Helminthological Days 2015

Charles University in Prague, Faculty of Science ______

22nd HELMINTHOLOGICAL DAYS 2015

Programme & Abstracts

Editors: Terezia Perháčová, Libuše Turjanicová & Vladimír Skála

Prague 2015

Conference organizers: Laboratory of Helminthology, Department of Parasitology, Faculty of Science, Charles University in Prague Helminthological Section Czech Society for Parasitology

Venue and date of the conference: 22nd Helminthological Days, Recreational facility FTVS UK, Dvorce near Stráž nad Nežárkou, Czech Republic, 4.-8. May 2015

Programme

PROGRAMME OF 22nd HELMINTHOLOGICAL DAYS

MONDAY, MAY 4

15.00 - 18.00 Arrival and registration of participants 18.00 - 18.30 Dinner 19.00 - 00.00 Get-together-evening

TUESDAY, MAY 5

08.00 - 09.00 Breakfast

09.00 - 09.10 Opening ceremony: H. Dvořáková

09.10 - 09.40 Invited talk: W. Haas: Strategies of parasitic worms to find, recognize and invade their hosts (Chairman: P. Horák)

Session I Interactions between Trichobilharzia spp. and their hosts (Chairman: P. Horák) 09.40 - 09.55 V. Skála, P. Horák: Haemocyte extracellular traps in the snail Lymnaea stagnalis (Lymnaeidae): do they occur and are they used as a defence response against trematodes? PhD 09.55 - 10.10 V. Krčmářová, J. Bulantová, P. Horák: Interactions of neurotropic larvae of bird schistosome Trichobilharzia regenti and immune cells of host central nervous system. BSc/MSc 10.10 - 10.25 T. Macháček, L. Panská, P. Horák: Antigens of the neuropathogenic fluke Trichobilharzia regenti elicit nitric oxide and proinflammatory cytokine secretion by glial cells in vitro. BSc/MSc 10.25 - 10.40 L. Vlčková, P. Horák: Interactions of the eggs and miracidia of Trichobilharzia regenti with the duck. BSc/MSc

10.40 - 11.00 Coffee break

Session II Biodiversity and variability of helmints (Chairman: R.Kuchta) 11.00 - 11.15 N. Kmentová, M. Gelnar, M. Mendlová, M.Van Steenberge, S. Koblmüller, M. P. M Vanhove: Intraspecific variation of Cichlidogyrus (Monogenea, Dactylogyridae) in Lake Tanganyika. BSc/MSc 11.15 - 11.30 T. A. Kuzmina, T. R. Spraker, E. T. Lyons, R. Kuchta, O. Lisitsyna, O. Kudlai: Biodiversity of gastrointestinal parasite community in northern fur seals (Callorhinus ursinus) on St. Paul Island, Alaska. 11.30 - 11.45 V. Michálková, M. Reichard, M. Ondračková: Metazoan parasites of killifish from Mosambique. PhD 11.45 - 12.00 M. Pravdová, R. Blažek, M. Gelnar, E. Řehulková: Diversity of monogeneans parasitizing freshwater fishes of the Nile River in Sudan: current state and prospects. BSc/MSc 12.00 - 12.15 K. Leštinová, R. Kuchta: Morphological and morphometrical analyses of the eggs of human-infecting diphyllobothriid cestodes. PhD 12.15 - 12.30 P. Zahradníčková, M. Barson, W. J. Luus-Powell, I. Přikrylová: An evaluation of different discriminative approaches used for discrimination of cryptic species. PhD

12.30 - 13.00 Lunch

Programme

Session III Environmental parasitology, epizootology (Chairman: M. Gelnar) 13.30 - 13.45 J. Bulantová, L. Turjanicová, M. Vaščiková, R. Leontovyč, M. Chanová, P. Horák: Velký Bolevecký pond: story of an outdoor laboratory for avian schistosomes. 13.45 - 14.00 J. S. Hernandes Orts, T.Scholz, J. Brabec, T. Kuzmina, R. Kuchta: High morphological plasticity and global geographical distribution of the Pacific broad tapeworm Adenocephalus pacificus (syn. Diphyllobothrium pacificum): molecular and morphological survey. 14.00 - 14.15 O. Kudlai: Morphological and molecular approaches to the study of digenean larvae from the Curonian lagoon in Lithuania. PhD 14.15 - 14.30 L. Raisingerová, Š. Mašová, A. Vetešníková Šimková, R. Šanda, J. Vukić, M. Gelnar: Basic epidemiological data of anisakid nematode larvae of gobiid fishes (Gobiidae, Actinopterygii) from northern Adriatic Sea. BSc/MSc 14.30 - 14.45 T. Sosnová, M. Kašný: Fascioloid flukes of livestock and wild animals in the Czech Republic. BSc/MSc

14.45 - 15.15 Coffee break

Session IV Host-parasite interactions and pathogenesis (Chairman: J. Brabec) 15.15 - 15.30 Z. Čadková, B. Horáková, V. Sloup, J. Száková, J. Vadlejch, I. Langrová: Are rodent tapeworms suitable accumulative indicators for their host´s risk element exposure? 15.30 - 15.45 V. Krasnovyd, A. Šimková: Influence of hybridization on the fish host conditions and parasite infection. 15.45 - 16.00 T. Pakosta, A. Vetešníková Šimková: Parasite load and immunity in diploid-polyploid complex of Carassius gibelio. PhD 16.00 - 16.15 T. R. Spraker, T. A. Kuzmina, E. T. Lyons, R. DeLong: Pathogenesis of hookworm/enteritis complex with bacteremia caused by Uncinaria spp. in California sea lions and Northern fur seals.

Poster session (Chairman: R. Leontovyč) 16.30 - 17.30 2 min presentations and individual poster discussion N. Bernardová, M. Chanová: Long-term murine cerebral toxocariasis. BSc/MSc K. Civáňová, L. Gettová, A. Gilles, A. Šimková: Effect of a co-existence of invasive Barbus barbus and local Barbus meridionalis on the composition of metazoan parasite communities. T. A. Kuzmina, V. A. Kharchenko, N. S. Zvegintsova: Strongylides (Nematoda; Strongylidae) of wild and domestic equids in Ukraine: biodiversity and parasite community structure. V. V. Pavliková, M. Chanová: Helminths and immune-mediated disorders (hypothesis and plans). BSc/MSc I. Přikrylová, I. Walter, W. J. Luus-Powell, M. Barson: Monogenean parasites from Pseudocrenilabraus philander (Weber, 1897) in the middle Limpopo River Basin (South African region). I. Přikrylová, Š. Mašová, M. M. Matla, M. Gelnar, W. J. Luus-Powell: The revision of the genus Afrodiplozoon (Khotenovsky, 1981.) (Monogenea: Diploziodae ). J. Sedláková, M. Borkovcová: Endoparasites of wild animals of vineyard and agricultural landscape in the South Moravia. PhD Y. Yu. Syrota, V. A. Kharchenko, T. A. Kuzmina: Study of the nematode fauna in waterfowls on the Ukrainian Polesie. PhD

18.00 - 18.30 Dinner

20.00 - 00.00 Campfire night

Programme

WEDNESDAY, MAY 6

08.00 - 08.45 Breakfast

08.45 - 09.15 Invited talk: J. P. Dalton: Understanding Fasciola hepatica relationship with it´s host using genomics, transcriptomics and proteomics, with basic biology. (Chairman: M. Kašný)

Session V Molecular biology (Chairman: M. Kašný) 09.15 - 09.30 H. Dvořáková, L. Jedličková, M. Kašný, P. Brož, J. Ilgová, M. Gelnar, L. Mikeš: Transcriptomic analyses of a monogenean: Next-Generation Sequencing techniques in hands of parasitologists. PhD 09.30 - 09.45 D. Jirsová, M. Gelnar, M. Kašný: Monogenea … what else can be done? 09.45 - 10.00 R. Leontovyč, N. Young, P. Horák, G. Robin, M. Kašný: Comparative study of transcriptome profiles of cercariae/schistosomula of bird schistosome Trichobilharzia regenti. PhD 10.00 - 10.15 R. Šašková, R. Lenontovyč, L. Ulrychová, J. Dvořák, P. Horák, M. Kašný: The expression of cathepsin L in developmental stages of Fascioloides magna. BSc/MSc 10.15 - 10.30 L. Škorpíková, B. Koudela, J. Ilgová, M. Gelnar, M. Kašný: Molecular characterization of cystatins of Trichinella spp. BSc/MSc

10.30 - 11.00 Coffee break

Session VI Peptidases and inhibitors (Chairman: L. Mikeš) 11.00 - 11.15 J. Ilgová, M. Gelnar, M. Kašný: Expression of a cysteine peptidase inhibitor from Eudiplozoon nipponicum (Monogenea). PhD 11.15 - 11.30 L. Jedličková, H. Dvořáková, M. Kašný, D. Potěšil, Z. Zdráhal, L. Mikeš: Peptidases detected in excretory/secretory products of the monogenean Eudiplozoon nipponicum. PhD 11.30 - 11.45 K. Skipalová, L. Jedličková, H. Dvořáková, P. Brož, M. Kašný, L. Mikeš: Anticoagulation factors and blood uptake by monogeneans of the family Diplozoidae. BSc/MSc 11.45 - 12.00 T. Perháčová, L. Mikeš: Cathepsin L-like peptidases in cercariae of Diplostomum pseudospathaceum. BSc/MSc 12.00 - 12.15 A. Leontovyč, L. Ulrychová, C. R. Caffrey, A. J. O'Donoghue, M. Mareš, M. Horn, J. Dvořák: Trypsin loaded worms: SmSP2, predominant serine protease in S. mansoni mammalian stages. PhD 12.15 - 12.30 P. Fajtová, S. Štefanić, M. Hradilek, J. Dvořák, J. Vondrášek, A. Jílková, L. Ulrychová, J. H. McKerrow, C. R. Caffrey, M. Mareš, M. Horn: Prolyl oligopeptidase from the blood fluke Schistosoma mansoni: from functional analysis to anti-schistosomal inhibitors. PhD 12.30 - 12.45 A. Jílková, M. Horn, P. Řezáčová, J. Brynda, J. H. McKerrow, C. R. Caffrey, M. Mareš: Structural and functional analysis of the Schistosoma mansoni cathepsin B1 drug target.

