CHARLES UNIVERSITY PRAGUE
1st FACULTY OF MEDICINE
Department of Tropical Medicine
HOST´S IMMUNE RESPONSES DURING
THE INFECTION OF MAMMALS BY
THE BIRD SCHISTOSOME TRICHOBILHARZIA REGENTI
Summary of Ph.D. Thesis
2004
Mgr. Pavlína Kouřilová
Supervisor: doc. RNDr. Libuše Kolářová, CSc.
Curriculum vitae:
Thesis supervisor: doc. RNDr. Libuše Kolářová, CSc.
Examination committee:
Born: Juli 2, 1976, Městec Králové, the Czech Republic
1994-1997
Bachelor study in Biology at Faculty of Science, Masaryk University in Brno
1999
Masters degree (Mgr.) in Systematic biology and ecology at Faculty of Science,
Prof. RNDr. Jaroslav Kulda, CSc.
Department of Parasitology, Masaryk University in Brno, the topic of the diploma
Prof. RNDr. Tomáš Scholz, CSc.
thesis: Spatial distribution of monogeneans on the gills of chub (Leuciscus cephalus
Prof. RNDr. Petr Volf, CSc.
L) in conditions of the environmental stress
Doc. MVDr. Iva Dyková, DrSc.
2000
Certificate of Pedagogy in Biology, Faculty of Science, Masaryk University in Brno
Doc. RNDr. Jan Kopecký, CSc.
1999-present
Ph.D. study at the Department of Tropical Medicine, 1 st Faculty of Medicine, Charles
University Prague
Work positions:
Reviewers:
2002-2003
Prof. Dr. Andreas Ruppel
Doc. MVDr. Iva Dyková, DrSc.
Assistant at the Department of Medical Microbiology, 3rd Faculty of Medicine, Charles
University Prague
2003-present
Doc. RNDr.Vladimír Holáň, DrSc.
Research assistant, Department of Parasitology, Faculty of Science, Charles University
Prague
Participation on grant projects:
The Wellcome Trust: Collaborative Research Initiative Grant: The biochemical and immunological
properties of Trichobilharzia proteases (2003-2006).
GA UK 118/2003/B-BIO/PrF): Migration of bird schistosomes in vertebrates (2003-2005)
Czech Ministry of Health (Grant No. NJ7545-3/2003): Bird schistosomes in water bodies as a risk factor
in the development of neurophysiological disorders (2003-2005)
Grant Agency of the Czech Republic (Grant No. GACR 524/03/1263): Interactions of bird schistosomes
with tissues and organs of vertebrates (2003-2005)
Czech Ministry of Education (Grant No. FRVS 1861/2004): Migration of bird schistosomes in
vertebrates (2004)
Czech Ministry of Health (Grant No. NJ/6718-3): Patogenesis of primoinfections and reinfections by
cercariae of bird schistosomes (period: 2001-2003)
Grant Agency of the Czech Republic (Grant No. GACR no. 524/00/0622): Migration of bird
schistosomes within hosts (2000-2002)
Ph.D. Thesis defence: January 10 (2005) at 10: 30h, Department of Parasitology, Viničná 7, Prague 2
Grant Agency of the Czech Republic (Grant No. GACR no. 204/00/1095): Penetration of trematode
larvae
through
surface
epithelia
of
vertebrate
and
invertebrate
hosts
(2000-2002)
Charles University Grant Agency (Grant No. 38/2000/1.LF): Development of the immune response
against bird schistosomes (2000-2002)
Introduction:
Brock, 1986; Haas and Pietsch, 1991; Horák et al., 1999). The larvae penetrating into the skin of humans
evoke usually allergic skin reaction – cercarial dermatitis. The spectrum of the species causing cercarial
Parasitic helminths comprise several diverse groups of metazoan parasites that infect more than
two billion people and animals worldwide (Colley et al., 2001). They afflict various tissues and organs of the
body causing frequently chronic infections and are responsible for malnutrition, fever, diarrhoea, hematuria,
blindness, and a variety of other clinical maladies, sometimes with life-threatening consequences. Among
dermatitis is considerably broad, currently covering the species belonging at least to 8 genera
(Heterobilharzia,
Orientobilharzia,
Schistosoma,
Schistosomatium,
Austrobilharzia,
Bilharziella,
Trichobilharzia and Gigantobilharzia) (Horák and Kolářová, 2001). It is not surprising then, that this disease
is occurring worldwide (except Antarctica), in Europe being not long ago reported from Poland (Żbikowska,
these parasites, schistosomes represent digenetic trematodes the adults of which live in the blood system of
2004; Żbikowska, 2003), France (Léger and Martin-Lœhr, 1999), Germany (Allgöwer and Matuschka,
vertebrates, predominantly of birds and mammals. At present, there are 14 genera in the family
1993), Italy (Nobile et al., 1996; Golo et al., 1998), Iceland (Kolářová et al., 1999), Norway (Thune, 1994),
Schistosomatidae; the most numerous and worldwide occurring is the genus Trichobilharzia covering more
