Nederlandse Vereniging voor Parasitologie
2009 Spring Meeting
Monday 22nd June, 2009
partially joint with
10th International Congress on
TOXOPLASMOSIS
Hotel & Conferentieoord Rolduc,
Kerkrade, the Netherlands
Program NVP Spring meeting 2009
Monday June 22nd, 2009, Rolduc, Kerkrade
10.00 Registration and coffee
10.30 Joint Morning session with 10th Int. Congress on Toxoplasmosis
David Roos, University of Pennsylvania, USA
Apicomplexan cell biology: Toxoplasma as a model for Plasmodium; Plasmodium as a model
for Toxoplasma.
Kurtis Straub, UCLA, Los Angeles, USA
Novel moving junction components and the emerging molecular model of apicomplexan
invasion
Karine Frenal, University of Geneva, Switzerland
Myosin A and D motor complexes are dually anchored in the inner membrane complex and
plasma membrane of Toxoplasma gondii
Stephen Matthews, Imperial College London, UK
Atomic resolution basis for host cell recognition and invasion by Toxoplasma gondii
12.30 Lunch
(joint with participants of 10th Int. Congress on Toxoplasmosis)
13.15 NVP spring meeting, 1st session (separate from 10th Int. Congress Toxoplasmosis)
Adrian Luty, Radboud Univ. Medical Centre, Nijmegen
The quantity and quality of African children’s IgG responses to Plasmodium falciparum asexual
stage antigens reflect protection against infection and disease.
Kwadwo Kusi, Biomedical Primate Research Centre, Rijswijk
Humoral Immune Response to Mixed PfAMA1 Alleles; Multivalent PfAMA1 vaccines induce
broad specificity.
Anne Teirlinck, Radboud Univ. Medical Centre, Nijmegen
T-cell memory responses in human volunteers protected against malaria by repeated
sporozoite inoculation under chloroquine prophylaxis.
Anne-Marie Zeeman, Biomedical Primate Research Centre, Rijswijk
In vitro cultivation of malaria hypnozoites.
Rob Hermsen, Radboud Univ. Medical Centre, Nijmegen
Quantitative determination of Plasmodium vivax gametocytes by real-time quantitative Nucleic
Acid Sequence Based Amplification in clinical samples.
Hein Sprong, RIVM, Bilthoven
The (not so) simple life cycle of Giardia.
Saskia deWalick, Erasmus Univ. Medical Centre, Rotterdam
The core proteome of the Schistosoma mansoni eggshell.
15.00 Tea break
15.30 NVP spring meeting, 2nd session
Merel Langelaar, RIVM, Bilthoven
Trichinella spiralis antigens suppress mouse macrophage and dendritic cell activation
Carmen Aranzamendi, RIVM, Bilthoven
A role for CD14 in the suppression of DC maturation by Trichinella spiralis ES
Gertie Bokken, Inst. Risk Assessment Sciences, Utrecht Univ.
The effect of T. spiralis and T. gondii on serologic responses in swine in concomitant infections.
16.15 Merial award (ceremony and lecture)
17.00 Algemene Leden Vergadering NVP (general assembly of society)
17.30 Drinks and diner
19.30 Closure
2
Apicomplexan cell biology: Toxoplasma as a model for Plasmodium; Plasmodium as
a model for Toxoplasma
David S. Roos, Daniel P. Beiting, Raj Chandramohandas, Zhongqiang Chen, Omar S. Harb,
Paul H. Davis, Doron C. Greenbaum, Ming Yeh Lee, Manami Nishi, Ina Ouologuem, Lucia
Peixoto, Dhanasekaran Shanmugam, and Bo Wu
Department of Biology and Penn Genomics Institute, University of Pennsylvania, Philadelphia PA
19104 USA
E-mail: droos@sas.upenn.edu
Toxoplasma and Plasmodium are both members of the phylum apicomplexa, but these
species diverged >108 years ago, and differ greatly in their biology, pathogenesis, and life
history strategies. Nevertheless, they also share many features, and comparative molecular
genetic, biochemical, cell biological, and genomic analysis offers both scientific opportunities
and informative surprises. From a cell biological perspective, all apicomplexans exhibit a
highly polarized organization, beginning with the specialized secretory organelles of the
apical complex that gives the phylum its name. Adhesive domain-containing proteins
secreted by the micronemes are critical for host cell attachment and invasion; comparative
analysis of available genomes identifies hundreds of probable microneme proteins, and
suggests candidate host cell ligands. Rhoptry secretion helps to define the host-parasite
interface, including the intracellular parasitophorous vacuole within which parasites replicate
and divide. Comparative genomics reveals strong selection and rapid evolution of rhoptry
proteins, including amplified families of secreted kinases that modulate the host cell
environment. Other notable features include the apicoplast, a secondary endosymbiotic
organelle acquired when an ancestral alveolate engulfed a eukaryotic alga, and retained the
algal plastid (along with its genome). Although no longer photosynthetic, ~10% of the