13.00 - 13.30 Lunch

14.00 - 18.00 Trip to the Regent brewery/ Třeboň castle

18.00 - 18.30 Dinner

19.00 - 00.00 Free evening

Programme

THURSDAY, MAY 7

08.00 - 09.00 Breakfast

09.00 - 09.30 Invited talk: I. Schabussova: Immunomodulation by Parasites: The big comeback? (Chairman: I. Vokřál)

Session VII Immunology and diagnostics (Chairman: I. Vokřál) 09.30 - 09.45 K. Melounová, J. Bulantová, J. Vadlejch, P. Horák, M. Kašný: Evaluation of qualitative and quantitative parametrs of interaction between Fascioloides magna. and definitive experimental host - rabbit. BSc/MSc 09.45 - 10.00 J. Novák, L. Panská, L. Kolářová, P. Horák: Immunological consequences of the infection of mice with Toxocara canis. PhD 10.00 - 10.15 N. Reslová, M. Kašný, M. Slaný, B. Koudela, P. Králík: Novel molecular diagnostic approach for detection of pathogens in meat. PhD 10.15 - 10.30 L. Turjanicová, L. Mikeš: Enolase: a suitable antigen for the immunodiagnostics of Trichobilharzia regenti infections in ducks. PhD 10.30 - 10.45 M. Vaščiková, L. Turjanicová, P. Horák: Molecular and serological diagnosis of infections caused by Trichobilharzia spp. BSc/MSc

10.45–11.15 Coffee break

Session VIII Development and physiology of helmints (Chairman: V. Skála) 11.15 - 11.30 J. Pankrác, M. Kašný, J. Bulantová, P. Horák: The role of stem cells in regenerative processes in trematodes. PhD 11.30 - 11.45 K. Vrbová, J. Pankrác, M. Kašný: In vitro cultivation of the trematode species Fascioloides magna and Trichobilharzia regenti. BSc/MSc 11.45 - 12.00 J. Peštová, P. Horák: Differentiation of totipotent germinal cells in larvae of T. regenti. BSc/MSc 12.00 - 12.15 I. Vokřál, Z. Seifertová, M. Veverková, L. Stuchlíková, L. Prchal, B. Szotáková, L. Skálová: Possible defence mechanisms against treatment in tapeworms.

12.30 - 13.00 Lunch

Session IX Molecular taxonomy and phylogenetics (Chairman: I. Přikrylová ) 13.30 - 13.45 R. Kuchta, J. Brabec, A. Waeschenbach, T. Scholz, D. T. J. Littlewood: Molecular phylogeny of the Bothriocephalidea (Cestoda): molecular data challenge morphological classification. 13.45 - 14.00 J. Brabec: First insights into the molecular diversity of the invasive Asian tapeworm Schyzocotyle acheilognathi. 14.00 - 14.15 S. Georgieva, Š. Mašová, A. Faltýnková: Far apart: Species of Macvicaria (Gigson & Bray, 1982) (Digenea: Opecoelidae) in the Mediterranean and Antartcic.

14.15 - 14.45 Coffee break

14.45 - 15.45 Invited talk: H. Bláhová: Paraziti: “Pravdu má ten, kdo vyléčil.” (Chairman: M. Kašný)

16.00 - 18.00 Presentations of sponsors

18.30 - 00.00 Closing ceremony, party with live music at Stráž nad Nežárkou

FRIDAY, MAY 8

08.00 - 09.00 Breakfast 09.00 - 11.00 Departure of participant

ABSTRACTS

Abstracts

LONG-TERM MURINE CEREBRAL TOXOCARIASIS

N. BERNARDOVÁ1,2, M. CHANOVÁ2

1Department of Parasitology, Faculty of Science, Charles University in Prague 2Institute of Immunology and Microbiology, 1st Faculty of Medicine, Charles University in Prague

Toxocara spp. are obligate endoparasitic worms with adults living in canine or feline digestive system. Their larvae can get into paratenic host in which they undergo visceral migration, clinically manifested as larval toxocariasis. Cerebral toxocariasis is a form of disease when larvae attack central nervous system. As a part of complex study on murine cerebral toxocariasis, a group of mice with long- term infections associated with neurological symptoms was in focus. Our goals were to characterize cerebral changes and damages, larvae presence, location and migratory routes through the brain and the cause of neurological symptoms. Mice BALB/c were infected by 1000 larvae per mouse. One of mice was reinfected by same dose 17 weeks post first infection (p.i). Each infected mouse showed neurological and physical changes as running in circles or ruffled coat in late phase of infection for several weeks. Between 26th and 31st week p.i. 4 mice died. Two mice with simple infection were then sacrificed in 31th week p.i., one was sacrificed in 32th week p.i. Reinfected mouse was sacrificed 44 weeks p.i. Serum of this mouse was frozen and prepared for immunological investigation. Spleen and liver of each mouse were measured and weighted. Livers were examined for larvae presence. Brain of each mouse was prepared for histological examination. Histopathological changes were observed. Larvae were found only in the brain of reinfected mouse in white matter of cerebrum in small amount. On the other hand, brains of all mice had local necrosis and atypical cell conglomerations. Mice sacrificed in 31th week had also unidentified structures with suspicion on granuloma. Even though larvae were not found in brains of mice with simple infection they may have caused local necrosis by migration or by activation of glial cells. Our next goal is to find out the cause of necrosis, discover larvae migratory routes more in detail and observe increased presence of glial cells or cell damages in brains.

The work is supported by Charles University in Prague (PRVOUK P25/LF1/2; UNCE 204017).

Abstracts

FIRST INSIGHTS INTO THE MOLECULAR DIVERSITY OF THE INVASIVE ASIAN TAPEWORM SCHYZOCOTYLE ACHEILOGNATHI

J. BRABEC

Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branišovská 31, 370 05 České Budějovice, Czech Republic

Schyzocotyle acheilognathi represents a recently resurrected species name for a fish pathogen formerly known as Bothriocephalus acheilognathi. Originally described in Japan by Yamaguti in 1934, S. acheilognathi is now globally distributed and infects an unrivalled diversity of freshwater fish (more than 200 species) with cyprinids being the most frequent hosts. It is considered an invasive species that has spread from East Asia by the means of the carp trade and managed to colonize a number of exotic fish taxa in the new areas. Despite being one of the most important fish pathogens, the phylogeography, population genetics and evolutionary ecology of S. acheilognathi remain significantly understudied. Here we use Illumina MiSeq next generation sequencing platform to shotgun sequence a fraction of the genomes of eight S. acheilognathi isolates as well as its closest known relative, S. nayarensis, from cyprinids in India. We de novo assemble and annotate abundant genomic loci, including complete mt genomes and nuclear ribosomal RNA operons, and on their basis gain the first insights into the molecular diversity and intraspecific relationships of this ubiquitous parasite.

Acknowledgement: Project Postdok_BIOGLOBE (CZ.1.07/2.3.00/30.0032) co-financed by the European Social Fund and the state budget of the Czech Republic.

Abstracts

VELKÝ BOLEVECKÝ POND: STORY OF AN OUTDOOR LABORATORY FOR AVIAN SCHISTOSOMES

J. BULANTOVÁ1, L. TURJANICOVÁ1, M. VAŠČIKOVÁ1, R. LEONTOVYČ1, M. CHANOVÁ2, P. HORÁK 1

1Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 00 Prague, Czech Republic 2Institute of Immunology and Microbiology, 1st Faculty of Medicine, Charles University in Prague, Studničkova 7, 128 00 Prague 2, Czech Republic

Natural water bodies are dynamic ecological systems with variable and often unpredictable environmental conditions. It is necessary to take it in an account particularly in the cases of field parasitological studies of avian schistosomes, their hosts and life cycles. To control as many factors of external environment as possible, it is usually necessary to transfer objects of our observation to more controlled conditions in a laboratory. Nevertheless, the primary source of material needs to be found in the field first, and this is just the point where our story begins. Velký Bolevecký pond is a water body on the northern outskirts of Pilsen, Czech Republic, with area of 43 ha, which is known as a famous locality for recreation (swimming, boating, etc.). In 2006, problems with water quality (occurrence of algae, cyanobacteriae) resulted in a long-term project of biomanipulation; majority of cyprinid fishes were continuously removed, bottom and littoral were planted by macrophytes, and the content of free phosphorus was modified chemically to reach clear water necessary for growth of submerged vegetation. Changed environment rich in water plants was quickly inhabited by numerous species of invertebrates including molluscs and, subsequently, the locality became important wintering place for many water birds, like Fulica atra, Cygnus olor, Anas platyrhynchos, Larus spp., Mergus merganser, Phalacrocorax carbo, Bucephala clangula, Aythya fuligula, Anas strepera, Podiceps cristatus, Tachybaptus ruficollis, and Gallinula chloropus. In spring 2013, people swimming in Velký Bolevecký pond experienced cercarial dermatitis, and ocellate furcocercariae from small alien planorbid snails of the species Gyraulus parvus were suspected of being the causative agent. During the next year, dissection of dead birds and examination of fresh feces of ringed individuals resulted in the finding of at least three other species of avian schistosomes. Molecular sequencing confirmed that in all cases, planorbid snails serve as intermediate hosts. Nevertheless, the role of any of those species of avian schistosomes as the agent of human cercarial dermatitis remains to be proved under laboratory conditions.

Acknowledgement: Czech Science Foundation (Grant No. 13-29577S) and Charles University in Prague (UNCE 204017, GAUK 502313) for financial support, DESOP Plzeň (Voluntary Association for Environment and Bird Protection) and Jindřich Duras for technical support.

Abstracts

ARE RODENT TAPEWORMS SUITABLE ACCUMULATIVE INDICATORS FOR THEIR HOST´S RISK ELEMENT EXPOSURE?

Z. ČADKOVÁ1, B. HORÁKOVÁ1, V. SLOUP1, J. SZÁKOVÁ2, J. VADLEJCH1, I. LANGROVÁ1

1Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 957, Prague 6, 165 21, Czech Republic 2Department of Agroenvironmental Chemistry and Plant Nutrition, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 957, Prague 6, 165 21, Czech Republic

Accumulative indicators are the organisms that store high quantities of a monitored element in their tissues. Suitable indicator need to be ubiquitous, sedentary, coupled with a high pollution tolerance and must live long enough to reveal risk element (RE) exposure. They have to be easily sampled and identified and they must provide sufficient biomass for chemical analyses. Based on their ecological characteristics, rodents are recognized as valuable indicators RE pollution. But - what about their tapeworms? This contribution discusses the practical issues concerning the use of rodent tapeworms as accumulative indicators for RE. The ability of tapeworms to accumulate and tolerate Cd, Pb and Zn was tested in laboratory experiment using a common host/parasite model R. norvegicus/H. diminuta. Pollutant exposure did not cause mortality in adult tapeworms and a 6 week exposure to contaminants leads to significant increase in pollutant levels in their tissue. Moreover, we detected an increase in toxic element concentrations after only a single dose. Since tapeworms usually live in their rodent hosts for at least several months, there is sufficient time to accumulate detectable RE levels. H. diminuta was showed to accumulate high RE levels in its tissue. However, BCF decreased in the following order: Pb >> Cd > Zn. To evaluate tapeworm ubiquity in our country, we examined 1184 small terrestrial mammals over the last 5 years and we detected 10-40 % tapeworm positive individuals, with an overall prevalence of 18 %. The intensity of tapeworm infection was as follows: insectivorous > omnivorous > herbivorous. In certain cases, a tapeworm biomass may be insufficient for conventional chemical analysis. However, the use of up-to-date analytical methods can sufficiently solve this issue. In their entire lifespan, adult tapeworms are strictly bound to one individual host and the maximum home range of a typical rodent is 2000 m2. Therefore, RE concentrations in parasite tissue should reflect contamination in a well-defined area. Based on above mentioned facts and considering all pros & cons, we assume that rodent tapeworm have promising potential to be suitable indicator of risk element contamination in the environment.