Sweden (Thors and Linder, 2001), Switzerland (Eklu-Natey et al., 1985; Chamot, 1998), and the Czech
than 40 species of bird parasites (Horák et al., 2002). These parasites cause trichobilharziasis of ducks and
Republic (Kolářová et al., 1989). The most frequent causative agents of cercarial dermatitis belong to the
geese (Wojcinski et al., 1987), and in humans, they are the most frequent causative agent of cercarial
genus Trichobilharzia (Horák et al., 2002), with at least two species occurring also in the Czech Republic –
dermatitis, an allergic cutaneous disease (Kolářová et al., 1997). Moreover, their ability to migrate into some
internal organs during experimental infections of mammals (e.g. Hrádková and Horák, 2002) revealed other
the visceral schistosome T. szidati (Kolářová et al., 1992) and the nasal schistosome T. regenti (Horák et al.,
1998a).
potential health risks connected with this infection. From a medical viewpoint, however, the most important
species are represented by specific parasites of humans belonging to the genus Schistosoma. Currently, over
200 million people in tropical and subtropical regions suffer from schistosomiasis (WHO estimation), the
disease being caused mainly by three species (Schistosoma mansoni, S. haematobium and S. japonicum).
Schistosomes, as many helminths, have a complex multistage life cycle. The larval stage called
cercaria is able to directly penetrate into the skin of a vertebrate and undergo a complex process of
transformation to another larval stage –schistosomulum. Cercaria/schistosomulum transformation connected
with ultrastructural and molecular changes of the larval surface as well as a conversion to an anaerobic
metabolism seems to be a crucial step for survival and following development of schistosomes within their
hosts. Providing that the specific (compatible) host is infected, schistosomes mature and lay eggs. These
very eggs are responsible for the most serious manifestation of infection and pathological changes in the
tissue being stimulators of cell immune responses which lead to a granuloma formation around the eggs
(Boros and Warren, 1970). Moreover, antigenic substances excreted from larval stages (miracidia) through
sub-microscopic pores in the egg shell elicit an acute inflammation and are hepatotoxic (Doenhoff et al.,
1981). When eggs enter fresh water, ciliated miracidia hatch from the eggs and penetrate into the body of
intermediate snail hosts. The development in the intermediate host goes through two generations of
sporocysts the second of which produces cercariae in the course of several weeks.
Infection of incompatible hosts, thus those who disable a long-term survival of the parasite
because of physiological, biochemical or immune responses incompatibility, does not lead to sexual
maturation of the parasites and consequently termination of the life cycle of schistosomes. However, during
primary infections partial schistosome migration in the body of an incompatible host is possible; the
migration has been documented in several studies of experimentally infected animals (e.g. Appleton and
Within their vertebrate hosts, schistosomes undergo a complex migratory route which is initiated
by the penetration of cercariae into the skin. Thus, the skin represents the primary target organ and the most
important barrier against the larvae penetration into an organism.
The time that the larvae spend in the skin and the speed of schistosomula migration though the skin layers
differ profoundly in various schistosome species and are probably dependent also on the host species that is
infected. While in a human skin vast majority of S. mansoni or S. haematobium schistosomula stays for 24h
in the epidermis and can be found in dermal vessels within 48-72 hours of infection, it seems likely that S.
japponicum larvae migrate faster being able to reach dermal vessels within 24h (He et al., 2003). In the
mouse, majority of S. mansoni schistosomula leaves the skin between 48h and 5 days (Wilson et al., 1986).
Concerning bird schistosomes, so far studied migration of the species has shown that the speed of migration
could be also faster in both compatible and incompatible hosts where schistosomula capable of further
migration usually leave the skin within 1.5 - 3 days of infection (Haas and Pietsch, 1991; Hrádková and
Horák, 2002).
