DepartmentDepartment of Tropical of Tropical ParasitologyParasitology

Institute of Maritime and Tropical MedicineInstitute of Maritime and Tropical Medicine

Head: Dr. Przemysław MyjakHead: Dr. Przemysław Myjak

Beata BiernatBeata Biernat

Staff membersStaff members

Maria RacewiczMaria Racewicz

Przemysław MyjakPrzemysław MyjakAlicja RostAlicja Rost

Joanna StańczakJoanna Stańczak

Beata SzostakowskaBeata Szostakowska

Agnieszka AdamczykAgnieszka Adamczyk

Halina PietkiewiczHalina Pietkiewicz

BalbinaBalbina

Halina PietkiewiczHalina Pietkiewicz

Beata BiernatBeata Biernat

and: Wiesława Kruminis-Łozowskaand: Wiesława Kruminis-Łozowska Maria PiesikMaria Piesik Ewa Zieliniewicz Ewa Zieliniewicz

Molecular diagnostics and epidemiology Molecular diagnostics and epidemiology of parasitic and arthropod-transmitted of parasitic and arthropod-transmitted

diseasesdiseases

Previous and current researchPrevious and current researchThe Department carries out research in the field of:The Department carries out research in the field of:

occurrence, biology, ecology and physiology of tropical and occurrence, biology, ecology and physiology of tropical and selected coselected cossmopolitan parasites of man and animals; mopolitan parasites of man and animals; medical acaroentomology, epidemiology of the parasitic medical acaroentomology, epidemiology of the parasitic and arthropod-borne diseases; immunology and and arthropod-borne diseases; immunology and experimental chemotherapy of the parasitic diseases, experimental chemotherapy of the parasitic diseases, biochemistry and genetics of parasites; ecto- and biochemistry and genetics of parasites; ecto- and endoparasites of rodents – reservoirs of infectious diseases.endoparasites of rodents – reservoirs of infectious diseases.

PROTOZOAPROTOZOA

Amoebiosis: Amoebiosis: Entamoeba histolytica-E. disparEntamoeba histolytica-E. dispar

The infection with The infection with E. histolyticaE. histolytica sensu lato is one sensu lato is one of the most common parasitic infections of the most common parasitic infections in humans worldwide and occurs in about 10% of in humans worldwide and occurs in about 10% of the world population.the world population.

Clinical symptoms are observed in 10% of the Clinical symptoms are observed in 10% of the infected people only and are related to infected people only and are related to E. histolyticaE. histolytica infection, while 90% of the cases infection, while 90% of the cases are asymptomaticare asymptomatic and most frequently attributed and most frequently attributed to to E. disparE. dispar infection. infection.

People infected with People infected with E. disparE. dispar do not require do not require medical treatment. It is therefore of vital medical treatment. It is therefore of vital importance to differentiate these two importance to differentiate these two amoebic species which are morphologically amoebic species which are morphologically identical. It is possible using molecular identical. It is possible using molecular methods.methods.

We demonstrated that in Poland, among the We demonstrated that in Poland, among the imported and indigenous strains of imported and indigenous strains of E. histolyticaE. histolytica s.l., s.l., E. disparE. dispar occurred most occurred most frequently (90%) and its phenotyp i.e. frequently (90%) and its phenotyp i.e. zymodeme I, was the most common one. zymodeme I, was the most common one. E. histolyticaE. histolytica occurred more rarely but also occurred more rarely but also in persons who did not travel abroad.in persons who did not travel abroad.(grant No 4 P05A 107 08)(grant No 4 P05A 107 08) Myjak Myjak et al.et al. 2000. 2000.

MalariaMalariaPlasmodium falciparum, P. vivax, P. ovale, Plasmodium falciparum, P. vivax, P. ovale,

P. malariaeP. malariae

According to WHO data, According to WHO data, from 300 to 500 millions of people from 300 to 500 millions of people worldwide are yearly newly worldwide are yearly newly infected with malaria.infected with malaria.