Acknowledgement: This study was supported by the GAČR 13-18154S and CIGA No. 20142030.

Abstracts

EFFECT OF A CO-EXISTENCE OF INVASIVE BARBUS BARBUS AND LOCAL BARBUS MERIDIONALIS ON THE COMPOSITION OF METAZOAN PARASITE COMMUNITIES

K. CIVÁŇOVÁ1, L. GETTOVÁ1, A. GILLES2, A. ŠIMKOVÁ1

1Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, Brno, 611 37, Czech Republic 2Aix-Marseille Université, IMBE, UMR CNRS 7263, Evolution Génome Environnement, Case 36, 3 Place Victor Hugo, 13331 Marseille Cedex 3, France

In France, rare and endemic Barbus meridionalis represents a native species while a widely distributed Barbus barbus is a recent immigrant, here. During the 2007-2013, Barbus individuals were sampled on several different localities in France. Allopatric populations for B. barbus and B. meridionalis were examined on the Rivers Allier and Varages, respectively. On the Rivers Durance and Ardeche, B. barbus individuals with a very low level of admixture from B. meridionalis individuals were found using microsatellite markers. Finally, individuals of both Barbus species were collected from the Argens River, where co-existence and hybridization between these two species is a result of a very recent introduction of B. barbus into the habitats of B. meridionalis. Examination of fish for metazoan revealed the presence of parasites belonging to both endoparasitic (Acanthocephala, Cestoda, Trematoda, Nematoda) and ectoparasitic (Monogenea, Crustacea, Mollusca) groups. In the allopatric populations of B. barbus and B. meridionalis, Monogenea and Acanthocephala, respectively, represent the most dominant groups of parasites. While monogeneans dominated in B. barbus populations on the Rivers Durance and Ardeche, both monogenean and acantocephalan parasites were highly abundant in host populations on the Argens River. Overall, higher abundance and species richness of metazoan parasites were detected in B. barbus allopatric population and populations on the Rivers Durance and Ardeche compared to the B. barbus individuals on Argens River. At the same time, significantly higher metazoan parasite species richness was revealed in the local B. meridionalis individuals compared with alien B. barbus individuals within the Argens River. These results are in congruence with general scenario of the parasite loss after the host introduction into the new environments where parasites are locally adapted. Moreover, Jaccard and Morisita indexes of parasite community similarity revealed the higher parasite similarity of the sympatric populations from the Argens River compared to the populations on the Rivers Durance and Ardeche. This might indicate that transmission of parasite infection between invasive B. barbus and local B. meridionalis is currently in a process.

Acknowledgement: This study was funded by the GACR project No. P505/12/0375.

Abstracts

UNDERSTANDING FASCIOLA HEPATICA RELATIONSHIP WITH IT'S HOST USING GENOMICS, TRANSCRIPTOMICS AND PROTEOMICS, WITH BASIC BIOLOGY

J. P. DALTON

Queen's University Belfast, Medical Biology Centre, School of Biological Sciences, 97 Lisburn Road Belfast BT9 7BL United Kingdom

Infection of the mammalian host with the trematode parasite Fasciola hepatica involves parasite activation from a dormant stage followed by penetration and migration through the intestine to the liver. As the parasite migrates it is continually developing and growing while encountering different host molecules, tissues and micro- environments that it must contend with, requiring strict gene regulation. Integration of the draft F. hepatica genome with the generation of extensive transcriptome datasets for the lifecycle stages present in the mammalian host, involved in the first 24 hours of invasion/infection; namely metacercariae, newly excysted juveniles, 1hr, 3hr and 24 hr post excystment. In particular our analysis has revealed that even within this short period of 24 hours remarkable differential gene expression is occurring within the parasite. Investigation into the gene transcription occurring within the infective stage, the metacercariae, has also revealed that several genes are "switched on" in contrast to the potential view of these stages being dormant. By integrating this data with the newly available F. hepatica draft genome, gene expression of multi-copy gene families such as the cathepsin cysteine proteases can be investigated. This study provides information on how the parasite prepares for infection and the changes that it must undergo once within the mammalian host, which can be exploited for control strategies to target this early stage of infection.

Abstracts

TRANSCRIPTOMIC ANALYSES OF A MONOGENEAN: NEXT-GENERATION SEQUENCING TECHNIQUES IN HANDS OF PARASITOLOGISTS

H. DVOŘÁKOVÁ1, L. JEDLIČKOVÁ1, M. KAŠNÝ1,2, P. BROŽ3, J. ILGOVÁ2, M. GELNAR2 AND L. MIKEŠ1

1Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 00 Prague, Czech Republic 2Department of Botany and Zoology, Faculty of Science, Masaryk University Brno, Kamenice 753/5, 625 00 Brno-Bohunice, Czech Republic 3Bioinformatician Institute of Applied Biotechnologies a.s., Služeb 4, 108 52 Prague 10, Czech Republic

Next-generation sequencing methods currently represent a robust tool enabling significant enrichment of our knowledge of biological systems at a level which has not been possible ever before. Unfortunately, the transcriptomic/genomic data on members of Monogenea are still extremely limited. Therefore we adopted the NGS techniques to generate a first high quality transcriptome of a representative of blood-feeding monogeneans - Eudiplozoon nipponicum (Diplozoidae: Heteronchoinea), the ectoparasite from the gills of common carp Cyprinus carpio. Using 454 sequencing technology (GS FLX System, Roche), a total of 324 941 reads were generated (average length 423 bp) and assembled into 6910 contigs. The second transcriptome analysis was performed by the MiSeq System (Illumina). A total of 74 823 255 input read pairs, a total of 142 813 all transcripts (median contig length 367 bp) and a total of 119 136 of genes (median contig length 334 bp) were obtained. Currently, the comprehensive bioinformatic analysis is running - the assemblies are in the process of evaluation and a pipeline for annotation of particular genes is being developed. Special attention was paid to peptidases that could be involved in digestion of host´s blood and other biological processes in E. nipponicum.

Acknowledgements: This research was supported by Czech Science Foundation (P506/12/1258, GBP505/12/G112), grants of the Masaryk University (MUNI/A/1484/2014) and grants of the Charles University in Prague (GAUK 502313, UNCE 204017, PRVOUK P41, SVV).

Abstracts

PROLYL OLIGOPEPTIDASE FROM THE BLOOD FLUKE SCHISTOSOMA MANSONI: FROM FUNCTIONAL ANALYSIS TO ANTI-SCHISTOSOMAL INHIBITORS

P. FAJTOVÁ1, S. ŠTEFANIĆ2, M. HRADILEK1, J. DVOŘÁK 3,4, J. VONDRÁŠEK1, JÍLKOVÁ A.1, L. ULRYCHOVÁ1,3, J. H. MCKERROW5*, C. R. CAFFREY5, M. MAREŠ1, M. HORN1

1Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic 2Institute of Parasitology, University of Zurich, Zurich, Switzerland, 3Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic 4Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic, Ceske Budejovice, Czech Republic 5Center for Innovation and Discovery in Parasitic Diseases, Department of Pathology, University of California San Francisco, San Francisco, California, USA * Current Address: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, California, USA

Blood flukes of the genus Schistosoma cause schistosomiasis, a parasitic disease that infects over 240 million people worldwide, and for which there is a need to identify new targets for chemotherapeutic interventions. Our research is focused on Schistosoma mansoni prolyl oligopeptidase (SmPOP) from the serine peptidase family S9, which has not been investigated in detail in trematodes. We demonstrate that SmPOP is expressed in adult worms and schistosomula in an enzymatically active form. By immunofluorescence microscopy, SmPOP is localized in the tegument and parenchyma of both developmental stages. The active site specificity of SmPOP was investigated using synthetic substrate and inhibitor libraries, and by homology modeling. SmPOP is a true oligopeptidase that hydrolyzes peptide (but not protein) substrates with a strict specificity for Pro at P1. Both the recombinant enzyme and live worms cleave host vasoregulatory, proline-containing hormones such as angiotensin and bradykinin. Finally, we designed nanomolar inhibitors of SmPOP that induce deleterious phenotypes in cultured schistosomes. Our results suggest that SmPOP plays a role in host-parasite interactions and is a potential target for the development of anti-schistosomal drugs.

Abstracts

FAR APART: SPECIES OF MACVICARIA GIBSON & BRAY, 1982 (DIGENEA: OPECOELIDAE) IN THE MEDITERRANEAN AND ANTARCTIC

S. GEORGIEVA1, Š. MAŠOVÁ2, A. FALTÝNKOVÁ1

1Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branišovská 31, 370 05 České Budějovice, Czech Republic 2Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic

Macvicaria Gibson & Bray, 1982 is a large digenean group of parasites in marine fishes throughout the world ocean. In a study on the diversity of digeneans in sparid fishes (Diplodus annularis, Lithognathus mormyrus, Oblada melanura, Sparus aurata and Spondyliosoma cantharus) in the western Mediterranean and in nototheniid fishes (Trematomus newnesi, T. hansoni, T. bernacchii and Notothenia coriiceps) in the western Antarctic we characterised morphologically and molecularly a large number of isolates of Macvicaria spp. The ITS1-5.8S-ITS2 gene cluster and partial fragments of the 28S rRNA gene were amplified for representative samples from each host species and 28S rDNA sequences were analysed together with sequences for related species of the family Opecoelidae Ozaki, 1925. Comparative morphological and molecular analyses revealed the presence of M. dubia (Stossich, 1905) and two new species of Macvicaria in the sparids of the western Mediterranean and of two species, M. antarctica (Kovaljova & Gaevskaya, 1974) and M. pennelli (Leiper & Atkinson, 1914) in the nototheniids from the Weddel Sea (Antarctica). The phylogenetic hypotheses estimated from Bayesian inference and maximum likelihood analyses revealed that species of Macvicaria collected from the two distant localities and from phylogenetically distant hosts do not represent a monophyletic assemblage. Our results, and especially the inclusion of novel molecular data for the Antarctic Macvicaria spp., highlight the need of obtaining sequences for more species, and especially for the type-species [M. alacris (Looss, 1901)] in order to resolve the boundaries of the genus Macvicaria and its relationships within the Opecoelidae.

Acknowledgement: This study was supported by the Czech Science Foundation (project P505/12/G112). We acknowledge the Czech Antarctic Station “J. G. Mendel” and its crew for their support.