The skin as the largest organ of the body represents also an important immunocompetent organ in
which components of both innate and acquired immune responses operate. The innate immune system
provides the first line of defence, allowing to identify and respond to infectious agents through the binding
of a number of receptors termed pattern recognition receptors (PRRs) to various pathogen-associated
molecular patterns (PAMPs) (Janeway and Medzhitov, 2002). These are often represented by pathogen´s
carbohydrates or lipid molecules (McGuinness et al., 2003). Moreover, this early recognition of pathogen´s
molecules is critically important for the stimulation, development and character of the acquired immune
response (Medzhitov and Janeway, 1997; Akira et al., 2001). Epidermal and dermal cells together with
incoming cells of the immune system release an array of mediators into the microenvironment that form a
also in the case of bird schistosome infection. Moreover, the secretion of S. mansoni schistosomula contains
network which is important for the initiation of an appropriate immune response against invading parasites
a 16.8 kDa anti-inflammatory factor which appears to have an immunomodulatory function during the skin
(Weiss et al., 2004).
phase of infection (Ramaswamy et al., 1995). The authors did not detect the mentioned molecule in the T.
Despite above mentioned facts, the importance of the skin immune response during a schistosome
infection has been underestimated in the past, perhaps particularly due to the findings that over 90% of S.
ocellata secretion and suggested that the absence of it could be the reason for more severe skin reaction in
the case of bird schistosome infection.
mansoni larvae successfully negotiate the skin barrier in their compatible hosts within first 5-7 days of
It appears that the interaction between schistosomula and cutaneous cells is crucial for further
infection (Mangold and Dean, 1983; Mountford et al., 1988). In contrast, majority of bird schistosomes
parasite existence within the host. In particular, keratinocytes, Langerhans cells, mast cells, tissue
during the infection of incompatible hosts dies in the skin (Macfarlane, 1949; Haemmerli, 1953, personal
macrophages, granulocytes, lymphocytes and fibroblasts belong to the major cellular components involved
observation), although the study on T. ocellata migration in incompatible hosts revealed that about 36% of
in the skin immune response (Weiss et al., 2004). The parasite larvae closely interact with cutaneous
schistosomula are able to leave the skin but usually do not survive longer than 5 days (Haas and Pietsch,
immunocompetent cells, however, the nature and immunological consequences of these interactions have not
1991). However, the key issue whether the larvae are killed by the immune system response, or whether they
yet been fully understood.
die because of other factors of incompatibility, is still not clearly determined.
Regulation of immune events in the skin during infection by schistosomes has been recently
Cercarial dermatitis
reviewed by Mountford and Trottein (2004). Schistosoma larvae rapidly induce oedema and dilatation of
Cercarial dermatitis is an allergic skin response developing after penetration of the larvae of the
peripheral blood vessels followed by neutrophil and later also by other cell influx into the dermis. The
family Schistosomatidae into the skin. Currently, the most frequently reported cases of this disease are
cellular infiltrates are coordinated by the secretion of pro-inflammatory cytokines and chemokines; within
caused by the species of the genus Trichobilharzia (Horák et al., 2002). In general, several contacts seem to
first 2 days of infection, production of macrophage inflammatory protein MIP-1α and MIP-1β, tumour
be necessary for its development but there are exceptions (Skirnisson, personal communication). Various
necrosis factor TNF-α, IL-1β, IL-6, IL-12 and IL-18 has been detected. These cytokines are not only
species of animal schistosomes usually evoke similar reactions (Brackett, 1940; Batten, 1956; Appleton and
involved in the skin infiltration by immune cells, but also promote their activation. The release of these
Lethbridge, 1979, Wiley et al., 1992) the intensity of which is dependent above all on sensitization of the
cytokines is accompanied also by the production of regulatory mediators IL-1ra, IL-10 and prostaglandins
host and the number of penetrating cercariae. Human schistosomes may cause milder skin reactions (Basch,
PGE2 and PGD2 (Mountford and Trottein, 2004; Angeli et al., 2001) which are likely to operate in
1991); the reason for this could be explained by the production of already alluded 16.8 kDa anti-
downregulation of inflammation coordinating the extent and character of immune responses. Other studies
inflammatory factor (Ramaswamy et al., 1995) which was described in excretory/secretory products of S.
revealed that Th2–type cytokine responses dominate transiently in the skin and draining lymph nodes of
mansoni, but was absent in T. ocellata. Nevertheless, in some cases, infection by both human and animal
mice early after schistosome entry into the skin (Kumar and Ramaswamy, 1999; Betts and Wilson, 1998). In
schistosomes cause indistinguishable severe hypersensitivity reactions (Kolářová, personal communication).
concrete terms, mRNA for IL-10 and IL-4 were increased in the skin within 16h after infection (Kumar and
Although not frequently reported, in some cases, repeated contacts with various species of cercariae might
Ramaswamy, 1999). Later, schistosomula evoke the production of both interferon-γ (IFN-γ) and IL-4 in the
lead to desensitization (Macfarlane, 1949; Berg and Reiter, 1960).
skin and skin draining lymph nodes (Hogg et al., 2003).
