transmission by cats. Science 188,
369-37 1.
Waters, A.P. (1994). The ribosomal RNA genes of Plasmodium. Advances in
Parasitology 34, 33-79.
Waters, A.P., Higgins, D.G. and McCutchan, T.F. (1991). Plasmodium falci-
parum appears to have arisen as a result of lateral transfer between avian
and human hosts. Proceedings of the National Academy of Sciences of the USA
88, 3140-3144.
Waters, A.P., Higgins, D.G. and McCutchan, T.F. (1993a). Evolutionary related-
ness of some primate models of Plasmodium. Molecular Biology and Evolution
10, 914-923.
Waters, A.P., Higgins, D.G. and McCutchan, T.F. (1993b). The phylogeny of
malaria: a useful study. Parasitology Today 9, 246-250.
Weiland, G. and Kiihn, D. (1970). Experimentelle Toxoplasma-Infektionen bei der
Katze. II. Entwicklungsstadien des Parasiten im Darm. Berliner und Miinchener
Tierdrztliche Wochenschrift 83, 128-132.
Wenyon, C.M. (1923). Coccidiosis of cats and dogs, and the status of the Isospora
of man. Annals of Tropical Medicine and Parasitology 17, 23 1-288.
PHYLOGENY OF THE TISSUE CYST-FORMING COCClDlA 139
Since this review was submitted, some major advances have been made that
have a significant bearing on knowledge of the phylogeny of the tissue cyst-
forming coccidia.
(i) A new method of phylogenetic analysis of 18s rRNA entitled ‘substitution
rate calibration’ finds the genus Sarcocystis to be monophyletic (Van de Peer, Y.,
Van der Auwera, G. and De Wachter, R., 1996. The evaluation of Stramenopiles
and Alveolates as derived by ‘substitution rate calibration’ of small ribosomal
subunit RNA. Journal of Molecular Evolution 42, 201-210).
(ii) Collaborative work by an Australian, a Canadian and a German group has
shown that a homoxenous and a facultatively hetroxenous Isospora species (i.e.
Isospora suis and I. felis, respectively) form a monophyletic clade, which is the
sister group to the Toxoplasma/lveosporaclade to the exclusion of the Sarcocystis
clade (Carreno, R.A., Schnitzler, B.E., Jeffries, A.C., Tenter, A.M., Johnson, A.M.
and Barta, J.R., Phylogenetic analysis of coccidia based on 18s rDNA sequence
comparison indicates that Isospora is most closely related to Toxoplasma and
Neospora; paper submitted for publication). This finding will have major implica-
tions for the classification of the genus Isospora, because it shows that homoxenous
as well and heteroxenous Isospora species should bot be classified in the family
Eimeriidae, but should be classified together with the tissue cyst-forming coccidia.
This would result in a family comprising all coccidia which have oocysts containing
two sporocysts, each with four sporozoites. Depending on the type genus of the
family, the family name would have to be either Sarcocystidae Poche, 1913 or
Isosporidae Minchin, 1903.
Biochemistry of the Coccidia
1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
2. Energy Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
2.1. Eimeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
2.2. Toxoplasrna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
2.3. Cryptosporidium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
2.4. Sarcocystis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3. Protein and Amino Acid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3.1. Protein synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
3.2. Protein catabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
3.3. Amino acid interconversions .................................. 171
4. Polyamine Metabolism ......................................... 171
5. Purine and Pyrimidine Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
5.1. Purine metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
5.2. Pyrimidine metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
6. Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
7. Lipid Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
8. Culturing Coccidia in vitro and Growth Factor Requirements . . . . . . . . . . . . 185
9. Antioxidant Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
10. The Oocyst Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188
I1 . Functional Surface Molecules .................................... 188
11.1 Glycosylation ............................................. 189
11.2 GPlanchors .............................................. 189
11.3 Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
11.4 Sialidase and other enzymes ................................. 190
11.5 Cell signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
1. INTRODUCTION
There are many parasites in the group known as the coccidia, but only
Toxoplasma gondii and Eimeria species have been investigated biochemi-
cally in any detail. Cryptosporidium parvum is now attracting attention,
although at present little is known of its biochemistry, whereas Sarcocystis,
Neospora and Zsospora have been studied in just a few aspects. This review
reflects the emphasis on studying ‘important’ parasites and thus most of the
content concerns them; the extent to which other species are similar
remains to be seen. It is to be expected, however, that closely related
organisms will share many features and one of the aims of this review is
to pin-point biochemical characteristics of the coccidia as a group. Such
common features, especially when they are significantly different from the
host, provide targets that potentially can be exploited with novel anti-
coccidial agents.
The advent of molecular methods for studying evolutionary relationships
has provided many new insights into the phylogeny of protozoa and
enforced a major re-assessment of previously accepted dogma (see Coombs
et af., 1997). Current evidence supports the view that the coccidia are a
related group, although there is increasing evidence that Cryptosporidium
may not be a true coccidian. For the purpose of this review, however, we
have included all organisms that have generally been accepted as coccidia
in recent years. Good knowledge of the phylogenetic relationships between
parasites will be valuable to parasite biochemists in enabling them to make
better predictions on the likely biochemical composition of a parasite and
helping to explain the presence and evolutionary origin of the more pecu-
liar features - such as the plastid-like organelles, mannitol cycle and
pyrophosphate-linked glycolytic enzymes of coccidia. Coccidia have
some components which are similar to their counterparts in plants, such
as dihydrofolate reductasehhymidylate synthase and also their micro-
tubules, but it is important to avoid making general conclusions about
similarities and differences from the very limited data available at present.
Current knowledge of the biochemistry of Toxopfasma and Eimeria is
fragmentary, so in this review we attempt not only to report the known
data but also to use these to hypothesize on possible common adaptations
BIOCHEMISTRY OF THE COCClDlA 143
2. ENERGY METABOLISM
Only the energy metabolism of Eimeria species has been studied in suffi-
cient detail to produce a reasonably coherent picture and so information on
this parasite dominates this section. The little that is known about other
coccidial parasites is either mentioned as appropriate in Section 2.1 or
detailed separately in Sections 2.2, 2.3 and 2.4.
2.1. Eimeria
Glycolysis
Hexokinase J1, 7 J1, 6 J1 J29,30, 31 J38 E l J42
Glucosephosphate
isomerase Ji’, 8 J6, 8, 25 J1 J21, 30, 31 J32, 33
Pyrophosphate-
phosphohctokinase J1 J1 J1 J1.22 J1
Aldolase J1, 3, 7 J6 (30 J38
Triose phosphate
isomerase Jl X6
Glyceraldehyde 3-phos-
phate dehydrogenase J6
Phosphoglycerate kinase J7
Phosphoglucose mutase JI, 9 J6. 25 J1 J 3 0 , 31 J34 J32, 33
Enolase J7 J6
Pyruvate kinase J1, 7 J1, 6 J1 J1 J38 J1
Lactate dehydrogenase J1, 2, I, 8 J1, 6, 8, 25 J 1 , 19 J30,31 J38 J1, 33, 42
TCA cycle
Citrate synthetase M
Aconitase J30
Iswitrate dehydrogenase $6 J 3 0 , 31 81
a-Ketoglutarate
dehydrogenase $6
Succinate CoA
synthetase E6
Succinate dehydrogenase J 4 . 15 E l 86, 1 J 4 J4 J4 84 84, 15 J1 E1.42
Fumarase 36 J29. 30 J33
Malate dehydrogenase J6 J42
Table 1B Enzymes of carbohydrate metabolism in coccidia"
Eimeria
Unsporulated Sporulated Sporozoites Merozoites Meronts Microgame- Macrogame- Toxoplasma Sarcocystis Cryptosporidium
oocysts oocysts tocytes tocytes
" J, Detected, Ir, apparently absent. Numbers indicate references: 1, Denton et al. (1994,1996aand unpublished observations); 2, Fransden and Cooper
(1972); 3, Mitchell and Daron (1982);4, Michael and Hodges (1973);5, Fransden (1976,1978);6, Smith, N.C. et al. (1994);7, Andrews et al. (1990); 8,
Shirley (1975); Rollinson et al. (1979); 10, Hosek et al. (1988); 11, Farooqui and Hanson (1988); 12, San-Martin Nunez et al. (1987); 13, Farooqui et al.
(1983); 14, Wang et al. (1979); 15, Beyer (1970); 16, Wang et al. (1975); 17, Karkhanis er al. (1993); 18, Heller and Scholtyseck (1970); 19, Fransden
(1970); 20, Schmatz et al. (1989); 21, Michalski et al. (1992); 22, Peng and Mansour (1992); 23, Sibley et al. (1994a); 25, Shirley et al. (1977); 26,
Vetterling and Waldrop (1976); 27, Darde et al. (1992); 28, Manafi et al. (1993); 29, Takeuchi et al. (1980); 30, Darde et al. (1988); 31, Barnert et al.
(1988); 32, Awad-El-Kariem et al. (1993,1995);33, Ogunkolade et al. (1993); 34, Atkinson and Collins (1981);35, Farooqui et al. (1987); 36, Chaudry
et al. (1985); 37, Chaudry et al. (1986b); 38, Gupta er al. (1992); 39, Gupta er al. (1993); 40,Fulton and Spooner (1960); 41, Metsis er al. (1995); 42,
Malek et al. (1996).
152 G.H. COOMBS ET AL.
energy production, at least for parts of their life cycle (Coombs and Muller,
1995). Like other micro-organisms containing PPi-utilizing glycolytic
enzymes, the coccidia lack cytosolic pyrophosphatase activity. The key
feature of type I PPi-PFKs is their lack of regulatory features. Since PFK is
a key enzyme in regulating glycolytic flux in most eukaryotes, organisms
which possess type I PPi-PFKs must utilize relatively unusual mechanisms
of glycolytic control.
In E. tenella and T. gondii it seems likely that glycolytic control is
exerted, at least partially, through the enzyme pyruvate kinase. Unlike
some other micro-organisms which have a PPi-PFK, the coccidia species
which have been investigated have an ADP-specific pyruvate kinase (PK)
rather than a PPi-specific activity (Denton et al., 1994, 1996a). The PKs of
E. tenella and T. gondii display sigmoidal saturation kinetics with respect
to their substrate phosphoenolpyruvate, but can be activated to hyperbolic
kinetics by certain compounds. The most potent of these activators are
glucose 6-phosphate and fructose 6-phosphate, which are relatively unu-
sual modulators. Fructose 1,6-bisphosphate, the major activator of most
eukaryotic PKs, is largely without effect. Unusually, the PK from C.
parvum shows no evidence of regulatory properties, presenting simple
Michaelis-Menten kinetics with respect to both its substrates (Denton et
al., 1996a). The only other PK so far reported not to be under allosteric
regulation is the type I enzyme from mammalian muscle (Fothergill-Gil-
more and Michels, 1993). Hexokinase, another regulatory enzyme in most
eukaryotes, appears to be unregulated, at least in Eimeria. Interestingly, the
parasite appears to contain only one hexokinase and this is capable of
phosphorylating both glucose (as in glycolysis) and fructose (as in the
mannitol cycle, see Section 2.1.2.(b)) (Schmatz et al., 1989; H. Denton
et al., unpublished observations). The presence of PPi-PFK is a distinct
difference between the coccidia and their hosts. The possibility that this
offers promise for rational chemotherapy is given credence by the report of
Peng et al. (1995) that some phosphonic acid analogues are capable of
specifically inhibiting PPi-PFK purified from T. gondii and also are selec-
tively toxic to parasites within their host cells in vitro.
All coccidia species which have been investigated contain high levels of
lactate dehydrogenase (LDH), the enzyme capable of mediating the oxida-
tion of reduced nicotinamide adenine dinucleotide (NADH) under anaero-
bic conditions. Once again, this might reflect a high dependence on
anaerobic energy generation within the coccidia. The enzyme was purified
from E. stiedai and characterized by Fransden and Cooper (1972). A single
enzyme was identified with an electrophoretic mobility corresponding to
the LDH4 of vertebrates. The enzyme was specific for L-lactate but was
capable of catalysing the reduction of both f3- and a-NAD (although the
rate with the latter was only 3% that with the former). The enzyme subunits
154 G.H. COOMBS EJAL.
Glucose 6-phosphate
t N
NAD(P)'
A
\
D P Mannitol
~ 1-phosphate wpi
5
ANt:
\
""k
Fructose 6-phosphate
Fructose 1,6-bisphosphate
Schmatz, 1989; 1997), although to date there is little direct evidence for or
against any of them. The following possibilities have been suggested:
1. NADH generated during the breakdown of mannitol may be used
directly for oxidative phosphorylation and so result in energy produc-
tion.
2. The first part of the pathway may act as an electron sink for replenish-
ment of NAD+ under anaerobic conditions.
3. Mannitol may act as an osmoregulator, keeping the oocyst wall rigid
during maturation.
4. Mannitol may have a protective effect against superoxide ions.
5 . Mannitol phosphate may be polymerized to act as a structural compo-
nent in the oocyst or sporocyst wall.
The mannitol cycle also appears to be present in both Toxoplasma and
Cryptosporidium and so it may be a common feature of all coccidia. Both
mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase have
been detected in C. parvum (see Schmatz, 1989) and specific antibodies
have apparently been used to show that mannitol 1-phosphate dehydrogen-
ase, but not its inhibitor, is present in the sexual stages of both Toxoplasma
and Cryptosporidium, whereas both proteins occur in other stages of the
life cycle (Schmatz, 1997).
(c) Tricarboxylic acid. It is currently not certain that Eimeria (or any of
the other coccidia) contains a fully functional tricarboxylic acid (TCA)
cycle at any stage of its life cycle. Of the classical TCA cycle enzymes,
Smith et al. (1994) were able to detect only malate dehydrogenase in
sporulated oocysts of E. tenella. Both phosphoenolpyruvate carboxykinase
(PEPCK) and malic enzyme were present, however, and the authors con-
cluded that Eimeria sporulated oocysts lack a conventional TCA cycle but
contain a PEPCK bypass similar to that in anaerobic protozoa such as
Giardia lamblia (syn. G. duodenalis) and Trichomonas vaginalis (see
Coombs and Muller, 1995). These results, however, conflict with the
assertion that ‘There is ample evidence indicating a functional tri-
carboxylic acid cycle in coccidia in general’ (Wang, 1982). The only
evidence quoted as justification for this statement was the detection of
isocitrate dehydrogenase and malate dehydrogenase in unsporulated
oocysts and the demonstration by cytochemical analysis of succinate
dehydrogenase and isocitrate dehydrogenase in proliferative Toxoplasma.
Succinate dehydrogenase was also apparently detected cytochemically in
several stages of Eimeria (see Beyer, 1970; Michael and Hodges, 1973).
However, we too have been unable to detect this enzyme or NAD+-specific
isocitrate dehydrogenase in oocysts or sporozoites by enzymatic analysis
(see Table 2).
Thus, there is evidence both for and against a functional TCA cycle. It
BIOCHEMISTRY OF THE COCClDlA 157
remains a possibility that the TCA cycle is operative in just some stages of
the parasite. Certainly, sporulation of eimerian oocysts occurs only under
aerobic conditions and can be inhibited by inhibitors of the respiratory
chain (see Section 2.1.2.(d)), suggesting that this form of the parasite may
also have a functional, but perhaps only partial, TCA cycle. Conversely,
however, the end products released by eimerian sporozoites and their lack
of sensitivity to respiratory inhibitors (see Section 2.1.2.(d)) are consistent
with the TCA cycle playing little part in their energy metabolism (see
Section 2.1.4). There are few data on other developmental stages.
(d) Respiratory chain. All developmental stages of Eimeria species
possess distinctive elongate, cristate mitochondria. The processes of spor-
ulation and excystation are associated with vigorous respiratory activity
including the consumption of oxygen and production of carbon dioxide
(see Section 2.1.4 for details). This is reversibly inhibited by cyanide and
other inhibitors of electron transport, implying that it is mediated, at least
partially, by a cytochrome-containing respiratory chain. In contrast, excys-
tation continues and released sporozoites remain viable when exposed to
these inhibitors of electron transport, suggesting that this stage does not
need a functional respiratory chain (Brown et al., 1996).
The detailed composition of the cytochrome chain is yet to be eluci-
dated but there is evidence that it differs from those found in mammalian
mitochondria. Mitochondria isolated from unsporulated oocysts of E.
tenella consumed oxygen in the presence of conventional respiratory
substrates, including NADH and succinate (Wang, 1975; Fry and Wil-
liams, 1984). The isolated mitochondria were uncoupled with respect to
oxidative phosphorylation: oxygen consumption was not dependent on
ADP, and the uncoupler carbonyl rn-chlorophenylhydrazone had no effect
(Fry and Williams, 1984). It remains unclear whether this lack of cou-
pling is an inherent property of the mitochondria or a result of damage
inflicted during the isolation procedure. Spectrophotometric analysis of
the mitochondria revealed absorbance maxima characteristic of type a and
b cytochromes but no clear indication of a type c cytochrome. Interaction
with carbon monoxide suggested that there might be two type a cyto-
chromes present, cytochrome a3 of cytochrome oxidase and an o-type
cytochrome. The mitochondria1 respiration was inhibited by cyanide,
azide, carbon monoxide (inhibitors of cytochrome oxidase) and also by
antimycin A, which blocks co-enzyme Q(Q)-cytochrome reductase.
However, rotenone and amytal were largely without effect, suggesting
that NADH-Q reductase was either absent or presented unusual proper-
ties. Similar findings have been reported for Plasmodium, which is
thought to lack the NADH-Q reductase and to use succinate dehydrogen-
ase as the major feed-in point (Fry, 1991).
The respiratory pathway in Eimeriu has attracted particular interest as
158 G.H. COOMBS ET AL.
phate and guanosine triphosphate: ATP, ITP and GTP) and the pyrimidine
triphosphates (cytidine triphosphate and uridine triphosphate: CTP and
UTP) were effective inhibitors of the enzyme at millimolar concentrations,
while phosphoenolpyruvate (a powerful inhibitor of some bacterial
enzymes) had no effect. There was, however, significant inhibition by oleic
and linoleic fatty acids. 6-Phosphogluconate dehydrogenase activity has
also been detected in starch gels of sporulated and unsporulated oocysts
(Shirley, 1975). From the presence of these enzymes, it seems likely that a
functioning pentose phosphate shunt exists in Eimeria species. It could be
envisaged that this pathway would be particularly important in the rapid
growth situations of merogony and gametogony where NADPH and ribose
requirements would be high. Indeed, James (1980) has presented circum-
stantial evidence that the pathway is very active in isolated meronts (see
Section 2.1.4.(c)).
(ii) Gluconeogenesis. Fructose 1,6-bisphosphatase and glucose 6-phos-
phatase have both been detected in extracts of Eimeria and so it would
seem that the parasites have gluconeogenic capabilities. This may relate to
the importance of amylopectin and possibly mannitol as energy reserves,
although at present there is no evidence that exogenous substrates other
than carbohydrates are used in their synthesis. Perhaps the endogenous
reserves of lipid can be converted to the carbohydrate stores, although this
would probably require a glyoxylate cycle (see below).
(iii) Glyoxylate cycle. No isocitrate lyase or malate synthetase activity
could be detected in crude extracts of E. tenella unsporulated oocysts
(Wang, 1982), implying that a glyoxylate pathway is not present.
acids from the environment, although these were mainly incorporated into
parasite proteins (Krylov and Svanbaev, 1980). There was a marked
decrease in the concentration of most free amino acids during sporulation
of oocysts (H. Denton et al., unpublished observations).
Sarcocystis fusiformis was analysed for aspartate aminotransferase and
alanine aminotransferase (Gupta et al., 1993). Both of these enzymes were
found, indicating that amino acids may be converted to keto acids which
could be catabolized as an energy source.
ATPases are an important group of enzymes that regulate intracellular
ATP and ion levels within cells (Pederson and Carafoli, 1987). ATPase
activity has been detected by histochemical means in most stages of
Eimeria (see Michael and Hodges, 1973; Vetterling and Waldrop, 1976).
The activity in Eimeria sporozoites has been characterized (K.-W. Thong,
unpublished observations), as has that of tachyzoites of T. gondii (see
Takeuchi et al., 1980). Both parasites appear unusual in lacking (or having
very low levels of) Na+/K+-ATPase,the enzyme responsible for maintain-
ing high potassium ion concentrations within most eukaryotic cells. The
apparent absence of this enzyme from Toxoplasma led Takeuchi et al.
(1980) to speculate that the parasite plasma membrane might be freely
permeable to Na+ and K+ and that transmembrane fluxes of these ions
occurring during transition between different environments (in particular
during invasion of host cells) might be important effectors of the parasite’s
metabolism. In apparent support of this conjecture, they demonstrated that
the parasite’s protein synthesis was markedly stimulated by K+ concentra-
tions up to 150 m~ and also showed that there were significantly higher
levels of Na+ than K+ in lysates of tachyzoites.
While lacking a Na+/K+ ATPase, both Eimeria and T. gondii contain
Mg2+-ATPaseactivity (probably the mitochondria1 variety which partici-
pates in oxidative phosphorylation). Ca2+-ATPaseactivity was also detected
in membrane preparations of the parasites. The Mg2+-and Ca2+-ATPasesof
Eimeria presented similar kinetic parameters and pH optima to the equiva-
lent ATPases in chick liver cells, but had significantly different inhibitor
sensitivities (K.-W. Thong, unpublished observations). In particular, the
parasite enzymes were much less affected by azide than their host cell
counterparts, while N-ethyl maleimide proved to be a potent inhibitor of
the eimerian Ca2+-ATPasebut had little effect on the host enzyme. The
activities of a number of ionophores and synthetic anticoccidial drugs were
investigated but they appeared not to inhibit the ATPases in Eimeria.
*SD, n in parentheses; n.d., not detectable, n.a., not assayed. Data from Denton et al., 1994, 1996a.b and unpublished observations. Key to enzymes:
PPi-PFK, pyrophosphate-phosphofructokinase;PK, pyruvate kinase; LDH, lactate dehydrogenase; ICDH, iswitrate dehydrogenase; SDH, succinate
dehydrogenase.
Maximum contribution of host-cell enzymes to activities measured in bradyzoite extracts (nmoVmidmg protein): PFK, 0; PK, 227; LDH, 0; NADP"-
ICDH, 2.8; SDH, 1.1.
BIOCHEMISTRY OF THE COCClDlA 163
granules from the cytoplasm, which suggests that these are the source of
the energy required.
Excystation under aerobic conditions was reported to be accompanied by
vigorous respiratory activity (Vetterling, 1968). This was initiated upon
breakage of the oocyst wall and reached a maximum 2-10 min after
excystation began, before declining to a constant low level which was
maintained until the sporozoites were stimulated to invade host cells. Ryley
(1973) compared the rates of respiration reported by Vetterling (1968) with
the amount of carbohydrate consumed during the equivalent period (Vet-
terling and Doran, 1969) and found that approximately 6 moles of oxygen
were consumed per mole of glucose. This ratio suggested that glucose was
being completely oxidized to carbon dioxide and water and implied that
both a TCA cycle and respiratory chain were operative in this develop-
mental form of the parasite.