Abstracts

STRATEGIES OF PARASITIC WORMS TO FIND, RECOGNIZE AND INVADE THEIR HOSTS

W. HAAS

Former Section Parasitology, Institute for Zoology, University Erlangen-Nuernberg, Germany

Many parasitic worms enter their hosts by active invasion and survival of these species fully depends on the invasion success of the infective stages. This is often based on a mass production of the invasive stages. However, most stages show in addition a highly specific host-finding behaviour. Information on host-finding mechanisms is available mainly for trematode miracidia and cercariae and for nematodes. The stages find and recognize their hosts, in some cases even with species-specificity, via complex sequences of behavioural patterns in which they respond successively to very different environmental and host cues (trematode cercariae perform 9 succeeding invasion steps). There is often a surprisingly high diversity of host-recognition strategies. Each species finds and identifies its host using mechanisms in response to host signals that differ from those of other species, even when these invade the same host genera. For example, 5 different species of schistosomes enter the human skin using different recognition-sequences. This diversity of recognition strategies is not yet understood. The hypothesis that this may reflect adaptations to distinct ecological conditions of transmission has still to be substantiated by experimental data. Another question is, how parasitic worms after invasion find their complex paths through their host’s tissues to their often very specific microhabitats. At least the migrating stages of the few species investigated so far can follow local chemical gradients of host’s tissue compounds. However, their navigation within the body over longer distances remains puzzling. The high complexity, specificity and diversity of host-recognition strategies suggest that host-finding and host-recognition are important determinants in the evolution of parasite life cycles.

Abstracts

HIGH MORPHOLOGICAL PLASTICITY AND GLOBAL GEOGRAPHICAL DISTRIBUTION OF THE PACIFIC BROAD TAPEWORM ADENOCEPHALUS PACIFICUS (SYN. DIPHYLLOBOTHRIUM PACIFICUM): MOLECULAR AND MORPHOLOGICAL SURVEY

J. S. HERNANDES ORTZ1,3, T. SCHOLZ1, J. BRABEC1, T. KUZMINA2, R. KUCHTA1

1Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Branišovská 31, 370 05 České Budějovice, Czech Republic 2Schmalhausen Institute of Zoology NAS of Ukraine, vul. B. Khmelnyts’kogo, 15, Kyiv 01601, Ukraine 3Present address: Departamento de Zoología, Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), Avenida Universidad 3000, Ciudad Universitaria, CP 04510, Distrito Federal, México

The most important causative agent of human diphyllobothriosis in South America, Diphyllobothrium pacificum, is transferred to the original genus Adenocephalus (Nybelin, 1931), revised and redescribed on the basis of the evaluation of an extensive material collected mainly from northern fur seal, Callorhinus ursinus, from St. Paul Island, Alaska. Detailed analysis of morphological and morphometrical data show a high variability in most of the characteristics traditionally used in diagnosis of diphyllobothriid tapeworms. Phylogenetic analyses based on newly characterised sequences of mitochondrial cytochrome c oxidase subunit 1 and nuclear large subunit ribosomal RNA genes consistently reveal A. pacificus as a sister lineage to the clade formed of the remaining Diphyllobothrium species and other genera (Digramma, Diplogonoporus, Ligula). Despite the generally similar morphology, A. pacificus can be differentiated from the closely related taxa by the presence of transverse papilla-like tegumental protuberances distributed anteriorly, separated by narrow semicircular grooves on the ventral surface of segments between their anterior margin and the anterior edge of the male gonopor, and relatively small eggs. Adenocephalus pacificus displays a relatively low host specificity (found in 9 of 15 otariids, and in accidental hosts such as man, dog and jackal, the latter representing a new host) and a uniquely wide geographical distribution on both hemispheres.

Abstracts

EXPRESSION OF A CYSTEINE PEPTIDASE INHIBITOR FROM EUDIPLOZOON NIPPONICUM (MONOGENEA)

J. ILGOVÁ1, M. GELNAR1, M. KAŠNÝ1, 2

1Department of Botany and Zoology, Faculty of Natural Sciences, Masaryk University Brno, Czech Republic 2Department of Parasitology, Faculty of Science, Charles University Prague, Czech Republic

Our research is focused primarily on expression and characterization of cystatin produced by platyhelminth Eudiplozoon nipponicum. This blood-feeding ectoparasite of Cyprinus carpio (common carp) is a representative of the family Diplozooidae (Monogenea). Cystatins – cysteine peptidase inhibitors are produced by a wide range of organisms and belong to bioactive molecules with immunomodulatory and inhibitory properties. The transcriptomic data of E. nipponicum were analyzed for the presence of cystatin sequences. The partial nucleotide sequence was identified and the whole gene sequence was obtained by PCR, 3’/5’ RACE PCR and sequencing using cDNA as a template. Gene coding 98 amino acid cystatin of E. nipponicum with predicted molecular weight 10.85 kDa and theoretical pI 6.27 was inserted into pET19b expression vector and the obtained construct was transferred into E. coli competent cells (BL 21 strain). Expression of recombinant cystatin molecule was induced by adding IPTG to the cultivation media. The success of production was confirmed by mass spectrometry in a dominant protein band after the fractionation of the E. coli derived protein mixture by SDS-PAGE. Recombinant protein was produced in the insoluble form incorporated into inclusion bodies. The cystatin from E. nipponicum will be subsequently solubilized and tested for its functional and structural properties.

Acknowledgements: Czech Science Foundation (P506/12/1258, GBP505/12/G112), grants of the Masaryk University (MUNI/A/1484/2014) and grants of the Charles University in Prague (UNCE 204017, PRVOUK P41, SVV).

Abstracts

PEPTIDASES DETECTED IN EXCRETORY/SECRETORY PRODUCTS OF THE MONOGENEAN EUDIPLOZOON NIPPONICUM

L. JEDLIČKOVÁ1, H. DVOŘÁKOVÁ1, M. KAŠNÝ1,2, D. POTĚŠIL3, Z. ZDRÁHAL3AND L. MIKEŠ1

1Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Prague 2, Czech Republic 2Department of Botany and Zoology, Faculty of Science, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic 3RG Proteomics, Central European Institute of Technology, Masaryk University, Brno, Kamenice 753/5(A31), 625 00 Brno Bohunice, Czech Republic

Monogeneans of the family Diplozoidae (Heteronchoinea) are blood-feeding ectoparasites occurring mainly on the gills of cyprinid fishes. Blood processing mechanism in monogeneans of this group are known only from ultrastructural and histochemical analyses. Only little information is available about the biochemistry of digestion and participating digestive enzymes (peptidases) which can be involved in degradation of blood proteins for nutrition. However, it is assumed that blood digestion proceeds similarly to other blood- feeding platyhelminths. The aim of our study was the biochemical and molecular characterization of main peptidases which can be involved in digestion in Eudiplozoon nipponicum. The presence of cysteine peptidases, mainly cathepsin(s) L, was confirmed in samples of excretory/secretory products (ESP) from adult E. nipponicum by using fluorogenic substrates, specific inhibitors, active site affinity probe DCG-04 and in-vitro hemoglobin degradation. ESP were separated by 1D electrophoresis and bands of relevant sizes (20-35 kDa) were excised from the gel and analysed by mass spectrometry. Resulting tryptic peptides corresponded to the sequences of cathepsins L of E. nipponicum, which were obtained by PCR methods: Degenerated primers were designed according to the consensus sequence of the active site of cysteine peptidases of several organisms. PCR and RACE PCR resulted in full length sequences of two cathepsins L termed EnCL1 and EnCL3. Both recombinant cathepsins L expressed in Pichia pastoris yeast are being biochemically characterized and antibodies will be produced for immunolocalization within the body of the worm. Based on the results of biochemical analyses, it seems that cathepsin(s) L may be the major peptidases responsible for hemoglobinolysis in freshwater monogeneans of the family Diplozoidae.

Acknowledgements: Acknowledgements: Czech Science Foundation (P506/12/1258, GBP505/12/G112), grants of the Masaryk University (MUNI/A/1484/2014), grants of the Charles University in Prague (GAUK 502313, UNCE 204017, PRVOUK P41, SVV) and “CEITEC - Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) project from the European Regional Development Fund.

Abstracts

STRUCTURAL AND FUNCTIONAL ANALYSIS OF THE SCHISTOSOMA MANSONI CATHEPSIN B1 DRUG TARGET

A. JÍLKOVÁ1, M. HORN1, P. REZÁČOVÁ1, J. BRYNDA1, J. H. MCKERROW2, C. R. CAFFREY2 AND M. MARES1

1Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 16610 Prague, Czech Republic 2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, San Diego, CA 92093, USA

Digestive protease cathepsin B1 (SmCB1) of the human blood fluke Schistosoma mansoni is a potential drug target for the treatment of schistosomiasis, a parasitic disease that afflicts over 200 million people worldwide. SmCB1 is biosynthesized in the form of inactive zymogen in which the propeptide operates as an intra-molecular inhibitor by blocking the active site. We investigated the proteolytic processing through which the propeptide is removed and identified a dual activation pathway for SmCB1 under the complex control of sulfated polysaccharides. The activation mechanisms were explained using crystal structures of three molecular forms of SmCB1 along the activation pathway. Further, we determined crystal structures of the mature SmCB1 complexed with two peptidomimetic vinyl sulfone inhibitors to describe their binding mode. These structural data and biochemical profiling provided insight into the specificity of SmCB1 inhibition. We demonstrated that the severity of phenotypes induced in the parasite by vinyl sulfone inhibitors correlated with inhibition of SmCB1 activity, thus confirming SmCB1 as a valuable drug target. Our data provide a footing for the rational design of anti-schistosomal protease inhibitors targeting SmCB1.

Abstracts

MONOGENEA … WHAT ELSE CAN BE DONE?

D. JIRSOVÁ1,2, M. GELNAR1, M. KAŠNÝ1,3

1Department of Botany and Zoology, Faculty of Science, Masaryk University Brno, Kamenice 753/5, 625 00 Brno Bohunice, Czech Republic. 2Department of Forest Botany, Dendrology and Geobiocoenology, Faculty of Forestry and Wood Technology, Mendel University in Brno, Zemědělská 3, 61300 Brno, Czech Republic. 3Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 00 Prague, Czech Republic. *Corresponding author, e-mail: [email protected]

Monogeneans are studied in depth by using various molecular techniques, which enable to obtain complex information concerning their morphology, biochemistry and genetics. Our study is focused on three model organisms Eudiplozoon nipponicum, Paradiplozoon homoion and Paradiplozoon gracile and four aims are realized; (i) compare the genetic diversity in populations of monogenean species, (ii) determine the level of genetic variability of the two permanently fused worms isolated from the same fish, (iii) reveal the intra- (population of worms parasitizing on the same fish) and inter-population patterns (population of worms originating from different fish) and (iv) evaluate the effect of different host species on genetic plasticity of monogenea with generalist life strategy. In order to answer these questions we would like to apply the whole genome scan method - amplified fragment length polymorphism technique. In addition we use the next generation sequencing methods as a powerful tool to obtain the whole genome and transcriptome. In this context the critical point is the achievement of high percentage of genome/transcriptome sequential coverage. The information of genome size is therefore very important. We would like to use the DNA fluorescent double staining method which represents simple and easy checking point for obtained bioinformatic data.