Although histopathological studies of cercarial dermatitis are relatively numerous, almost nothing
Cercariae and schistosomula, migrating through the skin, are also capable of modulating host
is known about the mechanism responsible for the induction of immunopathology of the skin. Initial
immune responses (Angeli et al., 2001; Ramaswamy et al., 2000; Ramaswamy et al., 1995). S. mansoni
manifestations may appear within several minutes following penetration of cercariae; the immune response
larvae possess an ability to produce and also induce production of several types of eicosanoids by skin cells
is usually mild during first contact of the host with the parasite. Petty maculopapular eruptions may appear
including PGE2 (Fusco et al., 1984) that stimulates IL-10 and inhibits IL-12 production (van der Pouw
most often within 1h post infection (p.i.), sometimes also oedematous reaction and erythema accompanied
Kraan et al., 1995; Harris et al., 2002). Comparison of eicosanoid production by human schistosome S.
by intensive itching can develop. Histologicaly, the reaction represents an acute inflammatory response with
mansoni and bird schistosome T. ocellata cercariae revealed that both schistosomes produce the same types
the influx of polymophonuclear leukocytes present in the skin by 6h p.i. (Augustine and Weller, 1949;
and similar quantities of prostaglandins, leukotrienes and hydroxyeicosatetranoic acids (Nevhutalu et al.,
Olivier, 1953). Severe allergic response develops in repeatedly infected hosts. Primary skin itching appears
1993), thus a similar regulation of immune responses within first hours of infection seems to be probable
within 4-20 min and is associated with transient development of maculae of 1-5 mm, which can be replaced
by papulae reaching 3-8 mm in diameter. The third and forth day vesicles can be formed. The papulae
present, covering one mammalian species – Schistosoma nasale (for review see Khalil, 2002) and eight
usually disappear by 10 days p.i., but in more severe cases, they can remain for 20 days (Bechtold et al.,
representatives of the genus Trichobilharzia (T. nasicola, T. rodhaini, T. spinulata, T. aurelani, T. duboisi,
1997) leaving pigmented spots which can outlast 1 month or longer (Olivier, 1949). Severe infections can
T. australis, T. arcuata and T. regenti; Horák et. al, 1998a). T. regenti is the only species in which the
also evoke fever, intensive itching of afflicted parts, skin draining lymph nodes enlargement, vomiting or
migration route has been studied in detail revealing marked differences from the migration of visceral
diarrhoea (Chamot et al., 1998; Kolářová et al., 1994).
schistosomes. After penetration into the host skin, schistosomula invade peripheral nerves, and migrate
Haemmerli (1953) describes three phases of cellular responses (leukocytic, lymphocytic and
further through the spinal cord and brain to the nasal area (Horák et al., 1999; Hrádková and Horák, 2002)
histiocytic) which lead to elimination of schistosomula of the genus Trichobilharzia from the human skin.
being able to reach it within 12 days of infection (Hrádková and Horák, 2002). Adult worms occur in the
During the first phase (3-9h p.i.), a damage of the parasite surface is not visible. After 24h, changes observed
nasal area by day 14 p.i. (Horák et al., 1999). Also this infection causes characteristic pathologies known for
as tegument degradation develop (lymphocytic phase) and during the histiocytic phase the parasites are
visceral schistosomes (inflammation and granuloma formation) (Kolářová et al., 2001), moreover, during
killed and destroyed (36-52h p.i.). Cellular infiltration of epidermis and dermis by polymorphonuclear
schistosomula migration through the CNS tissue, neuromotory complications such as leg paralysis and
leukocytes and lymphocytes is replaced within 48-50h p.i. by influx of eosinophils (Macfarlane, 1949).
balance/orientation disorders have been observed (Horák et al., 1999).
