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Control of Coccidiosis into the


Next Millennium
N.C. Smith, A. Lunden, F.J. Conraths and H.D. Chapman

Oxford, UK the prospects for such a vaccine has remarkably few intraspecific variants
September 1997 seem little better than when these worldwide (with the possible excep-
promises were first set forth. tion of Eimeria maxima) and induces a
Every four years, people interested in very effective natural immunity.
that group of Apicomplexan parasites Dennis Schmatz (Merck, NJ, USA), also The impeccably organized pro-
loosely termed the coccidia gather being loyal to the cause of chemo- gramme used plenary sessions to focus
together to discuss their obsessions. therapy, echoed these sentiments, the delegates on a new topic each day
The main obsession, traditionally, has drawing into the picture the spectre of and these were, most logically, followed
been with Eimeria, and Martin Shirley the, thus far, lack of success in the by vibrant, cutting-edge contributed
(Institute for Animal Health, Compton, quest for a malaria vaccine as reason paper sessions on the same theme. A
UK) gave insight as to why with some for pessimism with regard to hopes summary of the main new findings is
startling statistics: there are 35310 9 of a vaccine against coccidiosis. He presented here; it would be impossible,
chickens consumed each year, each went on to describe how, and probably inappropriate, to attempt
of these harbouring perhaps 5310 6 to cover all the contributions made at
Eimeria parasites, giving a staggering The discovery of novel anticoccidials the meeting, which was attended by
total of 17531015 parasites, surely has also been hindered by a shift almost 400 people from 40 countries.
making this one of the most numerous in focus during the 1980s toward The main themes were: Chemotherapy;
organisms on the planet. The scope coccidiosis vaccines. Although some Phylogeny, Life cycles, Epidemiology and
of the problem of dealing with this live attenuated vaccines have been diagnosis; Molecular and cell biology;
pathogen is hinted at in the mutation developed, they have had limited Immunology; and Vaccination.
rate; the frequency of pre-existing mu- applications and an effective subunit
tants in Eimeria is one in 10 9, therefore vaccine for coccidiosis has not been Chemotherapy
there are 175310 6 new mutants every achieved. As a result of the de-
year, which must create headaches for creased effort in anticoccidial drug A consequence of the widespread
chemotherapists and vaccinologists alike. discovery, resistance to available use of drugs for the control of coc-
Despite the traditional obsession with anticoccidials has become a serious cidiosis is the development of drug
Eimeria, there has been a significant in- issue. resistance. It is therefore not surprising
crease in interest in other coccidia, es- that many of the papers were con-
pecially Neospora, Cryptosporidium and This was exactly the start the meeting cerned with evaluation of the efficacy
Toxoplasma, and this was reflected in the needed, and thereafter everyone was of anticoccidial drugs and practical ap-
contributions at the most recent meeting. focused on the question of the future proaches to monitoring and managing
A further strength of this International for coccidiosis control. Of course, at the problem of resistance. However,
Coccidiosis Conference was that it this stage, discussion among the immu- there were also papers describing tech-
brought together individuals with diverse nologists and molecular biologists in niques for identifying much-needed
interests and expertise, including those the bar was animated, most being un- new chemical means of controlling
from universities, government agencies der the impression that the push for coccidiosis. Chuck Smith (Eli Lilly, IN,
and commercial organizations. This was vaccines was the result rather than the USA) suggested that rational mechanism
fitting, since the conference organizers, cause of increasing incidences of failure based approaches to drug discovery
Martin Shirley and Fiona Tomley had, in of anticoccidials. (directed at specific cellular/molecular
an inspired decision, made the theme of Unfortunately for the vaccinologists, targets) could be facilitated by the use
the conference Control of Coccidiosis one of their own, Martin Shirley, noting of novel high-throughput screening
into the Next Millennium. Another that the Eimeria genome was roughly methods that would permit large num-
venue for European coccidiologists is 1/20th the size of that of the chicken bers of compounds (.200 000 in
the annual COST820 Workshop on and therefore much larger than any three months) to be investigated. Such
Vaccines against Animal Coccidioses. other pathogen of poultry and substan- screens could test synthetic organic
This years workshop was integrated tially larger than that of the malaria substances, natural products and com-
into the Coccidiosis Conference. parasite, also implied that the prospects pounds of unique combinatorial chemi-
The meeting was opened in a suit- for a subunit vaccine were bleak. The cal structure. Potential targets are
ably provocative way by Tom Jeffers continued references to malaria began enzymes involved in intermediate
(Elanco Animal Health, IN, USA) who to rankle the molecular people: malaria, metabolism, molecules involved in para-
asserted that, after all, is a far more complex disease, sitehost communication, transport
using two hosts, undergoing schizogony molecules and molecules that can
With the advent of molecular im- in two different organs and being famous selectively inhibit transcription.