Abstracts

INTRASPECIFIC VARIATION OF CICHLIDOGYRUS (MONOGENEA, DACTYLOGYRIDAE) IN LAKE TANGANYIKA

N. KMENTOVÁ1, M. GELNAR1, M. MENDLOVÁ1, M. VAN STEENBERGE2, S. KOBLMÜLLER4, M. P. M. VANHOVE1,2,3

1Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic 2Biology Department, Royal Museum for Central Africa, Leuvensesteenweg 13, 3080 Tervuren, Belgium 3Capacities for Biodiversity and Sustainable Development, Operational Directorate Natural Environment, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B- 1000 Brussels, Belgium 4Department of Zoology, Karl-Franzens University of Graz, Universitätsplatz 2, A-8010 Graz, Austria

Lake Tanganyika figures as an exquisite study system for biologists because of its unique diversity throughout various fish and invertebrate taxa. Research on monogeneans has been intensified in the past few years there. Members of Cichlidogyrus showed a relatively strong host specificity in littoral cichlids while a reduction of this specificity was suggested for deepwater species. The main purpose of our study was testing the host range of deepwater representatives of Cichlidogyrus to check for ongoing speciation or decrease in host specifity. In total, 12 cichlid species from three different tribes were examined for monogenean infection. The morphometric part of the study was based on the measurements of parasite sclerotized structures; geomorphometric analysis provided a visualization of the complex shape variation as an additional view to classical morphology. Genetic characterization was performed by means of established markers with different rates of molecular evolution. Finally, 883 parasite individuals from 34 host fish were collected. Four different species belonging to the genus Cichlidogyrus were distinguished by both morphometric and genetic characterization in all obtained ribosomal DNA regions. The low host specificity of the deepwater species C. casuarinus was confirmed. Although no indication of ongoing speciation processes were observed in the genetic part of the study, significant intraspecific variation in the morphology of parasite sclerotized structures influenced by host preference and geographical origin was documented. This phenotypic plasticity is therefore probably correlated with specific host characteristics together with environmental factors.

Abstracts

INFLUENCE OF HYBRIDIZATION ON THE FISH HOST CONDITIONS AND PARASITE INFECTION

V. KRASNOVYD AND A. ŠIMKOVÁ

Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlařská 2, Brno, 611 37, Czech Republic

Frequent hybridization cases have been described between different species of the Cyprinidae family. Hybrids of roach and common bream have been repeatedly studied in terms of their biology, ecology and behavior. However, information on parasite load and hybrid condition in cyprinid hybrids are very limited. The presence and infection parameters of metazoan parasites of common bream (Abramis brama), roach (Rutilus rutilus) and their respective hybrids from Hamry reservoir (Czech Republic) were analyzed. Fish were sampled in spring and autumn seasons in three consecutive years. All individuals were identified using molecular markers (partial cyt b gene and 9 microsatellite loci) and morphological characteristics. The aim of this study was to analyze the influence of hybridization on parasite infection and host fish condition. Total parasite abundance and prevalence was higher in parental species when, compared to the hybrid specimens, nevertheless parasite species richness was higher in hybrids. The significant effects of season, locality and year of collection on the composition of the metazoan parasite communities were found. We confirmed the presence of a dominative ancestral maternal line of the Abramis brama in interspecific hybridization. Presence of host-specific monogenean parasites in hybrids of the both maternal origins, likely suggests the broken system of co-adaptation genes in hybrids.

Abstracts

INTERACTIONS OF NEUROTROPIC LARVAE OF BIRD SCHISTOSOME TRICHOBILHARZIA REGENTI AND IMMUNE CELLS OF HOSTS CENTRAL NERVOUS SYSTEM

V. KRČMÁŘOVÁ, J. BULANTOVÁ AND P. HORÁK

Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Prague 2, Czech Republic

Trichobilharzia regenti is a neurotropic fluke belonging to family Schistosomatidae. Although it is a bird schistosome, free-living larvae - cercariae are able to penetrate skin and develop in schistosomula also in accidental mammalian hosts including human. There they can cause inflammatory skin reaction called cercarial dermatitis or swimmer’s itch. In natural bird host (duck Anas platyrhynchos) schistosomula continue from skin to peripheral nerves and through central nervous system (CNS) to the nasal mucosa, where they reach sexual maturity, copulate and lay eggs. In experimentally infected mice, schistosomula can be captured by host immune response already in the skin. Nevertheless, some of them are able to survive and migrate to spinal cord where they activate immune response, particularly astrocytes and microglial cells. Despite of this fact, schistosomula can be found in murine spinal cord approximately for 3 weeks post primary infection. Repeated infections of mice lead to more intensive immune reaction and larvae are damaged already in early phase of infection. Cells responsible for destruction of schistosomula in murine spinal cord can be identified and visualized by immunohistochemistry using specific fluorescent labeled antibodies anti-Iba1 and anti-MHC II for microglia and anti-GFAP for astrocytes. For better understanding of interactions between host immune cells and parasite in situ, it was necessary to examine site of infection at the ultrastructural level. Data from our preliminary studies proved presence of host immune cells with noticeably flattened nuclei and numerous vacuoles in close contact with migrating larvae. Therefore, transmission electron microscopy will be performed in combination with immunolabeling by gold particles to identify these cells reliably and characterize their vacuolar content.

Acknowledgements: Czech Science Foundation (Grant No. 13-29577S).

Abstracts

MOLECULAR PHYLOGENY OF THE BOTHRIOCEPHALIDEA (CESTODA): MOLECULAR DATA CHALLENGE MORPHOLOGICAL CLASSIFICATION

R. KUCHTA, J. BRABEC, A. WAESCHENBACH, T. SCHOLZ, D. TIMOTHY, J. LITTLEWOOD

Institute of Parasitology, Biology Centre of the Academy of Sciences of the Czech Republic, 370 05 České Budějovice, Czech Republic

In this study, the relationships of the cestode order Bothriocephalidea, parasites of marine and freshwater fish, were assessed using multi-gene molecular phylogenetic analyses. The dataset included 60 species, covering about 70% of currently recognised genera, a sample of bothriocephalidean biodiversity gathered through an intense 15-year effort. The order, whilst being monophyletic, includes three non-monophyletic and one monophyletic family. Bothriocephalidae is monophyletic and forms the most derived lineage of the order, comprising a single freshwater and several marine clades. Biogeographic patterns within the freshwater clade are indicative of past radiations having occurred in Africa and North America. The earliest diverging lineages of the order are formed of a paraphyletic Triaenophoridae. The Echinophallidae, consisting nearly exclusively of parasites of pelagic fish, was also resolved as paraphyletic to the inclusion of the Bothriocephalidae. Philobythoides sp., the only representative of the Philobythiidae, a unique family of parasites of bathypelagic fish, formed the sister group to the genus Eubothrium, the latter forming one of the lineages of the paraphyletic Triaenophoridae. Because of the weak statistical support for most of the basal nodes of the Triaenophoridae and Echinophallidae, as well as the lack of obvious morphological synapomorphies shared by taxa amongst well-supported lineages, the current family-level classification is provisionally retained, with the exception of the family Philobythiidae, which is now recognised as a synonym of the Triaenophoridae.

Abstracts

MORPHOLOGICAL AND MOLECULAR APPROACHES TO THE STUDY OF DIGENEAN LARVAE FROM THE CURONIAN LAGOON IN LITHUANIA

O. KUDLAI

Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branišovská 31, 370 05 České Budějovice, Czech Republic

A combination of morphological and molecular approaches is being increasingly used to solve difficulties in identifying larval stages of digeneans. This study was carried out to identify the digenean larval stages by incorporating morphological and molecular data using specimens collected from snails in the Curonian Lagoon in Lithuania. Examination of 350 snails of seven species (Bithynia tentaculata, Lymnaea palustris, Planorbarius corneus, Planorbis planorbis, Radix auricularia, Valvata sp. and Viviparus viviparus) revealed infections with 17 digenean species, representatives of eleven families. The identity of two species of cercariae previously described as Cercaria helvetica XII and C. helvetica XIX was established based on sequences of the ITS2 region and partial 28S gene of the nuclear rDNA. Cercaria helvetica XII clustered with representatives of the genus Lecithodendrium (Lecithodendriidae), being very close, but not identical, to Lecithodendrium linstowi. Sequences C. helvetica XIX fell within a clade together with species of the genus Sphaeridiotrema (Psilostomidae). Additionally, cercariae of six species were identified (Haematoloechus asper, Metaleptophallus gracilimus, Moliniella anceps, Notocotylus imbricatus, Paryphostomum radiatum, Posthodiplostomum cuticola) and six species were identified to the genus level (Apatemon sp., Echinochasmus sp., Ichthyocotylurus sp., Plagiorchis sp., Psilotrema sp., Trichobilharzia sp.). Identification of cercariae of two species (Cyathocotylidae gen. sp. I and Cyathocotylidae gen. sp. II) and of metacercariae of one species (Strigeidae gen. sp.) was achieved to the family level only. Most of the species found (76%) mature in birds, which is primarily due to the fact that the Curonian Lagoon is an important bird area that supports enormous numbers of migrant and resident birds on the Baltic coast.

Acknowledgements: Charles University in Prague (Project Nos. UNCE 204017, PRVOUK P41, SVV 267210/2013, GAUK 502313).

Abstracts

STRONGYLOIDES (NEMATODA; STRONGYLIDAE) OF WILD AND DOMESTIC EQUIDS IN UKRAINE: BIODIVERSITY AND PARASITE COMMUNITY STRUCTURE.