Detection of Detection of PlasmodiumPlasmodium DNA is alternative DNA is alternative to standard microscopy examinations.to standard microscopy examinations.For For the the most cases the same results are most cases the same results are obtained obtained by by both microscopic examinations both microscopic examinations and PCR. and PCR. However, for cases with parasitemia of less However, for cases with parasitemia of less than 0.1%, parasites can not be speciated by than 0.1%, parasites can not be speciated by microscopy or diagnostic findings are microscopy or diagnostic findings are doubtful. Then, only PCR gives correct doubtful. Then, only PCR gives correct diagnosis.diagnosis.Moreover, PCR is useful in detection of Moreover, PCR is useful in detection of Plasmodium Plasmodium sppspp.. in patients with fever of in patients with fever of unknown originunknown origin..

(grant No 4 P05A 008 14)(grant No 4 P05A 008 14)

Myjak Myjak et alet al. 2002. 2002

The most dangerous among 4 species of The most dangerous among 4 species of human malaria is human malaria is P. falciparumP. falciparum. .

Very serious problem is increasing drug Very serious problem is increasing drug resistance of this parasite against common resistance of this parasite against common medicines like chloroquine, pyrimethamine medicines like chloroquine, pyrimethamine and sulfadoxine.and sulfadoxine.

We investigate, using DNA sequencing and PCR-RFLP methods, We investigate, using DNA sequencing and PCR-RFLP methods, drug-resistance of drug-resistance of P. falciparumP. falciparum isolates to these three isolates to these three medicaments.medicaments.

(grant No 6 P05B 046(grant No 6 P05B 046 20) 20)

Myjak Myjak et al. et al. 20022002

ToxoplasmosisToxoplasmosis

This is the most worldwide This is the most worldwide parasitic invasionparasitic invasion caused by caused by

Toxoplasma gondiiToxoplasma gondii..

IIn healthy individuals, generally is n healthy individuals, generally is clinically asymptomatic.clinically asymptomatic.

However, it may cause severe complications in pregnant womenHowever, it may cause severe complications in pregnant women (cause severe damage to the fetus) and immunocompromised (cause severe damage to the fetus) and immunocompromised patientspatients

In most cases, infection with this parasite is causedIn most cases, infection with this parasite is caused by consumption of meat containing cysts and consistsby consumption of meat containing cysts and consists 90% of 90% of per osper os infections. infections.

10% of 10% of per osper os infections is caused by oocysts infections is caused by oocysts originatedoriginatedfrom cat’s faeces.from cat’s faeces.

Congenital cases of toxoplasmosis take place rarely Congenital cases of toxoplasmosis take place rarely but are very dangerous for new-born babies.but are very dangerous for new-born babies.

Diagnosis of acute infection generally relies on serological Diagnosis of acute infection generally relies on serological methods and results depend on antigens used.methods and results depend on antigens used.

Most commercial serological assays detect antibodies by Most commercial serological assays detect antibodies by means of native antigens originating from means of native antigens originating from T. gondiiT. gondii grown grown in vivoin vivo or or in vitroin vitro. The production of these antigens is . The production of these antigens is rather expensive and constant quality of the antigen rather expensive and constant quality of the antigen preparations cannot be easily guarpreparations cannot be easily guarrranted. anted.

Recombinant antigens could overcome these drawbacks since Recombinant antigens could overcome these drawbacks since they can be produced inexpensively and free from host they can be produced inexpensively and free from host contamination.contamination.

We assess the usefulness of recombinant antigens We assess the usefulness of recombinant antigens (proteins SAG1, GRA1, GRA7 and mixture of (proteins SAG1, GRA1, GRA7 and mixture of these tree recombinant proteins) produced by these tree recombinant proteins) produced by Technical University of Gdańsk, Department of Technical University of Gdańsk, Department of Microbiology in serodiagnostics of toxoplasmosis.Microbiology in serodiagnostics of toxoplasmosis.

Results obtained indicate that Results obtained indicate that these these antigents can be antigents can be used in practical diagnostic of thused in practical diagnostic of thee disease in disease in ELISA-IgG and ELISA-IgG-avidity tests. ELISA-IgG and ELISA-IgG-avidity tests.

(grant No 4 P05A 103 18) (grant No 4 P05A 103 18)

Hiszczyńska-Sawicka Hiszczyńska-Sawicka et alet al. 2003.. 2003.

CryptosporidiosisCryptosporidiosis

Cryptosporidiosis is caused by Cryptosporidiosis is caused by

CryptosporidiumCryptosporidium spp., spp., widespread protozoans that widespread protozoans that primarily infects the intestinal primarily infects the intestinal epithelium of vertebrates.epithelium of vertebrates.