The fact that E. tenella sporozoites can excyst and remain motile in the
absence of oxygen, however, suggests that they are facultative anaerobes. It
should be remembered that they exist in the gut lumen, where oxygen
concentrations are low. Ryley (1973) monitored the anaerobic endogenous
metabolism of E. tenella sporozoites and identified lactate as the major end
product (1.49 moYmol glucose); there was also some carbon dioxide and
glycerol production (0.50 and 0.28 moYmol glucose, respectively). These
products accounted for 97% of the amylopectin consumed over the experi-
mental period. Thus it seemed that, under conditions of limited oxygen,
Eimen'a sporozoites obtain energy through glycolytic substrate level phos-
phorylation and depend largely on lactate production for regenerating the
intracellular pool of NAD. The origin of the C 0 2 was unclear, although
some may have been produced via the pentose phosphate pathway or via
pyruvate decarboxylation during the production of acetate.
More recent studies have confirmed these findings with both nuclear
magnetic resonance and enzymatic analyses showing that sporozoites of
E. tenella catabolized glucose to lactate, glycerol and acetate (but not
malate or succinate) under aerobic and anaerobic conditions (H. Denton
et al., unpublished observations). Although somewhat more acetate was
produced when oxygen was present, the total amount of fermentative
products was rather similar under both gaseous conditions - suggesting
that sporozoites have a low capacity for fully oxidative energy metabolism.
These findings are at variance with the conclusions arising from earlier
studies on respiratory rates. The oxygen consumed by sporozoites may
have a role other than energy metabolism.
It has been reported that the invasion rate of E. tenella sporozoites into
host cells in vitro was significantly greater under anaerobic conditions than
when oxygen was present (Wrede et al., 1993). A similar situation has also
been reported for C. parvum (see Upton et al., 1994b). These observations
164 G.H. COOMBS ETAL.
suggest that the sporozoites function better at low oxygen tension, perhaps
because they are specifically adapted towards anaerobiosis.
(c) Zntracellular stages. The metabolic activities of the intracellular
stages of Eimeria are difficult to monitor directly and interpretation of
data is complicated since it is hard to know whether observed changes are
caused by activity of the parasite or alterations in host cell metabolism.
Several groups have attempted to compare the rates of respiration or
anaerobic glycolysis in normal and parasitized caecal tissue, but these
experiments have yielded unreproducible and contradictory results (see
Ryley, 1973). However, a common feature of chicken coccidiosis is a
markedly lowered intestinal pH (van der Horst and Kouwenhoven,
1973). This might well reflect high levels of lactate production by para-
sitized cells, which would imply that there are significant periods of
fermentative metabolism during this phase of the life cycle.
James (1980) showed that isolated meronts of E. tenella were able to
take up and catabolize radiolabelled glucose to carbon dioxide (other
products were not investigated). The rate of production was approximately
three times higher when [l-'4C]glucose was used as opposed to [6-14C]-
glucose, suggestive of high pentose phosphate pathway activity.
Beyer (1970) used cytochemical techniques to investigate the levels of
succinate dehydrogenase and a-glycerophosphate dehydrogenase in the
intracellular stages of E. magma. Growing macrogametes appeared to
have high levels of a-glycerophosphate dehydrogenase but no detectable
succinate dehydrogenase, while the zygote immediately after fertilization
showed high levels of both enzymes. Beyer (1970) interpreted this as
reflecting changes in mitochondria1 activity during the life cycle of the
parasite and considered that, given the ample nutritional means provided
by the host cells, glycolysis can easily cover the energy requirements of the
developing parasites. Later, as the host substrate becomes exhausted and
the merozoites or zygote prepare for an extracellular phase of life, there is a
switch to more efficient aerobic metabolism using the TCA cycle. An
interesting alternative hypothesis is that oxygen becomes limited from
fairly early in the intracellular phase (the parasite grows and multiplies
very rapidly and thus would soon consume the available oxygen) and that
the switch to the potential for aerobic metabolism represents a preadaption
to extracellular existence in an aerobic environment. It seems highly likely
that there are marked changes in energy metabolism during the life cycle of
Eimeria, but more extensive studies are necessary to provide details of the
extent and significance of the variations.
BIOCHEMISTRY OF THE COCClDlA 165
2.2. Toxoplasma
Table 1). The glycolytic pathway is unusual (but similar to that of Eimeria)
in that it contains a phosphofructokinase specific for pyrophosphate rather
than ATP and an allosterically-regulated pyruvate kinase (see Section
2.1.2.(a) for details of both).
Ultrastructural studies have revealed mitochondria1 profiles with cristae
in all developmental stages of T. gondii, suggesting that the organelle is
functional. Some TCA cycle enzymes have been reported (see Tables 1 and
2) and there is evidence of a respiratory chain containing cytochrome
oxidase as well as cytochromes b and c. Apparent evidence that the
respiratory chain is indeed functional in energy production comes from
experiments by Werk and Bommer (1980), working on host cell invasion.
Using an in vitro system, they found that preincubating T. gondii tachy-
zoites with cyanide decreased their invasion efficiency by more than 80%.
The invasion rate was, however, restored to normality when glucose was
present during the invasion process. These results suggest that energy
production is far less efficient under the enforced fermentative metabolism
induced by cyanide, and that the cells consequently consume their finite
energy reserves too quickly for cell invasion to occur in most cases.
Recent evidence suggests that bradyzoites may differ from tachyzoites in
relying more heavily on fermentative metabolism and that the respiratory
chain and TCA cycle may be of little significance to bradyzoites (Denton et
al., 1996b; see Table 2). This could partially explain why tachyzoites
differentiate to bradyzoites (Tomavo and Boothroyd, 1995; Gross et al.,
1996). Interestingly, the two forms express different isoenzymes of lactate
dehydrogenase (Yang and Parmley, 1995; see Section 2.1.2.(a)).
An interesting possibility is that tachyzoites utilize host cell ATP in a
way that provides usable energy for the parasite. The evidence is some-
what circumstantial but includes the ‘gathering’ of the host cell mito-
chondria around the parasitophorous vacuole (and the slower growth of
the parasites in host cells lacking mitochondria), the presence of a pore in
the parasitophorous vacuole membrane which allows passage of materials
of molecular mass <1 m a , the formation of a network within the para-
sitophorous vacuole which may act as a conduit for materials from the
host cell, the stimulation of tachyzoite motility by exogenously supplied
ATP, and the release of a highly active nucleoside triphosphate hydrolase
by the parasite from its dense granules into the parasitophorous vacuole
(see Section 5.1.1).
2.3. Cryptosporidium
2.4. Sarcocystis
is important to identify and characterize the precise steps that are the key
target sites of these inhibitors so that more effective agents can be
designed.
One of the limitations in studying protein synthesis in the intracellular
stages of the parasites has been the problem of distinguishing between
protein synthesis in the parasite and in the host cell. This has now been
overcome by the use of ricin to inhibit selectively the synthetic process in
the host cell (Gurnett et al., 1995; Carrington et al., 1996). Development of
this procedure should allow studies that previously were not possible and
so, hopefully, will stimulate research on this important topic.
Little detail is known of the mechanisms of protein synthesis. The gene
encoding elongation factor-2, a protein essential for protein synthesis, has
been cloned from C. parvum (Jones et al., 1995). Toxoplasma has been
shown to contain a cyclophilin (High et al., 1994; Page et al., 1995). This is
thought to be involved in protein folding and to be at least one of the
targets of cyclosporins, compounds with anticoccidial activity (see
Section 13).
proteinases of these two classes may also be involved in aiding the para-
site’s penetration through the mucous layer lining the gut epithelium
(S.M.A. Brown et al., unpublished observations).
It has been reported that Eimeria has a gene with high homology with
those encoding aspartic proteinases in other organisms (Laurent et al.,
1993). Immunolocalization studies suggested that the enzyme was asso-
ciated with the refractile bodies of the sporozoite. It is yet to be determined
if the expression of the gene is developmentally regulated and the activity
of the enzyme encoded by the gene has not been described to date. Anti-
bodies raised against the recombinant protein inhibit invasion of a host cell
by sporozoites.
An interesting observation is that there is a ‘collagenase’ associated with
the second generation meronts of Eimeria. It is not certain whether the
enzyme is of parasite or host origin, but it appears that the activity is
important in enabling the meront to migrate through the epithelial layer.
Three leucine aminopeptidase activities were detected when extracts of
unsporulated oocysts of E. tenella were subjected to electrophoretic ana-
lysis (Wang and Stotish, 1978). These activities gradually declined during
sporulation and a different isoenzyme appeared, which was the only activ-
ity that could be detected in sporulated oocysts. It was shown to be located
primarily in the cytoplasm surrounding the sporocysts, but not found in
either sporozoites or merozoites. All four aminopeptidase activities had
similar substrate specificities and were unaffected by inhibitors of serine
proteinases. The enzymes present in unsporulated oocysts had pH optima
in the range 8.0 to 8.5, while that in sporulated oocysts was found to be
more active at pH 8.5-9.0. The latter activity differed from the others in
that it was greatly reduced in the absence of metal ions (Mg2+or Mn2+)and
was inhibited by chelating agents; thus it had characteristics of a metallo-
proteinase. As there is limited protein synthesis occurring during the late
stages of sporulation, it was suggested that the leucine aminopeptidase
activity appearing at that stage may result from the processing of pre-
existing activities. The rather different properties of the isoenzymes, how-
ever, make this unlikely and their presence at different stages during
sporulation suggests that the isoenzymes may perform specific roles in
this process and excystation.
The proteinases of Toxoplasma and Cryptosporidium have been studied
even less extensively than those of Eimeria. Two proteinase activities were
separated from tachyzoites of T. gondii (see Choi et al., 1989). One had
characteristics of a cysteine proteinase and was optimally active at pH 6.0,
the other was an ATP-dependent serine proteinase optimally active at pH
8.5. Several aminopeptidases were detected in T. gondii, using a range of
chromogenic substrates (Manafi et al., 1993). This parasite was also
reported to contain a cystatin-like inhibitor of cysteine proteinases (Irvine
BIOCHEMISTRY OF THE COCClDlA 171
et al., 1992). This is likely to play a role in protecting the parasite from its
own enzymes, although an involvement in aiding survival in the host cell
cannot be ruled out.
Membrane-associated aminopeptidase activity was detected in C. par-
vum sporozoite lysates (Okhuysen er d.,1994) and a role in excystation
was suggested for this enzyme (Okhuysen et al., 1996). A metal-dependent
surface-located cysteine proteinase of C. parvum sporozoites has also
recently been reported (Nesterenko et al., 1995). This finding is consistent
with the observation that E64 inhibits both mucus penetration and host cell
invasion by this parasite (S.M.A. Brown et al., unpublished observations).
The interaction of human natural inhibitors of serine proteinases with C.
parvum has also been studied (Forney et al., 1996a), and it has been
reported that serine proteinase inhibitors affect both oocyst excystation
and host cell invasion (Forney et al., 1996b,c).
The role of proteinases in host cell invasion has been studied in S. muris
by Strobe1 et al. (1992). The results suggested that a cysteine proteinase is
involved and it was postulated that it may enhance parasite invasion by
modifying the parasitophorous vacuole.
4. POLYAMINE METABOLISM
The polyamines spermidine and spermine, and the diamines putrescine and
cadaverine, are widely distributed and have been implicated in a number of
biological functions. The molecules appear to have important roles in cell
division, although their precise mode of action is unclear. In general, the
pathways of polyamine biosynthesis appear relatively similar throughout
nature, with the enzymes ornithine decarboxylase (ODCase) and S-adenosyl-
methionine decarboxylase (SAMDCase) catalysing the rate-limiting steps.
Inhibitors of these enzymes have proved to be effective in inhibiting the
replication of many cell types and have been extensively investigated as
potential antiparasite agents. To date, the effects of such inhibitors provide
172 G.H. COOMBS ETAL.
Eimeria
Guanine - I0
Xanthine Hypoxanthine a Adenine
If
Toxoplasma
I Guanosine
4 I
I Xanthosine I lnosine Adenosine I
suggested that the anticoccidial agent arprinocid may act through inhibiting
hypoxanthine transport (Wang, 1982); if so this unusual transporter could
be the target.
There has been no report on purine metabolism in Cryptosporidium.
obtain better inhibitors (Gangjee et al., 1993, 1995, 1996; Martinez et al.,
1996; Piper et al., 1996).
Enzymes of de novo folate synthesis were searched for in T. gondii
(Kovacs et al., 1989). The parasite was shown to use para-aminobenzoic
acid and convert it into reduced folates, of which 5-formyltetrahydrofolate
was the most abundant. This confirmed the importance of folate biosynth-
esis in the parasite. This had been deduced previously from the parasite’s
sensitivity to sulphonamides (see Section 13), which are thought to target
dihydropteroate synthetase. Better inhibitors are being pursued (Allegra et
al., 1990; Chi0 et al., 1996). The presence of a folate biosynthetic pathway
in Eimeria has also been demonstrated by the parasite’s inability to use
exogenous folate and sensitivity to sulphonamides (see Section 13),
although none of the enzymes has been investigated.
6. NUCLEIC ACIDS
stages. Progress has been made in some cases. For instance, it was shown that
different lactate dehydrogenase genes are expressed in tachyzoites and
bradyzoites of Toxoplasma (see Parmley et al., 1995; Yang and Parmley,
1995). The application of molecular techniques, including the development
of successful methods of reverse genetics, has progressed most rapidly with
Toxoplasma amongst the coccidia and they are now very amenable for such
studies (Sibley et al., 1993, 1994b; Donald and ROOS,1994; Messina et al.,
1995; Soldati et al., 1995; Seeber and Boothroyd, 1996). There has recently
also been some progress with the development of transfection procedures for
Eimeria. Full coverage of these advances in molecular biology of coccidia
would be inappropriate in this article; other recent reviews have summarized
the situation (Bonnin et al., 1995b; Boothroyd et al., 1995; Savira, 1995).
A most exciting advance has been the finding in coccidia of extranuclear
circular deoxyribonucleic acid (DNA) closely related to that of plastids
(Egea and Lang-Unnasch, 1995; McFadden et al., 1996). Similar DNA
appears to be a characteristic of the Apicomplexa and that in Plasmodium
has been studied most extensively (Feagin, 1994; Williamson et al., 1994;
Wilson et al., 1996; Wilson, 1997). It appears, however, that there is high
conservation between the different groups. Toxoplasma contains eight
copies of the circular DNA and it has now been demonstrated that the
DNA is contained within a distinct organelle located close to the nucleus
(McFadden et al., 1996). The name ‘apicoplast’ has been suggested for the
organelle. The DNA includes genes encoding RNA polymerase, transfer
RNA and various ribosomal proteins. Evidence has also been presented for
the presence of a chlorophyll a-Dl complex in Toxoplasma by Hackstein et
al. (1995). As this is a key component of the respiratory chain of plastids,
this is highly suggestive of electron transport occurring in the organelle. The
importance of this is uncertain (see Section 2.1.2.(d)) and the functional
significance of the plastid is still a matter of debate (Hackstein et al., 1997;
Wilson 1997). One possibility, in addition to energy metabolism, is the
provision of essential proteins. The findings that some herbicides thought to
act by inhibiting the components of the respiratory chain encoded by the
plastid have anticoccidial activity (Hackstein et al., 1995) and that malaria
parasites are sensitive to inhibitors of plastid metabolism (see McFadden et
al., 1996) have led to an explosion of interest in this aspect of the parasites’
biochemistry and attempts to exploit it as a drug target. Recent findings (F.
Roberts, C. Roberts, L. Mets, J. Johnson and R. McLeod, personal commu-
nication) from studies using specific inhibitors have suggested that the
plastid may be involved in haem biosynthesis and also that it may contain
the shikimate pathway, which in plants is central to the biosynthesis of
aromatic amino acids, para-aminobenzoic acid and ubiquinone. The plastid
DNA does not have genes for any of the proteins of these pathways and so
they would have to be nuclear-encoded, as is the case in plants.
BIOCHEMISTRY OF THE COCClDlA 181
It has been reported that some Eimeria species may contain viral nucleic
acids. Sporulated oocysts of E. nieschulzi were found to have an RNA-
dependent RNA polymerase activity which was absent from both E. tenella
and E. acervulina. This activity correlated with an unknown nucleic acid
species which may represent the genomic RNA of a possible virus (Sepp et
al., 1991; Roditi et al., 1994). Another study performed on E. necatrix and
E. maxima isolated RNA molecules, possibly of viral origin and thought to
be located in the cytoplasm of the parasite (Ellis and Revets, 1990).
The DNA content of S. muris was analysed using a DNA-specific Feul-
gen stain to measure the overall DNA content of the various developmental
stages. All the stages studied had haploid DNA except for early zygotes
which were diploid. These zygotes later underwent nuclear meiosis to
produce two haploid daughter cells (Mackenstedt et al., 1990).
The ways in which nucleic acids are synthesized, processed and catabo-
lized in coccidia have been little studied. Ribonuclease P of T. gondii has
been partially characterized (Mack et al., 1994), a type I1 topoisomerase
gene has been identified in C. pawum by Christopher and Dykstra (1994),
and it has been reported that DNA polymerase activity of T. gondii corre-
lated with virulence (Makioka and Ohtomo, 1995).
Studies on the mechanisms controlling gene expression in coccidia are in
their infancy. A most exciting recent finding, however, is that an inhibitor
of histone deacetylase has potent anticoccidial activity (Darkin-Rattray et
al., 1996; Singh et al., 1996). The compound, a natural product isolated
from fungi growing in a rain forest in Costa Rica, is a tetrapeptide called
‘apicidin’ - the name chosen to reflect the finding that the compound is
active against all members of the Apicomplexa tested. Histone deacetylase,
which is active towards acetylated lysine residues, is responsible for con-
trolling the binding of histones to DNA and so modulating gene expression.
This discovery suggests that this and other transcription factors may be
good targets for chemotherapeutic attack.
7. LIPID METABOLISM
C 14:O 1 2 9 0 5
C 16:O 12 8 17 31 27
C 16:l 1 3 n.d. trace 3
c 18:O 10 17 11 16 20
c 18:l 75 68 31 22 27
C 18:2 trace trace 20 29 15
c 20:o n.d. n.d. 6 trace 1
9. ANTIOXIDANT MECHANISMS
The oocyst wall of E. tenella has two layers, an outer layer 10 nm thick and
an inner 90 nm layer (Stotish et al., 1978). Studies using gas-liquid
chromatography have revealed that glucose is the predominant neutral
sugar, with small amounts of mannose and galactose (Stotish et al.,
1978). Unsporulated oocyst walls were found to be 67% protein, 14% lipid
and 19% carbohydrate. They contain an unusual polysaccharide, initially
thought to be chitin but which, on further analysis, was found to differ from
it in detail (Ryley, 1973). One of the protein components has been char-
acterized (Eschenbacher et al., 1996).
There appear to be two types of C. pawurn oocyst, designated ‘thin
walled’ (comprising 20% of the total number) and ‘thick walled’ (Cur-
rent, 1989). The thin-walled oocysts are thought to be auto-infective,
hence the initial infection is recycled (a unique feature of Cryptospor-
idium among coccidia), whereas the thick-walled oocysts are passed out
in the faeces to infect other hosts. It has been reported that these thick-
walled oocysts have three membranes and an outer wall of two chitinous
layers, while the thin-walled oocysts have a single unit membrane (Ster-
ling and Arrowood, 1993). More recently, Cryptosporidium oocysts were
reported to be similar to those of Eimeria in having two layers - an
outer 10 nm layer and a thicker inner one (Petersen, 1993). The oocyst
wall composition of three species of Cryptosporidiurn has been examined
using surface labelling and antibodies (Tilley et al., 1990; Nina et al.,
1992). The oocysts of C. baileyi, C. muris and C. pawum could all be
distinguished, but C. parvum and C. baileyi were shown to be more
similar to each other than either was to C. muris.
The structure of the oocyst wall of T. gondii has not been studied in
detail.
11.l.Glycosylation
Many molecules on cell surfaces are glycosylated both for protection and
as one mechanism of implementing and regulating interactions with the
environment and other cells. Glycosylation is not limited to surface mole-
cules and such processing also occurs with, for example, lysosomal pro-
teins. There have been several reports of the presence of glycoproteins in
Eimena and Toxoplasma (Schwarz et al., 1993), but in most cases the
subcellular location of the molecules has not been established and very few
structural or mechanistic details are known. Dieckmann-Schuppert el al.
( 1992) reported that exogenous dolichol pyrophosphoryl oligosaccharides
were utilized by T. gondii for glycosylation of its proteins. It was not until
1993 that biochemical evidence was obtained showing that T. gondii
tachyzoites synthesized N-linked glycoproteins and the lipid-linked glycan
precursor for N-linked oligosaccharides (Odenthal-Schnittler et al., 1993).
Many surface molecules are attached to the plasma membrane via GPI
anchors. Such anchors occur widely in eukaryotic cells but seem to be
particularly important in parasitic protozoa (McConville and Ferguson,
1993). However, this is an area in which coccidia have been investigated
in much less detail than some other protozoan parasites, notably kineto-
plastid flagellates. The major surface proteins on the tachyzoites of T.
gondii do have GPI anchors (Nagel and Boothroyd, 1989; Tomavo el al.,
1992a) and the parasite is capable of synthesizing GPI and also N- and
0-glycans (Tomavo et al., 1992b; Schwarz and Tomavo, 1993). Some
undefined antigens on the surface of Eimeria sporozoites are also attached
via GPI anchors (Gurnett et al., 1990).
190 G.H. COOMBS ETAL.
11.3. Lectins
The ways in which the parasite interacts with its host cell is one of the most
fascinating and important aspects of coccidian biology, but unfortunately at
present it is also one of the least well understood. Information is available
on the events that occur during the invasive process itself (Dubremetz and
Entzeroth, 1993; Dubremetz and Schwartzman, 1993; Kasper and Mineo,
1994; Sibley et al., 1994a; Dubremetz and McKerrow, 1995; Morisaki et
al., 1995; Sibley, 1995; Sam-Yellowe, 1996), but we have only a rudimen-
tary understanding of how the parasite interacts with the cell in which it is
growing. Because little is known of the mechanisms at the biochemical
level, these aspects do not fall within the remit of this review and it will
192 G.H. COOMBS ETAL.
refer only to some of the more recent studies and include a few comments
on major areas that warrant more detailed biochemical study.
All coccidia inhabit a parasitophorus vacuole when intracellular. Cryp-
tosporidiurn differs from most other coccidia, however, in that the para-
sitophorous vacuole remains at the surface of the host cell rather than lying
deep within the cytoplasm (Lumb et al., 1988; Sterling and Arrowood,
1993). The parasite is therefore said to be extracytoplasmic. The signifi-
cance of this difference in intracellular location is totally unknown. Much
remains to be discovered concerning the environments within the parasi-
tophorous vacuoles, but current evidence suggests that they may differ
quite considerably and recently it was reported that the vacuole resulting
from the invasion of Toxopfasma sporozoites differs distinctly from that
which contains tachyzoites. It will be interesting to discover if there is
similar stage specificity with other coccidia.
