Department of Tropical Parasitology Institute of Maritime and Tropical Medicine Head: Dr. Przemysław Myjak

1. Department of Tropical ParasitologyInstitute of Maritime and Tropical MedicineHead: Dr. Przemysław Myjak

2. Staff members Beata Biernat Beata Szostakowska Alicja Rost Przemysław Myjak Maria Racewicz Halina Pietkiewicz Joanna Stańczak Agnieszka Adamczyk Balbina and: Wiesława Kruminis-Łozowska Maria Piesik Ewa Zieliniewicz Halina Pietkiewicz Beata Biernat

3. Previous and current research The Department carries out research in the field of: occurrence, biology, ecology and physiology of tropical and selected cosmopolitan parasites of man and animals; medical acaroentomology, epidemiology of the parasitic and arthropod-borne diseases; immunology and experimental chemotherapy of the parasitic diseases, biochemistry and genetics of parasites; ecto- and endoparasites of rodents – reservoirs of infectious diseases. Molecular diagnostics and epidemiology of parasitic and arthropod-transmitted diseases

4. PROTOZOA Amoebiosis: Entamoeba histolytica-E. dispar The infection with E. histolytica sensu lato is one of the most common parasitic infections in humans worldwide and occurs in about 10% of the world population. Clinical symptoms are observed in 10% of the infected people only and are related to E. histolytica infection, while 90% of the cases are asymptomaticand most frequently attributed to E. dispar infection.

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

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

7. MalariaPlasmodium falciparum, P. vivax, P. ovale, P. malariae According to WHO data, from 300 to 500 millions of people worldwide are yearly newly infected with malaria.

8. Detection of Plasmodium DNA is alternative to standard microscopy examinations. For the most cases the same results are obtained by both microscopic examinations and PCR. However, for cases with parasitemia of less than 0.1%, parasites can not be speciated by microscopy or diagnostic findings are doubtful. Then, only PCR gives correct diagnosis. Moreover, PCR is useful in detection of Plasmodium spp. in patients with fever ofunknown origin. (grant No 4 P05A 008 14) Myjak et al. 2002

9. The most dangerous among 4 species of human malaria is P. falciparum. Very serious problem is increasing drug resistance of this parasite against common medicines like chloroquine, pyrimethamine and sulfadoxine. We investigate, using DNA sequencing and PCR-RFLP methods, drug-resistance of P. falciparum isolates to these three medicaments. (grant No 6 P05B 046 20) Myjak et al. 2002

10. Toxoplasmosis This is the most worldwide parasitic invasioncaused by Toxoplasma gondii. In healthy individuals, generally is clinically asymptomatic.

11. However, it may cause severe complications in pregnant women (cause severe damage to the fetus) and immunocompromised patients In most cases, infection with this parasite is caused by consumption of meat containing cysts and consists 90% of per os infections. 10% of per os infections is caused by oocysts originated from cat’s faeces. Congenital cases of toxoplasmosis take place rarely but are very dangerous for new-born babies.

12. Diagnosis of acute infection generally relies on serological methods and results depend on antigens used. Most commercial serological assays detect antibodies by means of native antigens originating from T. gondii grown in vivo or in vitro. The production of these antigens is rather expensive and constant quality of the antigen preparations cannot be easily guarranted. Recombinant antigens could overcome these drawbacks since they can be produced inexpensively and free from host contamination.

13. We assess the usefulness of recombinant antigens (proteins SAG1, GRA1, GRA7 and mixture of these tree recombinant proteins) produced by Technical University of Gdańsk, Department of Microbiology in serodiagnostics of toxoplasmosis. Results obtained indicate that these antigents can be used in practical diagnostic of the disease in ELISA-IgG and ELISA-IgG-avidity tests. (grant No 4 P05A 103 18) Hiszczyńska-Sawicka et al. 2003.

14. Cryptosporidiosis Cryptosporidiosis is caused by Cryptosporidium spp., widespread protozoans that primarily infects the intestinal epithelium of vertebrates. Parasites cause acute diarrhea in immunocompetent humans or fatal illness in immunocompromised or immunosupressed individuals such as patients with AIDS. Transmission of the parasite occurs by the faecal-oral route.

15. In our Department various molecular methods are used in the research on epidemiology of cryptosporidiosis. We study the role of cattle and rats as a reservoirs of Cryptosporidium in rural and urban environment in Poland, as well as role of synanthropic flies as transport hosts for this pathogen. (grant No 6 P04C 024 21)

16. As the occurrence of Cryptosporidium outbreaks in drinking water (USA, Canada, UK, the Netherlands) have brought an increased need for detection at levels necessary to protect human health, we carry on investigation on the contamination of surface water and water supply by oocysts of different species of this parasite. (grant NATO LST.CLG.979765) Cryptosporidium parvum is considered as a biological weapon!!!

17. HELMINTHES Cestodes. Echinococcosis: Echinococcus multilocularis – E. granulosus E. multilocularis, a parasite of foxes occurring in the northern regions of Eurasia and North America, is causative agent of alveolar echinococcosis (AE).

18. This life threatening disease is caused by the larva (metacestode) of E. multilocularis that develops in the liver and forms a sponge-like mass proliferating through the tissue. Sometime it is difficult to differentiate between E. multilocularis and E.granulosus or liver cancer. Different molecular techniques help do differentiation or confirmation of E. multilocularis infection.