T. A. KUZMINA1, V. A. KHARCHENKO1, N. S. ZVEGINTSOVA2

1Institute of Zoology NAS of Ukraine, 15, B. Khmelnitskogo str., Kyiv, 01601, Ukraine; [email protected] 2Askania Nova Biosphere reserve UAAS, Frunze str., 13, Askania-Nova, Kherson region, Ukraine

In Ukraine, more then 500,000 of domestic horses (Equus caballus) and several thousands of donkeys (E. asinus) are kept in different types of horse farms. Besides, four species of wild equids – wild Przewalski’s horses (E. ferus przewalskii), Turkmenian kulans (E. hemionus), zebras (E. burchelli and E. grevyi) are kept in the Askania Nova Biosphere reserve. Strongylid nematodes are the main and the most pathogenic group of horse parasites in Ukraine and worldwide; nowadays there are 65 species of these nematodes are registered in equids. The aim of our work to compare the biodiversity and structure of strongylid nematode community in different equid species by in vivo method. Totally, 238 equids (162 domestic horses, 24 Przewalski’s horses, 13 kulans, 16 donkeys, and 23 zebras) of various ages were included into studies. All animals were treated with anthelmintic drug “Univerm” (0.2% aversectin C, Russia). Fecal samples (200 g each) were collected from each animal after 24, 36, 48 and 60 hours after treatment. All strongylid expelled (> 80,000) were collected and identified. Totally, 36 species of strongylids (9 species of subfamily Strongylinae and 25 – of Cyathostominae) were found in six equid species. In domestic horses, 33 strongylid species were found; from 4 to 23 species (average 11.0±4.1) parasitized per one horse. In donkeys, 26 strongylid species were observed; from 6 to 16 species (12.7±2.4) parasitized per donkey. In wild Przewalski’s horses, 31 strongylid species were collected; from 9 to 18 species (14.5±2.5) per horse. In kulans, 25 species were found; from 7 to 19 species (14±3.6) per one kulan. In plains zebras, 21 species were found; from 3 to 14 species (8.5±3.2) per host. In Grevy’s zebras, 18 species were found; from 4 to 14 species (9.0±3.3) per host. General structure of strongylid community of all equids, except of Grevy’s zebras and domestic horses from brood horse farms was multimodal with dominant, subdominant, background and rare species. Grevy’s zebras and these horses had bimodal structure of strongylid community (“core – satellite” mode) which is typical for animals which are undergone frequent dewormings. Bray–Curtis Cluster analysis reveals similarity of strongylid communities in the both zebra species with donkeys, and similarity of strongylid community of Turkmenian kulans and domestic horses.

Abstracts

BIODIVERSITY OF GASTROINTESTINAL PARASITE COMMUNITY IN NORTHERN FUR SEALS (CALLORHINUS URSINUS) ON ST. PAUL ISLAND, ALASKA.

T. A. KUZMINA1, T. R. SPRAKER2, E. T. LYONS3, R. KUCHTA4, O. LISITSYNA1, O. KUDLAI1,4

1Institute of Zoology NAS of Ukraine; 15, Bogdan Khmelnitsky street, Kiev, 01601, Ukraine; e-mail: [email protected] 2Diagnostic Medical Center, Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA 3University of Kentucky, Department of Veterinary Science, Gluck Equine Research Center, Lexington, KY, USA 4Institute of Parasitology, Biology Centre, Czech Academy of Sciences, Branišovská, 31, 37005 České Budějovice, Czech Republic

The northern fur seals (Callorhinus ursinus) (NFS) serve as hosts for more than 25 species of helminths. Several studies documenting parasites of NFSs were performed in the 1960’s through the 1980’s in the USA and the former USSR. No furter parasitological studies of NFSs were carried out in this region during last decades. The aim of our studies performed at St. Paul Island, Alaska, was to examine the biodiversity of the helminth community parasitizing NFSs and extend the knowledge of the parasites in this host. The studies were carried out between July–August of 2011–2014 on St. Paul Island, Alaska. Gastrointestinal tracts of 756 subadult NFS males were collected during the annual Aleut subsistence harvests. All helminths (27,169) were collected, fixed in 70% ethanol and identified. Gastrointestinal helminthes were found in all NFS’s examined. Totally, 18 species were found: 5 species of nematodes (Anisakis simplex, Contracaecum osculatum, Pseudoterranova decipiens, P. azarazi and Phocascaris cystophorae), 3 cestodes (Diphyllobothrium (Adenocephalus) pacificum, Diplogonoporus tetrapterus and Anophryocephalus cf. ochotensis), 7 acanthocephalans (Bolbosoma nipponicum, Corynosoma strumosum, C. alaskensis, C. semerme, C. similis, C. validum, C. villosum) and 3 digeneans (Phocitrema fusiforme, Pricitrema zalophi and Stictodora sp.). Nematodes were found in 90.9% of NFSs; the intensity was 1 to 467 nematodes (average 11.2±21.6) per host; median intensity (MI) = 7. Prevalence of NFS infection with cestodes was 98.6%; with intensity from 1–107 (19.6±16.6) per host; MI = 16. Acanthocephalans were observed in 43.5% of NFSs; the intensity of NFS infection was 1–29 (3.6±4.4) acanthocephalans per host; MI = 2. Digeneans were found in 32.3% of NFSs; with intensity from 1–1540 (18.4±111.1) per host; MI = 2. Comparison of our data with data collected from the 1960’s and 1980’s revealed significant changes in NFS parasite community structure during the last 50 years, which, in our opinion, is connected with a declining NFS population and overfishing in the Bering Sea and North Pacific which has led to a decline in the intermediate hosts for these gastrointestinal helminths.

Abstracts

TRYPSIN LOADED WORMS: SMSP2, PREDOMINANT SERINE PROTEASE IN S. MANSONI MAMMALIAN STAGES

A. LEONTOVYČ1, L. ULRYCHOVÁ1,2, C. R. CAFFREY3, A. J. O'DONOGHUE3, M. MAREŠ1, M. HORN1, J. DVOŘÁK1

1 Institute of Organic Chemistry and Biochemistry ASCR, Flemingovo nám. 2. 166 10 Praha 6, Czech Republic. 2 Department of Parasitology, Faculty of Science, Charles University, Viničná 7, 12844 Praha 2, Czech Republic. 3 Center for Discovery and Innovation in Parasitic Diseases, University of California, 1700 4th Street, San Francisco, USA

Schistosomiasis is a parasitic disease caused by several blood fluke species of the genus Schistosoma. It is considered the second most important parasitic infection after malaria with more than 200 million people infected worldwide and many more at risk. In our research we focused on newly discovered trypsin-like protease SmSP2 [1]. Levels of SmSP2 expression in developmental stages parasitizing humans (schistosomula and adults) are stunning. Biological role of SmSP2 is unknown but due to the high expression level we suppose its important role in parasite physiology and potential impact on parasite/host interactions. In order to prepare fully active reocmbinat enzymes, we tested expression system based on Escheria coli and Pichia pastoris; both using differently modified SmSP2 sequences and under various expression conditions. Despite, E. coli production led to the high yield of the recombinant protein, we failed to produce it in properly folded form. Therefore, this recombinant was used solely for production of polyclonal antibodies. Later, after numerous optimizations we successfully expressed fully processed and activated SmSP2 enzyme in Pichia pastoris. This recombinant protease was purified using combination of size exclusion chromatography and ion-exchange chromatography and used for biochemical characterizations. SmSP2 has a trypsin activity in a broad pH range with optimum around pH 8. Substrate specificity screening revealed typical trypsin preference for arginine and lysine in P1 position and no significant preference in P2 and P3 positions. However, unusual preference for proline in P4 position was found. Localization of SmSP2 is currently under progress using RNA in situ hybridization method and immunohistochemistry. This study represents now solid base for further research of the biological role of SmSP2 in S. mansoni mammalian stages.

[1] M. Horn, P. Fajtová, L. Rojo Arreola, L. Ulrychová, P. Bartošová-Sojková, Z. Franta, A. V. Protasio, D. Opavský, J. Vondrášek, J. H. McKerrow, M. Mareš, C. R. Caffrey, and J. Dvořák, “Trypsin- and Chymotrypsin-Like Serine Proteases in Schistosoma mansoni - ‘The Undiscovered Country‘, PLoS Negl. Trop. Dis., vol. 8, no. 3, 2014.

Abstracts

COMPARATIVE STUDY OF TRANSCRIPTOME PROFILES OF CERCARIAE/SCHISTOSOMULA OF BIRD SCHISTOSOME TRICHOBILHARZIA REGENTI

R. LEONTOVYČ1, N. YOUNG2, P. HORÁK1, R. GASSER2, M. KAŠNÝ 1,3

1Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Prague 2, Czech Republic 2Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia 3Department of Botany and Zoology, Faculty of Science, Masaryk University Brno, Kamenice 753/5 625 00 Brno Bohunice, Czech Republic

The bird fluke Trichobilharzia regenti is a member of the largest genus within the medically and veterinary important trematode family Schistosomatidae. Trichobilharzia regenti is a unique among trematodes due to its affinity to nervous tissue. Free living cercariae can penetrate the skin of definitive host – waterfowl and migrating schistosomula of T. regenti are the causative agents of serious neuromotoric disorders not only of birds but also of accidentally infected mammals. The knowledge concerning the molecules involved into these processes as well as the the molecular level of parasite-host interactions are still not completely understood. Here we present the first bird schistosoma transcriptomic data, which reflects the moment of preparation of cercariae to invasion, and the late phase of migration of schistosomula in the duck as definitive host. Millions of sequences were generated, thousands of genes were annotated and the differences in their expression estimated for both stages – cercariae and schistosomula. The essential metabolic pathways and protein classes were identified. At the molecular level the principles of transition of free-living to parasitic stage were revealed.

Acknowledgements: Czech Science Foundation (Grant No. GA13-29577S), Charles University in Prague (UNCE 204017, PRVOUK P41, SVV) and Masaryk University (MUNI/A/1484/2014).

Abstracts

MORPHOLOGICAL AND MORPHOMETRICAL ANALYSES OF THE EGGS OF HUMAN-INFECTING DIPHYLLOBOTHRIID CESTODES

K. LEŠTINOVÁ, ROMAN KUCHTA

Institute of Parasitology, Biology Centre of the Czech Academy of Sciences and Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic

Tapeworms of the order Diphyllobothriidea are well-known parasites of tetrapodes, including man. Identification of the members of the genus Diphyllobothrium is difficult because of their morphological uniformity. Around 14 species of this genus have been reported from man, but their differentiation is almost impossible based on clinical samples. Clinical samples include almost exclusively only eggs or pieces of decomposed strobila. The eggs of diphyllobothiid cestodes possess a hard shell; their measurements were tested as potential discriminative characters for most common human-infecting species. Fifty eight samples of the eggs of 8 species were measured (length and width) and their surface was studied using scanning electron microscopy (SEM). It has been found that D. cameroni and D. pacificum possess smallest, most rounded eggs, whereas D. cordatum has the largest eggs. The eggs of D. latum and D. nihonkainese are most similar to each other, which makes their differentiation based on egg size impossible. Numerous pits were observed on the surface of the eggs of marine species, whereas those of freshwater species have smooth surface devoid of any pits.

Acknowledgment: This study was supported by the Grant Agency of the Czech Republic (project No. P506/12/1632).