In all coccidia studied, three types of organelles have been reported to be
involved in host cell invasion, namely rhoptries, micronemes and dense
granules. These are distinct types of organelles, although we are only
beginning to obtain an insight into the part that each plays in the invasion
process. It is thought that the contents of the organelles are secreted during
host cell invasion and that this results in changes to the host cell membrane
which aid entry of the parasite. Rhoptries are thought to contain mainly
enzymes, although they also contain unusually large amounts of lipid.
Some of the molecules present in micronemes and dense granules have
also been identified, but the precise way in which they function is yet to be
determined.
Laminin on the surface of T. gondii tachyzoites has been suggested to
enhance host cell entry (Hall and Joiner, 1991; McLeod et al., 1991;
Furtado et af., 1992). Tachyzoite binding to host cells was inhibited by
antibodies to both laminin and a-6-p-1 integrins. It has been suggested that
laminin may facilitate host cell invasion by creating a bridge between the
tachyzoite and host cell. A fibronectin-like molecule has been identified on
E. tenefla (see Lopez-Bernad et uf., 1996).
Invasion of host cells was inhibited when E. tenefla sporozoites were
preincubated with phopholipase A2, possibly due to the sporozoite surface
membrane being modified (Crane and McGaley, 1991). Yet, when T.
gondii tachyzoites were treated in the same way, the infection rate of
host cells increased. It appears that T. gondii possesses a calcium-requiring
phospholipase activity which is involved in some way with host-cell
penetration (Saffer and Schwartzman, 1991; Marin et af., 1996). The
surface-located sialidase activity present on Eimeria (see Section 11.4) is
likely to play a part in host cell invasion (Pellegrin et af., 1993) and
parasite proteinases also seem to play a role in the invasion process (see
Section 3.2).
BIOCHEMISTRY OF THE COCClDlA 193
In general, agents that are effective against the genus Eimeria are also
effective for Toxoplasma, Sarcocystis and Isospora (see Stuart and Lindsay,
1986; Kirkpatrick and Dubey, 1987; Cawthorn and Speer, 1990; Johnson,
1990; McDougald, 1990), but until recently the vast majority of research
effort had been devoted to the economically important avian parasites. The
realization that toxoplasmosis and cryptosporidiosis are important infec-
tions related to the acquired immune deficiency syndrome (AIDS) resulted
in increased efforts to obtain better chemotherapy against these infections;
with promising results concerning the former (see below), but little success
so far with the latter. Although many different compounds, including those
active against Eimeria, have been tested against Cryptosporidium (see, for
example, Woods et al., 1996), only halofuginone (Current and Blagburn,
1990; Naciri et al., 1993; Khaw and Panosian, 1995) was found to be
effective and it has yet to be shown to be useful clinically. More recently,
paromomycin has been reported to have some activity against Cryptospor-
idium (see Wallace et al., 1993; Scaglia et al., 1994; Tzipori et al., 1995;
Verdon et al., 1995; Flanigan et al., 1996), as have the ionophore lasalocid
(Rehg, 1993) and benzimidazoles (Fayer and Fetterer, 1995). There is little
information on chemotherapy for Caryospora or Hammondia (see Kirkpa-
trick and Dubey, 1987; Upton and Sundermann, 1990; Dubey, 1993) and
only one report on Besnoitia (see Shkap et al., 1987). Infections with
Neospora are difficult to treat; clindamycin and sulphadiazine can be
effective if used early in infection, and trimethoprim and pyrimethamine
can also produce cures (Dubey and Lindsay, 1993). Therefore, the main
focus of this section is on Eimeria and Toxoplasma.
The drugs of choice for avian Eimeria are polyether ionophores (Dutton
et al., 1995; see Table 4),which have captured approximately 80% of the
current market (worth in total about US$350X lo6 per year). It is expected
that these compounds will dominate the market for many years to come,
because there has been no commercial launch of any novel class of syn-
thetic or semisynthetic avian anticoccidial drug since the early 1980s. This
is a major concern as resistance problems have arisen with current agents
(Chapman, 1993; Edgar, 1993; Vertommen and Peek, 1993). Some
vaccines are now available and perhaps they will set the trend for control
BIOCHEMISTRY OF THE COCClDlA 195
of newborn infants (Georgiev, 1994). These two agents, and other macro-
lides and azalides, are known to inhibit parasite protein synthesis (Blais et
al., 1993a, b). Interestingly, recent evidence suggests that the target site is
the plastid ribosomes rather than cytoplasmic or mitochondrial ribosomes
(Pfefferkorn and Borotz, 1994; Beckers et al., 1995). Eimeria also contains
similar plastid-like DNA, and so it is possible that the anti-Eimeria activity
of macrolides and azalides also operates through targeting plastid
ribosomes.
Another promising drug for the treatment of toxoplasmosis is the hydroxy-
naphthoquinone atovaquone (Georgiev, 1994). This is thought to act by
inhibiting the ubiquinol-cytochrome c reductase component (complex 111)
of the mitochondrial respiratory chain (Hudson, 1993). The role of this in
the parasite is not entirely clear as current evidence suggests that both
tachyzoites and bradyzoites are largely fermentative (see Section 2). It is
possible that the electron transport chain is primarily involved in pyrimi-
dine biosynthesis, as appears to be the case for the asexual bloodstream
forms of P. falciparum.
There have been several recent reports of novel experimental compounds
with anticoccidial activities. Some of these interesting anticoccidial
'leads' and their putative biochemical action are listed in Table 5. It is
apparent that macrolides/azalides are well represented, suggesting that
protein synthesis provides an important coccidial target. On the other
hand, nothing is known about the identity of the molecular targets of
many of the compounds. The recent discovery that a histone deacetylase
inhibitor has good anticoccidial activity (see Section 6) suggests that
transcription factors may be exploitable drug targets. It remains to be
seen whether any of these compounds or their analogues will become
viable commercial products.
animal but live within cells in different parts of the gut. Why? How similar/
different are the species that invade different hosts (e.g. E. bovis in cattle
compared with E. tenella in chickens and C. pamum in humandcattle and
C. muris in mice) and what determines the host specificity? Comparative
studies on different species are one way of pin-pointing the key adaptations
of the parasites for their particular host. Similarly, comparative studies of
the different developmental stages during the life cycle of a single parasite
species can help to elucidate the special adaptations of the stages. One area
requiring urgent attention with Toxoplusma is to determine how the brady-
zoites differ from the tachyzoites, and so explain the differing sensitivity to
drugs of the two forms. How do the sporozoites find an appropriate host
cell and penetrate the mucus lining the gut? It seems likely that the parasite
excretes some materials to disrupt the mucous layer, but this requires
further study. The oocyst of C. muris excysts in the stomach, how does
it avoid the lethal effects of the acidity? Perhaps only those oocysts that
excyst in the mucous layer can produce viable sporozoites or maybe the
parasite has a mechanism for neutralizing the acid in its microenvironment,
for instance by secreting urease in a similar fashion to Helicobacter pylori.
There are also several areas of biochemistry that have attracted, and are
attracting, considerable interest in other parasites but which are yet to be
seriously addressed in coccidia. These include cell signalling, the cell
cycle, cell differentiation and the subcellular organization of metabolism.
To be able to exploit rationally the biochemical peculiarities of a para-
site, an extensive amount of data on the putative target is usually required
(see Coombs and Croft, 1997). Such data have been obtained for coccidia
in very few instances to date, although the recent interest in protein
synthesis, mannitol metabolism and pyrophosphate-linked enzymes is
encouraging. Obtaining new drugs by empirical means requires adequate
methods of screening for activity. Screens which simulate conditions in
vivo are crucial for progress; unfortunately the in vitro methods currently
available are far from ideal.
What should be the priorities for the future?
The use of anticoccidial drugs is shadowed by resistance development.
Understanding the mechanisms of resistance is always useful, but it
becomes essential when there is no alternative drug that can be used.
Knowledge of the resistance mechanisms may enable the design of resis-
tance reversers that can overcome the immediate problem (see Coombs and
Croft, 1997). This is the strategy that has been pursued with some success
in malaria and cancer chemotherapy, but as yet little is known about the
mechanisms underlying resistance to anticoccidial compounds.
Advances in parasitological procedures are required, whether one wishes
to pursue the rational or the empirical route to drug discovery. One of the
most urgent needs for future biochemical work on many stages of several
BIOCHEMISTRY OF THE COCClDlA 20 1
ACKOWLEDGEMENT
REFERENCES
Abrahamsen, M.S., Johnson, R.R., Clark, T.G. and White, M.W. (1994). Devel-
opmental regulation of an Eimeria bovis mRNA encoding refractile body-
associated proteins. Molecular and Biochemical Parasitology 68, 25-34.
Abrahamsen, M.S., Johnson, R.R., Hathaway, M. and White, M.W. (1995). Iden-
tification of Eimeria bovis merozoite cDNAs using differential mRNA display.
Molecular and Biochemical Parasitology 71, 183-19 1.
Adams, J.H. and Bushell, G.R. (1988). The effect of protease inhibitors on Eimeria
vermiformis invasion of cultured cells. International Journal for Parasitology
18, 683-685.
Aguirre-Cruz, L., Calderon, M. and Sotelo, J. (1996). Colchicine decreases the
infection by Toxoplasma gondii in cultured glial cells. Journal of Parasitology
82, 325-327.
Allegra, C.J., Boarman, D., Kovacs, J.A., Morrison, P., Beaver, J., Chabner, B.A.
and Mansour, H. (1990). Interaction of sulfonamide and sulfone compounds with
Toxoplasma gondii. Journal of Clinical Investigation 85, 311-379.
Andrews, R., O’Donoghue, P.O., Adams, M. and Prowse, S. (1990). Enzyme
markers for genetic characterisation of Eimeria spp. Parasitology Research
76, 627-629.
Aomine, M. (1981). Carbohydrate transport and utilization in protozoa. Compara-
tive Biochemistry and Physiology 68A, 131-148.
Araujo, F.G. and Remington, J.S. (1992). Recent advances in the search for new
drugs for treatment of toxoplasmosis. International Journal of Antimicrobial
Agents 1, 153-164.
Araujo, F.G., Shepard, R.M. and Remington, J.S. (1991). In vivo activity of the
macrolide antibiotics azithromycin, roxithromycin and spiramycin against Toxo-
plasma gondii. European Journal of Clinical Microbiology and Infectious
Diseases 10, 519-524.
Araujo, F.G., Slifer, T. and Remington, J.S. (1994). Rifabutin is active in murine
models of toxoplasmosis. Antimicrobial Agents and Chemotherapy 38,570-575.
Araujo, F.G., Suzuki, Y. and Remington, J.S. (1996a). Use of rifabutin in combi-
nation with atovaquone, clindamycin, pyrimethamine, or sulfadiazine for treat-
ment of toxoplasmic encephalitis in mice. European Journal of Clinical
Microbiology and Infectious Diseases 15, 394-397.
Araujo, F.G., Khan, A.A. and Remington, J.S. (1996b). Rifapentine is active in
vitro and in vivo against Toxoplasma gondii. Antimicrobial Agents and Che-
motherapy 40, 1335-1337.
Archbarou, A., Mercereau-Puijalon, O., Autherman, J.M., Fortier, B ., Camus, D.
and Dubremetz, J.F. (1991). Characterization of microneme proteins of Toxo-
plasma gondii. Molecular and Biochemical Parasitology 47, 223-234.
Arrowood, M.J., Mead, J.R., Xie, L.T. and You, X.D. (1996). In vitro anti-
BIOCHEMISTRY OF THE COCClDlA 203
Buxton, D. and Innes, E.A. (1995). A commercial vaccine for ovine toxoplasmosis.
Parasitology 110, Supplement, SI 1-S16.
Carrington, M., Shochat, Y. and Gurnett, A.M. (1996). Selective metabolic
labelling of intracellular parasite proteins using ricin. Parasitology Today 12,
492-495.
Catterall, J.R., Sharma. S.D. and Remington, J.S. (1986). Oxygen-independent
killing by alveolar macrophages. Journal of Experimental Medicine 163,
1 1 13-1 131.
Cawthorn, R.J. and Speer, C.A. (1990). Sarcocystis: infections and disease of
humans, livestock and other hosts. In: Coccidiosis of Man and Domestic Animals
(P.L. Long, ed.), pp. 91-120. Boston: CRC Press.
Cesbron-Delauw, M.-F. ( 1994). Dense-granule organelles of Toxoplasma gondii:
their role in the host-parasite relationship. Parasitology Today 10, 293-296.
Cesbron-Delauw, M.-F., Tomavo, S., Beauchamps, P., Fourmaux, M.-P., Camus,
D., Capron, A. and Dubremetz, J.-F. (1994). Similarities between the primary
structures of two distinct major surface proteins of Toxoplasma gondii. Journal
of Biological Chemistry 269, 16211-16222.
Chang, H.R. ( 1993). Toxoplasma gondii: chemotherapy. In: Toxoplasmosis (J.E.
Smith, ed.), pp. 191-198. Berlin: Springer.
Chang, H.R., Arsewijevic, D., Comte, R., Polak, A., Then, R.L. and Pechere, J.C.
(1994). Activity of epiroprim (Ro-1 1 -8958), a dihydrofolate reductase inhibitor,
alone and in combination with dapsone against Toxoplasma gondii. Antimicro-
bial Agents and Chemotherapy 38, 1803-1807.
Chapman, H.D. (1993). Resistance to anticoccidial drugs in fowl. Parasitology
Today 9, 159-162.
Chaudry, R.K., Kushwah, H.S. and Shah, H.L. (1985). Biochemistry of the sarco-
cyst of Sarcocystis fusiformis of buffalo Bubalus bubalis. Veterinary Parasitol-
ogy 17, 295-298.
Chaudry, R.K., Shah, H.L. and Guru, V.K. (1986a). Oxygen consumption by the
sarcocyst of Sarcocystis fusiformis of buffalo. Indian Journal of Animal Sciences
56, 53.
Chaudry, R.K., Kushwah, H.S. and Shah, H.L. (1986b). Biochemical and histo-
chemical studies of the sarcocyst of Sarcocystis fusiformis of buffalo Bubalus
bubalis. Veterinary Parasitology 21, 211-274.
Chio, L.C., Bolyard, L.A., Nasr, M. and Queener, S.F. (1996). Identification of a
class of sulfonamides highly active against dihydropteroate synthase from Toxo-
plasma gondii, Pneumocystis carinii, and Mycobacterium avium. Antimicrobial
Agents and Chemotherapy 40,121-733.
Choi, W.-Y., Nam, H.-W. and Youn, J.-H. (1989). Characterization of proteases of
Toxoplasma gondii. Korean Journal of Parasitology 27, 16 1-1 70.
Christopher, L.J. and Dykstra, C.C. (1994). Identification of a type I1 topoisome-
rase gene from Cryptosporidium parvum. Journal of Eukaryotic Microbiology
41, 28s
Clark, D.P. and Sears, C.L. (1996). The pathogenesis of cryptosporidiosis. Para-
sitology Today 12, 221-225.
Clark, T.G., Abrahamsen, M.S. and White, M.W. (1996). Developmental expres-
sion of heat shock protein 90 in Eimeria bovis. Molecular and Biochemical
Parasitology 78, 259-263.
Conway, D.P., Johnson, J.K., Guyonnet, V., Long, P.L. and Smothers, C.D. (1993).
Efficacy of semduramicin and salinomycin against different stages of Eimeria
tenella and E. acervulina in the chicken. Veterinary Parasitology 45, 215-229.
206 G.H. COOMBS ETAL.
Coombs, G.H. and Croft, S.L., eds (1997). Molecular Basis of Drug Design and
Resistance. Cambridge: Cambridge University Press.
Coombs, G.H. and Mottram, J.C. (1997). Parasite proteinases and amino acid
metabolism: possibilities for chemotherapeutic exploitation. In: Molecular Basis
of Drug Design and Resistance (G.H. Coombs and S.L. Croft, eds). Cambridge:
Cambridge University Press, in press.
Coombs, G.H. and Muller,M. (1995). Energy metabolism in anaerobic protozoa.
In: Biochemistry and Molecular Biology of Parasites (J.J. Marr and M. Muller,
eds), pp. 33-47. London: Academic Press.
Coombs, G.H., Vickerman, K., Sleigh, M.A. and Warren, A., eds (1997) Evolu-
tionary Relationships among Protozoa. London: Chapman & Hall, in press.
Cote, S., Morency, M.-J. and Levesque, R. C. (1992). Isolation, cloning and
primary structure of the hypoxanthine-guanine phosphoribosyltransferase
(HGPRT) gene from the parasite Toxoplasma gondii. Antimicrobial Agents
and Chemotherapy 32, 129.
Crane, M.S.J. and McGaley, C.J. (1991). Eimeria tenella: inhibition of host cell
invasion by phospholipase treatment of sporozoites. Experimental Parasitology
72. 219-222.
Current, W.L. (1989). Cryptosporidium spp. In: Parasitic Infections in the
Immunocompromised Host (P.W. Walzer and R.M. Genta, eds), pp. 281-341.
New York: Marcel Dekker.
Current, W.L. and Blagburn, B.L. (1990). Cryptosporidium: infections in man and
domestic animals. In: Coccidiosis of Man and Domestic Animals (P.L. Long,
ed.), pp 155-187. Boston: CRC Press
Current, W.L. and Garcia, L.S. (1991). Cryptosporidiosis. Clinical Microbiology
Reviews 4, 325-358.
Darde, M.L., Bouteille, B. and Pestre-Alexandre, M. (1988). Isoenzyme character-
isation of 7 strains of Toxoplasma gondii by isoelectrofocusing in polyacryla-
mide gels. American Journal of Tropical Medicine and Hygiene 39, 551-558.
Darde, M.L., Bouteille, B. and Pestre-Alexandre, M. (1992). Isoenzyme analysis of
35 Toxoplasma gondii isolates and the biological and epidemiological
implications. Journal of Parasitology 78, 786-794.
Darkin-Rattray, S.J., Gurnett, A.M., Myers, R.W., Dulski, P.M., Crumley, T.M.,
Alloco, J.J., Cannova, C., Meinke, P.T., Colletti, S.L., Bednarek, M.A., Singh,
S.B.,Goetz,M.A.,Dombrowski,A.W., Polishool, J.D. andSchmatz,D.M. (1996).
Apicidin: a novel antiprotozoal agent that inhibits parasite histone deacetylase.
Proceedings of the National Academy of Sciences of the USA 93, 13143-13147.
Daszak, P., Ball, S.J., Pittilo, R.M. and Norton, C.C. (1991). Ultrastructural studies
of the effects of the ionophore lasalocid on Eimeria tenella in chickens.
Parasitology Research 77,224-229.
De Carvalho, L. and De Souza, W. (1989). Cytochemical localization of plasma
membrane enzyme markers during interiorization of tachyzoites of Toxoplasma
gondii by macrophages. Journal of Protozoology 36, 164-170.
Denton, H., Thong, K.-W. and Coombs, G.H. (1994). Eimeria tenella contains a
pyrophosphate-dependent phosphofructokinase and a pyruvate kinase with
unusual allosteric regulators. FEMS Microbiology Letters 115, 87-92.
Denton, H., Brown, S.M.A., Roberts, C.W., Alexander, J., McDonald, V., Thong,
K.-W. and Coombs, G.H. (1996a). Comparison of the phosphofructokinase and
pyruvate kinase activities of Cryptosporidium parvum, Eimeria tenella and
Toxoplasma gondii. Molecular and Biochemical Parasitology 76,23-29.
Denton, H., Roberts, C.W., Alexander, J., Thong, K.-W. and Coombs, G.H.
BIOCHEMISTRY OF THE COCClDlA 207
Dutton, C.J., Banks, B.J. and Cooper, C.B. (1995). Polyether ionophores. Natural
Product Reports 12, 165- 18I.
Edgar, S.A. (1993). How to prevent long-lasting resistance of coccidia to drugs.
Misset World Poultry, pp. 12-13.
Edgar, S.A., Herrick, C.A. and Fraser, L.A. (1944). Glycogen in the lifecycle of the
coccidium Eimeria tenella. Transactions of the American Microscopical Society
63, 199-202.
Edlind, T.D. (1991). Protein synthesis as a target for antiprotozoal drugs. In:
Biochemical Protozoology (G.H. Coombs and M.J North, eds), pp. 569-586.
Basingstoke: Science Press.
Edlind, T., Visvesvara, G., Li, J. and Katiyar, S. (1994). Cryptosporidium and
microsporidial P-tubulin sequences: predictions of benzimidazole sensitivity and
phylogeny. Journal of Eukaryotic Microbiology 41, 38s.
Egea, N. and Lang-Unnasch, N. (1995). Phylogeny of the large extrachromosomal
DNA of organisms in the phylum Apicomplexa. Journal of Eukaryotic Micro-
biology 42, 679-684.
El Kouni, M.H., Naguib, F.N.M., Panzica, R.P., Otter, B.A., Chu, S.H., Gosselin,
G., Chu, C.K. Schinazi, R.F., Shealy, Y.F., Goudgaon, N., Ozerov, A.A. Ueda, T.
and Iltzsch, M.H. (1996). Effects of modifications in the pentose moiety and
conformational changes on the binding of nucleoside ligands to uridine phos-
phorylase from Toxoplasma gondii. Biochemical Pharmacology 51, 1687-1 700.
El-Moukdad, A.R. (1976). Uber die Wirkung verschiedener Desinfektionsmettel auf
parasitare Entwicklungsstadien. Wiener Tierarztliche Monatsschrift 63, 399-532.
Ellis, J. and Revets, H. (1990). Eimeria species which infect the chicken contain
virus-like RNA molecules. Parasitology 101, 163-169.
Ellis, J. and Thurlby, T. (1991). Changes in the messenger RNA population during
sporulation of Eimeria maxima. Parasitology 102, 1-8.
Endo, T., Yagita, K., Yasuda, T. and Nakamura, T. (1988). Detection and localiza-
tion of actin in Toxoplasma gondii. Parasitology Research 75, 102-106.
Eschenbacher, K.H., Eggli, P., Wallach, M. and Braun, R. (1996). Characterization
of a 14 kDa oocyst wall protein of Eimeria tenella and E. acervulina. Para-
sitology 112, 169-176.
Farooqui, A.A. and Hanson, W.L. (1983). Changes in acid phosphatase activity
during sporulation of Eimeria tenella oocysts. Comparative Biochemistry and
Physiology 75B, 185-187.
Farooqui, A.A. and Hanson, W.L. (1988). Partial purification and characterization
of acid phosphatase from sporulated oocysts of Eimeria tenella. Experientia 44,
437-440.
Farooqui, A.A., Lujan, R. and Hanson, W.L. (1983). Acid hydrolases of the
coccidian Eimeria tenella. Experientia 39, 1368-1 370.
Farooqui, A.A., Adams, D.D., Hanson, W.L. and Prestwood, A.K. (1987). Studies
on the enzymes of Sarcocystis suicanis: purification and characterization of an
acid phosphatase. Journal of Parasitology 73, 68 1-688.