Parasites cause acute diarrhea in immunocompetent Parasites cause acute diarrhea in immunocompetent humans or fatal illness in immunocompromised or humans or fatal illness in immunocompromised or immunosupressed individuals such as patients with immunosupressed individuals such as patients with AIDS. AIDS.

Transmission of the parasite occurs by the faecal-oral Transmission of the parasite occurs by the faecal-oral route.route.

In our Department various molecular In our Department various molecular methods are used in the research on methods are used in the research on epidemiology of cryptosporidiosis.epidemiology of cryptosporidiosis.

We study the role of cattle and rats as a reservoirs We study the role of cattle and rats as a reservoirs of of CryptosporidiumCryptosporidium in rural and urban in rural and urban environment in Poland, as well as role of environment in Poland, as well as role of synanthropic flies as transport hosts for this synanthropic flies as transport hosts for this pathogen.pathogen.

(grant No 6 P04C 024 21)(grant No 6 P04C 024 21)

As tAs the occurrence he occurrence of of Cryptosporidium Cryptosporidium outbreaks in outbreaks in drinking water (USA, Canada, UK, the drinking water (USA, Canada, UK, the Netherlands) have brought an Netherlands) have brought an increased need for detection at levels increased need for detection at levels necessary to protect human healthnecessary to protect human health,, we carry on investigation on the we carry on investigation on the contamination of surface water and contamination of surface water and water supply by water supply by oocysts of oocysts of different different species of species of this parasite.this parasite.

(grant NATO LST.CLG.979765)(grant NATO LST.CLG.979765)

Cryptosporidium parvumCryptosporidium parvum is considered as a biological weapon!!!is considered as a biological weapon!!!

HELMINTHESHELMINTHES

CestodesCestodes..

Echinococcosis: Echinococcosis: Echinococcus multilocularis – E. granulosusEchinococcus multilocularis – E. granulosus

E. multilocularisE. multilocularis, a parasite of foxes occurring in the northern , a parasite of foxes occurring in the northern regions of Eurasia and North America, is causative agent of alveolar regions of Eurasia and North America, is causative agent of alveolar echinococcosis (AE).echinococcosis (AE).

This life threatening disease is caused This life threatening disease is caused by the larva (metacestode) by the larva (metacestode) of of E. multilocularisE. multilocularis that develops in that develops in the liver and forms a sponge-like mass the liver and forms a sponge-like mass proliferating through the tissue.proliferating through the tissue.

Sometime it is difficult to differentiate between Sometime it is difficult to differentiate between E. multilocularisE. multilocularis and and E.granulosusE.granulosus or liver cancer. or liver cancer.

Different molecular techniques help do differentiation or Different molecular techniques help do differentiation or confirmation of confirmation of E. multilocularisE. multilocularis infection. infection.

To exactly determine the causative agent of Polish AE To exactly determine the causative agent of Polish AE cases we studcases we studyy a panel of specimens comparatively by a panel of specimens comparatively by histology, serology and DNA analysis. To this end we histology, serology and DNA analysis. To this end we have have developed two developed two E. multilocularisE. multilocularis-specific microsatellite -specific microsatellite markers and sequenced a fragment of mitochondrial 12S markers and sequenced a fragment of mitochondrial 12S rDNA.rDNA.

Our results definitively prove the occurrence of Our results definitively prove the occurrence of autochthonous human AE in Poland.autochthonous human AE in Poland.

(grant No 4 P05D 042 12 )(grant No 4 P05D 042 12 )

Myjak Myjak et alet al. 2003 (in press). 2003 (in press)

Nematodes -Nematodes -from the family from the family AnisakidaeAnisakidae – parasites of fish, sea mammals, – parasites of fish, sea mammals, fish-eating birds and, accidentally, also humans.fish-eating birds and, accidentally, also humans.