parasite’s nuclear genome encodes proteins destined for the apicoplast, which synthesizes
isoprenoids (via the xylulose pathway), fatty acids (using a type II fatty acyl synthase), and
heme (sequestering three steps in the C4 biosynthetic pathway). The apicoplast is essential
for parasite survival, and a possible target for therapeutics. Apicomplexan parasites employ
the classical eukaryotic secretory pathway, using the ER/Golgi to traffic proteins to
micronemes and rhoptries, as well as the dense granules responsible for constitutive
secretion. Fusing a secretory signal sequence to a plastid transit peptide provides an
ingenious mechanism for targeting proteins across the four membranes surrounding the
apicoplast. The secretory pathway is probably also responsible for producing the inner
membrane complex: a membrane-cytoskeletal scaffold upon which daughter parasites are
assembled during endodyogeny (in Toxoplasma) or schizogony (in Plasmodium). This
distinctive mode of replication – more analogous in concept to a viral burst than typical
eukaryotic division – maintains strict polarity during daughter parasite assembly, and permits
an usual mechanism for waste disposal. Following the assembly of mature daughter
merozoites (tachyzoites) assembly, host cell egress is a rapid event. Remarkably, both
Toxoplasma and Plasmodium appear to co-opt host cell calpain proteases to facilitate
escape from the host cell.
3
Novel Moving Junction Components and the Emerging Molecular Model of
Apicomplexan Invasion
Kurtis Straub, Stephen Cheng, Catherine Sohn and Peter Bradley.
UCLA, Los Angeles, USA
E-mail: kurt@ucla.edu
Toxoplasma gondii and other apicomplexan parasites employ a distinctive mechanism of
active invasion that involves the formation of a tight moving junction between parasite and
host cell membranes. This moving junction anchors the parasite to the host cell during
invasion and is likely also responsible for sieving out host transmembrane proteins that
would otherwise target the parasite to degradation by host lysosomes, making this structure
crucial for parasite survival. The duct-shaped necks of the rhoptries store multiple proteins
that act in concert with micronemal AMA1 to form the moving junction, yet functional roles for
these proteins remain unknown. We have recently discovered two novel moving junction
components in Toxoplasma, RON5 and RON8. RON5 is processed into N and C-terminal
portions that traffic to the moving junction and is conserved across the Apicomplexa, like
most moving junction proteins. In contrast, RON8 is a unique moving junction component
that appears to be conserved in Neospora and Eimeria, but not Plasmodium. RON8
coimmunoprecipitates RON5 and other moving junction components from extracellular
parasites, indicating a preformed complex exists prior to invasion. Intriguingly, RON8 and
RON4 are secreted to the cytoplasmic face of the host membrane during invasion, where
they can engage in anchoring or molecular sieving. To explore RON8 interactions with the
host cell, we exogenously expressed this protein in mammalian cell lines and show that it
consistently traffics to its site of action at the cell periphery mediated by a necessary and
sufficient portion of its C-terminus. This peripheral localization mirrors that of the host cortical
cytoskeleton, and chemical disruption of the cytoskeleton does not appear to affect RON8
targeting. We are currently refining RON8’s peripheral targeting domain and attempting to
identify its binding partner within the host. Identifying the host link to RON8 will greatly
illuminate its functional role in the moving junction and define its contribution to Toxoplasma’s
ability to invade nearly any nucleated host cell in vitro and many different vertebrate species
in vivo.
4
Myosin A and D motor complexes are dually anchored in the inner membrane complex
and plasma membrane of Toxoplasma gondii
Karine Frenal, Dominique Soldati-Favre
University of Geneva, Switzerland
E-mail: karine.frenal@unige.ch
The apicomplexans share a unique form of actin-based gliding motility. The glideosome is
known as the conserved molecular machinery that drives parasite motility and contributes to
invasion and egress. One essential component of the glideosome is the myosin motor
complex composed of the myosin heavy chain MyoA, the myosin light chain MLC1 and the
associated integral protein GAP50 and acylated protein GAP45. Among the Apicomplexa,
Toxoplasma possesses the largest and most diverse repertoire of myosin motors
encompassing 11 myosin heavy chains and 6 myosin light chains (TgMLC1 to 6).