Fayer, R. and Ellis, W. (1993). Paromomycin is effective as prophylaxis for
cryptosporidiosis in dairy calves. Journal of Parasitology 79, 77 1-774.
Fayer, R., and Fetterer, R. (1995). Activity of benzimidazoles against crypto-
sporidiosis in neonatal BALBJc mice. Journal of Parasitology 81, 794-795.
Feagin, J.E. ( 1994). The extrachromosomal DNAs of apicomplexan parasites.
Annual Review of Microbiology 48, 81-104.
Ferguson, D.J.P. and Hutchison, W.M. (1987). An ultrastructural study of the early
BIOCHEMISTRY OF THE COCClDlA 209
Hill, B., Kilsby, J., Rogerson, G.W., McIntosh, R.T. and Ginger, C.D. (1981). The
enzymes of pyrimidine biosynthesis in a range of parasitic protozoa and
helminths. Molecular and Biochemical Parasitology 2, 123-1 34.
Holfels, E., McAuley, J., Mack, D., Milhous, W.K. and McLeod, R. (1994). In vitro
effects of artemisinin ether, cycloguanil hydrochloride (alone and in combina-
tion with sulfadiazine), quinine sulfate, mefloquine, primaquine phosphate,
trifluoperazine hydrochloride and verapamil on Toxoplasma gondii. Antimicro-
bial Agents and Chemotherapy 38, 1392-1 396.
Hosek, J.E., Todd, K.S. and Kuhlenschmidt, M.S. (1988). Demonstration of acid
phosphatase in Eimeria spp. - partial characterization. Journal of Protozoology
35, 531-532.
Hudson, A.T. (1993). Atovaquone - a novel broad-spectrum anti-infective drug.
Parasitology Today 9, 66-68.
Hughes, H.P.A., Boik, R.J., Gerhardt, S.A. and Speer, C.A. (1989). Susceptibility
of Eimeria bovis and Toxoplasma gondii to oxygen intermediates and a math-
ematical model for parasite killing. Journal of Parasitology 75, 489-497.
Hupe, D.J., Azzolina, B.A. and Behrens, N.D. (1986). IMP dehydrogenase from the
intracellular parasitic protozoan Eimeria tenella and its inhibition by mycophe-
nolic acid. Journal of Biological Chemistry 261, 8363-8369.
Iltzsch, M. H. (1993). Pyrimidine salvage pathways in Toxoplasma gondii. Journal
of Eukaryotic Microbiology 40, 24-28.
Iltzsch, M.H. and Klenk, E.E. (1993). Structure-activity relationship of nucleobase
ligands of uridine phosphorylase from Toxoplasma gondii. Biochemical Phar-
macology 46, 1849-1 858.
Iltzsch, M.H. and Tankersley, K.O. (1994) Structure-activity relationship of
ligands of uracil phosphoribosyltransferase from Toxoplasma gondii. Biochem-
ical Pharmacology 48, 78 1-792.
Iltzsch, M.H., Uber, S.S., Tankersley, K.O. and El Kouni, M.H. (1995). Structure-
activity relationship for the binding of nucleoside ligands to adenosine kinase
from Toxoplasma gondii. Biochemical Pharmacology 49, 1501-15 12.
Irvine, J.W., Coombs, G.H. and North, M.J. (1992). Cystatin-like cysteine protein-
ase inhibitors of parasitic protozoa. FEMS Microbiology Letters 96, 67-72.
Ivanetich, K.M. and Santi, D.V. ( 1990). Bifunctional thymidylate synthase-
dihydrofolate reductase in protozoa. FASEB Journal 4, 1591-1597.
Jackson, H.C., Biggadike, K., McKilligin, E., Kinsman, O.S., Queener, S.F., Lane,
A. and Smith, J.E. (1996). 6,7-Disubstituted 2,4-diaminopteridines: novel inhi-
bitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase.
Antimicrobial Agents and Chemotherapy 40, 137 1-1375.
James, S. (1980). Isolation of second-generation schizonts of avian coccidia and
their use in biochemical investigations. Parasitology 80, 301-3 12.
Joe, A., Hamer, D.A., Kelley, M.A., Pereira, M.E.A., Keusch, G.T.. Tzipori, S. and
Ward, D.H. (1994). Role of a GaUGalNAc-specific sporozoite surface lectin in
Cryptosporidium parvum - host cell invasion. Journal of Eukaryotic Micro-
biology 41, 44s.
Johnson, A.M. (1990). Toxoplasma: biology, pathology, immunology and treat-
ment. In: Coccidiosis of Man and Domestic Animals (P.L. Long, ed.), pp.
121-153. Boston: CRC Press.
Johnson, A.M., Illana, S., McDonald, P.J. and Asai, T. (1989). Cloning, expression
and nucleotide sequence of the gene fragment encoding an antigenic portion of
the nucleoside triphosphate hydrolase of Toxoplasma gondii. Gene 85, 2 15-220.
BIOCHEMISTRY OF THE COCClDlA 213
Morisaki, J.H., Heuser, J.E. and Sibley, L.D. (1995). Invasion of Toxoplasma
gondii occurs by active penetration of the host cell. Journal of Cell Science
108, 2457-2464.
Murray, H.W. and Cohn, Z.A. (1980). Macrophage oxygen-dependent antimicro-
bial activity. 111. Enhanced oxidative metabolism as an expression of macro-
phage activation. Journal of Experimental Medicine 152, 1596-1609.
Murray, H.W., Nathan, C.F. and Cohn, Z.A. (1980). Macrophage oxygen-depen-
dent antimicrobial activity. IV. Role of endogenous scavengers of oxygen
intermediates. Journal of Experimental Medicine 152, 1610-1624.
Murray, H.W., Rubin, B.Y., Carriero, S.M., Harris, A.M. and Jaffee, E.A. (1985).
Human mononuclear phagocyte antiprotozoal mechanisms: oxygen-dependent
versus oxygen-independent activity against intracellular Toxoplasma gondii.
Journal of Immunology 134, 1982-1988.
Naciri, M., Mancassola, R., Yvore, P. and Peeters, J. E. (1993). The effect of
halofuginone lactate on experimental Cryptosporidium parvum infections in
calves. Veterinary Parasitology 45, 199-207.
Nagel, S.D. and Boothroyd, J C. (1989). The major surface antigen, P30, of
Toxoplasma gondii is anchored by a glycolipid. Journal of Biological Chemistry
264, 5569-5574.
Naguib, F.N.M., Iltzsch, M.H., El Kouni, M.M., Panzica, R.P. and El Kouni, M.H.
(1995). Structure-activity relationships for the binding of ligands to xanthine or
guanine phosphoribosyl transferase from Toxoplasma gondii. Biochemical Phar-
macology 50, 1685-1693.
Nakai, Y. and Ogimoto, K. (1983a). Relationship between amylopectin and viabi-
lity of Eimeria tenella sporozoites. Japanese Journal of Veterinary Science 45,
127- 129.
Nakai, Y. and Ogimoto, K. (1983b). Utilization of carbohydrate by Eimeria tenella
sporozoites. Japanese Journal of Veterinary Science 45, 501-506.
Nakai, Y. and Ogimoto, K. (1983~).Amylopectin synthesis of the sporozoite of
Eimeria tenella. Japanese Journal of Veterinary Science 45, 673-677.
Nam, H.W. and Choi, W.Y. (1992). Physiological changes of host cell types after
challenge with Toxoplasma gondii. Journal of Catholic Medical College 45,
41 1-423.
Nesterenko, M.V., Tilley, M. and Upton, S.J. (1995). A metallo-dependent cysteine
proteinase of Cryptosporidium parvum associated with the surface of
sporozoites. Microbios 83, 77-88.
Ng, H.C., Singh, M. and Jeyaseelan, K. (1995). Identification of two protein serine/
threonine kinase genes and molecular cloning of an SNFl type protein kinase
gene from Toxoplasma gondii. Biochemistry and Molecular Biology Interna-
tional 35, 155-165.
Nina, J.M.S., McDonald. V., Dyson, D.A., Catchpole, J., Uni, S., Iseki, M.,
Chiodini, P.L. and McAdam, K.P.W.J. (1992). Analysis of oocyst wall and
sporozoite antigens from 3 Cryptosporidium spp. Infection and Immunology
60, 1509-1513.
North, M.J. and Lockwood, B.C. (1995). Amino acid and protein metabolism. In:
Biochemistry and Molecular Biology of Parasites (J.J. M a n and M. Muller, eds),
pp. 67-88. London: Academic Press.
North, M.J., Mottram, J.C. and Coombs, G.H. (1990). Cysteine proteinases of
parasitic protozoa. Parasitology Today 6, 270-275.
Odberg-Feragut, C., Soete, M., Engels, A., Semyn, B., Loyens, A., van Beeumen,
J., Camus, D. and Dubremetz, J.F. (1996). Molecular cloning of the Toxoplasma
218 G.H. COOMBS EJAL.
gondii sag4 gene encoding an 18 kDa bradyzoite specific surface protein. Mole-
cular and Biochemical Parasitology 82, 237-244.
Odenthal-Schnittler, M., Tomavo, S., Becker, D., Dubremetz, J.-F. and Schwartz,
R.T. (1993). Evidence for N-linked glycosylation in Toxoplasma gondii.
Biochemical Journal 291, 7 13-721.
O’Donoghue, P.J. (1995). Cryptosporidium and cryptosporidiosis in man and
animals. International Journal for Parasitology 25, 139-195.
Ogunkolade, B.W., Robinson, H.A., McDonald, V., Webster, K. and Evans, D.A.
(1993). Isoenzyme variation within the genus Cryptosporidium. Parasitology
Research 79, 385-388.
Okhuysen, P.C., Dupont, H.L., Sterling, C.R. and Chappell, C.L. (1994). Arginine
aminopeptidase, an integral membrane protein of the Cryptosporidium parvum
sporozoite. Infection and Immunity 62, 46674670.
Okhuysen, P.C., Choppen, C.L., Kettner, C. and Sterling, C.R. (1996). Crypto-
sporidium parvum metalloaminopeptidase inhibitors prevent in vitro excystation.
Antimicrobial Agents and Chemotherapy 40, 278 1-2784.
Olliaro, P., Gorini, G., Jabes, D., Regazzetti, A., Rossi, R., Marchetti, A. Tinelli, C.
and Della-Bruna, C. (1994). In vitro and in vivo activity of rifabutin against
Toxoplasma gondii. Journal of Antimicrobial Chemotherapy 34, 649-657.
Omura, S., Tsuzuki, K., Iwai, Y., Kishi, M., Watanbe, S. and Shimizu, H. (1985).
Anticoccidial activity of fenolicin B and its derivatives. Journal of Antibiotics
38, 1447-1448.
Oshaka, A., Yoshikawa, K. and Hagiwara, T. (1982). Proton NMR spectroscopic
study of aerobic glucose metabolism in Toxoplasma gondii harvested from the
peritoneal exudate of experimentally infected mice. Physiological Chemistry and
Physics 14, 381-384.
Ossorio, P.N., Dubremetz, J.F. and Joiner, K.A. (1994). A soluble secretory protein
of the intracellular parasite Toxoplasma gondii associates with the parasitophor-
ous vacuole membrane through hydrophobic interactions. Journal of Biological
Chemistry 269, 15350-15357.
O’Sullivan, W.J., Johnson, A.M., Finney, K.G., Gero, A.M., Hogan, E., Holland,
H.M. and Smithers, G.W. (1981). Pyrimidine and purine enzymes in Toxoplasma
gondii. Australian Journal of Experimental Biology and Medical Science 59,
763-767.
Ovington, K.S. and Smith, N.C. (1992). Cytokines, free radicals and resistance to
Eimeria. Parasitology Today 8, 422-426.
Page, A.P., Kumar, S. and Carlow, C.K.S. (1995). Parasite cyclophilins and anti-
parasite activity of cyclosporin A. Parasitology Today 11, 385-388.
Parmley, S.F., Weiss, L.M. and Yang, S.M. (1995). Cloning of a bradyzoite-
specific gene of Toxoplasma gondii encoding a cytoplasmic antigen. Molecular
and Biochemical Parasitology 73, 253-257.
Patillo, W.H. and Becker, E.R. (1955). Cytochemistry of Eimeria brunetti and
Eimeria acervulina of the chicken. Journal of Morphology 96, 61-96.
Pedersen, P.L. and Carafoli, E. (1987). Ion motive ATPases. I. Ubiquity, properties
and significance to cell function. Trends in Biochemical Sciences 12, 146-150.
Pellegrin, J.-L.J., Ortega-Barria, E., Prioli, R.P., Buerger, M., Strout, R.G., Alroy,
J. and Pereira, M.E.A. (1993). Identification of a developmentally regulated
sialidase in Eimeria tenella that is immunologically related to the Trypanosoma
cruzi enzyme. Glycoconjugate Journal 10, 57-63.
Peng, Z.Y.and Mansour, T.E. (1992). Purification and properties of a pyropho-
BIOCHEMISTRY OF THE COCClDlA 219
John M. Kelly
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
2. Transfection of Leishmania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
2.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
2.2. Stable transformation of Leishmania by episomal vectors . . . . . . . . . . . . 229
2.3. Gene targeting in Leishmania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
2.4. Functional analysis of Leishmania genes . . . . . . . . . . . . . . . . . . . . . . . . . 233
2.5. Analysis of gene expression in Leishmania ....................... 237
3. Transfection of Trypanosoma brucei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
3.1. Development of stable transformation vectors ..................... 239
.
3.2. Analysis of gene expression in T brucei . . . . . . . . . . . . . . . . . . . . . . . . . 242
3.3. Transfection as a tool for functional analysis in T. brucei . . . . . . . . . . . . .246
4. Transfection of Trypanosoma cruzi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
4.1. Development of vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
4.2. Transfection as a tool for functional analysis of T. cruzi genes . . . . . . . . . 250
4.3. Analysis of gene expression in T. cruzi . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
5. Transfection of the Apicomplexa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
5.1. Development of transfection techniques for Toxoplasma gondii . . . . . . . 252
5.2. Development of transfection techniques for Plasmodium . . . . . . . . . . . . . 256
6. Transfection of Intestinal Protozoan Parasites ......................... 257
6.1. Transfection of Entamoeba histolytica . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
6.2. Transfection of Giardia lamblia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
7 . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
1. INTRODUCTION
2. TRANSFECTION OF LEISHMANIA
2.1. Background
vectors using flanking sequences derived from the genes encoding a-tubu-
lin (Laban et al., 1990) and the bifunctional enzyme dihydrofolate reduc-
tase-thymidylate synthetase (dhfr-ts) (Kapler er al., 1990). These
transfection vectors contained a bacterial origin of replication, an ampi-
cillin-resistance (amp) gene and had the respective Leishmania sequences
placed on either side of the neomycin phosphotransferase (neo) gene. This
gene, which is encoded by the bacterial transposon Tn5, confers resistance
to the aminoglycoside G418 (Southern and Berg, 1982). After electropora-
tion, transformed Leishmania cells selected on the basis of G418 resistance
were obtained. These transformants were found to contain multiple extra-
chromosomal copies of the plasmids, which in some cases were organized
in large circular episomes composed of head-to-tail repeats of the vector.
Integration into the parasite genome was not demonstrated. The neo RNA
transcribed from these vectors was found to be correctly processed in terms
of polyadenylation and spliced leader addition. Although the mechanisms
by which transcription of these transfected genes are regulated remain to be
defined, it is clear that promoter elements, as recognized in other eukar-
yotic systems, are not involved. The spliced leader acceptor site and the
associated upstream polypyrimidine stretch both seem to be important for
efficient expression (Curotto de Lafaille et al., 1992).
These first-generation constructs have since been modified to function as
Leishmania expression vectors (LeBowitz et al., 1990; Tobin et al., 1993).
In the case of the dhfr-ts derived vectors (the pX family) it was found that
they could also be used to confer transfection-mediated G418 resistance on
several Leishmania species, and on the genera Endotrypanum and Crithi-
dia, but not on T. cruzi or T. brucei (Coburn et al., 1991). More recently,
several other Leishmania expression vectors have been developed, includ-
ing one which also functions in T. cruzi (Kelly et al., 1992) (Figure l), and
one which is completely deficient in Leishmania-derived sequences (Papa-
dopoulou et al., 1994a). This latter finding suggests that in Leishmania
specific signals may not be required for episomal replication or for the
initiation of transcription. The range of drug-selectable markers available
for Leishmania transfection experiments has also been extended to include
the hygromycin phosphotransferase (hyg) gene (Cruz et al., 1991), the N-
acetylglucosamine-1-phosphate transferase (nagt) gene (Lui and Chang,
1992a), the streptothricin acetyltransferase (sat) gene (Joshi et al., 1995)
and the phleomycin (ble)and puromycin (pac) resistance genes (Freedman
and Beverley, 1993). LeBowitz et al. (1992) have also reported the con-
struction of a Leishmania expression vector in which the herpes simplex
virus thymidine kinase gene (HSVtk) functions as a negative selectable
marker. Expression of HSVtk renders transformants susceptible to nucleo-
side analogues such as ganciclovir, enabling cells which have lost the
construct to be rapidly selected from a parasite population.
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 231
500 bp
H
Figure I The pTEX vector which facilitates the expression of transfected genes
in Trypanosoma cruzi and Leishmania (Kelly et al., 1992). Both the multiple
cloning site (MCS) and the neor gene are flanked by the 5'-upstream and 3'-
downstream regions of the T. cruzi gapdh genes (thin line). The thick line,
including the amp' gene, represents sequences derived from the bacterial plasmid
pBluescript. Genes cloned into the MCS can be expressed at high levels in
transfected parasites. Other protozoan episomal expression vectors conform to
this general design.
Eco RI
Bam HI
pcosTL Sma I
8.5kb
Sac II cos Eco RI
Bst XI
Sac I
ADa I
Kpn i
Integration was favoured when low amounts of linearized vector DNA was
used for electroporation. In the first successful targeted integration, the
dhfr-ts genes of the avirulent L. major strain CC-1 were replaced with the
neo drug-selectable marker after simultaneous integration at both alleles
(Cruz and Beverley, 1990). In the construct used in this experiment, the
dhfr-ts flanking regions were placed on either side of the neo gene. As
expected from the role of DHFR-TS in folate biosynthesis, homozygous
‘knockouts’ could be isolated when electroporated cells were selected in
the presence of a thymidine supplement. Similar results were obtained
when the dhfr-ts genes were replaced consecutively with neo then hyg
(Cruz et al., 1991). In contrast, when this experiment was repeated with
a virulent L. major strain (LV39), auxotrophic mutants could not be iso-
lated (Cruz et al., 1993). Replacement of a single dhfr-ts gene with neo was
achieved, but it was then not possible to obtain dhfr-ts mutants using a
second replacement vector containing the hyg gene, even when selection
was carried out in the presence of thymidine. Instead, the transformed lines
were found to have undergone alterations in chromosome number, either
aneuploidy or tetraploidy, to accommodate the double replacement of dhfr-
ts with neo and hyg. This experiment, while demonstrating the plasticity of
the Leishmunia genome, also suggests that in this virulent L. major strain,
DHFR-TS performs an additional essential function which is independent
of its role in thymidine synthesis.
The a-tubulin genes of L. enriettii are organized in two allelic clusters
containing a total of 30 genes (Landfear et al., 1983), a common type of
organization for trypanosomatid genes. The applicability of gene targeting
techniques to study such multicopy arrays was demonstrated when the L.
enriettii a-tubulin genes were replaced with drug-selectable markers (Cur-
Otto de Lafaille and Wirth, 1992). The vectors were constructed such that
the neo and hyg genes were positioned between sequences derived from the
flanking regions of the tubulin clusters. In cases where replacement of both
clusters was observed, a-tubulin genes could be found within extrachro-
mosomal elements which were presumably generated as part of the deletion
process.
questions now being addressed using these techniques. Many of the initial
functional studies have been aimed at elucidating the mechanisms of drug
resistance.
In Leishmania, drug resistance has often been associated with gene
amplification (Beverley, 1991; Ouellette and Papadopoulou, 1993). For
example, exposure of cells to a number of apparently unrelated drugs has
been correlated with amplification of the H locus (Beverley et al., 1984) in
the form of large extrachromosomal circles. Using plasmid shuttle vectors
it has been possible to dissect the 40 kb H locus and to identify the genes
which confer drug resistance when overexpressed. Thus the gene that
confers arsenite resistance was identified after fragments of the L. major
H region were ligated into a Leishmania expression vector and introduced
by transfection back into the parasite at high copy number (Callahan and
Beverley, 1991). Constructs which produced resistance to both arsenite and
trivalent antimonials were shown to contain a P-glycoprotein-related gene,
ImpgpA. The mechanism of antimonial resistance conferred by overexpres-
sion of lmpgpA has been shown to result from decreased influx, possibly
caused by the interaction of PGPA with other Leishmania proteins (Call-
ahan er al., 1994). In an independent study with L. tarentolae, a similar
resistance phenotype was observed (Papadopoulou et al., 1994b). In addi-
tion, these authors also found an association between overexpression of the
P-glycoprotein-related gene and resistance to pentavalent antimonials.
The pattern of drug resistance observed in Leishmania cells that over-
express 1mpgpA does not conform to the multi-drug-resistant (MDR) phe-
notype that is found in mammalian cells as a result of overexpression of P-
glycoproteins. However, an MDR phenotype has been identified in L.
donovani. It results from overexpression of a Leishmania mdr homologue
(ldmdr I ) , either by gene amplification or by transfection (Henderson et al.,
1992). This gene, which is not localized in the H region, is associated with
cross-resistance to vinblastine, puromycin and the anthracyclines. In L.
enriettii, stepwise selection with vinblastine has been shown to result in
the generation of V-circles, 40 kb extrachromosomal elements which con-
tain an mdr homologue (lemdr I ) (Chow et al., 1993). Using a homologous
recombination approach to disrupt specific sites within the V-circle, it was
demonstrated that vinblastine resistance results only from overexpression
of the lemdr I gene (Wong et al., 1994).
Transfection procedures have been used in the functional analysis of
another novel gene associated with the H region, ltdh, a short chain
dehydrogenase (Papadopoulou et al., 1994~).Plasmid-mediated overex-
pression of this gene was shown to confer resistance to methotrexate, an
inhibitor of DHFR. The mechanism of resistance is thought to involve
increased production of reduced folates by a pathway that involves pterin
and which can compensate for the inhibition of DHFR by methotrexate.
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 235
Lee and Van der Ploeg, 1990; Eid and Sollner-Webb, 1991). In each of
these cases, transformation was mediated by integration which was found
to occur exclusively by homologous recombination, with the formation of
episomes being rare (ten Asbroek et al., 1993). Transfection with linear
DNA is considerably more efficient than with circular DNA and transfor-
mation efficiencies levels as high as 1 X have been reported (ten
Asbroek et al., 1993). Less than 1 kb of homologous flanking sequence was
found to be necessary for targeted insertion into the tubulin locus. Other
selectable markers including the hyg (Lee and Van der Ploeg, 1991) and ble
(Jefferies et al., 1993) resistance genes have now also been used success-
fully in T. brucei transformation experiments.