Following tissue repair is accompanied by parakeratosis of adjacent tissue and cell melanotic pigmentation
Laboratory infections of incompatible mammalian hosts by bird schistosomes revealed that also
(100h p.i.). The disease may be aggravated by a secondary bacterial infection due to the skin damage during
in this type of the host, some parasites are able to escape from the skin and migrate into other organs.
scraping of intensively itching areas (Kolářová et al., 1989).
Migrating parasites were recorded during primary infections caused by various species of visceral
Diagnosis of the disease is usually based on immunological diagnostic methods; a direct
schistosomes: immature T. ocellata, T. szidati, T. stagnicolae, T. physellae, T. brevis, Trichobilharzia sp.,
detection of schistosomula in skin biopsies can be successful only short time after exposure (usually by 24-
Bilharziella polonica and Ornithobilharzia sp. were detected in the lungs of mice, hamsters, gerbils, rabbits
72h) (Haemmerli, 1953). Several methods such as the ”Cercarienhűllenreaktion”, ELISA or IFAT based on
and rhesus monkeys (Olivier, 1953; Appleton and Brock, 1986; Haas and Pietsch, 1991; Horák and
detection of antibody responses against cercarial antigens have been developed. For infections by both
Kolářová, 2000; Bayssade-Dufour et al., 2001), and additionally in the liver, kidney, heart and intestines
human and bird schistosomes, the main antigenic structure seems to be carbohydrate-rich glycocalyx of
(Haas and Pietsch, 1991). Experimental murine infections by T. regenti also revealed that this species is able
cercariae (Samuelson and Caulfield, 1985; Horák et al., 1998b). Although these methods are sensitive and
to exit the skin and migrate into the CNS of mice (Horák et al., 1999; Hrádková and Horák, 2002). In the
easily to perform, they can not be used for differential diagnosis between cercarial dermatitis caused by
mouse spinal cord, living schistosomula were detected during a period of 2 days till 21-24 days p.i.
animal schistosomes and infection by human schistosomes which are causative agents of schistosomiasis
(depending on the mouse strain); in the mouse brain from 3 days p.i until 24 days p.i. (Hrádková and Horák,
due to the cross-reactivity of anti-parasite antibodies.
2002). Contrary to infection of birds, the parasites never mature in mice and schistosomula die during
various intervals p.i. (Horák et al., 1999).
Migration into other organs
Clinical symptoms and pathologies caused by schistosome infection of incompatible hosts may
To reach the final sites of parasitism, schistosomula migrate into other organs via the vascular
reflect migratory routes of the larvae in the body as well as host responses in afflicted organs. The
circulation having the lungs as the first organ afflicted by the vast majority of schistosomes (Basch, 1991).
histological studies of the CNS tissue of mice with primary T. regenti infection showed both intact and
After the lung phase which proceeds within 2-10 days of infection depending on the schistosome and the
destroyed worms on day 10 p.i. (Kolářová et al., 2001). Cross sections of various parts of the spinal cord and
host species, schistosomula are carried through the left heart into the systemic circulation. Most schistosome
brain showed the parasites located in the white and grey matters as well as in meninges of mice. Histological
species mature in the visceral organs where the adults usually occur in portal or mesenterial veins or venous
examination of Kolářová et al. (2001) showed that the establishment of T. regenti in the mammalian CNS
plexus of urinary bladder. The females lay eggs some of which leave the host and reach the outside
with subsequent pathologies (a cellular infiltration of a spongy tissue around the immature parasites,
environment. However, some are trapped in the tissue causing inflammatory reactions and being the source
dystrophic and necrotic changes of neurons) may lead to changes in neurobehavioral reactions similar to
of the most serious pathologies accompanying the infection (Doenhoff et al., 1986).
those described for human and animal neuroinfections caused by the genus Schistosoma (e.g. Scrimgeour
A smaller group of schistosomes is represented by flukes the adults of which occur in the nasal
area of final hosts. This unusual location of adult schistosomes has been described only for nine species till
1991; Lambertucci, 1993; Fiore et al., 1996). In certain cases, the T. regenti infections of mammals can be
also manifested by neuromotor symptoms known in bird infections (Horák et al., 1999).