Anisakid nematodes occur in a larval Anisakid nematodes occur in a larval stage in many species of marine fish stage in many species of marine fish (in the Baltic Sea mainly herring but (in the Baltic Sea mainly herring but also cod, flatfish and other). Adult also cod, flatfish and other). Adult are parasites of sea mammals. are parasites of sea mammals. Humans became infected after Humans became infected after consumption of raw or wrongly consumption of raw or wrongly processed fishprocessed fish

Worldwide, more than twelve Worldwide, more than twelve thousand of documented cases of thousand of documented cases of anisakiasis in man have been anisakiasis in man have been reported so far, mainly with larvae of reported so far, mainly with larvae of Anisakis simplexAnisakis simplex and and Pseudoterranova decipiensPseudoterranova decipiens

Since the beginning Since the beginning of 90. of 90. we have been we have been conducting the following studies:conducting the following studies:

•Estimation of infection of fish from the Southern Estimation of infection of fish from the Southern Baltic Sea with anisakid nematodes Baltic Sea with anisakid nematodes..

The prevalence, intensity and seasonal The prevalence, intensity and seasonal character of fish infection were estimated. character of fish infection were estimated.

grants: No 4 411 92 03 and No 6 P04G 053 11grants: No 4 411 92 03 and No 6 P04G 053 11

Investigation of anisakid with molecular methods Investigation of anisakid with molecular methods (multilocus allozyme electrophoresis and PCR-based (multilocus allozyme electrophoresis and PCR-based techniques like PCR-RFLP and DNA sequencing)techniques like PCR-RFLP and DNA sequencing)

The method of identification of anisakid species occurring in the Baltic The method of identification of anisakid species occurring in the Baltic Sea using molecular techniques was elaborated (as it is difficult or Sea using molecular techniques was elaborated (as it is difficult or sometime impossible to distinguish species of Anisakidae on the basis sometime impossible to distinguish species of Anisakidae on the basis of their morphology)of their morphology)

It was established that It was established that Hysterothylacium actumHysterothylacium actum from different fish from different fish species and place of occurrence belongs to one speciesspecies and place of occurrence belongs to one species

(grants No 6 P04C 084 10 and (grants No 6 P04C 084 10 and No No 6 P04C 041 16)6 P04C 041 16)

Szostakowska Szostakowska et alet al. 2001; Szostakowska . 2001; Szostakowska et alet al. 2002.. 2002.

At present, we continue molecular investigations At present, we continue molecular investigations of anisakid nematodes by studying of population of anisakid nematodes by studying of population structure of structure of Contracaecum rudolphiiContracaecum rudolphii,, parasite of cormorants nesting in Northeastern Polandparasite of cormorants nesting in Northeastern Poland

In this field we collaborate with University of Gdańsk, Department of In this field we collaborate with University of Gdańsk, Department of Genetics and Cytology and Agricultural University in Warsaw, Genetics and Cytology and Agricultural University in Warsaw,

Department of ZoologyDepartment of Zoology

(grant No 3 P04C 099 23)(grant No 3 P04C 099 23)

Arthropod-borne diseasesArthropod-borne diseases

In In many parts of the world a group many parts of the world a group of the smaller arthropod of the smaller arthropod animals, the mites, ticks, bugs, animals, the mites, ticks, bugs, lice, fleas, and flies, has always lice, fleas, and flies, has always been and still is a most serious been and still is a most serious factor in the dissemination of factor in the dissemination of diseases.diseases.

Molecular techniques are widely Molecular techniques are widely used for identification of vectors used for identification of vectors of different infectious diseases of different infectious diseases as well as for detection and as well as for detection and differentiation of the etiologic differentiation of the etiologic agents of these diseases.agents of these diseases.

Identification of vectorsIdentification of vectors

Molecular taxonomy can improve the quality of ecological Molecular taxonomy can improve the quality of ecological and epidemiological studies because:and epidemiological studies because:

Members of species complexes can be identifiedMembers of species complexes can be identified

Morphologically similar species can be identified at any Morphologically similar species can be identified at any life stageslife stages

Current technology allows visualization of biochemical and Current technology allows visualization of biochemical and protein markers in small arthropodsprotein markers in small arthropods

The remainder of specimen homogenate can be used for The remainder of specimen homogenate can be used for identification of associated microorganisms, whether they identification of associated microorganisms, whether they be pathogens or potential biological control agents, and for be pathogens or potential biological control agents, and for estimation of genetic variation in population genetic estimation of genetic variation in population genetic studiesstudies

A diagnostic PCR assay has been developed that enables A diagnostic PCR assay has been developed that enables identification sibling mosquito species by means of the identification sibling mosquito species by means of the length of their PCR product.length of their PCR product.