munology, promises of an effective for its antigenic variability and the poor Dennis Schmatz presented exam-
recombinant vaccine were voiced development of protective immunity. ples of two exciting leads obtained by
by a number of investigators, yet Conversely, Eimeria has the simplest of (1) biochemical analyses of Eimeria in an
after years of work and millions of Apicomplexan life cycles, infects a host effort to identify parasite-specific path-
dollars being expended in this quest, whose environment is strictly controlled, ways, and (2) screening of compounds
Parasitology Today, vol. 14, no. 6, 1998 Copyright 1998, Elsevier Science Ltd All rights reserved 01694758/98/$19.00 PII: S0169-4758(98)01238-1 215
News
that can kill the parasite in vitro. The man- Astrid Tenter (Tierrztliche Hochschule, organelles seems to play a crucial role.
nitol cycle is a unique pathway found in Hannover, Germany) described studies First, microneme proteins (eg. MIC 1,
Eimeria but not in the host. It was orig- done in collaboration with John Barta 2 and 3 of Toxoplasma gondii) contrib-
inally identified in the oocyst and dis- (University of Guelph, Canada) and ute to host cell recognition. Usually they
covered to be a major energy source Alan Johnson (University of Technology, contain thrombospondin-like motifs
for sporulation but, initially, it was con- Sydney, Australia) showing that, based on likely to be involved in binding sul-
ceptually difficult to know how this in- 18S rRNA, the genus Isospora is the sis- phated glycoconjugates or represent
formation could be used to advantage in ter group to the Toxoplasma/Neospora EGF-like proteins and are attached to
the formulation of a control programme. clade, to the exclusion of Sarcocystis. the cell membrane by GPI anchors.
However, the more recent discovery Alan Johnson (University of Tech- Rhoptry proteins are involved in the
that the mannitol cycle begins to operate nology, Sydney, Australia) described formation of the parasitophorous vacu-
during the sexual cycle of the parasite how in T. gondii, at least two clonal lin- ole (PV), may be incorporated into the
(ie. as early as 56 days post-infection in eages can be recognized that correlate parasitophorous membrane (PM) and
chickens) suggested a possible strategy: with their virulence in mice and several could be implicated in the formation of
a transmission-blocking approach. That molecular markers. Whether or not pores in the PM (ROP1, 2). Three se-
such an approach is a feasible means of these differences justify a separation quenced members of the ROP2 family
control is suggested by work with ga- into different species is controversial; contain a transmembrane domain. Fi-
metocyte antigens (see below) and, thus, however, Johnson put forward a com- nally, dense granule proteins are se-
the elucidation of the mannitol cycle was pelling hypothesis that the virulent lin- creted into the PV or integrated into
used to develop biochemical assays to eage may undergo an asexual life cycle, the PM. Dense granules are thought to
identify potential inhibitors. Indeed, it was similar to that of the closely related remodel the PV into a metabolically
discovered that an old anticoccidial, N. caninum, with vertical transmission active compartment.