Aims of the thesis:
Summary of the results:
The skin represents the first line of defence against the schistosome infection, and therefore, the
Penetration of T. regenti cercariae into the skin of an incompatible host had an immediate effect
first place of interaction between the parasite antigens and the host´s immune system. Bird schistosomes,
on inflammatory reactions at this site. Substantial skin oedema has been recorded within 1h of each
when infecting mammals, are not able to mature, however, a few parasites can escape from the skin and
infection, with dramatic increases seen after the third and fourth infections. Cercariae, present at this time in
migrate into other organs, where they become a potential source of health complications in certain cases. At
the upper layers of epidermis starting the penetration process, release the content of penetration glands, and
present, very little is known about the immune response against these parasites in the skin and their eventual
this secretion is likely to affect significantly the initiation of inflammatory processes. Moreover, the surface
pathological effect in affected organs such as the lungs or CNS. It has not been known yet what is the major
glycocalyx, which is shed within a few hours of infection, may serve as other source of antigens and
reason for the parasite death in an incompatible host and what is the role of immune system components by
allergens. The larvae evoked a fast reaction also during the first infection of mice. Within first few hours of
this event.
infection, T. regenti schistosomula in the skin stimulated the production of acute phase cytokines IL-1ß and
The presented thesis proposes the analysis of the immune response during the infection of
IL-6. This was accompanied by progressively increasing amounts of IL-12. The amount of IFN-γ produced
mammals by bird schistosomes with a particular emphasis on the skin immune response. A murine model of
by cutaneous cells was rather low in all culture supernatants. Cytokines associated with Th2-type immune
infection by bird nasal species Trichobilharzia regenti (Horák et al., 1998a) was chosen for most
responses were scarce in culture supernatants of 1x infected skin, in contrast, re-infections of the skin
experimental studies. In the case of its escape from the skin, this highly neurotropic parasite may be a source
evoked a release of a large amount of IL-4 and IL-10 already within 1h of infection, which subsided after
of balance/orientation disorders and even leg pareses of infected mammals (Horák et al., 1999).
48h. This early IL-10 production in the affected skin may have both the positive regulatory effect on
reduction of inflammation and the effect on modulating the tissue response towards Th2. We conclude that
The particular aims of the thesis:
IL-10 profoundly regulates the skin inflammation and, as in other systems, protects the host against
1. characterization of cellular components involved in innate and adaptive immune responses in the
excessive pathology and suppresses the expression of allergic symptoms. Although the immune response in
skin during primary and repeated infections
IL-10 deficient mice after bird schistosome infections has not been studied yet, we could expect much wilder
2. detection of inflammatory mediators, secreted by cells in the skin and in the skin draining lymph
reaction in this type of the host leading to severe immunopathological complications.
nodes, which are responsible for the development of cercarial dermatitis and polarization of the
Histamine, secreted from activated mast cells and basophils, was one of the first mediators
adaptive immune response
detected mainly after repeated infections, but also after first infection. Similarly, the number of mast cells
3. antibody response analysis against various developmental stages of bird schistosomes
was increased also in 1x but more in 4x infected skin, in which most of them were in the process of
4. evaluation of the parasite survival and migration into other organs of an incompatible host during
degranulation. Histamine is preformed and stored in mast cell secretory granules, therefore, it can be
primary and repeated infections
released immediately after stimulation. As the production of histamine in 1x infected mice occurred in the
5. description of pathological changes and clinical symptoms of the CNS infection of
absence of parasite-induced Ab response, we infer that mast cells, in response to a primary infection with
immunocompetent and immunodeficient hosts.
Trichobilharzia, are activated and degranulate in an IgE-independent mechanism. However, the quantity of
histamine produced by skin biopsies was substantially greater in 4x compared with 1x infected mice and
therefore, the IgE-dependent mast cell degranulation probably plays the dominant role in the skin
hypersensitivity reaction.
As a consequence of histamine and other inflammatory mediators release, we observed
vasodilatation (erythema) and later recruitment of inflammatory cells into the sites of infection. Histological
observation showed that the infection by T. regenti lead to a rapid development of an acute inflammatory
reaction and causes severe pathological changes (oedema, parakeratosis, perineuritis and neuritis, vasculitis
and foliculitis) in the tissue, especially in the re-infected skin. More detailed immunohistochemical analysis
of the dermal site of infection confirmed the presence of 7/4 + cells (neutrophils), Gr-1+ cells (granulocytes),
CD4+ T cells, F4/80+ cells (macrophages) and I-a+ cells (MHC II) in both primoinfected and re-infected skin.