In our Laboratory it has been used to identify three cryptic In our Laboratory it has been used to identify three cryptic species of native anophelinae mosquitoes species of native anophelinae mosquitoes of of Anopheles Anopheles maculipennismaculipennis complex: complex:

An. atroparvusAn. atroparvus

An. maculipennis An. maculipennis s.s.s.s.

An. messeae, An. messeae,

potential vectors of potential vectors of PlasmodiumPlasmodium spp., the etiologic agent of spp., the etiologic agent of malariamalaria..

(grant No 6 P04C 019 18)(grant No 6 P04C 019 18)

Kubica-Biernat Kubica-Biernat et alet al. 2001.. 2001.

AnophelesAnopheles maculipennis maculipennis complexcomplex

Detection and differentiation Detection and differentiation of tick-borne pathogensof tick-borne pathogens

Borrelia burgdorferi Borrelia burgdorferi s.ls.l.: .: including including B. burgdorferi B. burgdorferi s.s., s.s., B. B. garinii garinii and and B. afzelii,B. afzelii, spirochetes spirochetes being etiologic agent of Lyme being etiologic agent of Lyme borreliosis, the disease that can borreliosis, the disease that can affect a variety of systems affect a variety of systems including the skin, heart, joints, including the skin, heart, joints, and nervous system. and nervous system. (grant no 6 P04C 037 10)(grant no 6 P04C 037 10)

Stańczak Stańczak et alet al. 1999, 2000, 2002. 1999, 2000, 2002

Anaplasma phagocytophilumAnaplasma phagocytophilum (formerly HGE (formerly HGE agent), the causative agent of human granulocytic agent), the causative agent of human granulocytic ehrlichiosis (HGE), an obligate intracellular ehrlichiosis (HGE), an obligate intracellular bacterium that targets mainly granulocytes of bacterium that targets mainly granulocytes of various mammalian species and manvarious mammalian species and man..

(grant No 6 P04C 047 17)(grant No 6 P04C 047 17)

Grzeszczuk Grzeszczuk et alet al. 2002, Stańczak . 2002, Stańczak et alet al. 2002, Stańczak . 2002, Stańczak et alet al. in print.. in print.

Babesia Babesia spp.,spp., intraerythrocytic protozoan parasites which intraerythrocytic protozoan parasites which cause babesiosis, a world wide haemolytic cause babesiosis, a world wide haemolytic disease disease of wild of wild and domestic animals, and humans. and domestic animals, and humans.

(grant No 6 P04C 047 17)(grant No 6 P04C 047 17)

Stańczak Stańczak et alet al. – in print.. – in print.

Moreover, PCR can serve as a confirmation test for Moreover, PCR can serve as a confirmation test for proper diagnosis of Lyme borreliosis, HGE and proper diagnosis of Lyme borreliosis, HGE and babesiosis by detection agents of diseases in tissue babesiosis by detection agents of diseases in tissue biopsies and body fluids.biopsies and body fluids.

(grant (grant NNo 3 P5D 097 25)o 3 P5D 097 25)

Mosquito biological control in the Vistula SpitMosquito biological control in the Vistula Spit

The application of chemical insecticides has been the most common and The application of chemical insecticides has been the most common and widely used method in Poland. However, the appearance of resistance to widely used method in Poland. However, the appearance of resistance to great number of them as well as their negative impact on the natural great number of them as well as their negative impact on the natural environment determined one’s attention to biological agents, especially environment determined one’s attention to biological agents, especially Bacillus thuringiensis israelensis Bacillus thuringiensis israelensis (B.t.i.). (B.t.i.).

In 2000, we started the first mosquito abatement program based on B.t.i. In 2000, we started the first mosquito abatement program based on B.t.i. preparations in Poland which included:preparations in Poland which included:

•Mapping of mosquito breeding placesMapping of mosquito breeding places

•Monitoring of mosquito populationsMonitoring of mosquito populations

•Applications of B.t.i. formulationsApplications of B.t.i. formulations

•Evaluation of Evaluation of the the efficacyefficacy

The overall efficiency of the control was 99.2%.The overall efficiency of the control was 99.2%.

(grant Leonardo da Vinci(grant Leonardo da Vinci of European Union of European Union and collaboration with and collaboration with KABS – German Mosquito Control AssociationKABS – German Mosquito Control Association))

Kubica-Biernat Kubica-Biernat et alet al. 2000, 2001.. 2000, 2001.