19. To exactly determine the causative agent of Polish AE cases we study a panel of specimens comparatively by histology, serology and DNA analysis. To this end we have developed two E. multilocularis-specific microsatellite markers and sequenced a fragment of mitochondrial 12S rDNA. Our results definitively prove the occurrence of autochthonous human AE in Poland. (grant No 4 P05D 042 12 ) Myjak et al. 2003 (in press)

20. Nematodes -from the family Anisakidae – parasites of fish, sea mammals, fish-eating birds and, accidentally, also humans. • Anisakid nematodes occur in a larval stage in many species of marine fish (in the Baltic Sea mainly herring but also cod, flatfish and other). Adult are parasites of sea mammals. Humans became infected after consumption of raw or wrongly processed fish • Worldwide, more than twelve thousand of documented cases of anisakiasis in man have been reported so far, mainly with larvae of Anisakis simplex and Pseudoterranova decipiens

21. Since the beginning of 90. we have been conducting the following studies: • Estimation of infection of fish from the Southern Baltic Sea with anisakid nematodes. • The prevalence, intensity and seasonal character of fish infection were estimated. • grants: No 4 411 92 03 and No 6 P04G 053 11

22. Investigation of anisakid with molecular methods (multilocus allozyme electrophoresis and PCR-based techniques like PCR-RFLP and DNA sequencing) The method of identification of anisakid species occurring in the Baltic Sea using molecular techniques was elaborated (as it is difficult or sometime impossible to distinguish species of Anisakidae on the basis of their morphology) It was established that Hysterothylacium actum from different fish species and place of occurrence belongs to one species (grants No 6 P04C 084 10 and No 6 P04C 041 16) Szostakowska et al. 2001; Szostakowska et al. 2002.

23. At present, we continue molecular investigations of anisakid nematodes by studying of population structure of Contracaecum rudolphii,parasite of cormorants nesting in Northeastern Poland In this field we collaborate with University of Gdańsk, Department of Genetics and Cytology and Agricultural University in Warsaw, Department of Zoology (grant No 3 P04C 099 23)

24. Arthropod-borne diseases In many parts of the world a group of the smaller arthropod animals, the mites, ticks, bugs, lice, fleas, and flies, has always been and still is a most serious factor in the dissemination of diseases. Molecular techniques are widely used for identification of vectors of different infectious diseases as well as for detection and differentiation of the etiologic agents of these diseases.

25. Molecular taxonomy can improve the quality of ecological and epidemiological studies because: Members of species complexes can be identified Morphologically similar species can be identified at any life stages Current technology allows visualization of biochemical and protein markers in small arthropods The remainder of specimen homogenate can be used for identification of associated microorganisms, whether they be pathogens or potential biological control agents, and for estimation of genetic variation in population genetic studies Identification of vectors

26. A diagnostic PCR assay has been developed that enables identification sibling mosquito species by means of the length of their PCR product. In our Laboratory it has been used to identify three cryptic species of native anophelinae mosquitoes of Anopheles maculipenniscomplex: An. atroparvus An. maculipennis s.s. An. messeae, potential vectors of Plasmodium spp., the etiologic agent of malaria. (grant No 6 P04C 019 18) Kubica-Biernat et al. 2001.

27. Anopheles maculipennis complex

28. Detection and differentiation of tick-borne pathogens • Borrelia burgdorferi s.l.: including B. burgdorferi s.s., B. garinii and B. afzelii, spirochetes being etiologic agent of Lyme borreliosis, the disease that can affect a variety of systems including the skin, heart, joints, and nervous system. (grant no 6 P04C 037 10) Stańczak et al. 1999, 2000, 2002

29. Anaplasma phagocytophilum (formerly HGE agent), the causative agent of human granulocytic ehrlichiosis (HGE), an obligate intracellular bacterium that targets mainly granulocytes of various mammalian species and man. (grant No 6 P04C 047 17) Grzeszczuk et al. 2002, Stańczak et al. 2002, Stańczak et al. in print.

30. Babesia spp., intraerythrocytic protozoan parasites which cause babesiosis, a world wide haemolytic disease of wild and domestic animals, and humans. (grant No 6 P04C 047 17) Stańczak et al. – in print. • Moreover, PCR can serve as a confirmation test for proper diagnosis of Lyme borreliosis, HGE and babesiosis by detection agents of diseases in tissue biopsies and body fluids. (grant No 3 P5D 097 25)

31. Mosquito biological control in the Vistula Spit • The application of chemical insecticides has been the most common and widely used method in Poland. However, the appearance of resistance to great number of them as well as their negative impact on the natural environment determined one’s attention to biological agents, especially Bacillus thuringiensis israelensis (B.t.i.). • In 2000, we started the first mosquito abatement program based on B.t.i. preparations in Poland which included: • Mapping of mosquito breeding places • Monitoring of mosquito populations • Applications of B.t.i. formulations • Evaluation of the efficacy • The overall efficiency of the control was 99.2%. • (grant Leonardo da Vinci of European Union and collaboration with KABS – German Mosquito Control Association) • Kubica-Biernat et al. 2000, 2001.