Abstracts

ANTIGENS OF THE NEUROPATHOGENIC FLUKE TRICHOBILHARZIA REGENTI ELICIT NITRIC OXIDE AND PROINFLAMMATORY CYTOKINE SECRETION BY GLIAL CELLS IN VITRO

T. MACHÁČEK, L. PANSKÁ, P. HORÁK

Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Prague 2, Czech Republic

Glial cells present in the central nervous system (CNS), particularly microglia and astrocytes, are known to participate in the immune response during CNS infections. These cells possess several mechanisms which can contribute to pathogen elimination. Two of them, nitric oxide production and proinflammatory cytokine secretion, were investigated in our experiments dealing with in vitro stimulation of glial cells by antigens of Trichobilharzia regenti (Digenea: Schistosomatidae). Trichobilharzia regenti is a pathogen of anatid birds showing high affinity to the nervous tissue. This results in migration through the host CNS which is accompanied by serious neurological disorders. Similar neurotropic behaviour was observed also in mice, however, the development of parasites was delayed and they died without maturation. It was hypothesised that the host immune response, including activation of glial cells, is responsible for parasite elimination. In order to elucidate the role of microglia and astrocytes in the immune response of mice to the infection by T. regenti, we prepared primary microglia and astrocyte cultures from neonatal mouse pups and exposed the cells to antigens of T. regenti. The influence of homogenate of transformed cercariae and recombinant cathepsins B 1.1 and B 2 on the production of nitric oxide (NO) and interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF) alpha secretion was evaluated. We observed that stimulation of microglia and astrocytes with both homogenate of transformed cercariae and recombinant cathepsins caused increase in NO, IL-6 and TNF alpha production. These data from in vitro experiments suggest that microglia and astrocytes might contribute to T. regenti elimination from murine CNS.

Acknowledgements: Czech Science Foundation (Project No. 13-29577S).

Abstracts

EVALUATION OF QUALITATIVE AND QUANTITATIVE PARAMETERS OF INTERACTION BETWEEN FASCIOLOIDES MAGNA AND DEFINITIVE EXPERIMENTAL HOST - RABBIT

K. MELOUNOVÁ1, J. BULANTOVÁ1, J. VADLEJCH3, P. HORÁK1 AND M. KAŠNÝ1,2

1Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 00 Prague, Czech Republic 2Department of Botany and Zoology, Faculty of Science, Masaryk University Brno, Kamenice 753/5 625 00 Brno Bohunice, Czech Republic. 3Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamycka 957, 165 21 Prague, Republic.

Fascioloides magna (giant liver fluke) is veterinary important endoparasite belonging to the class Trematoda, subclass Digenea. It can infect a wide range of definitive hosts to which it causes serious health problems sometimes leading to death. The typical final hosts of F. magna are primarily members of free living cervids (Cervus elaphus, Dama dama, Capreolus capreolus), but it can also occasionally occurs in domestic bovids. The adult flukes are within the definitive host usually localized in the liver - mostly in pseudocyst (fibrous capsule); they induce severe damage of the liver tissue. The fascioloidosis is accompanied by reaction of the host organism, e.g. the immune system is stimulated for elimination of the pathogen and some other host´s physiological processes are also influenced, these can result in the increase of liver enzymes and blood proteins quantity. We experimentally infected rabbits with 50 – 300 pieces of F. magna metacercariae and monitored the changes in body weight, haematocrit and production of specific antibodies. We observed also the blood count (WBC differential), biochemical parameters of liver enzymes and presence of metabolites in the blood reflecting the level of the liver tissue damage. In parallel the regular coprology was performed and finally the isolated trematodes were histologically processed. Among the most significant changes the increase in quantity of IgG and eosinophils was recorded.

Acknowledgements: Charles University in Prague (UNCE 204017, PRVOUK P41, SVV, GAUK 502313) and Masaryk University (MUNI/A/1484/2014).

Abstracts

METAZOAN PARASITES OF KILLIFISH FROM MOSAMBIQUE

V. MICHÁLKOVÁ1,2, M. REICHARD2, M. ONDRAČKOVÁ2

1Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 267/2, 611 37 Brno, Czech Republic 2Institute of Vertebrate Biology, v.v.i., Academy of Sciences of the Czech Republic, Květná 8, 603 65 Brno, Czech Republic

In this study, we examined 205 specimens of four annual killifish species (Nothobranchius orthonothus, N. furzeri, N. kadleci and N. rachovii) from 14 different localities in south part of Mosambique to study their metazoan parasites and to find factors responsible for differences in intra-specific and inter-specific variation of parasite infection. Killifish of the genus Nothobranchius (Cyprinodontiformes, Nothobranchiidae) are small annual fish inhabiting ephemeral rain-fed savannah pools of east Africa. Their life span is very short. It is limited to several months until the pool dries. In that time the fish have to grow, mature and reproduce. The only stage surviving the dry season is the dormant egg buried in the dry substratum, waiting for the onset of the annual rains that fill the pool and enable hatching. The populations are strictly separated, young fish thus cannot obtain parasites directly from parental population. We recorded 17 parasite taxa. Except for two individuals of leech all parasites were endoparasites. Trematode larval stages, metacercariae, were the most abundant parasites, with 75 % prevalence. They infected mainly muscle of fish, often in high abundances (up to 807 individuals per host). Also cestodes and nematodes were often found in killifish. Except the trematode Emoleptalea sp. in the intestine, all parasites were larvae, indicating the killifish may be the important intermediate hosts. There was no difference in parasite species richness and total abundance between killifish species. The species do not differ in spatial distribution and trophic demands, the exposure to parasites is thus similar. The parasite species richness was positively associated with size of the pool and, to a smaller extent, with the amount of grass littoral vegetation. Larger pools enable the existence of different habitats attractive for a variety of parasite´s intermediate hosts serving as a source of killifish infection. Larger sites may also attract bird definitive hosts that can excrete the eggs of parasites.

This study was supported by the Czech Science Foundation P505/12/G112.

Abstracts

IMMUNOLOGICAL CONSEQUENCES OF THE INFECTION OF MICE WITH TOXOCARA CANIS

J. NOVÁK1, L. PANSKÁ2, L. KOLÁŘOVÁ1, AND P. HORÁK2

1Institute of Immunology and Microbiology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Studničkova 7, Praha 2, 128 00, Czech Republic 2Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, Praha 2, 128 44, Czech Republic

Infection of mouse, a paratenic host, with Toxocara canis larvae has a wide range of consequences from immunological point of view. For some parasitic helminths, there were described both protective and/or exacerbating effects of infection on allergy and autoimmune diseases of their hosts. Semicontinuous way of infection of mice with a low amount of larvae was developed, as it probably resembles more natural way of infection. Humoral and cellular immunity against T. canis will be compared in the groups of mice conventionally infected once or twice by high quantity of larvae, and the groups infected by larvae using the new semicontinuous method. T-cell subpopulations and antibody response to components of T. canis excretory-secretory antigens (TES) will be characterized, and potentially protective role of antigens in allergy and autoimmune conditions will further be evaluated.

Acknowledgements: The study was supported by the Charles University in Prague, project GAUK No 32915.

Abstracts

PARASITE LOAD AND IMMUNITY IN DIPLOID-POLYPLOID COMPLEX OF CARASSIUS GIBELIO

T. PAKOSTA, A. VETEŠNÍKOVÁ ŠIMKOVÁ

Department of Botany and Zoology, Faculty of Science, Masaryk University, Kamenice 753/5, 625 00 Brno

Carassius gibelio (Cyprinidae) is a diploid-polyploid cyprinid fish species, a member of Carassius auratus complex. This species is morphologically and ecologically very similar and phylogenetically closely related to other three species of C. auratus complex i.e. C. auratus, C. landsdorfi and so-called “M line”. Gibel carp is a unique cyprinid fish species, which is characterized by a dual form of reproduction - gynogenetic (with large proportion of triploid females and small proportions of triploid males and tetraploid individuals) and sexual reproduction. The coexistence of both forms in the same habitats is well documented. As a non-native species, originating in Asia, C. gibelio was introduced into the waters of the Czech Republic in the 80s of last century. The aim of this study was to analyse the selected aspects of fish physiology (including the condition and fitness parameters) and immunology (including the measures of specific and non-specific immunity) as well as parasite load in order to explain evolutionary strategy of diploid-polyploid complex of gibel carp. First, we focussed on the molecular determination of gibel carp specimens using mitochondrial DNA in order to distinguish between C. gibelio specimens and the representatives of other lines. Next, we evaluated the levels of metazoan parasite infections in diploid (sexual) form (separating males and females) and triploid (gynogenetic) form of C. gibelio. The analyzed specimens of the same age were sampled from the selected localitiy situated in the Lednice-Valtice area, representing a tributary of the river Dyje. Gibel carp was investigated in the august of three consecutive years. Our analyses revealed no difference in non-specific immunity between two reproductive forms but the difference in IgM production between gynogenes and sexuals was found. High investment in the reproduction was found for both gynogenetic and sexual females when compared to sexual males. No obvious difference was found in the abundance of different parasite group between diploid sexuals and triploid gynogens.

This study will be funded by the Czech Science Foundation, Project No. P505/12/0375.

Abstracts

THE ROLE OF STEM CELLS IN REGENERATIVE PROCESSES IN TREMATODES

J. PANKRÁC1, M. KAŠNÝ1,2, K. VRBOVÁ1, J. BULANTOVÁ1, P. HORÁK1

Many members of phylum Platyhelminthes (parafyletic freeliving Turbellaria [planarians] and parasitic Neodermata) are able to regenerate damaged tissues and missing parts of their bodies. During the regenerative processes totipotent (or pluripotent) stem cells called neoblasts play important role. Their progeny migrates and replaces damaged cells or forms undifferentiated tissue (blastema) from which the missing part of the body is subsequently formed. The successfully application of experimental methods where the analogues of thymidine have been used for labeling of replicating cells enabled the realization of detailed studies on population dynamics of neoblasts. Recently these methods were successfully applied also for well investigated trematode species - Schistosoma mansoni. We suppose that methods of specific labeling of neoblasts might help us to understand the importance of cellular regenerative processes in defense of trematodes against effector mechanisms of the vertebrate immune response. For the purpose of investigation of these processes we chose two trematode species as experimental models – Trichobilharzia regenti (Schistosomatidae) and Fascioloides magna (Fasciolidae). The main aims of our research are: 1) to characterize the influence of selected humoral and cell immune response on differentiation of neoblasts of trematodes, 2) to describe the pathological changes in tissues of trematodes related to the immune response of the vertebrate hosts, 3) to evaluate the regeneration ability of different trematode tissues. Interactions between parasite and immune system of the vertebrate host will be assayed by using in vitro systems. Up to now the cultivation of T. regenti in schistosoma culture medium 169 (SCM 169) containing duck´s erythrocytes was already successfully implemented. Several schistosomula have been already used for optimization of labeling of neoblasts by thymidine analogues. The histological methods and electron microscopy will be used for characterization of tissue damage.

Acknowledgements: Czech Science Foundation (Grant No. GA13-29577S), Charles University in Prague (UNCE 204017, PRVOUK P41, SVV, GAUK 502313) and Masaryk University (MUNI/A/1484/2014).