In these initial reports, transformation of bloodstream form T. brucei was
not described. However this has now been achieved (Carruthers et al.,
1993), largely as a result of improvements in culturing methods, particu-
larly the development of a technique for obtaining high plating efficiency
of colonies on agarose plates (Carruthers and Cross, 1992). In these blood-
ation experiments, constructs were targeted to the
For reasons that are still unclear, transformation in
these and other experibents with bloodstream parasites (Kelly et al., 1995;
Rudenko et al., 1995) *as 1000-fold less efficient than with the procyclic
forms. Despite this, th$ development of bloodstream form transfection
represents a significant step forward which has greatly extended the poten-
tial applications of these techniques to the African trypanosome.
Unlike Leishmania and other trypanosomatids, stable transformation of
T. brucei with episomal vectors has proved difficult to achieve routinely.
Integration is the favoured mechanism of transformation and the require-
ments for autonomous replication appear to be more stringent than in
other trypanosomatids. For example, after transfection with a circular
construct containing the neo gene flanked by the tubulin intergenic
regions, ten Asbroek et al. (1993) obtained only a single transformant
containing extrachromosomal plasmid. This was in the form of a circular
pentamer and was lost soon after drug pressure was removed. In contrast,
however, Patnaik et al. (1993) have reported an episomal vector which
exhibits greater stability in T. brucei procyclics in the absence of drug
pressure, and which replicates as a single copy element. This vector
(pT13-11) contains the neo gene flanked by the 5’- and 3’-regions of
the procyclic acidic repetitive protein (parp) gene, including the promo-
ter. Stable episomes were isolated after random fragments of T. brucei
DNA had been introduced into the construct prior to ‘shotgun’ transfec-
tion and selection of drug-resistant transformants. Since the ability of
pT13-11 to replicate and segregate in T. brucei resulted from the inser-
tion of a single DNA fragment, one possibility is that both these proper-
ties were conferred by closely linked genomic elements. Further studies
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 241
have also shown a close linkage between elements responsible for repli-
cation and transcription (Patnaik er al., 1994a); a region encompassed by
the parp promoter seems to play a crucial role in plasmid replication.
Transfection experiments with pT 13-1 1 and derivatives thereof have
demonstrated that these episomes can exist in a broad range of trypano-
somatids. However, there is variation in the requirements for stable
replication (Patnaik et al., 1994b) between trypanosomatids with the
requirements of T. brucei being the most stringent.
An analogous approach was used by Metzenberg and Agabian (1994) to
study episome-mediated transformation in T. brucei. Xba I fragments of T.
@
eucbrF&+w
.a e cloned randomly into a plasmid which contained the parp
N m o t e r placed in'front of the hyg gene. After electroporation of procyclics
with the resultant libraw of molecules, drug-resistant cells in which trans-
formation was episomally mediated were isolated. The vector was found to
contain a 1 kb insert thar was derived from a kinetoplast DNA (kDNA)
minicircle. Vector molechles were confined to the nucleus and were not
detected in the mitochondrion. If these kDNA sequences function by pro-
viding an origin of replication, it will imply a common mechanism of
episome maintenance in the nucleus and mitochondrion of T.brucei. Simi-
lar to observations described in a previous report (ten Asbroek et al., 1993),
the vector molecules were present in several copies per cell and were
maintained as supercoiled extrachromosomal elements composed of head-
to-tail repeats (7-9 copies). The formation of large episomes composed of
tandemly linked vector molecules has also been reported by Lee (1995). The
construct used in this transfection experiment contained the parp promoter
linked to the neo gene and was found to exhibit mitotic stability in procyclic
trypanosomes, although the mechanism involved is unclear. At the moment,
therefore, there is no consensus on the absolute requirements for episomal
stability, replication and segregation in T. brucei.
More recently, the construction of trypanosome artificial chromosomes
(TACs) has been reported (Lee et al., 1995). These vectors contain theparp
promoter, use the hyg gene as a selectable marker, and have telomere
repeats and subtelomeric sequences at the termini of the linear molecule.
In transfected cells, the molecules were mitotically stable and remained
linear, but were found to have increased in size due to the addition of new
sequences at internal sites. These additional sequences were derived from
the mini-chromosomes and from a subset of larger chromosomes. Although
the development of TACs is still at a early stage, and further work will be
required, they could have applications in the study of gene expression,
particularly the switching events involved in antigenic variation (see
below).
To extend the range of tools available for functional analysis, Wirtz and
Clayton (1995) have recently reported the development of an inducible
2 42 J.M. KELLY
In T. brucei, the parp genes are arranged in arrays of two or three copies
and are transcribed polycistronically under the control of a single upstream
prorp6(P=., 1994). Transient transfection assays coupled with
M e r scanning analysh have been used to delineate the upstream regula-
tory sequences and to evhluate their contribution to transcriptional control
(Sherman et al., 1991; Bro\wn et al., 1992). These experiments revealed the
presence of a bipartite core control element situated just upstream of the
transcription initiation site and other features characteristic of a RNA
polymerase I promoter. In the bloodstream form of the parasite, parp
mRNA is completely absent even though transcription of the parp genes
still occurs at about 10% of the level measured in procyclics (Pays et al.,
1990). This suggests that other mechanisms in addition to control of
promoter activity must play a significant role in the stage-specific expres-
sion of PARP. To investigate this further, Vanhamme et al. (1995) targeted
a reporter construct into the parp promoter region. They confirmed the
presence of transcriptional activity in the bloodstream form and obtained
evidence for regulation at the level of RNA elongation. Using a variety of
transfection based techniques, Berberof et al. (1995) have demonstrated
that sequences in the 3'-UTR of parp mRNA also play an important part in
regulation, presumably acting at the level of RNA maturation andor
stability. Hehl et al. (1994) have identified an additional regulatory element
in the 3'-UTR of parp mRNA. This 16-nucleotide sequence has the poten-
tial to form a stable hairpin loop structure, does not affect mRNA stability,
but is required for efficient translation.
The major surface antigen of procyclics in the related parasite Trypano-
soma congolense is the protein GARP (glutamic acidalanine rich protein).
Although it shares no sequence similarity, this molecule appears to be a
functional analogue of PARP. Indeed, when GARP is expressed in T.
brucei procyclics following stable transformation, it can be localised on
the cell surface (Hehl et al., 1995). Like the parp genes, those expressing
GARP are also arranged in tandem repeats. In a series of experiments,
which utilized transient transfection assays and nuclear run-ons (Graham et
al., 1996), the garp promoter has been localized to a region about 500 bp
upstream of the start codon of the 5'-proximal gene; this compares to less
than 100 bp in the case of parp. In addition, it was found that transcription
of the garp genes is a-amanitin sensitive, suggesting that the process may
be mediated by RNA polymerase 11. This could be related to the observa-
tion that the garp genes are constitutively active like other protein coding
genes transcribed by RNA polymerase 11. In T. congolense bloodstream
forms, the garp mRNA is present but the protein is not detectable implying
that GARP expression is controlled predominantly at the translational a n d
or post-translational level.
In bloodstream form T. brucei, the actively expressed vsg gene is
244 J.M. KELLY
fection (Graham and Barry, 1995). The genes encoding the M-VSG seem to
be distinct from other trypanosome genes in that they are transcribed as
monocistronic transcripts and their stage-specific expression is regulated
exclusively at the level of transcription. This distinct mechanism of control
is thought to facilitate the random activation of m-vsg genes which could
enhance transmission by presenting the host immune system with an anti-
genically diverse parasite population.
Several studies have shown that post-transcriptional regulation is an
important feature of gene expression in the trypanosomatids (Graham,
1995). This is a consequence of polycistronic transcription; linked genes
are probably co-transcribed at equivalent rates and any differences in the
level of steady state mRNA must therefore result from control exerted at
the post-transcriptional level. In bloodstream form T. brucei, such mechan-
isms account for the greater than 100-fold difference in the abundance of
the mRNAs encoding vsg and some of the upstream ESAGs. Sequences in
the 3’-UTR of vsg mRNA have been shown to mediate stage-specific
expression (Berberof et al., 1995). A 97-nucleotide region, immediately
upstream of the vsg mRNA polyadenylation site, was found to confer a
three-fold stimulation of reporter gene expression in bloodstream parasites
and to downregulate by the same extent in procyclics. This nine-fold
difference between the two stages suggests that elements within this 3’-
UTR sequence make a significant contribution to the stage-regulated
expression of vsg.
Downstream of trypanosomatid genes consensus signals for polyadeny-
lation such as the AAUAAA sequence have not been identified. In Leish-
mania, for example, it has been shown that the site of polyadenylation is
specified not by a consensus signal sequence but by the position of the
splice acceptor site of the downstream gene (above) (LeBowitz et al.,
1993). Consistent with this, analysis of transcription using permeabilized
cells has shown that in T. brucei, trans-splicing of the downstream gene
must precede polyadenylation during the synthesis of transcripts derived
from the tubulin array (Ullu et al., 1993). To identify signals required for
polyadenylation of T. bmcei tubulin mRNA, Matthews et al. (1994) pro-
duced an expression construct in which the intergenic region of the p- and
a-tubulin genes was placed between an upstream cat and downstream luc
gene. The intergenic region was then mutagenized with a series of block
substitutions. After introduction of the resulting constructs into T. brucei
by electroporation, the RNA expressed by the reporter genes was analysed.
It was inferred from the results that pyrimidine-rich elements located
upstream of the a-tubulin splice acceptor site were required for accurate
polyadenylation of the upstream p- and for trans-splicing of the downstream
a-tubulin genes. Similar conclusions were drawn from an analogous series
of experiments aimed at investigating polyadenylation of the parp mRNA
246 J.M. KELLY
(Schurch et al., 1994; Vassella et al., 1994). This coupled reaction of trans-
splicing and polyadenylation of co-transcribed genes may be an important
site of gene regulation in trypanosomes.
RNA polymerase I1 promoters have not been characterized in T. brucei
and several studies have implicated the 3’-UTR as the major mediator of
stage-specific regulation of pol I1 transcribed genes. For example, aldolase
mRNA is present at increased levels in bloodstream parasites, although
there is no change in transcription rate (Hug et al., 1993). To test whether
or not the aldolase 3’-UTR could influence the level of expression it was
placed downstream of the luc gene in a transfection construct. After
electroporation of bloodstream and procyclic forms, the stage specificity
of luc expression was found to be similar to that shown by the aldolase
genes. In other experiments, it was also demonstrated that 3’-UTRs derived
from the hexose transporter genes could confer stage specificity on reporter
genes after stable transformation (Hotz et al., 1995). Although the mechan-
isms remain to be defined, it appears that control operates at several levels,
including alterations in RNA stability.
flagellar pocket as in bloodstream parasites, but was spread over the entire
surface. In addition, the reconstituted complex did not mediate transferrin
uptake. These results suggest that other bloodstream form-specific proteins
could be required for correct localization of the complex and to facilitate
transferrin uptake.
The ability to confer transfection-mediated drug resistance on T. brucei
has been exploited to investigate the mechanisms of genetic exchange. T.
brucei genetic hybrids can be isolated after co-transmission of mixed
parasite populations through tsetse flies (Jenni et al., 1986). However,
genetic exchange is a rare event and is a non-obligatory part of the life
cycle. To improve the efficiency of hybrid selection, transformation has
been used to confer either neo or hyg resistance on parasites prior to
passage through the fly. Hybrids were then successfully selected on the
basis of double drug resistance (Gibson and Whittington, 1993; Gibson and
Bailey, 1994). This technique has great potential and should be a useful
additional approach for those seeking to understand the mechanisms of
genetic exchange in T. brucei.
The mechanisms of gene expression in T. cruzi have been less well studied
than those in Leishmania or T. brucei. However, transfection techniques
are now being applied to this area and steady progress can be expected. As
yet, no RNA polymerase 11-dependent promoters have been characterized
and, similar to the situation in Leishmania, it is thought that they are not
required for episomal expression. Transient transfection assays have, how-
ever, been used to identify and characterize the rRNA promoter in T. cruzi
(Tyler-Cross et al., 1995). In the presence of a suitable 3’-splice site, the
rRNA promoter was shown to mediate expression of the cat gene, at a level
100-fold greater than previously obtained with the mini-exon gene promo-
ter (Lu and Buck, 1991). Unexpectedly, the promoter did not function in all
T. cruzi isolates, indicating. a restricted host range. This may reflect the
extensive diversity of the T. cruzi species and can be correlated with the
observation that pol I promoters have been less well conserved during
evolution than pol I1 or pol I11 promoters. It will be interesting to determine
whether there is a relationship between promoter activity in various strains
and other taxonomic parameters such as isoenzyme profiles.
252 J.M. KELLY
As has been reported for Leishmania and T. brucei (see above), recent
studies with T. cruzi suggest that the 3’-UTRs of genes play an important
role in the control of gene expression (Nozaki and Cross, 1995). In a series
of stable transformation experiments using episomal vectors containing the
luc gene as a reporter, it was shown that the 3’-UTRs of the stage-specific
genes gp72, gp82 and amastin (ama) could mediate a considerable down-
regulation in the level of luc mRNA in transformed epimastigotes, com-
pared with the control situation in which the 3’-UTR was derived from the
constitutively expressed gapdh gene. In amastigotes, the ama 3 ’-UTR was
shown to confer upregulation, such that the overall stage-specific effect
compared with epimastigotes was greater than 100-fold. Since previous
studies have shown that the ama gene is transcribed at the same rate in
epimastigotes and amastigotes (Teixeira et al., 1994), these findings imply
that regulation at the level of mRNA stability is a major point of control for
stage-specific expression in T. cruzi.
ionic composition to the cytosol (Van den Hoff et al., 1992) and CAT
activity was detected after 8 h. When the sagl 5’-flanking fragment was
replaced with the corresponding regions from the genes encoding a-tubulin
and rhoptry protein 1 (ropl), the amount of CAT expressed was eight- and
four-fold higher respectively. Remarkably, in these transient experiments
up to 15% of electroporated cells expressed the transfected gene product.
To characterize the upstream sequences that govern expression of sagl,
transient transfection experiments coupled with deletion analysis were
performed (Soldati and Boothroyd, 1995). These experiments identified
six repeated sequence elements situated in the region between 35 and
190 bp upstream of the first of two transcription start sites that were
essential for transcription initiation.
The sagl-based vector used in the transient experiments was modified
prior to being utilized in experiments aimed at conferring stable trans-
formation using the cat gene as a selectable marker (T. gondii are
susceptible to chloramphenicol under conditions in which host cells are
unaffected). The modified vector (SBCATl) contained the 5‘- and 3’-
flanking sequences of sagl in addition to a copy of the B l gene, which is
highly re-iterated in T. gondii (Kim et al., 1993). Both linearized and
circular forms of SBCATl were found to mediate stable transformation
after electroporation, with linear DNA being more efficient. In all cases,
SBCATl became integrated into the B l locus. Attempts were then made
to delete the single copy sagl and ropl genes using vectors designed for
gene replacement. Knockout of the ropl gene was achieved, although in
the majority of transformants the gene was not deleted and the vector
DNA had integrated by non-homologous recombination. Transformants
in which the sagl gene was deleted were not isolated, suggesting that
loss of this gene could confer a selective disadvantage. However, the
precise function of SAGl remains unclear since mutant T. gondii that do
not express the antigen can still invade and proliferate in mammalian
cells.
To study the role of SAGl, Kim and Boothroyd (1995) used transfection
to complement these mutants and a novel approach to isolate the transfor-
mants. The sag1 gene was introduced into the parasite by electroporation
and transformants that expressed SAGl on the cell surface were selected
directly using a fluorescence-activated cell sorter (FACS). The success of
this rapid selection method suggests that it can be readily adapted to allow
direct isolation of recombinant parasites that express other cell surface
proteins encoded by transfected genes. Restriction enzyme-mediated inte-
gration is another technique that has been used to improve transformation
procedures in T. gondii (Black et al., 1995). This technique, which simply
involves performing the electroporation step in the presence of a restriction
enzyme, is thought to enhance random integration by inducing breaks in
254 J.M. KELLY
the genomic DNA. In experiments with T. gondii it was found that Not1
increased the efficiency of stable transformation by almost 50-fold; indeed
the enhancement of integration occurred even if the vector DNA was not
linearized prior to electroporation. This technique has been used to facil-
itate the co-transfection of T. gondii with four different constructs, only one
of which expressed a drug-selectable marker (CAT).
As an alternative approach, stably transformed T. gondii have also been
generated on the basis of pyrimethamine resistance conferred by expression
of a modified dhfr-ts gene (Donald and Roos, 1993). The gene was mod-
ified by introduction of point changes to the sequence by analogy with the
mutant gene associated with pyrimethamine resistance in malaria. This
modified gene was cloned into a plasmid containing the endogenous
dhfr-ts flanking sequences and was found to confer pyrimethamine resis-
tance after transfection. Integration was mediated by non-homologous
recombination in these experiments and gene replacement was not
observed. However, when other constructs containing larger fragments of
contiguous genomic DNA sequence (up to 16 kb) were used, targeted
integration occurred in half the cases (Donald and Roos, 1994). In these
experiments circular plasmids were found to be more efficient than linear
DNA at undergoing integration by homologous recombination. Reciprocal
cross-over resulting in duplication of the dhfr-ts locus appeared to be the
most common mechanism involved, although complete allelic replacement
also occurred at low frequency. One drawback with using pyrimethamine
resistance for selection of recombinant T. gondii is the possibility of
accidentally acquired infection, since the drugs of choice used to treat
toxoplasmosis are normally anti-folates.
Another T. gondii transformation system that has been developed is
based on the naturally occurring tryptophan auxotrophy of the parasite
(Sibley et al., 1994). The transformation vector was constructed such
that the E. coli trpB gene (which encodes the fJ-subunit of tryptophan
synthase, an enzyme that catalyses the synthesis of tryptophan from indole
and serine) was placed between the 5’- and 3’-flanking sequences of the
sag1 gene. After electroporation, transformants were selected by growth in
tryptophan-deficient medium supplemented with indole. In these experi-
ments, transformants were obtained using both circular and linear con-
structs, and integration occurred at distinct non-homologous sites normally
resulting in tandemly repeated copies of the vector.
Two recent reports (Messina et al., 1995; Soldati et al., 1995) have
described the use of phleomycin to select transformed T. gondii. Messina
et al. constructed vectors in which the ble gene was placed between
sequences derived from the flanking regions of the sagl or the gral or
gra2 genes. After transfection, the tachyzoites were allowed to invade host
cells without drug selection. Following lysis of the cell monolayers, the
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 255
ments, the highest level of expression was obtained with a construct con-
taining a gdhnuc fusion gene in which the first 18 codons of gdh preceded
the Zuc sequence. Luciferase activity was considerably reduced in parasites
transfected with constructs in which either the 5’- or 3’-flanking regions of
gdh had been removed.
In a novel approach, Yu ef aZ. (1995) used a viral vector to mediate
transient expression of luciferase in transfected G. Zamblia. This followed
on from an earlier study (Furfine and Wang, 1990) which demonstrated that
the 7 kb double-stranded RNA G. ZambZia virus (GLV) (Wang and Wang,
1986) can be introduced into the parasite by electroporation in its single-
stranded form. A cDNA clone was constructed in which the 5’- and 3’-
UTRs of GLV cDNA were placed either side of the Zuc gene. After
electroporation of G. Zamblia trophozoites with an in vitro transcript
derived from this cDNA, it was shown that the RNA could be replicated
and transcribed to produce anti-sense RNA. In addition, luciferase activity
could be detected up to 120 h after transfection. Luciferase expression was
dependent on the presence of the UTRs and of a putative packaging site.
This system has obvious scope for development, and the authors suggest
that it may be possible to use G. ZambZia infected with recombinant GLV
that express a toxic product as a therapeutic approach to treatment of
giardiasis.
7. CONCLUDING REMARKS
the form of expressed sequence tags (ESTs). It is clear that the traditional
approaches to the cloning and sequencing of parasite genes are becoming
redundant and that, in future, the major research effort will be aimed at
functional analysis of protein sequences derived from the genome projects.
Transformation techniques will allow us to investigate these questions in
the context of the parasite itself.
ACKNOWLEDGEMENTS
I thank David Baker, Anita Skinner and Martin Taylor for critical reading
of this manuscript. I also thank Martin Taylor for producing Figure 3. The
financial assistance of the Leverhulme and Wellcome Trusts, the Medical
Research Council and the UNDPNorld B a n W H O Special Programme
for Research and Training in Tropical Diseases (TDR) is gratefully
acknowledged.
REFERENCES
Agami, R., Aly, R., Halman, S. and Shapira, M. (1994). Functional analysis of cis-
acting DNA elements required for expression of the SL RNA gene in the
parasitic protozoan Leishmania amazonensis. Nucleic Acids Research 22,
1959-1965.
Argaman, M., Aly, R. and Shapira, M. (1994). Expression of the heat shock protein
83 in Leishmania is regulated post-transcriptionally. Molecular and Biochemical
Parasitology 64, 95-1 10.
Avery, O.T., MacLeod, C.M. and McCarty, M. (1944). Studies on the chemical
nature of substances inducing transformation of pneumococcal types. Journal of
Experimental Medicine 79, 137-158.
Bellofatto, V. and Cross, G.A.M. (1989). Expression of a bacterial gene in a
trypanosomatid protozoan. Science 244, 1167-1 169.
Bellofatto, V., Torres-Munoz, J.E. and Cross, G.A.M. (1991). Stable transforma-
tion of Leptomonas seymouri by circular extrachromosomal elements. Proceed-
ings of the National Academy of Science of the USA 244, 6711-6715.
Berberof, M., Vanhamme, L., Tebabi, P., Pays, A., Jefferies, D., Welburn, S. and
Payes, E. (1995). The 3’-terminal region of the mRNAs for VSG and procyclin
can confer stage specificity to gene expression in Trypanosoma brucei. European
Molecular Biology Organisation Journal 14, 2925-2934.
Beverley, S.M. (1991). Gene amplification in Leishmania. Annual Reviews of
Microbiology 45, 417-444.
Beverley, S.M., Coderre, J.A., Santi, D.V. and Schimke, R.T. (1984). Unstable
DNA amplification in methotrexate-resistant Leishmania consists of extrachro-
mosomal circles which relocalize during stabilization. Cell 38, 43 1-439.
Black, M., Seeber, F., Soldati, D., Kim, K. and Boothroyd, J.C. (1995). Restriction
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 261
Kapler, G.M., Coburn, C.M. and Beverley, S.M. (1990). Stable transfection of the
human parasite Leishmania major delineates a 30-kilobase region sufficient for
extrachromosomal replication and expression. Molecular and Cellular Biology
10, 1084-1094.
Kaye, P.M., Coburn, C., McCrossan, M. and Beverley, S.M.(1993). Antigens
targeted to the Leishmania phagolysosome are processed for CD4+ T cell
recognition. European Journal of Immunology 23, 231 1-23 19.
Kelly, J.M., Ward, H.M., Miles, M.A. and Kendall, G. (1992). A shuttle vector
which facilitates the expression of transfected genes in Trypanosoma cruzi and
Leishmania donovani. Nucleic Acids Research 20,3963-3969.