Assessment of the subcellular localization of these light chains revealed that TgMLC2, like
TgMLC1, is associated to the IMC. Further analyses uncovered the identification of a second
motor complex composed of TgMyoD-TgMLC2-TgGAP70, which is only present in
Coccidia.We have undertaken a detailed dissection of the components of these complexes
that led to the identification of the determinants that are implicated in the assembly and the
anchoring of the motor in the inner membrane complex and plasma membrane.The Nterminal extension on TgMLC1 and TgMLC2 as well as the N-terminal acylation of TgGAP45
and TgGAP70 and their conserved C-terminus are critical for the dual anchoring of these two
motors in the pellicle.
Atomic resolution basis for host cell recognition and invasion by Toxoplasma gondii
Stephen Matthews
Imperial College, London
E-mail: s.j.matthews@imperial.ac.uk
Apicomplexan parasites possess adhesion-protein complexes that play essential roles in
targeting host cells and in propagating infection. Using X-ray crystallography and the latest
NMR methodology we have embarked on several structural studies of several adhesion
complexes. We demonstrate our approach on important microneme protein complexes from
T. gondii. Not only do we provide high-resolution structural information but we reveal new
insights into binding interfaces and stoichiometry. We have also combined newly solved 3D
structures with microarrays and functional assays, and uncovered new features regarding
pathogen-receptor interactions. We are now in a position to begin constructing robust
models that will reveal the structural basis for assembly, architecture and host recognition.
New unpublished results and conclusions will be discussed.
5
The quantity and quality of African children’s IgG responses to Plasmodium
falciparum asexual stage antigens reflect protection against infection and disease
David Courtina, c, Mayke Oesterholta, Harm Huissmana, Kwadwo Kusib, Jacqueline Milet c,
Cyril Badaut c, Oumar Gayed, Will Roeffen a, Ed Remarque b, Robert Sauerwein a, André
Garcia c and Adrian J.F. Luty a.
a
Radboud University Nijmegen Medical Centre, Medical Parasitology, PO Box 9101 6500 HB,
Nijmegen, The Netherlands
b
Biomedical Primate Research Centre, Postbox 3306, 2280 GH Rijswijk, The Netherlands
c
Institut de Recherche pour le Développement (IRD), Unité de Recherche (UR) 010 « Santé de la
mère et de l’enfant en milieu tropical », Laboratoire de Parasitologie, Faculté de Pharmacie, 4 avenue
de l’Observatoire, 75006 Paris, France
d
Laboratoire de Parasitologie et de Mycologie, Département de Biologie et d'Explorations
fonctionnelles, Faculté de Médecine, Université Cheikh Anta Diop (UCAD), Dakar, Sénégal
Background
Immunoglobin G (IgG) antibody responses, and particularly those of the cytophilic
subclasses, directed to specific asexual blood stage antigens, are thought to play an
important protective role in acquired immunity to malaria caused by Plasmodium falciparum.
The evaluation of such responses to candidate antigens in longitudinal sero-epidemiological
field studies, allied to increasing knowledge of the immunological mechanisms associated
with anti-malarial protection, will help in the development of malaria vaccines.
Methods and Findings
We conducted a detailed epidemiological follow-up of 305 Senegalese children over 1 year in
order to identify those resistant or susceptible to malaria. The IgG antibody responses to six
leading candidate malaria vaccine antigens were then compared between groups of
individuals with defined, distinctly different clinical and parasitological histories with respect to
infection with P. falciparum. In age-adjusted analyses, children resistant to both malaria and
high-density parasitaemia had significantly higher IgG responses to GLURP and MSP2 than
their susceptible counterparts. Cytophilic IgG1 anti-GLURP and IgG3 anti-MSP2 antibodies
were specifically associated with this protection. Among those resistant to malaria, the levels
of IgG1 with specificity for MSP1 were associated with protection against high parasitaemia.