skin of primary infected animals. These parasites migrated in a large proportion into the CNS being able to
Parasite residues within cellular infiltrates were in some cases present in the dermis up to day 8 p.i. In a
reach it within 2-3 days. It was also confirmed that T. regenti (at least in a mouse model) does not utilize the
sharp contrast, the skin of immunodeficient SCID mice was not heavily inflamed even in the case of
migration through the lungs as there was detected only approx.1% of total number of the parasites. We
challenge infections.
concluded that the vast majority of the larvae (approx. 90%) do not migrate beyond the mouse skin. Re-
In response to soluble cercarial antigen, the sdLN cells from 1x infected mice showed higher
infection experiments in which mice were infected three times by normal cercariae and the last infection was
proliferation than cells from 4x infected mice. The excessive production of IL-4 and IL-10 by cutaneous
performed by radio-labelled parasites showed that the migration of larvae from the skin is scarce (less than
cells in re-infected skin was accompanied by elevated production of IL-4, IL-5 and IL-10 in sdLN. This was
1%) and the larvae are killed faster than in the case of primoinfection. These results were confirmed by the
in contrast to cytokine production of sdLN in primoinfected animals where cytokine INF-γ was highly
histological study with another strain of immunocompetent mice (hairless mice of the hr/hr strain). It seems
abundant. The high level of IL-4 had an obvious effect on antibody production with a clear dominance of
therefore, that a severe reaction and extensive inflammatory deposits around the larvae in the re-infected
Th2-associated antibody isotypes IgG1 and IgE. The profoundly enhanced production of total IgE after the
skin of immunocompetent animals are host-protective against subsequent parasite migration. On the
fourth infection completed the main hallmarks of type I/allergic hypersensitivity response and we believe
contrary, the infection of immunodeficient SCID mice led to the migration of schistosomula into the CNS
that IgE dependent mast cell degranulation plays a pivotal role in pathogenesis of cercarial dermatitis.
regardless of the number of infections.
Histopathological analysis of the CNS tissue of primary infected immunocompetent mice
revealed the presence of schistosomula in both white and grey matters of the spinal cord and brain with the
A comparison of the antibody response against various developmental stages of bird and human
thoracic spinal cord as the most affected area. The subarachnoidal localization of the larvae was less
schistosomes in compatible and incompatible hosts revealed the cross-reactivity of anti-glycocalyx
frequent. Migrating schistosomula evoked an influx of inflammatory cells into the CNS, and in some cases
antibodies in all sera of individuals or mice infected with a schistosome with various species of cercariae.
inflammation of the central canal with neutrophils as dominant cells. Re-infections were manifested by
First antibodies recognizing the surface antigens of all species were detected at day 9 p.i. Also the surface of
granuloma formation in the CNS tissue with an occurrence of various cell types, above all eosinophils and
very young skin schistosomula (4h p.i.) was recognized by the serum antibodies of various schistosome
macrophages. While we did not observe any leg pareses or noticeable neuromotor abnormalities during
infected animals and individuals, however, 12h old schistosomula showed no reactivity. This result
infections of immunocompetent mice, infections of immunodeficient mice led to a severe partial or complete
confirmed the loss of the glycocalyx within first hours of infection. The most important finding is that T.
leg paralysis in 25% of primoinfected and 83% of re-infected mice. In contrast to the immunocompetent
regenti infected birds produce antibodies against gut associated excretory-secretory antigens (GAA) of adult
mice, the CNS tissue of SCID mice was not infiltrated by immune cells and the parasites seem not to be
worms which cross-react with the GAA of S. mansoni adults, however, in incompatible hosts, any reactivity
damaged by the host reaction. In addition, a large number of schistosomula occurred in the cerebellum of
with GAA of both species has not been observed. Thus, only compatible hosts (e.g. mice and humans in the
SCID mice.
case of S. mansoni infection; ducks in the case of Trichobilharzia infection) produce antibodies against the
complex of GAA. This conclusion enables us to use the anti-GAA reactivity for differential diagnosis of
cercarial dermatitis and distinguish patients with human schistosomiasis from those infected by bird
schistosomes when a patient visited areas that are endemic for schistosomiasis.