nitrophenyl disulphide, affects mannitol- either by passage to the foetus in preg- The micronemes of Eimeria have
1-phosphate dehydrogenase (M1PDH) nancy or by transmission in milk to the been the subject of intense investig-
and shuts off the mannitol cycle in gut neonate (see also Ref. 1). ation. Out of five microneme proteins
parasites, without really affecting the Fittingly, much of the discussion of E. tenella characterized thus far,
viability of these parasites but resulting concerning epidemiology and diag- three (Etmic-1, -3 and -4) have the ap-
in the shedding of mannitol-deficient nosis focused on the newest coccidian, pearance of type I integral membrane
oocysts, which fail to mature normally. N. caninum. John Ellis (University of proteins whereas the remaining two
The 14-3-3 protein family, which inhibits Technology, Sydney, Australia) de- lack predicted membrane spanning re-
M1PDH, may be a modern-day lead scribed the various techniques for gions. Etmic-1, -4 and -5 contain motifs
for a novel chemotherapeutic. Apicidin detecting N. caninum, ranging from his- characteristic for adhesins (Fiona Tomley,
is an example of a compound identified tological and immunohistological detec- Institute for Animal Health, Compton,
by in vitro screening. Once identified, tion of parasites in lesions in various UK). Upon induction by host cell factors,
Apicidin was subsequently found to be organs of the aborted foetus to more microneme proteins undergo rapid re-
an inhibitor of histone deacetylase, a recent innovations such as an enzyme- localization and exocytosis from the
key enzyme in the regulation of nuclear linked immunosorbent assay (ELISA) apical end and can be detected on the
transcription, and so it is no great sur- based on immunostimulatory complexes surface of both parasites and host cells;
prise that it is active against many Api- (ISCOMs) and a nested PCR (polym- as the parasite moves over the surface
complexan parasites. The compound is erase chain reaction), which can detect of host cells, it leaves a trail of micro-
not just active in vitro but demonstrates as little as 10 fg of Neospora DNA. In- neme proteins (Janene Bumstead, Insti-
good anti-Plasmodium berghei activity in triguingly, through the evaluation of the tute for Animal Health, Compton, UK).
mice and modest anticoccidial activity in specificity of serological and PCR-based The host factor responsible for trigger-
chickens. However, it is also an inhibitor diagnostic tests for N. caninum, the pres- ing microneme release is associated
of mammalian histone deacetylase and, ence of T. gondii DNA has also been de- with the albumin fraction of foetal calf
therefore, modified versions of Apicidin tected in the brains of aborted foetuses serum. Interestingly, microneme secre-
are being tested in the hope of finding a (Bruno Gottstein and Brigitte Hentrich, tion is inhibited by staurosporine, a pro-
more specifically antiparasitic compound. University of Bern, Switzerland). Although tein kinase C substrate inhibitor, and this
it was not clear whether this was the inhibits parasite development. This led
Phylogeny, Life Cycles, cause of the abortions, the finding is of to speculation about possible control
Epidemiology and Diagnosis interest since Toxoplasma-induced abor- strategies based on microneme pro-
tion in cattle is considered to be ex- teins, eg. blocking of release, disruption
Investigations on the molecular phy- tremely rare and the implication is that of ligand binding and induction of novel
logeny of coccidia showed that, with the abortion storms associated with N. cani- immune responses.
exception of Cryptosporidium, cladograms num may be multifactorial phenomena. Samantha Brown (University of
based on 18S rDNA sequences are Glasgow, UK) introduced the possibility
reasonably congruent with taxonomic Molecular and Cell Biology that proteases are important partici-
ideas (David Morrison, University of pants in cell invasion by the coccidia.
Technology, Sydney, Australia). Data Discussion in the molecular and cell Observations that an E. tenella aspartyl
from Joakim Holmdahl (University of biology sessions was dominated by ques- proteinase can be found at the apical
Technology, Sydney, Australia/Swedish tions related to cell invasion. Recent tip (among other locations) of the para-
University for Agricultural Sciences, progress in the understanding of host- site and that parasite invasion can be
Uppsala, Sweden) concurred with this cell invasion by Apicomplexa was sum- inhibited by either pepstatin or mono-
assertion and showed that Hammondia, marized by Jean-Franois Dubremetz clonal antibodies to this protease
Besnoitia, Neospora caninum and Toxo- (INSERM, France). After initial triggering added further fuel to this idea (Letitia
plasma gondii form a monophyletic clade. steps, successive exocytosis of secretory Jean, INRA, Jouy-en-Josas, France).