To assess the functional activity of antibodies, we used an in vitro parasite growth inhibition
assay with purified IgG. Samples from individuals with high levels of IgG directed to MSP1,
MSP2 and AMA1 gave the strongest parasite growth inhibition, but a marked age-related
decline was observed in these effects. Affinity-purified anti-GLURP IgG showed no such
direct growth inhibitory effect, but it did exhibit parasite growth inhibitory effects in vitro in
cooperation with monocytes.
Conclusion
Our data are consistent with the idea that protection against P. falciparum malaria in children
depends on acquisition of a constellation of appropriate, functionally active IgG subclass
responses directed to multiple asexual stage antigens. Our results suggest at least two
distinct mechanisms via which antibodies may exert protective effects. Although declining
with age, the growth inhibitory effects of purified IgG measurable in vitro reflected levels of
anti-AMA1, -MSP1 and -MSP2, but not of anti-GLURP IgG. The monocyte-dependent growth
inhibitory effects of the latter indicate the existence of at least one indirect parasiticidal
pathway.
6
Humoral Immune Response to Mixed PfAMA1 Alleles; Multivalent PfAMA1 Vaccines
Induce Broad Specificity
Kwadwo A. Kusi, Bart W. Faber, Alan W. Thomas and Edmond J. Remarque
Department of Parasitology, Biomedical Primate Research Centre, Postbox 3306, 2280GH, Rijswijk,
The Netherlands.
Apical Membrane Antigen 1 (AMA1) is a merozoite protein essential for erythrocyte invasion
and a candidate malaria vaccine component. AMA1 immune responses can protect in
experimental animal models and antibodies from AMA1-vaccinated or malaria-exposed
humans can inhibit parasite multiplication in vitro. AMA1 is polymorphic, primarily due to
selective immune pressure, and anti-AMA1 antibodies more effectively inhibit strains carrying
homologous ama1 genes, suggesting polymorphism may compromise vaccine efficacy.
Here, we analyse induction of broad strain inhibitory antibodies with a three-allele
Plasmodium falciparum AMA1 (PfAMA1) vaccine in rabbits, and determine the relative
importance of cross-reactive and strain-specific IgG fractions by competition ELISA and in
vitro parasite growth inhibition.
Immunisation with a three-allele PfAMA1 mixture yielded a higher proportion of antibodies to
epitopes common to all vaccine alleles compared with a single allele. About 80% of antiPfAMA1 antibodies that were cross-reactive between two alleles (FVO and 3D7), also
reacted with other PfAMA1 alleles in ELISA. For either one of the two PfAMA1 alleles (FVO
or 3D7) the cross-reactive fraction alone, on a weight basis, had the same functional capacity
on homologous parasites as the total affinity-purified IgGs.
These findings warrant further clinical investigation of multi-allele vaccination approaches.
7
T CELL MEMORY RESPONSES IN HUMAN VOLUNTEERS PROTECTED AGAINST
MALARIA BY REPEATED SPOROZOITE INOCULATION UNDER CHLOROQUINE
PROPHYLAXIS
Anne Teirlinck1, Matthew McCall1, Meta Roestenberg1, Geert-Jan van Gemert1, Marga van
de Vegte-Bolmer1, Joost Hopman1, Theo Arens1, André van der Ven2, Rob Hermsen1, Adrian
Luty1, Robert Sauerwein1
Departments of 1Medical Microbiology and 2Internal Medicin, Radboud University Nijmegen Medical
Centre, Nijmegen, The Netherlands
Background: Repeated inoculation of healthy malaria-naïve adult volunteers with intact
sporozoites under chloroquine (CQ) prophylaxis induces complete sterile protection against
subsequent challenge. Here we have explored the specificity and longevity of cellular
immune responses induced in these volunteers by immunisation and challenge.
Methods: Peripheral blood mononuclear cells were collected from volunteers prior to
immunisation and prior to, during & post-challenge. These cells were re-stimulated ex vivo
with whole sporozoites or schizont-infected erythrocytes (PfRBC).
Results: Sporozoite inoculation under CQ prophylaxis induced detectably increased cellular
responses to sporozoites and robust anti-PfRBC responses compared to controls.
Challenge infection further boosted cellular responses in these protected volunteers. In
unprotected naive control volunteers, challenge infection and subsequent treatment induced
equally robust anti-PfRBC, but not anti-sporozoite, responses. Cellular responses in both
groups were long-lived, being still detectable at 14 months post-challenge. Effects were most
pronounced in pluripotent effector memory cells.
Conclusions: Both pre-erythrocytic and blood-stage cellular responses are induced in
sporozoite-immunised volunteers and may contribute to protection against challenge.