Our interest was focussed also on the evaluation of the real number of the parasites that stay and
die in the skin, and that are capable of further migration into the body of an incompatible host. This was
performed by use of radio-labelled cercariae for infection of mouse skin, and confirmed by the histological
study. Although low dose of infection (30 parasites/pinna) did not lead to further migration of the larvae
(with one exception) and over 90% of skin schistosomula died within first 8 days of infection, higher
infection dose (300 penetrating parasites/mouse) led to an escape of approx. 11% of schistosomula from the
In conclusion, our study shows important features of the immune responses after infection of
mammals with bird schistosomes and completes histopathological knowledge of cercarial dermatitis on
immunological bases. We document mast cell degranulation and histamine production immediately after the
infection. The dominance of IL-6, IL-4, and over-expression of IL-10 in the re-infected skin together with
the production of IL-4, IL-5 and IL-10 in sdLN and elevated specific IgG1 and total IgE in serum show
highly polarized Th2 response. The establishment of Th2-type responses seems to prevent further parasite
migration, however, we showed that also during first infection not more than 12% of parasites escaped from
the skin. Thus, it is likely that innate immune responses contribute to a protection. We believe that early
events in the skin are critical for the nesting of the parasites in the host and for the effective immune
response against them. Further studies are needed to elucidate which components of this complex immune
response are dominant for parasite killing mainly in the case of first infection and which parasite molecules
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Original papers published:
Kouřilová P, Hogg KG, Kolářová L and Mountford AP (2003). Type I hypersensitivity response is a major
Kouřilová P and Kolářová L (2002). Variations in immunofluorescent antibody response against
mechanism of the skin immune response during cercarial dermatitis caused by bird schistosomes. 2nd
Trichobilharzia and Schistosoma antigens in compatible and incompatible hosts. Parasitol Res 88, 513-21
workshop on bird schistosomes, Annecy, Juni 16-18
Kouřilová P, Hogg KG, Kolářová L and Mountford AP (2004). Cercarial dermatitis caused by bird
Lichtenbergová L, Kolářová L and Kouřilová P (2004). Trichobilharzia regenti – původce
schistosomes comprises both immediate and late phase cutaneous hypersensitivity reactions. J Immunol 172,
histopatologických změn nervové tkáně ptačích a savčích hostitelů. Sborník abstraktů konference České a
3766-74
Slovenské parazitologické dny, May 17-21
Kouřilová P, Syrůček M and Kolářová L (2004). The severity of mouse pathologies caused by the bird
Kouřilová P, Kolářová L and Mountford A (2004). Alergické kožní reakce působené larválními stádii
schistosome Trichobilharzia regenti in relation to the host immune status. Parasitol Res 93, 534-42
parazitů z čeledi Schistosomatidae. XXI. sjezd českých a slovenských alergologů a klinických imunologů.
Brno, November 3-6
Abstracts from conferences:
Dvořáková P and Hrádková K (2000). Protilátková odpověď a vývoj ptačích schistosom. České a slovenské
parazitologické dny Srní, May 22-25
Dvořáková P and Kolářová L (2000). Character of humoral response of nonspecific host infected with
cercariae of the genus Trichobilharzia. Helminthologia 37,3,180
Kolářová L, Kouřilová P and Horák P (2001). Trichobilharzia regenti, infection of nonspecific hosts as a
source of CNS pathologies. Proceedings of the XX Symposium of the Scandinavian Society for
Parasitology, Stockholm. Sweeden, October 4-7. Bulletin of the Scandinavian Society for Parasitology 11(12), 19
Kouřilová P and Kolářová L (2001). Histopathological changes in the mouse skin infected with
Trichobilharzia regenti. Helminthologia 38,3,167
Kouřilová P (2001). Histopathological observations of the mouse skin and CNS during the primo- and
reinfections by bird schistosome Trichobilharzia regenti. Helminthologia 38 (4)
Kouřilová P, Kolářová L and Mountford AP (2002). Imunitní odpověď a histopatologie savce při infekci
ptačí nazální schistosomou Trichobilharzia regenti. 5. slovenské a české parazitologické dny, Stará Lesná,
Vysoké Tatry , May 28-31
Skirnisson K, Hrádková K, Kouřilová P and Kolářová L (2002). The recently found Trichobilharzia
cercaria in Iceland is a nasal schistosome. ICOPA X, „Parasitology in a New World' Vancouver, Canada, .
Vancouver, Canada, August 4-9
Kouřilová P, Hogg KG, Kolářová L and Mountford AP (2003). Cercarial dermatitis induced by
Trichobilharzia regenti is a type I hypersensitivity response. BSP spring meeting and malaria meeting,
Manchester, April 6-9
Kouřilová P, Hogg KG, Kolářová L and Mountford AP (2003). New knowledge in the skin immune
response of mammals infected with the bird schistosome Trichobilharzia regenti. Helmintologické dny,
Dolní Věstonice, May 5-8