Kelly, J.M., Taylor, M.C., Smith, K., Hunter, K.J. and Fairlamb, A.H. (1993).
Phenotype of recombinant Leishmania donovani and Trypanosoma cruzi which
over-express trypanothione reductase: sensitivity towards agents that are thought
to induce oxidative stress. European Journal of Biochemistry 218, 29-37.
Kelly, J.M., Das, P. and Tomas, A.M. (1994). An approach to functional comple-
mentation by introduction of large DNA fragments into Trypanosoma cruzi and
Leishmania using a cosmid shuttle vector. Molecular and Biochemical
Parasitology 65, 5 1-62.
Kelly, J.M., Taylor, M.C., Rudenko, G. and Blundell, P.A. (1995). Transfection of
the African and American trypanosomes. In: Electroporation Protocols for
Microorganisms. Methods in Molecular Biology, Vol. 47 (J.A. Nickoloff, ed.),
pp. 349-359. New Jersey: Humana Press.
Kendall, G., Wilderspin, A.F., Ashall, F., Miles, M.A. and Kelly, J.M. (1990).
Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase
does not conform to the ‘hotspot’ topogenic signal model. European Molecular
Biology Organisation Journal 9, 275 1-2758.
Kim, K. and Boothroyd, J.C. (1995). Toxoplasma gondii: Stable complementation
of sag 1 (p30) mutants using SAG1 transfection and fluorescence-activating cell
sorting. Experimental Parasitology 80, 46-53.
Kim, K., Soldati, D. and Boothroyd, J.C. (1993). Gene replacement in Toxoplasma
gondii with chloramphenicol acetyltransferase as selectable marker. Science 262,
911-914.
Laban, A. and Wirth, D.F. (1989). Transfection of Leishmania enriettii and expres-
sion of the chloramphenicol acetyl transferase gene. Proceedings of the National
Academy of Science of the USA 86, 91 19-9123.
Laban, A., Tobin, J.F., Curotto de Lafaille, M.A. and Wirth, D.F.(1990).
Stable expression of the bacterial neo gene in Leishmania enriettii. Nature
343, 572-574.
La Flamme, A.C., Buckner, F.S., Swindle, J., Ajioka, J.A. and Van Voorhis, W.C.
(1995). Expression of mammalian cytokines by Trypanosoma cruzi indicates
unique signal sequence requirements and processing. Molecular and Biochem-
ical Parasitology 75, 25-3 1.
Landfear, S., McMahon-Pratt, D. and Wirth, D.F. (1983). Tandem arrangement of
tubulin genes in the protozoan parasite Leishmania enriettii. Molecular and
Cellular Biology 3, 1070-1076.
LeBowitz, J.H., Coburn, C.M., McMahan-Pratt, D. and Beverley, S.M. (1990).
Development of a stable Leishmania expression vector and application to the
study of parasite surface antigen genes. Proceedings of the National Academy of
Science of the USA 87, 9736-9740.
LeBowitz, J.H., Cruz, A. and Beverley, S.M. (1992). Thymidine kinase as a
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 265
Ramamoorthy, R., Swihart, K.G., McCoy, J.J., Wilson, M.E. and Donelson, J.E.
(1995). Intergenic regions between tandem gp63 genes influence the differential
expression of gp63 RNAs in Leishmania chagasi. Journal of Biological
Chemistry 270, 12133-12139.
Ribeiro de Jesus, A., Cooper, R., Espinosa, M., Gomes, J.E.P.L., Garcia, E.S., Paul,
S. and Cross, G.A.M. (1993). Gene deletion suggests a role for Trypanosoma
cruzi surface glycoprotein GW2 in the insect and mammalian stages of the life
cycle. Journal of Cell Science 106, 1023-1033.
Roos, D.S., Donald, R.G.K., Morrissette, N.S. and Moulton, A.L.C. (1995). Mole-
cular tools for genetic dissection of the protozoan parasite Toxoplasma gondii.
In: Methods in Cell Biology, Vol. 45 (D. Russell, ed.), pp. 28-63. San Diego:
Academic Press.
Rudenko, G., Le Blancq, S., Smith, J., Lee, M.G.-S., Rattray, A. and Van der
Ploeg, L.H.T.(1990). Procyclic acidic repetitive protein (PARP) genes located in
an unusually small a-amanitin-resistant transcription unit: PARP promoter activ-
ity assayed by transient DNA transfection of Trypanosoma brucei. Molecular
and Cellular Biology 10, 3492-3504.
Rudenko, G., Blundell, P.A., Taylor, M.C., Kieft, R. and Borst, P. (1994). VSG
gene expression site control in insect form Trypanosoma brucei. European
Molecular Biology Organisation Journal 13, 5470-5482.
Rudenko, G., Blundell, P.A., Dirks-Mulder, A., Kieft, R. and Borst, P. (1995). A
ribosomal DNA promoter replacing the promoter of a telomeric VSG gene
expression site can be efficiently switched on and off in T. brucei. Cell 83,
547-553.
Ryan, K.A., Dasgupta, S. and Beverley, S.M. (1993a). Shuttle cosmid vectors for
the trypanosomatid parasite Leishmania. Gene 131, 145-150.
Ryan, K.A., Garraway, L.A., Descoteaux, A., Turco, S.J. and Beverley, S.M.
(1993b). Isolation of virulence genes directing surface glycosylphosphatidylino-
sitol synthesis by functional complementation. Proceedings of the National
Academy of Science of the USA 90, 8609-8613.
Sadick, M.D., Heinzel, F.P., Holaday, B.J., Pu, R.T., Dawkins, R.S. and Locksley,
R.M. (1990). Cure of murine leishmaniasis with anti-interleukin-4 monoclonal
antibody. Journal of Experimental Medicine 171, 115-127.
Saito, R.M., Elgort, M.G. and Campbell, D.A. (1994). A conserved upstream
element is essential for transcription of the Leishmania tarentolae mini-exon
gene. European Molecular Biology Organisation Journal 13, 5460-5469.
Schell, D., Evers, R., Preis, D., Ziegelbauer, K., Lottspeich, F., Cornelisson,A.W.C.A.
and Overath, P. (1991). A transferrin-binding protein of Trypanosoma b m e i is
encoded by one of the genes in the variant surface glycoprotein gene expression site.
European Molecular Biology Organisation Journal 10, 1061-1066.
Schurch, N., Hehl, A., Vassella, E., Braun, R. and Roditi, I. (1994). Accurate
polyadenylation of procyclin W A S in Trypanosoma brucei is determined by
pyrimidine-rich elements in the intergenic regions. Molecular and Cellular
Biology 14, 3668-3675.
Sherman, D.R., Janz, L., Hug, M. and Clayton, C. (1991). Anatomy of the parp
gene promoter of Trypanosoma brucei. European Molecular Biology Organisa-
tion Journal 10, 3370-3386.
Sibley, L.D., Messina, M. and Niesman, I.G. (1994). Stable DNA transformation of
the obligate intracellular parasite Toxoplasma gondii by complementation of
tryptophan auxotrophy. Proceedings of the National Academy of Science of
the USA 91,5508-5512.
268 J.M. KELLY
Soldati, D. and Boothroyd, J.C. (1993). Transient transfection and expression in the
obligate intracellular parasite Toxoplasma gondii. Science 260, 349-352.
Soldati, D. and Boothroyd, J.C. (1995). A selector of transcription initiation in
the protozoan parasite Toxoplasma gondii. Molecular and Cellular Biology 15,
87-93.
Soldati, D., Kim, K., Kampmeier, J., Dubremetz, J.-F. and Boothroyd, J.C. (1995).
Complementation of a Toxoplasma gondii ROPl knock-out mutant using phleo-
mycin selection. Molecular and Biochemical Parasitology 74, 87-97.
Sommer, J.M. and Wang, C.C. (1994). Targeting of proteins to the glycosomes of
African trypanosomes. Annual Review of Microbiology 48, 105-138.
Sommer, J.M., Cheng, Q.L., Keller, G.A. and Wang, C.C. (1992). In vivo import of
firefly luciferase into the glycosomes of Trypanosoma brucei and mutational
analysis of the C-terminal targeting signal. Molecular and Cellular Biology 3,
749-759.
Sommer, J.M., Peterson, G., Keller, G.A., Parsons, M., and Wang, C.C. (1993).
The C-terminal tripeptide of glycosomal phosphoglycerate kinase is both neces-
sary and sufficient for import into the glycosomes of Trypanosoma brucei.
Federation of European Biochemical Societies Letters 316, 53-58.
Southern, P.J. and Berg, P. (1982). Transformation of mammalian cells to anti-
biotic resistance with a bacterial gene under the control of the SV40 early region
promoter. Journal of Molecular and Applied Genetics 1, 327-341.
Souza, A.E., Bates, P.A., Coombs, G.H. and Mottram, J.C. (1994). Null mutants for
the lmcpa cysteine proteinase gene in Leishmania mexicana. Molecular and
Biochemical Parasitology 63, 2 13-220.
Taylor, M.C., Kelly, J.M., Chapman, C.J., Fairlamb, A.H. and Miles, M.A. (1994).
The structure, organisation, and expression of the Leishmania donovani gene
encoding trypanoihione reductase. Molecular and Biochemical Parasitology 64,
293-301.
Teixeira, S.M.R., Russell, D.G., Kirchhoff, L.V. and Donelson, J.E. (1994). A
differentially expressed gene family encoding ‘amastin’, a surface protein of
Trypanosoma cruzi amastigotes. Journal of Biochemical Chemistry 269,
20509-205 16.
ten Asbroek, A.L.M.A., Ouellette, M. and Borst, P. (1990). Targeted insertion of
the neomycin phosphotransferase gene into the tubulin gene cluster of Trypano-
soma brucei. Nature 348, 174-175.
ten Asbroek, A.L.M.A., Mol, C.A.A.M., Kief, R. and Borst, P. (1993). Stable
transformation of Trypanosoma brucei. Molecular and Biochemical Parasit-
ology 59, 133-142.
Tibbetts, R.S., Klein, K.G. and Engman, D.M. (1995). A rapid method for protein
localisation in trypanosomes ( 1995). Experimental Parasitology 80, 572-574.
Titus, R.G., Gueiros-Filho, F.J., De Freitas, L.A.R. and Beverley, S.M. (1995).
Development of a safe live Leishmania vaccine line by gene replacement.
Proceedings of the National Academy of Science of the USA 92, 10267-10271.
Tobin, J.F., Reiner, S.L., Hatam, F., Zheng, S., Leptak, C.L., Wirth, D.F. and
Locksley, R.M. (1993). Transfected Leishmania expressing biologically active
IFN-y. Journal of Immunology 150, 5059-5069.
Tomas, A.M. and Kelly, J.M. (1996). Stage regulated expression of cruzipain, the
major cysteine protease of Trypanosoma cruzi is independent of the level of
RNA. Molecular and Biochemical Parasitology 76, 9 1-103.
Tyler-Cross, R.E., Short, L.S., Floeter-Winter, L.M. and Buck, G.A. (1995). Tran-
GENETIC TRANSFORMATION OF PARASITIC PROTOZOA 269
Patricia S . Coulson
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
1.1. Thedisease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
1.2. The need for a vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
1.3. Laboratory animals as hosts for schistosomes ..................... 274
2. Parasite Migration and Development in the Naive Animal . . . . . . . . . . . . . . . . 275
2.1. Migration in mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
2.2. Elimination of parasites during a primary infection . . . . . . . . . . . . . . . . . 278
2.3. Migration and development in other laboratory animals . . . . . . . . . . . . .280
3. The Radiation-attenuated Vaccine in Mice ............................ 282
3.1. Protection involves a specific immune response . . . . . . . . . . . . . . . . . . .282
3.2. Parameters of parasite attenuation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
4. lnductionoflmmunio/ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
4.1. The effect of radiation on the parasite ........................... 284
4.2. Persistence of attenuated schistosomula ......................... 286
4.3. Immunogenic potential of attenuated schistosomula . . . . . . . . . . . . . . . .289
5. Immune Responses During Priming ................................ 290
5.1. Lymph nodes draining sites on the migration route . . . . . . . . . . . . . . . . . 290
5.2. The lungs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
.
6 The Immune Effector Mechanism .............. .................... 295
6.1. The site of challenge parasite elimination ......................... 295
6.2. The nature of the immune response ............................. 296
6.3. Events in the immune lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
6.4. Potential mechanisms of parasite elimination . . . . . . . . . . . . . . . . . . . . . . 303
6.5. The central role of IFNy in the effector mechanism . . . . . . . . . . . . . . . . . .305
7 . The Irradiated Vaccine in Other Laboratory Hosts ...................... 307
7.1. The rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
1. INTRODUCTION
Infection
++I
rLungs
the liver and migrated, via the hepatic portal vessels, to the mesenteric
veins where egg laying commences.
The fate of the 50-70% of penetrants that fail to mature in the naive mouse
has received considerable attention. It was originally thought that most
schistosomula died in the skin shortly after penetration, as only a small
number could be recovered when this tissue was minced and subsequently
incubated in culture medium for several hours (Clegg and Smithers, 1968).
However, fundamental to interpretation of these data is the assumption,
now shown to be erroneous, that all viable parasites are recovered by the
assay (Miller and Wilson, 1980; Coulson and Wilson, 1988). Whilst noting
an inflammatory response in the skin, numerous investigators carrying out
histological studies failed to find dead or dying parasites in this tissue (e.g.
Stirewalt, 1959; von Lichtenberg et al., 1976; Mastin et al., 1983).
Autoradiographic tracking showed that the vast majority of labelled para-
sites penetrating the skin of naive mice eventually left this tissue and
arrived in the lungs (Mangold and Dean, 1983; Dean et al., 1984; Wilson
et al., 1986). In addition, if the skin was bypassed by intravenous injection
of lung schistosomula directly to the lungs, the level of maturation did not
differ significantly from that which followed percutaneous infection
(McLaren et al., 1985; Mangold et al., 1986). There would thus appear
to be no major elimination of schistosomula in the skin of naive mice.
Since the eventual number of parasites maturing in the hepatic portal
system was much lower than the peak number detected in the lungs by
autoradiography, Georgi et al. (1983) concluded that elimination occurred
either in the latter organ, en route to the liver, or in the liver itself. The last
possibility was shown to be unlikely by the injection of lung schistosomula
directly to the hepatic portal system (Mangold et al., 1986; Coulson and
Wilson, 1988). The outcome was a high level of maturation (70-85%)
which, given the efficiency of trapping in the liver, would suggest that this
organ provides a safe haven for migrating parasites.
The exact site and timing of elimination can only be defined by establish-
ing whether a decline in parasite numbers in a tissue represents death and
clearance or exit to another organ on the migration route. In order to
achieve this all mouse tissues must be sampled to permit the construction
of an accurate balance sheet of parasite numbers. In the study where this
was carried out (Wilson et al., 1986), it transpired that no decline in the
total population occurred until later than 14 days after infection. Recruit-
ment of parasites to the hepatic portal system was complete by day 21, at
which time schistosomula in other locations, and hence destined to die,
RADIATION-AlTENUATED VACCINE AGAINST SCHISTOSOMES 279
were distributed in the ratio of 3.9: 1.9: 1 in the lungs, systemic organs and
skin infection area. The lungs were therefore considered to be the prevalent
site of parasite elimination in the naive mouse.
Autoradiographic tracking of isotopically labelled schistosomula,
extracted from the lungs of donor animals and injected as a pulse at various
points on the migration route, has provided estimates of organ transit times
(Wilson and Coulson, 1986). A mean transit time of 30-35 h was required
for a schistosomulum to traverse the pulmonary capillary bed, whilst
passage through non-splanchnic systemic organs was more rapid taking
only 16 h. Therefore, despite the morphological changes which are thought
to facilitate their migration through capillaries, schistosome larvae appear
to find the lungs a difficult organ to traverse.
Histological and ultrastructural examination of schistosomula in the
lungs of naive mice revealed that newly arrived parasites were intravas-
cular and did not attract an inflammatory response (Mastin et al., 1983; von
Lichtenberg et al., 1985; Crabtree and Wilson, 1986). However, inflam-
matory foci developed in the vicinity of schistosomula with time postinfec-
tion (von Lichtenberg et al., 1985), particularly at later sampling points
when a substantial proportion of the parasites was located partly or com-
pletely in alveoli (Crabtree and Wilson, 1986). Passage through the lungs is
thought to be an active process involving muscular exertion by the schis-
tosomulum; this could lead to rupture of the thin blood-air barrier causing
the larva to enter an alveolus. It was concluded that the intra-alveolar
parasites observed in the above study accounted for a large proportion of
the non-maturing population of a primary infection. Corroborating evi-
dence for this hypothesis was provided by transfer experiments in which
schistosomula were extracted from the lungs of donor mice and delivered,
via the trachea, to the alveoli of naive recipients (Coulson and Wilson,
1988). Such parasites had a limited capacity to recover from alveolar sites
and complete their migration, unlike their cohorts introduced directly into
the vasculature. The alveolus would thus appear to represent a cul-de-sac
on the migration route; the fate of a parasite in this inhospitable location is
most likely death due to starvation. It has, however, been suggested that
schistosomula in alveoli could be expelled after passing via the trachea into
the gastrointestinal tract (Crabtree and Wilson, 1986; Dean and Mangold,
1992). In this context, radiolabelled larvae have been detected by auto-
radiography in the trachea, and lumina of the eosophagus, stomach and
intestines (Georgi et al., 1986).
Less systematic application has been made of parasite recovery and
autoradiographic tracking techniques to investigate the migration and site
of elimination of S. haematobium and S. juponicum. Whereas the limited
data point to similarities in the pattern of migration for the three schisto-
some species, differences are highlighted in the kinetics of the process.
280 P.S. COULSON
Thus, S. haematobium larvae are slower than S. mansoni in their exit from
the skin, as judged by autoradiography (Georgi et al., 1986); their peak
accumulation in the lungs is also later, with an apparent retention of
parasites in this organ. Conversely, S. japonicum undergo a rapid migra-
tion, with maximal yields of parasites being obtained from the lungs on
days 3 4 , in comparison to day 6 for S. mansoni larvae (Gui et al., 1995).
Petechial haemorrhages, visible to the naked eye, also appear on the lungs
during the first week of infection (Mitchell et al., 1991; Gui et al., 1995),
but are seldom seen in S. mansoni-infected mice, suggesting a more
aggressive migration on the part of the former parasite. The rapidity of
S. japonicum migration is also borne out by the recovery of a small number
of worms from the liver as early as day 3, with the peak occurring around
days 7-10, approximately 2 weeks sooner than that for S. mansoni (Gui et
al., 1995). Autoradiographic tracking has confirmed that virtually all S.
japonicum larvae leave the skin infection site by day 4, the time when peak
numbers are detected in the lungs (Laxer and Tuazon, 1992; P.S. Coulson,
N.A. Moloney and R.A. Wilson, unpublished data). Both studies confirmed
that parasite numbers reach a maximum in the liver between days 6 and 10,
but Laxer and Tuazon (1992) noted a reduction thereafter, leading them to
suggest that this organ represents a major site of attrition. However, no
evidence for this was found by Coulson et al. (unpublished data) nor by
Mitchell et al. (1991) who recovered parasites from the hepatic portal
system at frequent intervals after infection.
Although the final level of maturation in the mouse depends to some
extent on the geographical strain of S. japonicum, it is usually in the region
of 45-75% (Mitchell et al., 1991), and hence considerably higher than for
S. mansoni. On the other hand, maturation of S. haematobium ranges from
only 7% (Dean et al., 1996) to 15% (Agnew et al., 1989). There would thus
appear to be a relationship between the number of worms establishing in
the mouse and the speed of migration through the lungs. Whether this is
due to differences in the migratory capacity of the schistosome species or
to the host’s response to their presence in the lungs remains to be dis-
covered.
from the lungs and portal tract of rats at various times after a primary
exposure (Perez et al., 1974; Miller and Wilson, 1978, 1980; Knopf et al.,
1983; Ford et al., 1984a). These studies established that schistosomula
spent 2-3 days in the skin before migrating to the lungs. Parasites were
detected in the hepatic portal system from day 9, with numbers rising to a
maximum between 3 and 4 weeks before declining as they were eliminated
from the liver.
Autoradiographic tracking (Knopf et al., 1986; Ward and McLaren,
1989; P.S. Coulson and R.A. Wilson, unpublished data) revealed that there
was a rapid and highly efficient migration out of the skin, so that less than
10% of penetrants remained in this location by day 6 whilst > 85% were
detected in the lungs. In the study where all tissues were sampled (P.S.
Coulson and R.A. Wilson, unpublished data), significant numbers of schis-
tosomula were located in systemic organs on days 9 and 12 postinfection.
All three studies confirmed that the first parasites reached the liver between
days 6 and 9, with numbers remaining virtually steady from days 10/12 to
perfusion at day 21. These data indicate that migration of S. mansoni in the
rat proceeds at a faster rate than in the mouse, with arrival in the lungs and
liver occurring approximately 2 days earlier. One other notable discrepancy
was the lack of a substantial number of larvae in the lymph nodes draining
the skin exposure site (P.S. Coulson and R.A. Wilson, unpublished data). A
maximum of only 2.5% of penetrants was detected on day 6, whereas a
comparable study in mice revealed a peak of 16% on day 5 (Mountford et
al., 1988).
Despite the greater speed of migration in the rat compared to the mouse,
maturation is much lower, reaching at best 25-30%. The situation is
complicated in the rat because it is difficult to ascertain whether the plateau
in parasite numbers in the liver is the result of a cessation in migration to
this organ. Although this is the most likely explanation, a turnover in the
worm population, in which gain is matched by loss as the parasites suc-
cumb to a destructive mechanism in the liver, cannot be ruled out. How-
ever, there is reason to believe that, as in the mouse, the lungs present a
significant obstacle to the migrating larvae. A histological investigation
revealed that the rat develops significant cellular reactions around larvae in
the lungs 5-7 days after exposure to a primary infection (von Lichtenberg
and Byram, 1980). Furthermore, when a balance sheet of parasite numbers
in all rat tissues was constructed (Coulson and Wilson, unpublished data),
it was found that four times as many larvae were detected in the lungs
compared to other systemic organs at a time when migration to the liver
appeared to be complete. It would therefore be reasonable to suggest that
the majority of non-maturing parasites are eliminated in the naive rat lung.
282 P.S. COULSON
2.3.2. Primates
Compared to the wealth of information on schistosome migration in the
mouse, little is known about the subject in man or in related primates. The
limited data available were obtained from an autoradiographic tracking
study in the olive baboon Papio anubis (Wilson et al., 1990). Exit of
schistosomula from the skin exposure site was rapid, with 72% of applied
S. mansoni parasites being detected in the lungs on day 5 . Schistosomula
made an equally rapid transit to the liver, since 60% of the adult worm
burden had arrived by day 9. No parasites could be detected in the brain or
kidneys at any sampling time, making it impossible to comment on the
systemic phase of migration; nor were they detected in lymph nodes
draining the exposure site. The final adult population, measured by portal
perfusion, indicated a maturation of 78%, a level similar to that recorded
by Sturrock et al. (1984a). The data suggest that, in comparison to rodents,
schistosome migration in baboons is faster and more successful, possibly
because the lungs do not represent a significant obstacle to migrating
larvae. One might postulate that a similar situation pertains in humans
but it remains an open question that is impossible to resolve.