Interestingly, a single patent malaria infection appears sufficient to induce equally robust and
long-lived cellular responses to blood-stage parasites in previously naive volunteers,
although it remains unknown whether these are subsequently protective.
8
In vitro cultivation of malaria hypnozoites
Anne-Marie Zeeman1, Annemarie Voorberg-van der Wel1, Jean-François Franetich2, Adrian
Luty3 Dominique Mazier2, Clemens Kocken1 and Alan Thomas1
1) BPRC, Department of Parasitology, PO-box 3306, 2280 GH Rijswijk, the Netherlands
2) INSERM/UPMC UMR S 945, Centre Hospitalier Universitaire Pitié-Salpêtrière
Faculté de Médecine Pierre et Marie Curie, 91 Bd de l'Hôpital, 75013 Paris, France
3) Medical Parasitology, Department of Medical Microbiology, University Medical Centre, St.
Radboud, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
Dormant liver stage malaria parasites (hypnozoites) cause relapse in Plasmodium vivax
infected people without new exposure to infected mosquitoes and are difficult to treat. There
are no diagnostics for infection and only primaquine provides radical cure, with risk of
complication in G6PD deficient patients. To help understand the underlying biology of
developmental arrest, persistence and activation and in order to generate a screen for
hypnozoiticidal drugs we have developed an in vitro liver stage system using P. cynomolgi, a
macaque monkey malaria. Of known parasites, P. cynomolgi has the MRCA with P. vivax. It
has very similar biology to P. vivax and is one of few other parasites that forms hypnozoites.
After P. cynomolgi sporozoite infection of primary hepatocyte cultures, small and large liver
forms were observed. The small forms remain stable for the lifetime of the culture and have a
differential drug sensitivity profile expected of hypnozoites. The larger forms mature to
release merozoites. This system can now be used to investigate parasite biology and to test
new drugs for their in vitro activity against liver stages of P. vivax type parasites, and in
particular to screen for those with activity against the hypnozoites.
9
Quantitative Determination of Plasmodium vivax Gametocytes by Real-Time
Quantitative Nucleic Acid Sequence Based Amplification in Clinical Samples
Martijn Beurskens1, Pètra Mens2, Henk Schallig2, Din Syafruddin3, Puji Budi Setia Asih3,
Rob Hermsen1 and Robert Sauerwein1
1) Dept. Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The
Netherlands,
2) KIT Biomedical Research, Koninklijk Instituut voor de Tropen (KIT) / Royal Tropical Institute,
Amsterdam, The Netherlands; Centre for Infection and Immunity Amsterdam (CINEMA), Division of
Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Centre, Amsterdam, The
Netherlands;
3) Eijkman Institute for Molecular Biology, Jakarta, Indonesia.
Plasmodium vivax accounts for over half of all malaria cases outside Africa, with an
estimated 130 to 435 million new infections annually and 75 million acute clinical episodes. It
is the predominant Plasmodium species in south and central Asia, north Africa, the Pacific
and the Americas and, contrary to what is generally assumed, P. vivax infections may not
always follow a benign course. Microscopical detection of Plasmodium vivax gametocytes,
the sexual life stage of this malaria parasite, is insensitive because P. vivax parasitaemia is
low. To detect and quantify gametocytes more sensitive, quantitative real-time Pvs25-QTNASBA based on Pvs25 mRNA was developed and tested in two clinical sample sets from
three different continents. Pvs25-QT-NASBA is highly reproducible with low inter-assay
variation and reaches a sensitivity approximately 800 times higher than conventional
microscopical gametocyte detection. Specificity was tested in 104 samples from P. vivax, P.
falciparum, P. malariae, P. ovale infected patients. All non-vivax samples were negative in
the Pvs25-QT-NASBA; out of 74 PvS18-QT-NASBA positive samples 69% were positive in
the Pvs25-QT-NASBA. In a second set of 136 P. vivax microscopically confirmed samples,
gametocyte prevalence was 8%, while in contrast 66% were positive by Pvs25-QT-NASBA.
The data suggest that the human P. vivax gametocyte reservoir is much larger when
assessed by Pvs25-QT-NASBA than by microscopy.
10
The (not so) simple life cycle of Giardia
Hein Sprong1, Simone M. Cacciò2, and Joke W. B. van der Giessen1
1) Laboratory for Zoonoses and Environmental Microbiology, National Institute for Public Health
and Environment (RIVM). e-mail: hein.sprong@rivm.nl
2) Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di
Sanità, Viale Regina Elena 299, Rome, 00161, Italy.