216 Parasitology Today, vol. 14, no. 6, 1998
News
Several technical breakthroughs in so hopes are high that the complex in mice that received monoclonal anti-
cell and molecular biology may prove network determining resistance to Ei- bodies directed against IFN-g, the ef-
extremely useful in the future and meria may be unravelled. It was es- fect of exogenous IL-12 was blocked. At
might be anticipated to be the basis for pecially pleasing that Elaine Roses faith least some of these observations appear
important breakthroughs to be pre- in the E. vermiformis model seems to relevant to cattle too: in calves, signifi-
sented at the next meeting in 2001: have been vindicated. cant increases in CD31 and CD81
Michelle Kelleher (Institute for Animal It is a distinct possibility that the were found in lamina propria and intra-
Health, Compton, UK) described a TNF, free oxygen radicals and nitric epithelial lymphocytes from C. parvum-
transient transfection technique for oxide metabolites produced in re- infected calves. Reverse transcriptase
Eimeria; Martin Shirley told of the sponse to Eimeria may account for PCR (RT-PCR) demonstrated increased
establishment of a yeast artificial chro- some of the pathology associated with levels of IFN-g and IL-12, raising specu-
mosome (YAC) library and work on an infection. An intriguing finding from lation about the potential use of bovine
RFLP (restricted fragment length poly- Adrian Haydays laboratory seemed to IL-12 in the prophylaxis of cryptosporidi-
morphism) linkage map of the Eimeria confirm the immune systems role in osis (Ron Fayer, USDA, MD, USA).
genome as a major thrust towards the causing at least some of the pathology
construction of a complete physical associated with coccidiosis. He re- Vaccination
map of the genome; and Anne Gurnett ported that although g/d T cells do not
(Merck, NJ, USA) described a novel appear to play a direct role in mediat- Rumours abounded that a revised
and elegant technique to look inside ing resistance to E. vermiformis, they version of Paracox would soon be
the host cell and see what the coccidia are important in preventing pathology. available for use in broilers but these
are really up to by selectively inhibiting Thus, in mice genetically depleted of could neither be confirmed nor denied.
host protein synthesis with ricin this g/d T cells, the course of infection is Mark Jenkins (USDA) opened official
technique could facilitate study of unaltered but intestinal haemorrhaging, proceedings by pointing out that there
post-translational events and identify extravasation and villous tip damage had been some errors in the past
important parasite-specific proteins. are exacerbated and these effects are perhaps using antisera to complex anti-
apparently mediated by a/b T cells. gens for screening of cDNA libraries
Immunology The implication is that g/d T cells play from the extracellular stages of the
a crucial role in downregulating the parasite was not the most rational ap-
The advent of gene knockout tech- exuberant a/b T-cell response. Hayday proach to identifying subunit vaccine
nology has spurred on research using the pointed out that this is perhaps not an candidates. However, Jenkins generated
murine model, E. vermiformis, and this isolated case; lupus-like syndrome (an a sense of optimism with his proposal
dominated discussion in the Immunol- autoimmune disease) is worse in mice that targeting antigens of the intra-
ogy Sessions. Workers in three groups lacking g/d T cells and examples of cellular stages of Eimeria to identify
(Karen Ovingtons at the Australian other pathologies in the absence of g/d vaccine candidates was now a real
National University, Hyun Lilllehojs at T cells may be apparent in Listeria, possibility. Furthermore, the technology
the USDA and Adrian Haydays at Yale Mycobacterium and Plasmodium in- for introducing genes into invasive,
University) have produced data that are fections. All in all, it appears that fur- non-pathogenic, attenuated vectors
remarkably cross-confirmatory. It appears ther research into the immunology of and for administering vaccines in ovo is
that resistance to Eimeria does not Eimeria infections will have profound now firmly established and encourages
obey the prevalent paradigm of Th1/ repercussions for T-cell and cytokine the belief that suitable means to pre-
Th2 cell dichotomy since all of inter- biology generally. sent vaccines to chickens already exist.