4. INDUCTION OF IMMUNITY
be taken to imply that antigens are shared between immunizing and normal
larvae. This is hardly surprising when one considers that the host’s immune
system primed by antigens from irradiated larvae has to be capable of
responding to those from normal parasites, in order to initiate the effector
mechanism.
Once it had been established that optimally irradiated parasites did not
reach maturity (e.g. Minard et al., 1978a), their survival pattern was an
obvious target for investigation. In early studies, only a small proportion
(5%) of optimally irradiated larvae could be recovered from the lungs of
mice, yet some appeared to persist in this organ from 6 to 21 days after
infection (Minard et al., 1978b; Bickle et al., 1979a). It was therefore
concluded that irradiated schistosomula died at the site of administration,
en route to, or in the lungs. However, both the above studies used recovery
of parasites from minced tissues as their assay. Apart from known ineffi-
ciency of this technique to retrieve all schistosomula present, it might be
anticipated that radiation-damaged parasites would be even less readily
extractable than normal ones.
Indeed, the extent of migration proved greater than these original studies
indicated. Using a quantitative histological technique (Mastin et al. (1983)
demonstrated that a large proportion of attenuated schistosomula were
located in the lungs (57% on day 7) and persisted there until at least 21
days after infection, although by this time most were dead. There was a
slight retardation of migration out of the skin but death at this site appeared
negligible. Attenuated schistosomula, and the residual inflammatory foci
resulting from their destruction, were also detected in the lungs of vacci-
nated mice in a study by von Lichtenberg et al. (1985); whilst most
parasites had died prior to day 14, resolution of residual foci was pro-
tracted. Using autoradiography tracking, Mangold and Dean (1984) con-
firmed that migration of attenuated larvae between skin and lungs
paralleled that of normal schistosomula except for the slight delay men-
tioned above. These authors also concluded that the majority of irradiated
larvae died in the lungs during the first 3 weeks of infection. An assessment
of the ultrastructure of attenuated schistosomula fixed in situ in the lungs
has revealed that, as far as could be seen, they had undergone the normal
developmental changes associated with this stage of migration (Mastin et
al., 1985). The only apparent difference between normal and irradiated
schistosomula seemed to be one of location since many of the latter were
detected in alveoli, rather than blood vessels, by day 13 postexposure.
However, it was subsequently demonstrated that a large number of normal
parasites also enter alveoli at later times during infection (Crabtree and
Wilson, 1986). It is difficult to quantify the numbers of parasites in alveolar
locations, but one might postulate that a larger proportion of irradiated than
normal schistosomula suffer this fate.
286 P.S. COULSON'
Figure 2 Differences between normal and irradiated larvae at the lung stage of
development. Scanning electron micrographs of schistosomula cultured for 8 days
in vitro. Scale bar = 10 pn. A. Normal schistosomulum showing the smooth
outline of the body and characteristic loss of midbody spines but presence of
anterior (a) and posterior (p) spines. B. Schistosomulum derived from an irradiated
cercaria showing constrictions of the body at random points along its length
(arrows), and presence of anterior (a) and posterior (p) spines (reproduced from
Harrop and Wilson, 1993a, with permission).
288 P.S. COULSON
There has been a great deal of controversy regarding the major site of
challenge parasite elimination, the main issues of which were addressed in
a pair of debate articles (Wilson and Coulson, 1989; McLaren, 1989).
Studies to date have provided conflicting results, with some implicating
the skin and others the lungs. Evidence in support of elimination in the skin
comes from several lines of investigation. Mincing and incubation of skin
and lungs of vaccinated mice yielded lower numbers of challenge schisto-
somula compared to normal animals (Miller and Smithers, 1980). Histo-
pathological studies of the skin of vaccinated mice showed damaged and
dead challenge parasites within subcutaneous inflammatory foci (Hsu et al.,
1983; Ward and McLaren, 1988). The level of resistance was reduced if the
skin was bypassed by direct injection of challenge larvae intravenously to
the lungs (McLaren et al., 1985). MAbs, which selectively deplete neu-
trophils (McLaren et al., 1987) or CD4+ T cells (Piper and McLaren, 1993),
diminished resistance most effectively if they were administered at the
time of challenge. Likewise, serum from multiply vaccinated mice con-
ferred partial protection only if transferred at early times after infection,
before schistosomula reached the lungs (McLaren and Smithers, 1988).
Finally, results from autoradiographic tracking have been interpreted to
mean that challenge parasites do not reach the lungs in similar numbers to
those in normal mice (Kamiya et al., 1987).
Running counter to this is the wealth of evidence in favour of elimina-
tion of parasites at a later stage in their migration. For example, similar
296 P.S. COULSON
numbers of challenge larvae were recovered from the lungs of normal and
vaccinated mice, by mincing and incubation (Minard et al., 1978b; Stek et
al., 198la). Histopathological studies revealed that, despite intense inflam-
matory responses in the skin, there was no evidence of challenge parasite
damage or elimination in this site (Mastin et al., 1983; von Lichtenberg et
al., 1985). Little or no reduction in the level of resistance was observed
when the skin was bypassed (Dean et al., 1981b; Mangold et al., 1986).
The most consistent level of protection was obtained when immune serum
was administered several days postinfection, coincident with parasite
migration through the lungs (Mangold and Dean, 1986). Furthermore,
autoradiographic tracking demonstrated that challenge parasites eventually
reached the lungs of vaccinated mice in similar numbers to those in normal
mice (Dean et al., 1984). However, these data do not establish the precise
site of challenge elimination, only that it occurs at some point after arrival
of schistosomula in the lungs.
In order to determine the site more accurately, an autoradiographic
tracking study was performed, in which all organs were sampled to provide
an estimate of the total number of challenge parasites in the mouse as well
as their location (Wilson et al., 1986). It was discovered that identical
proportions reached the lungs of normal and vaccinated mice but in the
latter there was a greatly reduced systemic phase of migration and hepatic
portal accumulation, indicating that fewer larvae travelled beyond this site.
When migration was complete in vaccinated mice a large fraction of
schistosomula was still detectable in the lungs compared to other tissues.
It was therefore concluded that the majority of parasite elimination took
place in this organ, although the process was extended, starting around 7-
12 days after challenge and continuing at least to day 35.
The disparity between observations of skin and lung elimination may
reside, for instance, in the different mouse strains and/or parasite isolate
used in the various laboratories, although there is little evidence to support
this notion (Dean et al., 1995). However, whilst it is difficult to reconcile
the data on the site of elimination, all workers agree that the target of the
immune response is the lung schistosomulum. Even when elimination was
considered to occur in the skin, the larvae trapped in inflammatory reac-
tions in the subcutaneous tissues have succeeded in developing from skin-
stage to lung-stage worms (Ward and McLaren, 1988).
Taken together, the data suggest that whilst Thl -dependent immune
mechanisms predominate in mice vaccinated once with irradiated cercar-
iae, protection after multiple exposures results from a mixture of Thl and
Th2 responses and involves antibodies. The reason for the switch towards
Th2 reactivity is presently unknown but could be influenced by, for exam-
ple, differences in antigen concentration, the antigen-presenting cell (APC)
types, or T-cell interactions. In this context, Pemberton and Wilson (1995)
recently observed that challenge of once-vaccinated mice enhanced pro-
duction of Th2 cytokines in antigen-stimulated cultures of lymphocytes
from the skin-draining lymph nodes, whilst depressing production of the
Thl cytokine IFNy. Induction of the counter-regulatory cytokine IL-10
appeared to be the key factor, possibly acting on APCs to modify their
immunostimulatory potential. This might explain why there is always a
ceiling on the level of protection that cannot be raised to 100% by further
vaccinations.
(In marked contrast, parasites in the lungs of normal mice did not attract a
focus unless they entered alveoli.) The inflammatory infiltrates in the lungs
of vaccinated mice were composed of greater than 85% mononuclear cells
(Crabtree and Wilson, 1986), and immunocytochemical analysis revealed
an abundance of macrophages and CD4+ T lymphocytes in both focal
aggregates and perivascular tissues (Kambara and Wilson, 1990). These
cellular reactions around challenge larvae display many of the character-
istics of DTH granulomas similar to those observed in Mycobacteriurn
tuberculosis infections (Crabtree and Wilson, 1986). An analogous
response has been described in the lungs of mice receiving three vaccina-
tions with irradiated cercariae prior to challenge (Kassim et al., 1992).
More recently, the sequence of events during focus formation around
challenge schistosomula in the lungs of once-vaccinated mice has been
characterized (Smythies et al., 1996). As migrating larvae arrive in the
lungs over an extended period after percutaneous challenge, lung-stage
schistosomula were administered directly to this organ via the intravenous
route to synchronize the process. One of the most striking findings of this
study was the time required for an effector focus to develop. Histopathol-
ogy established that a small number of mononuclear cells had accumulated
around each larva in the lungs of vaccinated mice within 24 h (Figure 3).
However, the main increment in leucocyte recruitment took place between
2 and 4 days, with foci reaching a maximum diameter on day 8. IFNy was
the major cytokine detected in the lungs, with the greatest increment in
production coinciding with peak cell infiltration and aggregation. In con-
trast to these findings, responses in challenge control animals were negli-
gible prior to day 12.
The time taken for an effector focus to develop may be crucial to the
elimination of a challenge parasite. Thus, the presence of schistosome-
reactive T cells in the lungs prior to the arrival of the first challenge larvae
is likely to be a major factor in the speed of the secondary response. It has
been suggested that the recruitment of inflammatory cells to the lungs
during priming serves to ‘pre-arm’ it against the arrival of challenge
parasites (Wilson and Coulson, 1989), and experiments with parabiotic
mice suggest that this is indeed the case (Coulson and Wilson, 1997).
When vaccinated mice were surgically joined to naive partners for a 28-
day period coincident with priming of the immune system, sensitized T
cells were detected in the spleens of both partners. However, the naive
parabiont, unlike its vaccinated partner, did not recruit lymphocytes to its
lungs during the priming period (most probably due to the absence of
irradiated larvae in this organ). After percutaneous challenge, schisto-
some-specific lymphocytes infiltrated the lungs of both separated para-
bionts, and the level of protective immunity transferred to naive partners
was approximately two-thirds that of their vaccinated counterparts. The
Figure 3 Photomicrographs of mouse lung tissues showing pulmonary foci around migrating S.mansoni larvae. Panel (a) shows a
challenge larvae in the naive mouse at day 1; panels (b-f) show the sequential development of foci in vaccinated mice on days 1, 2,
4, 8 and 12 postchallenge, respectively. ( X 200) Schistosomulum (S); infiltrating leucocytes (small arrows). CC, naive mouse, VC,
vaccinated mouse (reproduced from Smythies et al., 1996, with permission from Academic Press Ltd).
RADIATION-ATTENUATED VACCINE AGAINST SCHISTOSOMES 303
explanation is that the inflammation does not bring about parasite death
directly, but effectively prevents onward progress thereby causing the larva
to be retained in the lungs even though it still has the potential to undertake
intravascular migration. In some instances, pulmonary inflammation
destroys the blood vessel along which a parasite is migrating such that it
is deflected into an alveolus (Crabtree and Wilson, 1986). This appears to
be a terminal event as parasites have a limited capacity to re-enter tissues,
which diminishes still further with age (Coulson and Wilson, 1988). The
lack of noticeable damage to schistosomula in the lungs of vaccinated mice
(von Lichtenberg et al., 1985; Kassim et al., 1992), even as late as 31 days
postchallenge (Crabtree and Wilson, 1986), coupled with the observations
that they disappear relatively slowly from this location (von Lichtenberg et
al., 1985; Dean and Mangold, 1992), suggests that parasite death and
disintegration is a far from rapid process. Understandably, this hypothesis
is not supported by advocates of cytotoxic killing as a mechanism for
elimination of challenge larvae.
The results point to a crucial role for IFNy in the DTH effector mechanism
operating in the irradiated vaccine model, and further proof has been
obtained by neutralization of the cytokine during the period when chal-
lenge elimination is taking place. Sher et al. (1990) administered anti-IFNy
mAb to vaccinated mice around the time of challenge and found it reduced
the level of immunity, but only by 28-34%. However, administration of
anti-IFNy mAb to vaccinated mice between days 4 and 16 postchallenge
resulted in an average of 90% abrogation of protective immunity from four
separate experiments (Smythies et al., 1992a). Variations in antibody
purification methods and administration schedules may account for the
disparity in results. A 50% increase in worm burdens, compared to
untreated controls, has recently been recorded for mice receiving neutraliz-
ing mAb over the whole vaccination and challenge period (Wynn et al.,
1994).
Paradoxically, there was a significant increase in the number of cells
recovered from the lungs of mAb-treated animals at day 14 postchallenge,
relative to intact vaccinated and challenged controls (Smythies et al.,
1992a). The increment was due mainly to a sharp rise in the number of
eosinophils, low levels of which would normally be expected in the lungs
of vaccinated mice after challenge (Smythies et al., 1992a; R.A. Wilson
and L.E. Smythies, unpublished data). The inverse relationship between
eosinophil numbers and protection suggests that these cells do not play a
vital role in the immune effector mechanism. This is also the opinion of
306 P.S. COULSON
Sher et al. (1990) who reported that in vivo depletion of IL-5 in vaccinated
and challenged mice decreased circulating and tissue eosinophils but had
no impact on immunity. (On this point, IL-5 transgenic mice, which
possess very high levels of peripheral blood eosinophils, do not manifest
augmented protection to the irradiated vaccine compared to wild-type
controls; Freeman et al., 1995.)
Histological examination of the lungs of IFNy-depleted animals at day
14 postchallenge established that the pulmonary foci were larger and more
numerous than those seen in control groups (Smythies et al., 1992a); these
features were also evident in lung tissue analysed at 9 days (Sher et al.,
1990). A significantly higher proportion of eosinophils was detected in the
pulmonary foci at 14 days (Smythies et al., 1992a) and was also noted by
Wynn et al. (1994). At this time, many multi-nucleated giant cells were
present in each focus; frequently they were very large and appeared to
disrupt the integrity of the focal structure, thereby creating a much looser
aggregate of cells than observed in untreated vaccinated mice (Smythies et
al., 1992a). Image analysis demonstrated a substantial reduction in cell
numbers in the pulmonary foci of treated compared to control mice. It was
suggested that the looser foci, containing fewer cells, may not be able to
prevent parasites from migrating through the pulmonary vasculature, thus
accounting for the abrogation of immunity (Smythies et al., 1992a).
The availability of mice with a targeted disruption of the IFNy receptor
gene (IFNyR-’- mice) has also enabled investigation of the role of IFNy in
the pulmonary immune response (Wilson et al., 1996). These mice can
synthesize the cytokine but lack the ability to transduce signals at the target
cell surface and are thus unresponsive to its actions (Huang et al., 1993).
The irradiated vaccine induced only a low level of protection in IFNyR-’-
mice, one half or less that of their wild-type counterparts, or C57BL/6 mice
(Wilson et al., 1996). As noted after neutralization of IFNy, the diminished
level of protective immunity was associated with a greater degree of cell
infiltration into the pulmonary parenchyma and airways in response to
challenge schistosomula, and a strong Th2 bias in the pattern of cytokine
mFWA and protein production in the lungs. Furthermore, the infiltrating
leucocytes in the gene-disrupted animals were only loosely aggregated, and
comprised many eosinophils and multi-nucleated giant cells. On the basis
of these findings, it was suggested that a major role for IFNy in the
pulmonary response is to promote intercellular adhesion between the leu-
cocytes which constitute an effector focus (Smythies et al., 1993).
Cell-cell adhesion is considered to result from interactions between
ligandheceptor pairs, such as leucocyte function associated (LFA)- Uinter-
cellular adhesion molecule- 1 (ICAM-1) and CD2/LFA-3 on the leucocyte
surface (Springer, 1990). In many cases, the genes determining the level of
expression of adhesion molecules are regulated by the actions of inflam-
RADIATION-ATTENUATEDVACCINE AGAINST SCHISTOSOMES 307
matory cytokines, including IFNy. The 1FNyR-I- mice have provided the
means to investigate the influence of this cytokine on adhesion molecule
expression on leucocytes recovered from the pulmonary interstitium at the
time of peak responses to challenge larvae (P.S. Coulson and R.A. Wilson,
unpublished data). The results so far suggest that IFNy regulates LFA-I/
ICAM-1 interactions since the majority of CD4+ T cells from WT and
C57BL/6 mice expressed ICAM-1 but only a small proportion from
IFNyR-” mice was positive for this inducible molecule. Moreover, whilst
virtually all CD4+ T cells from the three groups of mice were positive for
LFA-1, the intensity of expression was substantially lower in the gene-
disrupted animals. In contrast, the lack of any differences in the level of
expression of CD2 and CD48 (the murine homologue of LFA-3) on pul-
monary T-helper cells from IFNyR-” and WT mice implies that this ligand/
receptor pair is not influenced by IFNy.
worm challenge introduced directly to the lungs as they were when the skin
was bypassed by percutaneous administration of cercariae.
Further confirmation comes from studies on the timing of passive trans-
fer of serum (Ford et al., 1984b; McLaren and Smithers, 1985). In both,
serum harvested from rats vaccinated with 20 h a d attenuated parasites
conferred a high level of protection when given 5 days after exposure of
the recipients to normal cercariae. In one study (Ford et al., 1984b), the
serum was also protective if given on the day of infection whereas in the
other (McLaren and Smithers, 1985) it was not. Nevertheless, these data,
together with the finding that vaccine serum was not effective if its admin-
istration was delayed until 8 or 10 days postinfection (Ford et al., 1984b),
provide sound evidence for the elimination of larvae during the lung phase
of migration. Parenthetically, the same is true for rats receiving serum from
donors infected with unirradiated cecariae (Knopf et al., 1986; Oshman et
al., 1986).
7.2. Primates
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the immune effector mechanism and also the site(s) at which it oper-
ates still need to be defined. On the basis of research in vaccinated
mice and rats, the lungs would be the favoured organ for immune-
mediated elimination of challenge larvae but, at this stage, one can
only speculate as to whether this is the case. The speed of migration
of S. rnansoni schistosomula through the baboon lung, coupled with the
high level of maturation (Wilson er al., 1990), would appear to warrant
RADIATION-ATTENUATED VACCINE AGAINST SCHISTOSOMES 315
along with unvaccinated animals (Majid et ul., 1980). The calves were
followed over 10 months of natural exposure, during which protection was
confirmed by a lower mortality rate and lower faecal egg counts in the
vaccinated group. At necropsy, the vaccinated animals displayed a 60-70%
reduction in worm burden and tissue egg counts compared to controls.
Success has also been achieved with an irradiated S. japonicum vaccine
in both cattle and pigs in China. Since S. juponicum is nowadays trans-
mitted in that country predominantly by domestic livestock, a veterinary
vaccine might contribute to the control of the disease in humans. Hsii et ul.
(1984) demonstrated that vaccination of cattle and buffaloes with gamma-
irradiated schistosomula was highly effective against both laboratory and
field challenge. More recently, UV light has been used to attenuate S.
juponicum larvae for vaccination of water buffaloes (Shi et ul., 1990)
and pigs (Shi et ul., 1993). In both instances, the animals received three
vaccinations with attenuated cercariae and developed approximately 90%
resistance to a laboratory challenge. The procedure did not appear to have
any detrimental effect on the animals’ health, and the experiments were
performed using portable and relatively cheap irradiation equipment in a
rural area of China. Thus whilst the irradiated vaccine is unsuitable for use
in the human population, it may prove effective in reducing zoonotic
transmission of S. juponicum.
8. FUTURE DIRECTIONS
antigen. The parasites in the live vaccine model are unusual in that they
deliver themselves to the requisite site of lymphocyte sensitization. This
appears to be an important feature for obtaining high levels of protection
and one that may be difficult to accomplish with a recombinant vaccine
unless some mechanism of prolonged antigen delivery into the lymph node
can be implemented. Appropriate adjuvants or live vectors may be the key
in this respect, especially if they have the potential to activate selectively
different arms of the immune system.
It has been suggested that the most important factor in T-helper subset
development is the cytokine milieu at the time of antigen stimulation
(Seder and Paul, 1994). In this respect, IL-12 appears to be a crucial
cytokine in the promotion of a Thl response (Heinzel, 1994). Secreted
by cells of the monocyte/macrophage lineage, it is a potent stimulator of
IFNy production by natural killer (NK) cells as well as T cells; it not only
stimulates the expansion of Thl cells but can suppress differentiation of the
Th2 subset. Thus, IL-12 may prove valuable as a form of immunological
adjuvant to potentiate host-protective Thl responses when delivered in
conjunction with recombinant antigens. The data on its effects so far
seem prohising. In an attempt to increase the efficacy of the irradiated
vaccine, Wynn et al. (1995) administered recombinant IL-12 to mice over
the first 2 weeks after priming with attenuated cercariae. Their efforts were
successful and in three experiments IL-12-treated, vaccinated mice dis-
played a significant decrease in worm burden when compared to those that
were only vaccinated (Figure 5). The enhanced immunity was accompanied
by an increase in Thl-associated cytokines at both the mRNA and protein
level during the priming period. In contrast, Th2 cytokine production and
related responses were markedly suppressed.
These researchers have extended their studies to include multiply vacci-
nated mice which usually display a more ThZdominant cytokine profile
and develop a protective response involving an antibody component (Wynn
et al., 1997). Somewhat surprisingly (since IL-12 is considered a potent
stimulator of cell-mediated immunity) multiply vaccinated, IL- 12-treated
mice displayed a marked increase in resistance to challenge infection, with
some animals demonstrating complete protection. The IL- 12-treated, vac-
cinated mice developed strongly polarized Thl responses but also showed
significant increases in parasite-specific IgG. Furthermore, passive transfer
experiments revealed that the sera from these animals possessed an
enhanced ability to protect naive recipients. The results from the two
studies suggest that IL-12 may be used to augment both humoral and
IFNy-dependent cell-mediated immunity when combined with vaccines.
If this were the case, then it might prove possible to elicit both types of
immune response against different parasite stages and ultimately create a
recombinant vaccine against schistosomes that is highly protective.
RADIATION-ATTENUATEDVACCINE AGAINST SCHISTOSOMES 319
ACKNOWLEDGEMENTS
REFERENCES
Agnew, A.M., Murare, H.M. and Doenhoff, M.J. (1989). Specific cross protection
between Schistosoma bovis and S. haematobium induced by highly irradiated
infections in mice. Parasite Immunology 11, 341-349.
Aitken, R., Coulson, P.S., Dixon, B. and Wilson, R.A. (1987). Radiation-resistant
acquired immunity of vaccinated mice to Schistosoma mansoni. American
Journal of Tropical Medicine and Hygiene 37, 570-577.
Aitken, R., Coulson, P.S. and Wilson, R.A. (1988). Pulmonary leucocytic
responses are linked to acquired immunity of mice vaccinated with irradiated
cercariae of Schistosoma mansoni. Journal of Immunology 140, 3573-3579.