Giardiasis is a gastrointestinal disease of humans and animals, which causes major public
and veterinary health concerns worldwide. The causative agent, Giardia duodenalis, is a
protozoan with a simple life cycle comprising rapidly multiplying trophozoites in the intestine,
and the production of cysts which are passed in the faeces and shed into the environment.
Transmission may occur from animals to humans or from humans to animals. However,
animals are also infected with host-adapted genotypes. A molecular epidemiological
database from G. duodenalis field isolates has been generated by ZoopNet, an European
network of public and veterinary health Institutions. Here, we performed an extensive genetic
characterization of 978 human and 1440 animal isolates, which together comprises 3886
sequences from 4 loci, allowing genotyping at different levels of resolution. The zoonotic
potential of G. duodenalis assemblage A and B is evident when studied at the level of
assemblages, sub-assemblages, and even at each single locus. However, multi-locus
sequence genotyping (MLG) using 3 loci identified only 2 MLGs within assemblage A with
zoonotic potential, and none within assemblage B. Interestingly, mixed genotypes in
individual isolates was repeatedly observed. Possible explanations are the simultaneous
uptake of genetically different cysts from an environmental source or subsequent exposure of
an already infected host with a different type of cysts. Other explanations are the presence of
substantial allelic sequence heterogeneity and sexual recombination, particularly among
assemblage B isolates. In conclusion, this powerful and unique molecular database has the
potential to tackle intricate epidemiological questions regarding protozoal diseases.
11
The core proteome of the Schistosoma mansoni eggshell
Saskia deWalick1, Bas W.M. van Balkom2, Ya-Ping Wu3,4, Michiel L. Bexkens1, Aloysius G.M.
Tielens1, Jaap J. van Hellemond1
1) Dept.Medical Microbiology & Infectious Diseases, Erasmus MC, Univ.Medical Center Rotterdam
2) Dept. Biomolecular Mass Spectrometry, Bijvoet Center For Biomolecular Research, Utrecht Univ.
3) Dept. Haematology, University Medical Centre Utrecht, Utrecht
4) Dept. Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht
Schistosomiasis is an important parasitic disease affecting over 200 million people
worldwide. Adult schistosomes are able to maintain themselves for decades in the veins of
their mammalian host. Despite its abundant exposure to the immune system of the host, this
parasite apparently prevents an adequate immune response. Schistosome eggs and their
secretions are very immunogenic. They skew the host immune response towards a Th2
response and initiate granuloma formation. Pathology due to schistosome infection is mainly
caused by the inflammatory response directed against the parasite’s eggs trapped in the host
tissue.
We previously unraveled the proteome of the tegument of the adult worms. We now
investigated the eggshell and identified its proteins by mass spectrometry.
Schistosoma mansoni eggs were isolated from livers of infected hamsters. After hatching
of the eggs, the eggshells were collected and crushed into small fragments in a microdismembrator. Attached cellular material was removed in five consecutive steps, after which
the remaining material of the eggshell was purified. These purified eggshell fragments were
used for protein identification by mass spectrometry.
This study identified a relatively small number of proteins, 37 in total, to be part of the
schistosomal eggshell. Among the identified eggshell proteins, expected schistosomal egg
antigens were identified, as well as expected structural proteins, but also glycolytic enzymes
and other non-structural proteins and some schistosome-specific proteins with no analogs in
other species. Some of the identified proteins are known to be immunogenic.
In conclusion, Schistosoma mansoni eggshell is constructed from a wide range of
proteins. It is not only produced from specific eggshell proteins, but also from proteins
randomly available at the time of eggshell formation. The relevance of egg deposition for the
immune reaction by the host will be discussed.
12
Trichinella spiralis antigens suppress mouse macrophage and dendritic cell
activation.
M. Langelaar, C.R. Aranzamendi, F. Franssen, N. Youssuf, P. van der Ley, J. van der
Giessen, H. Sprong and E. Pinelli.
Centre for Infectious Disease Control Netherlands, National Institute of Public Health and the
Environment (RIVM), 3720 BA Bilthoven, The Netherlands.