feron gamma (IFN-g), tumour necrosis Investigations into the immune Michael Wallach (Abic Pharmaceuti-
factor (TNF), interleukins IL-4, IL-5 and response to Cryptosporidium are also cals, Israel) gave us all even more rea-
IL-6, free oxygen radicals and nitric oxide proceeding rapidly, with mouse models son for optimism with his summary of
metabolites are produced with broadly again being prominent. The importance the work performed to validate the
similar kinetics and peak at the same of cryptosporidiosis as an emerging in- feasibility of utilizing maternal immunity
time that mice appear to gain control fectious disease, particularly for AIDS to protect broilers from coccidiosis
of the parasite. Intriguingly, however, patients and other immunocompro- and to use maternal antibodies to iden-
apart from IFN-g, all of these factors mised individuals, was underlined by tify candidate antigens (see Ref. 2).
either play no role in resistance (eg. IL-4 determining 1.8 oocysts as the ID50 in Wallach described work done in Israel,
and IL-6) or actually exacerbate infec- immunosuppressed C57BL/6N mice the USA and Switzerland that estab-
tion (eg. TNF, free oxygen radicals and (Shiguang Yang, Utah State University, lished maternal immunization using
nitric oxide metabolites). Thus, mice USA). Adoptive transfer of intra- gametocyte antigens of 56, 82 and
depleted of the genes for IFN-g or the epithelial lymphocytes showed that 230 kDa derived from E. maxima as
IFN-g receptor are more susceptible CD41 T cells and production of IFN-g the currently most promising approach
to infection with E. vermiformis, excret- were required for a local immunity to immunoprophylactic control for
ing substantially more oocysts for a sig- in SCID (severe combined immuno- coccidiosis in broilers. Even more en-
nificantly longer period than do wild-type deficiency) mice (Vince McDonald, couraging are recent results obtained
mice. However, mice that are unable London School of Hygiene and Tropi- in floor pen trials designed to mimic
to use TNF, free oxygen radicals or ni- cal Medicine, UK). Furthermore, admin- field conditions. In these trials, hens
tric oxide metabolites actually harbour istration of recombinant IL-12 to neo- were immunized with the gametocyte
fewer parasites than do wild-type mice. natal SCID or BALB/c mice prevented antigens and their progeny kept in floor
Hence, exactly how IFN-g mediates or greatly reduced the severity of infec- pens seeded with sporulated oocysts
resistance is mysterious but, encourag- tion if the cytokine was given prior to of E. maxima, E. tenella and E. acervulina.
ingly, the data now being collected in infection with Cryptosporidium parvum, The buildup of oocysts in the litter
chickens parallels that collected in mice, this effect being dependent on IFN-g; of the floor pens was followed for a
Parasitology Today, vol. 14, no. 6, 1998 217
News
7-week period (a typical grow-out time abortion rate after challenge with N. skills of Martin Shirley and Fiona Tomley,
for broilers) and demonstrated that the caninum tachyzoites during pregnancy. but it would be remiss not to acknowledge
level of oocyst contamination in flocks In contrast, immunization with the live the great generosity of the sponsors of the
derived from immunized hens was T. gondii vaccine Toxovax (Intervet BV) meeting: F. Hoffman-La Roche; Bayer AG;
around 30% of that found in flocks did not prevent abortion associated Biopharm; Elanco Animal Health; Embrex
Inc.; Hoechst Roussel Vet; Intervet Inter-
derived from unimmunized hens. This with N. caninum. national bv; Janssen Animal Health; Merck,
validated an earlier mathematical pre- Sharpe & Dohme; Merial; Pharmacia &
diction, and demonstrated that mater- Conclusion Upjohn; Pfizer Inc.; Schering-Plough Animal
nal immunization with gametocyte anti- Health; Vetech Laboratories; and the Euro-
gens could achieve exactly the same Thus, the conference ended on a pean Union, through support of COST820.