Amiri, P., Locksley, R.M., Parslow, T.G., Sadick, M., Rector, E., Ritter, D. and
McKerrow, J.H. (1992). Tumor necrosis factor-a restores granulomas and induces
parasite egg laying in schistosome-infected SCID mice. Nature 356,604-609.
Amory-Soisson, L., Reid, G.D.F., Farah, I.O., Nyindo, M. and Strand, M. (1993).
Protective immunity in baboons vaccinated with a recombinant antigen or
radiation-attenuated cercariae of Schistosoma mansoni is antibody-dependent.
Journal of Immunology 151,4782-4789.
Bergquist, N.R. (1995). Controlling schistosomiasis by vaccination: a realistic
option? Parasitology Today 11, 191-194.
Bickle, Q.D. (1982). Studies on the relationship between the survival of Schisto-
soma mansoni larvae in mice and the degree of resistance produced. Parasitol-
ogy 84, 111-122.
Bickle, Q.D., Dobinson, J. and James, E.R. (1979a). The effects of gamma-
irradiation on the migration and survival of Schistosoma mansoni schistosomula
in mice. Parasitology 79, 223-230.
Bickle, Q.D., Taylor, M.G., Doenhoff, M.J. and Nelson, G.S. (1979b). Immuniza-
tion of mice with gamma-irradiated, intramuscularly injected schistosomula of
Schistosoma mansoni. Parasitology 79, 209-222.
Bickle, Q.D., Andrews, B.J., Doenhoff, M.J., Ford, M.J. and Taylor, M.G. (1985).
Resistance against Schistosoma mansoni induced by highly irradiated infections:
studies on species specificity of immunization and attempts to transfer
resistance. Parasitology 90, 301-3 12.
Boros, D.L. and Warren, K.S. (1970). Delayed hypersensitivity type granuloma
formation and dermal reaction induced and elicited by soluble factor isolated
from Schistosoma mansoni eggs. Journal of Experimental Medicine 132,
488-507.
Budd, R.C., Cerottini, J.-C. and MacDonald, H.R. (1987). Selectively increased
production of interferon-gamma by subsets of Lyt.2+ and L3T4+ T cells identi-
fied by expression of Pgp-1. Journal of Immunology 138, 3583-3586.
Bushara, H.O., Hussein, M.F., Saad, A.M., Taylor, M.G., Dargie, J.D., Marshall, T.F.
de C. and Nelson, G.S. (1978). Immunisation of calves against Schistosoma bovis
using irradiated cercariae or schistosomula of S. bovis. Parasitology 77,303-3 11.
Butterworth, A.E. (1992). Vaccines against schistosomiasis: where do we stand?
Transactions of the Royal Society of Tropical Medicine and Hygiene 86, 1-2.
Butterworth, A.E. (1994). Human immunity to schistosomes: some questions.
Parasitology Today 10, 378-380.
Butterworth, A.E., Taylor, D.W., Weith, M.C., Vadas, M.A., Dessein, A., Sturrock,
R.F. and Wells, E. (1982). Studies on the mechanisms of immunity in human
schistosomiasis. Immunological Reviews 61, 1-39.
324 P.S. COULSON
Butterworth, A.E., Capron, M., Cordingley, J.S., Dalton, P.R., Dunne, D.W.,
Kariuki, H.C., Kimani, G., Koetch, D., Mugamba, M., Ouma, J.H., Prentice,
M.A., Richardson, B.A., Arap-Siongok, T.K., Sturrock, R.F. and Taylor, D.W.
(1985). Immunity after treatment of human schistosomiasis mansoni: II. Identi-
fication of resistant individuals and analysis of their immune responses. Trans-
actions of the Royal Society of Tropical Medicine and Hygiene 79, 393408.
Butterworth, A.E., Fulford, A.J.C., Dunne, D.W., Ouma, J.H., and Sturrock, R.F.
(1988). Longitudinal studies on human schistosomiasis. Proceedings of the
Royal Society. London B , 321,495-51 1.
Capron, A., Dessaint, J.P., Capron, M., Joseph, M. and Pestel, J. (1980). Role of
anaphylactic antibodies in immunity to schistosomes. American Journal of
Tropical Medicine and Hygiene 29, 849-857.
Caulada-Benedetti, Z., Al-Zamel, F., Sher, A. and James, S. (1991). Comparison of
Thl-and Th2-associated immune reactivities stimulated by single versus multi-
ple vaccination of mice with irradiated Schistosoma mansoni cercariae. Journal
of Immunology 146, 1655-1660.
Cheever, A.W. (1969). Quantitative comparison of the intensity of Schistosoma
mansoni infections in man and experimental animals. Transactions of the Royal
Society of Tropical Medicine and Hygiene 63, 78 1-795.
Cheever, A.W., Hieny, S., Duvall, R.H. and Sher, A. (1983). Lack of resistance
to Schistosoma iavonicum in mice immunized with irradiated S. mansoni
cercariae. Trans&iions of the Royal Society of Tropical Medicine and Hygiene
77. 812-814.
Chen, M.G. and Mott, K.E. (1988). Progress in assessment of moribidity due to
Schistosoma mansoni infection. Tropical Diseases Bulletin 85, R1.
Clegg, J.A. and Smithers, S.R. (1968). Death of schistosome cercariae during
penetration of the skin. 11. Penetration of the mammalian skin by Schistosoma
mansoni. Parasitology 58, 111-128.
Connell, N.D., Medina-Acosta, E., McMaster, W.R., Bloom, B.R. and Russell, D.G.
(1993). Effective immunization against cutaneous leishmaniasis with recombi-
nant bacille Calmette-GuCrin expressing the Leishmania surface proteinase gp63.
Proceedings of the National Academy of Science of the USA 90, 11473-1 1477.
Constant, S.L. and Wilson, R.A. (1992). In vivo lymphocyte responses in the
draining lymph nodes of mice exposed to Schistosoma mansoni: Preferential
proliferation of T cells is central to the induction of protective immunity.
Cellular Immunology 139, 145-161.
Constant, S.L., Mountford, A.P. and Wilson, R.A. (1990). Phenotypic analysis of
the cellular responses in regional lymphoid organs of mice vaccinated against
Schistosoma mansoni. Parasitology 101, 15-22.
Correa-Oliveira, R. and Sher, A. (1985). Defective immunoglobulin M responses to
vaccination or infection with Schistosoma mansoni in xid mice. Infection and
Immunity 50, 409414.
Correa-Oliveira, R., Sher, A. and James, S.L. (1984). Mechanisms of protective
immunity against Schistosoma mansoni infection in mice vaccinated with
irradiated cercariae. V. Anamnestic cellular and humoral responses following
challenge infection. American Journal of Tropical Medicine and Hygiene 33,
26 1-268.
Coulson, P.S. and Mountford, A.P. (1989). Fate of attenuated schistosomula admi-
nistered to mice by different routes, relative to the immunity induced against
Schistosoma mansoni. Parasitology 99, 3 9 4 5 .
Coulson, P.S. and Wilson, R.A. (1988). Examination of the mechanisms of pul-
RADIATION-AlTENUATED VACCINE AGAINST SCHISTOSOMES 325
Dessein, A.J., Begley, M., Demeure, C., Caillol, D., Fueri, J., Dos Reis, M.G.,
Andrade, Z.A., Prata, A. and Bina, J.C. (1988). Human resistance to Schistosoma
mansoni is associated with IgG reactivity to a 37-kDa larval surface antigen.
Journal of Immunology 140, 2727-2730.
Dunne, D.W., Jones, F.M., Cook, L. and Moloney, N.A. (1994). Passively transfer-
able protection against Schistosomajaponicum induced in the mouse by multiple
vaccination with attenuated larvae: the development of immunity, antibody
isotype responses and antigen recognition. Parasite Immunology 16, 655-668.
Finkelman, F.D., Holmes, J., Katona, I.M, Urban, J.F., Beckman, M.P., Park, L.S.,
Schooly, K.A., Coffman, R.L., Mosmann, T.R. and Paul, W.E. (1990). Lympho-
kine control of in vivo immunoglobulin isotype selection. Annual Review of
Immunology 8, 303-334.
Firestein, G.S., Roeder, W.D., Laxer, J.A., Townsend, K.S., Weaver, C.T., Hom, J.T.,
Linton, J., Torbett, B.E. and Glasebrook, A.L. (1989). A new murine CD4+ T cell
subset with an unrestricted cytokine profile. Journal of Immunology 143,518-525.
Ford, M.J., Bickle, Q.D. and Taylor, M.G. (1984a). Immunisation of rats against
Schistosoma mansoni using irradiated cercariae, lung schistosomula and liver
stage worms. Parasitology 89, 327-334.
Ford, M.J., Bickle, Q.D., Taylor, M.G. and Andrews, B.J. (1984b). Passive transfer
of resistance and the site of immune-dependent elimination of challenge infec-
tion in rats vaccinated with highly irradiated cercariae of Schistosoma mansoni.
Parasitology 89, 461-482.
Ford, M.J., Bickle, Q.D. and Taylor, M.G. (1987a). Immunity to Schistosoma
mansoni in congenitally athymic, irradiated and mast cell-depleted rats. Para-
sitology 94, 313-326.
Ford, M.J., Dissous, C., Pierce, R., Taylor, M.G., Bickle, Q.D. and Capron, A. (1987b).
The isotypes of antibody responsible for the ‘late’ passive transfer of immunity in
rats vaccinated with highly irradiated cercariae. Parasitology 94,509-522.
Freeman, G.L. Jr, Tominaga, A., Takatsu, K., Secor, W.E. and Colley, D.G. (1995).
Elevated innate peripheral blood eosinophilia fails to augment irradiated cercar-
ial vaccine-induced resistance to Schistosoma mansoni in IL-5 transgenic mice.
Journal of Parasitology 81, 1010-1011.
Georgi, J.R. (1982). Schistosoma mansoni: quantification of skin penetration and
early migration by differential external radioassay and autoradiography. Para-
sitology 84, 263-28 1.
Georgi, J.R., Dean, D.A. and Mangold, B.L. (1983). Schistosoma mansoni: tem-
poral distribution of radioselenium-labelled schistosomula in lungs of mice
during the first two weeks of infection. Parasitology 86, 31-36.
Georgi, J.R., Wade, D.E. and Dean, D.A. (1986). Attrition and temporal distribu-
tion of S. mansoni and S. haematobium schistosomula in laboratory mice.
Parasitology 93, 55-70.
Gryseels, B. (1994). Human resistance to Schistosoma infections: age or experi-
ence? Parasitology Today 10, 380-384.
Gui, M., .Kusel, J.R., Shi, Y.E. and Ruppell, A. (1995). Schistosomajaponicum and
S. mansoni: comparison of larval migration patterns in mice. Journal of
Helminthology 69, 19-25.
Hagan, P. and Gryseels, B. (1994). A perspective on schistosomiasis research.
Parasitology Today 10, 166-170.
Harrison, R.A., Bickle, Q.D., Kiare, S., James, E.R., Andrews, B.J., Sturrock, R.F.,
Taylor, M.G. and Webbe, G. (1990). Immunization of baboons with attenuated
schistosomula of Schistosoma haematobium: levels of protection induced by
RADIATION-AlTENUATED VACCINE AGAINST SCHISTOSOMES 327
James, S.L., Natovitz, P.C., Farrar, W.L. and Leonard, E.J. (1984). Macrophages as
effector cells of murine schistosomiasis: macrophage activation in mice vacci-
nated with radiation-attenuated cercariae. Infection and Immunity 44,569-575.
James, S.L., Deblois, L.A., Al-Zamel, F., Glaven, J. and Langhorne, J. (1986).
Defective vaccine-induced resistance to Schistosoma mansoni in P strain mice.
111. Specificity of the associated defect in cell-mediated immunity. Journal of
Immunology 137, 3959-3967.
Jwo, J. and LoVerde, P.T. (1989). The ability of fractionated sera from animals
vaccinated with irradiated cercariae of Schistosoma mansoni to transfer immu-
nity to mice. Journal of Parasitology 75, 252-260.
Kambara, T. and Wilson, R.A. (1990). In situ pulmonary cellular responses of T
cell and macrophage subpopulations to a challenge infection in mice vaccinated
with irradiated cercariae of Schistosomula mansoni. Journal of Parasitology 76,
365-372.
Kamiya, H., Smithers, S.R. and McLaren, D.J. (1987). Schistosoma mansoni:
autoradiographic tracking studies of isotopically labelled challenge parasites in
naive and vaccinated CBAlCa mice. Parasite Immunology 9, 5 15-529.
Kassim, O.O., Dean, D.A., Mangold, B.L. and von Lichtenberg, F. (1992).
Combined microautoradiographic and histopathologic analysis of the fate of
challenge Schistosoma mansoni schistosomula in mice immunized with irra-
diated cercariae. American Journal of Tropical Medicine and Hygiene 47,
23 1-237.
Kelly, E.A. and Colley, D.G. (1988). In viva effects of monoclonal anti-L3T4
antibody on immune responsiveness of mice infected with Schistosoma mansoni.
Reduction of irradiated cercariae-induced resistance. Journal of Immunology
140, 2737-2745.
Kloetzel, K.S. and Da Silva, J.R. (1967). Schistosomiasis mansoni acquired in
adulthood: behaviour of egg counts and the intradermal test. American Journal
of Tropical Medicine and Hygiene 16, 167-169.
Knopf, P.M., Mangold, B.L. and Makari, G.J. (1983). Recovery of parasites at
different stages of migration following infection of rats with Schistosoma man-
soni. Parasitology 86, 3 7 4 9 .
Knopf, P.M., Cioli, D., Mangold, B.L. and Dean, D.A. (1986). Migration of
Schistosoma mansoni in normal and passively immunized laboratory rats.
American Journal of Tropical Medicine and Hygiene 35, 1173-1 184.
Laxer, M.J. and Tuazon, C.U. (1992). Migration of '%e-methionine-labeled Schis-
tosoma japonicum in normal and immunized mice. Journal of Infectious
Diseases 166, 1133-1 138.
Lewis, F.A. and Wilson, E.M. (1982). Regional and splenic lymphocyte prolif-
erative responses of mice exposed to normal or irradiated Schistosoma mansoni
cercariae. American Journal of Tropical Medicine and Hygiene 31, 505-5 13.
Mahmoud, A.A.F., Peters, P.A.S., Civil, R.H. and Remington, J.S. (1979). In vitro
killing of schistosomula of Schistosoma mansoni by BCG and C. parvum-
activated macrophages. Journal of Immunology 122, 1655-1657.
Mahmoud, A.A.F., Siongok, T.A., Ouma, J., Houser, H.B. and Warren, K.S.
(1983). Effect of targeted mass treatment on intensity of infection and morbidity
in schistosomiasis mansoni: three year follow up of a community in Machakos,
Kenya. Lancet i, 849-859.
Majid, A.A., Bushara, H.O., Saad, A.M., Hussein, M.F., Taylor, M.G., Dargie,
J.D., Marshall, T.F. de C. and Nelson, G.S. (1980). Observations on cattle
schistosomiasis in the Sudan, a study in comparative medicine. 111. Field testing
RADIATION-AlTENUATED VACCINE AGAINST SCHISTOSOMES 329
with irradiated cercariae. IV. Analysis of the role of IgE antibodies and mast
cells. Journal of Immunology 131, 1460-1465.
Sher, A., James, S.L., Correa-Oliveira. R., Hieny, S. and Pearce, E. (1989).
Schistosome vaccines: current progress and future prospects. Parasitology 98,
S61-S68.
Sher, A., Coffman, R.L., Hieny, S. and Cheever, A.W. (1990). Ablation of eosi-
nophil and IgE responses with anti-IL-5 or anti-IL-4 antibodies fails to affect
immunity against Schistosoma mansoni in the mouse. Journal of Immunology
145, 3911-3916.
Shi, Y.E., Jiang, C.F., Han, J.J., Li, Y.L. and Ruppel, A. (1990). Schistosoma
japonicum: an ultraviolet-attenuated cercarial vaccine applicable in the field for
water buffaloes. Experimental Parasitology 71, 100-106.
Shi, Y.E., Jiang, C.F., Han, J.J., Li, Y.L. and Ruppel, A. (1993). Immunization of
pigs against infection with Schistosoma japonicum using ultraviolet-attenuated
cercariae. Parasitology 106, 459-462.
Simpson, A.J.G., Hackett, F., Walker, T., Payares, G., De Rossi, R. and Smithers,
S.R. (1985). Antibody response against schistosomula surface antigens and
protective immunity following immunization with highly irradiated cercariae
of Schistosoma mansoni. Parasite Immunology 7, 133-1 52.
Smithers, S.R. and Terry, R.J. (1969). Immunity in schistosomiasis. Annals of the
New York Academy of Science 160, 826-840.
Smythies, L.E., Coulson, P.S. and Wilson, R.A. (1992a). Monoclonal antibody to
interferon-gamma modifies pulmonary inflammatory responses and abrogates
immunity to Schistosoma mansoni in mice vaccinated with attenuated
cercariae. Journal of Immunology 149, 3654-3658.
Smythies, L.E., Pemberton, R.M., Coulson, P.S., Mountford, A.P. and Wilson,
R.A. (1992b). T cell-derived cytokines associated with pulmonary immune
mechanisms in mice vaccinated with irradiated cercariae of Schistosoma man-
soni. Journal of Immunology 148, 1512-1518.
Smythies, L.E., Coulson, P.S. and Wilson, R.A. (1993). Immunity to Schistosoma
mansoni in mice vaccinated with irradiated cercariae: cytokine interactions in
the pulmonary protective response. Annals of Tropical Medicine and Parasitol-
ogy 87, 653-657.
Smythies, L.E., Betts, C., Coulson, P.S., Dowling, M.-A. and Wilson, R.A. (1996).
Kinetics and mechanism of effector focus formation in the lungs of mice
vaccinated with irradiated cercariae of Schistosoma mansoni. Parasite Immunol-
ogy 18, 359-369.
Springer, T.A. (1990). Adhesion receptors of the immune system. Nature 346,
425-434.
Stek, M., Dean, D.A. and Clark, S.S. (1981a). Attrition of schistosomes in an
irradiation attenuated cercarial immunization model of Schistosoma mansoni.
American Journal of Tropical Medicine and Hygiene 30, 1033-1038.
Stek, M., Minard, P., Dean, D.A. and Hall, J.E. (1981b). Immunization of baboons
.with Schistosoma mansoni cercariae attenuated by gamma irradiation. Science
212, 1518-1520.
Stirewalt, M.A. (1959). Chronological analysis, pattern, and route of migration of
cercariae of Schistosoma mansoni in body, ear and tail skin of mice. Annals of
Tropical Medicine and Parasitology 53, 400-4 13.
Stover, C.K. (1994). Recombinant vaccine delivery systems and encoded vaccines.
Current Opinion in Immunology 6, 568-57 1.
Sturrock, R.F., Cottrell, B.J. and Kimani, R. (1984a). Observations on the ability of
334 P.S. COULSON
Webbe, G., Sturrock, R.F., James, E.R. and James, C. (1982).Schistosoma hae-
matobium in the baboon (Papio anubis): effect of vaccination with irradiated
larvae on the subsequent infection with percutaneously applied cercariae. Trans-
actions of the Royal Society of Tropical Medicine and Hygiene 76, 354-361.
Wei, X.,Charles, I.G., Smith, A., Ure, J., Feng, G., Huang, F., Xi, D., Muller, W.,
Moncada, S. and Liew, F.Y. (1995).Altered immune responses in mice lacking
inducible nitric oxide synthase. Nature 375, 408-41 1.
Wheater, P.R. and Wilson, R.A. (1979).Schistosoma mansoni: a histological study
of migration in the laboratory mouse. Parasitology 79, 49-62.
Wilkins, H.A., Blumenthal, U.J., Hagan, P., Hayes, R.J. and Tulloch, S. (1987).
Resistance to reinfection after treatment of urinary schistosomiasis. Transactions
of the Royal Society of Tropical Medicine and Hygiene 81, 29-35.
Wilson, R.A. (1987).Cercariae to liver worms: development and migration in the
mammalian host. In: The Biology of Schistosomes (D. Rollinson and A.J.G.
Simpson, eds), pp. 115-146. London: Academic Press.
Wilson, R.A. (1990).Leaky livers, portal shunting and immunity to schistosomes.
Parasitology Today 6 , 354-358.
Wilson, R.A. and Coulson, P.S. (1986).Schistosoma mansoni: dynamics of migra-
tion through the vascular system of the mouse. Parasitology 92, 83-100.
Wilson, R.A. and Coulson, P.S. (1989).Lung-phase immunity to schistosomes: a
new perspective on an old problem. Parasitology Today 5, 274-279.
Wilson, R.A. and Sher, A. (1992).Vaccines against schistosomes: an alternative
view. Transactions of the Royal Society of Tropical Medicine and Hygiene 79,
237.
Wilson, R.A., Draskau, T., Miller, P. and Lawson, J.R. (1978). Schistosoma
mansoni: the activity and development of the schistosomulum during migration
from the skin to the hepatic portal system. Parasitology 77, 57-73.
Wilson, R.A., Coulson, P.S. and McHugh, S.M. (1983).A significant part of the
‘concomitant immunity’ of mice to Schistosoma mansoni is a consequence of
a leaky hepatic portal system, not immune killing. Parasite Immunology 5,
595-601.
Wilson, R.A., Coulson, P.S. and Dixon, B. (1986).Migration of the schistosomula
of Schistosoma mansoni in mice vaccinated with radiation attenuated cercariae,
and normal mice: an attempt to identify the timing and site of parasite death.
Parasitology 92, 101-1 16.
Wilson, R.A., Coulson, P.S., Sturrock, R.F. and Reid, G.D.F. (1990).Schistosome
migration in primates: a study in the olive baboon (Papio anubis). Transactions
of the Royal Society of Tropical Medicine and Hygiene 84, 80-83.
Wilson, R.A., Coulson, P.S., Betts, C., Dowling, M.-A. and Smythies, L.E. (1996).
Impaired immunity and altered pulmonary responses in mice with a disrupted
interferon-y receptor gene exposed to the irradiated Schistosoma mansoni
vaccine. Immunology 87, 275-282.
Wynn, T.A., Oswald, I.P., Eltoum, I.A., Caspar, P., Lowenstein, C.J., Lewis, F.A.,
James, S.L. and Sher, A. (1994).Elevated expression of Thl cytokines and nitric
oxide synthase in the lungs of vaccinated mice after challenge infection with
Schistosoma mansoni. Journal of Immunology 153, 5200-5209.
Wynn, T.A., Jankovic, D., Hieny, S.,Cheever, A.W. and Sher, A. (1995).IL-12
enhances vaccine-induced immunity to Schistosoma mansoni in mice and
decreases T helper 2 cytokine expression, IgE production, and tissue
eosinophilia. Journal of Immunology 154, 4701-4709.
Wynn, T.A., Reynolds, A., James, S.L., Cheever, A.W., Caspar, P., Hieny, S.,
336 P.S. COULSON