The beneficial as well as the detrimental effect of helminth infections on allergy and autoimmune diseases has been previously reported. However, the mechanisms underlying this
association are not fully understood. Here we aim at evaluating the effect of antigens derived
from Trichinella spiralis (T.spiralis) on the initial events of the immune response. To activate
macrophages of the J774 cell line or bone marrow derived dendritic cells (DC) we used
E.coli-LPS (LPSEc). We measured nitric oxide produced by macrophages and cell surface
molecule expression and cytokines produced by DC. Nitric oxide production, as a hallmark of
macrophage activation, was significantly suppressed when cells were incubated with T.
spiralis antigen in combination with LPSEc .Both excretory/secretory (ES) antigen as well as
crude larval antigen suppressed APC maturation. Maturation of DC as expressed by the upregulation of the surface molecules MHCII, CD40, CD80 and CD 86 and cytokine production
(IL-1alpha, IL-6, IL-10, IL-12p70 and TNF-alpha) was also significantly inhibited when cells
were incubated with T. spiralis ES and LPSEc. Our results suggest that T. spiralis antigens
interfere with APC maturation via TLR4 in a specific way as a means to prevent APC
activation induced by LPSEc.
A role for CD14 in the suppression of DC maturation by Trichinella spiralis ES
C.R. Aranzamendi, M. Langelaar, F. Franssen, P. van der Ley, J. van Putten and E. Pinelli.
Centre for Infectious Disease Control Netherlands, National Institute of Public Health and the
Environment (RIVM), 3720 BA Bilthoven, The Netherlands.
Maturation of Dendritic Cells (DC) is an important process required for initiating the adaptive
immune response. In this process the activation of TLRs play a pivotal role. We have
previously shown that excretory/secretory (ES) antigens derived from Trichinella spiralis
(TspES) suppress LPS-induced DC maturation in vitro. However, suppression of surface
molecule expression and cytokine production was observed when Escherichia coli LPS but
not when Neisseria meningitidis LPS was used to activate the DC. The present study aims at
studying the molecules and mechanisms involved in suppression of DC maturation by
TspES. Considering that E. coli LPS but not N. meningitidis LPS requires CD14 to activate
DC, we decided to compensate the expression of CD14 by adding CD14 transfected
HEK293 cells. As a result, partial recovery of DC maturation induced by E. coli LPS was
observed. Our results indicate that the suppressive effect of TspES on DC
maturation depends on the nature of the TLR4 ligand used. In addition, we show that CD14
plays an essential role in this process.
13
The effect of Trichinella spiralis and Toxoplasma gondii on serologic responses in
swine in concomitant infections.
G. Bokken1, M. Opsteegh3, E. van Eerden1, M. Augustijn2, L. Graat4, A. Tenter5, J. van der
Giessen3, F. van Knapen1 and A. Bergwerff1
1 - Institute for Risk Assessment Sciences (IRAS), Division of Veterinary Public Health, Utrecht
University, Utrecht, The Netherlands
2 – Departement Gezondheidszorg Landbouwhuisdieren, Utrecht University, Utrecht, The Netherlands
3 – National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
4 - Quantitative Veterinary Epidemiology Group, Animal Sciences Group, Wageningen University,
Wageningen, The Netherlands.
5 – Institut für Parasitologie, Zentrum für Infektionsmedizin, University of Veterinary Medicine (TIHO),
Hannover, Germany
A common transmission route of Trichinella spiralis and Toxoplasma gondii in humans runs
through the consumption of infected undercooked pork. In the Netherlands, prevalence of
Trichinella in pigs is negligible, whereas Toxoplasma prevalence is up to 5.6%.
Paradoxically, monitoring of muscle Trichinella larvae in pigs at slaughter is obligatory,
whereas the control of toxoplasmosis is not regulated. Another method used to determine
Trichinella status is detection of immune responses to the parasite. A possible alternative,
the prediction of Trichinella status, may be based on the serological assessment of the
Toxoplasma status of a herd, which is substantiated by the similar transmission routes of the
parasites in pigs, i.e. through eating of infected rodents. However, co-infections of T .gondii
and T. spiralis might influence their serological responses and consequently question the
reliability of a T. gondii response as indicator in the assessment. To study this possible
influence, pigs were simultaneously and serially infected and serologic responses were
analyzed with ELISAs.
Results suggested no significant effect of T. spiralis and T. gondii on humoral IgG responses
of the animals against the parasites in concomitant infections. This finding forms the basis for
further investigations on Trichinella status assessment through detection of anti-Toxoplasma
antibodies.
14