results as Paracox or monensin, both of suitably optimistic note for the pros-
which also aim to reduce oocyst con- pects of controlling these parasites in References
tamination of litter by around 70% (Ray the next millennium. There appear to be 1 Johnson, A.M. (1997) Parasitol. Today 13,
393397
Williams, Schering-Plough, Uxbridge, UK). excellent candidates for basing control 2 Wallach, M. et al. (1995) Parasitol. Today 11,
These apparently cross-species con- strategies around both chemotherapy 262265
served and protective gametocyte anti- and vaccination; intriguingly, perhaps
gens can now be produced in large the best prospects in both camps lie in Nick Smith is at the Department of Cell and
quantities to GMP (good manufactur- the concept of controlling transmission Molecular Biology, University of Technology,
ing practice) standards, and an enor- of oocysts rather than controlling the Sydney, PO Box 123, Broadway, NSW 2007,
mous field trial involving several tens of internal parasite per se. It is perhaps Australia; Anna Lunden is at the National
thousands of hens and many millions of this level of lateral thinking that will Veterinary Institute Laboratory of Parasitology,
their progeny is currently proceeding in lead to further breakthroughs. PO Box 7073, S-75007 Uppsala, Sweden;
poultry houses all over Israel. Franz Conraths is at the Federal Research
The prospect of vaccination against Centre for Virus Diseases of Animals, Institute
Acknowledgements for Epidemiological Diagnostics, Seestrasse 55,
abortion associated with N. caninum
The success of this, the Seventh Inter- D-16868 Wusterhausen, Germany; and Dave
was addressed in a study presented national Coccidiosis Conference and Euro- Chapman is at the Department of Poultry
by David Buxton (Moredun Research pean Union COST820 Workshop, held Science, University of Arkansas, Fayetteville,
Institute, Edinburgh, UK). Inoculation of 15 September 1997 at Keble College, AR 72701, USA. Tel: +61 2 9514
sheep with live N. caninum tachyzoites Oxford, UK, was first and foremost due to 4013, Fax: +61 2 9514 4026, e-mail:
prior to tupping resulted in reduced the incredible energy and organizational Nick.Smith@uts.edu.au

Comment

The SPf66 Malaria Vaccine:


What is the Evidence for Efficacy?
P. Graves, H. Gelband and P. Garner

SPf66 is a synthetic malaria vaccine and continuously updated on CD-ROM searched. This register was compiled
containing peptides derived from three in the Cochrane Library (http://www. through an international effort by re-
asexual blood-stage proteins of Plas- cochrane.co.uk). searchers who have hand-searched the
modium falciparum1. In Parasitology Today, A systematic review is an overview complete archives of more than 200
Gilbert and Hill2 stated that recently, of primary studies that has clear objec- journals for controlled trials. In addition,
extensive testing of the candidate vac- tives and uses explicit and reproducible an electronic search of Medline was con-
cine SPf66 led to the disappointing methods for identifying studies and ducted up to May 1997, and research-
conclusion that SPf66 does not protect applying inclusion criteria5. Such a review ers in the field were contacted to iden-
against clinical malaria, and that further may (or may not) contain a meta- tify any unpublished or ongoing trials.
efficacy trials are not warranted. The analysis of primary studies, which has Only randomized, placebo-controlled
authors cite one trial of SPf66 with a the aim of increasing the precision of a trials of malaria vaccines were included.
negative result as support for their result such as vaccine efficacy. The To date, two types of vaccine have
statement3. However, the vaccine has Cochrane Collaboration is a large inter- been tested in such trials: (1) SPf66
been tested in eight published trials national undertaking to promote and derived from asexual stages; and (2)
in malaria-endemic areas, with varying maintain electronic publication of sys- vaccines composed of an antigen from
results. We re-examine the evidence tematic reviews. Systematic reviews car- the circumsporozoite protein (NANP
for Gilbert and Hills statement, based ried out using rigorous methods lead vaccines). The pre-specified outcomes
on a systematic review and meta-analysis to at least the possibility of an unbiased were: incidence of first episode of clinical
(mathematical synthesis) of all the avail- synthesis of existing research. malaria or parasitaemia, total number of
able unbiased evidence from malaria Systematic review strategy. The episodes of clinical malaria, admission to
vaccine trials4. The review is published Cochrane Controlled Trials Register was hospital, and death.
218 Copyright 1998, Elsevier Science Ltd All rights reserved 01694758/98/$19.00 PII: S0169-4758(98)01242-3 Parasitology Today, vol. 14, no. 6, 1998

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