Abstracts

HELMINTHS AND IMMUNE-MEDIATED DISORDERS (HYPOTHESIS AND PLANS)

V. V. PAVLÍKOVÁ1,2, M. CHANOVÁ2

The number and spectrum of immune-mediated disorders in western developed world seems to rise up during last decades. The key initial factors and/or co-factors remain unclear for lot of them. Some of many theories emphasize the influence of helminthic infections on these diseases development. E.g. "hygienic hypothesis" nowadays called also “old friends hypothesis” points towards the correlation of increase in immune-mediated diseases prevalence with eradication of parasitic infections in high-income countries. This is explained by the ability of helminths to modulate the host immune response and thus regulate an inflammatory reaction. The potential use of helminths or their products in anti-inflammatory treatments is recently widely discussed. On the other hand, hypothesis that particular helminthic infections directly or indirectly participate in autoimmune disease development also occur. The present study is focused on modified cytokine production in the host infected with selected helminths. Particularly, cytokines that are commonly associated with autoimmune disease (e.g. IFNγ, TNF-α, IL-12, IL-17, IL-21) are in focus. Two approaches are planned: wide cytokine spectra measurement in sera of patients previously diagnosed as highly positive for helmithiases, and subsequent measurement of selected cytokines during experimental infections/stimulation of mice. Human sera, as well as sera of experimentally infected mice collected in desired intervals during infection will be investigated by sandwich ELISA technique. Than mice will be sacrifices and further investigation of cytokine production will follow, including flow cytometry measurement of whole blood and splenocytes. Besides infected mice, selected cytokine production will be measured also for in vitro cultured murine splenocytes stimulated by antigens of helminths.

The work is supported by Charles University in Prague (PRVOUK P25/LF1/2; UNCE 204017).

Abstracts

CATHEPSIN L-LIKE PEPTIDASES IN CERCARIAE OF DIPLOSTOMUM PSEUDOSPATHACEUM

T. PERHÁČOVÁ AND L. MIKEŠ

Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Prague 2, Czech Republic

Cercariae of the widespread bird trematode Diplostomum pseudospathaceum are able to invade the skin of fish, the second intermediate host, by active penetration. After transformation to diplostomulum they migrate through tissues and blood circulation to the eye lenses. For this purpose they are equipped with penetration glands and their dilated ducts serving as reservoirs of secretory material. Secretions of the well developed gut might take part in histolysis and tissue migration, too. We tried to purify and characterize cysteine endopeptidases from cercarial soluble protein extracts. Tests with relevant inhibitors and fluorogenic substrates confirmed that the purified proteins are cathepsin L-like peptidases in nature. Besides the differences in pI, the peptidase activities in particular FPLC fractions (cation exchange chromatography) slightly differed in pH optima. Degenerate PCR primers based on consensual sequences of active sites of cysteine peptidases were employed for amplification of genes by PCR with cDNA from sporocysts/developing cercariae as a template. Following cloning and RACE PCR we obtained sequences of three different cathepsin L-like isoforms. So far, one of these enzymes was expressed as a recombinant protein and further biochemically characterized. Production of antibodies for localization by imunohistochemistry is under way.

Acknowledgements: This work was supported by institutional grants of the Charles University in Prague (PRVOUK P41, SVV260074/2014).

Abstracts

DIFFERENTIATION OF TOTIPOTENT GERMINAL CELLS IN LARVAE OF T. REGENTI

J. PEŠTOVÁ AND P. HORÁK

Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Prague 2, Czech Republic

Trichobilharzia regenti is a bird fluke, intermediate hosts of which are Radix peregra, R. labiata and R. lagotis. Adult female of this parasite produces eggs in the bird nasal cavity. The first larval stage, miracidium, hatches there. Then the miracidium leaves the bird and penetrates head-foot or mantle regions of the intermediate host where it transforms to mother sporocyst. Inside the mother sporocyst there are many dividing germinal cells that give rise to other larval stages called daughter sporocysts which migrate to hepatopancreas. These larvae also contain germinal cells, next larval generation of daughter sporocysts or cercariae. Cercariae escape from the intermediate host and penetrate the skin of a definitive host. There is no detailed information about germinal cells, their differentiation and parasite development in the intermediate host. Therefore, the goal of our research is to characterize larval development in detail, and determine the onset of sporocystogenesis and cercariogenesis within daughter sporocysts. Individual larval stages were studied as whole cercarial embryos or in histological sections, stained by different histological stains and probes. We have defined seven developmental stages of daughter sporocysts and ten developmental cercarial stages. The first stage of both larvae is the germinal cell. It divides and gives rise to a cell aggregate. Then an envelope (primitive epithelium) is formed around the embryo and subsequently, the embryo elongates. We can distinguish daughter sporocyst from cercaria in the phase when the tegument is completed. As a new structure, we disclosed two gland types, type I and type II. In hepatopancreas of some snails we observed an unusual type of sporocystogenous sporocysts that were characterized by a thicker wall and conspicious spine-like projections. In the future we are going to observe the development of musculature, glands, nervous and excretory systems of developing cercariae using phalloidin, lectins and antibodies.

Abstracts

DIVERSITY OF MONOGENEANS PARASITIZING FRESHWATER FISHES OF THE NILE RIVER IN SUDAN: CURRENT STATE AND PROSPECTS

M. PRAVDOVÁ, R. BLAŽEK, M. GELNAR, E. REHULKOVÁ

Department of Botany and Zoology, Faculty of Science, Masaryk University Brno, Kamenice 753/5 625 00 Brno Bohunice, Czech Republic

During a systematic survey of monogeneans parasitizing freshwater fishes from the Nile River in Sudan, 44 monogenean species were recorded on/in 26 species of host fishes. Eleven of them probably represent species new for science. Thirty three species represent previously described taxa and all of them are reported from Sudan for the first time. The monogenean species found were represented by the following 18 genera: Annulotrema (2 species), Bagrobdella (4 species), Bouxiella (1 species), Cichlidogyrus (7 species), Dactylogyrus (8 species), Diplectanum (2 species), Dogielius (2 species), Enterogyrus (2 species), Gyrodactylus (2 species), Heteronchocleidus (1 species), Heterobothrium (1 species), Characidotrema (1 species), Onchobdella (3 species), Protoancylodiscoides (2 species), Scutogyrus (1 species), Schilbetrema (1 species), Synodontella (2 species), Quadriacanthus (2 species). Oreochromis niloticus was the host species with the highest parasite species richness (3 species of Cichlidogyrus, 1 species of Scutogyrus, and 1 species of Gyrodactylus on the gills, and 2 species of Enterogyrus in the stomach. The majority of the monogenean species found were situated on gills; the only monogeneans recorded from the stomach were 2 species of Enterogyrus from Oreochromis niloticus.

Acknowledgements: This study was supported by the Czech Science Foundation (project No. P505/12/G112).

Abstracts

THE REVISION OF THE GENUS AFRODIPLOZOON (KHOTENOVSKY, 1981) (MONOGENEA: DIPLOZIODAE)

I. PŘIKRYLOVÁ1,2, Š. MAŠOVÁ1, M. M. MATLA2, M. GELNAR1 AND W. J. LUUS-POWELL2

1Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic 2Department of Biodiversity, School of Molecular and Life Sciences, University of Limpopo, Sovenga, South Africa.

The genus Afrodiplozoon was proposed by Khotenovsky in 1981 when Afrodiplozoon polycotyleus (Paperna, 1963) has been exluded from the genus Neodiplozoon Tripathi, 1959 based on the number of clamps on the attachment apparatus. Since then, the inconsistency in the use of the name for this parasite can be seen in the literature. Specimens of A. polycotyleus were collected during a fish survey carried out in April and July 2014 in the Venda region, Limpopo Province, South Africa, from Labeobarbus maraquensis (n=36; mean total length=7.9 cm) and Barbus paludinosus (n=1; total length=6.6 cm). A prevalences of 64% and 59% were recorded for A. polycotyleus during April and July, respectively. Morphological analysis of the composition of the internal organs and attachment clamps using different microscopic methods (light microscopy of stained/unstained specimens and scanning electron microscopy, SEM) provided details for the redescription of the genus. The type material was also studied. Molecular characterization based on the variabily of the second internal transcribed spacer (ITS2) rDNA showed the taxonomic relationship to other representatives of Diplozoidae. Parasites can bear asymetrically from 7 up to 10 clamps in one row on each side of the attachment apparatus, with the first clamp is significantly smaller. The connection sclerite of posterior end of the central plate is wide in all its length. The anterior end of the central plate has short processes. The SEM observations showed the presence of numerous papilae around the mouth of the worm. Molecular characterization and subsequent analysis revealed as a sister species to A. polycotyleus recently described Paradiplozoon bingolensis (Civáňová, Koyun, Koubková, 2013) both positioned far from African representatives of the genus Paradiplozoon (Achmerov, 1974).

Acknowledgements: Funding from the project no. GBP505/12/G112 from the Czech Science Foundation and Biodiversity Research Chair, University of Limpopo.

Abstracts

MONOGENEAN PARASITES FROM PSEUDOCRENILABRAUS PHILANDER (WEBER, 1897) IN THE MIDDLE LIMPOPO RIVER BASIN (SOUTH AFRICAN REGION)

I. PŘIKRYLOVÁ1,3, I. WALTER1, W. J. LUUS-POWELL1, M. BARSON2

1Department of Biodiversity, School of Molecular and Life Sciences, University of Limpopo, Sovenga, South Africa 2Department of Biological Sciences, University of Zimbabwe, Harare, Zimbabwe 3Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic

The Southern mouthbrooder, P. philander, with its opalescent blue and pale yellow colours could become an important ornamental fish. Its distribution in southern African region covers the Orange River and southern KwaZulu-Natal Province northwards throughout the region and extends to southern Congo tributaries and Lake Malawi. In South Africa, P. philander has been spread unnaturally since its introduction in 1941, as a consequence of the transportantion beyond limit of native range and direct releasing into novel environments. Fish were collected using an electroshocker during two seasons, July 2013 (winter, W) and January 2014 (summer, S), from two sampling spots, representing tributaries of Limpopo River from both Zimbabwean (ZIM) and South African (SA) sides where 20 specimens from each locality and sampling season were sampled. The localities were Nwanedi River, Nwanedi Nature Reserve (SA) and Bubi River, Bubiana Conservatory (ZIM). The fish were examined in a field laboratory and recovered monogeneans were fixed in glycerin ammonium picrate solution. Morphometric analysis of the hard structures of the attachment organs and male copulatory organs reaveled presence of monogenean parasites representing three genera. The only species of viviparous parasites of the genus Gyrodactylus (von Nordmann, 1832) was recorded from the fins during the summer in SA and from only one fish. In the stomach, the infections of endoparasitic monogeneans of the genus Enterogyrus (Paperna, 1963) were recorded in S-SA and W-ZIM with a prevalence of 35% in both cases, and identified as Enterogyrus coronatus Pariselle, Lambert & Euzet, 1991. Four species of the genus Cichlidogyrus (Paperna, 1960) were recorded from the gills. The species composition and prevalences differed significantly between the localities and sampling seasons.

Acknowledgements: Funding from the project no. GBP505/12/G112 from the Czech Science Foundation and Biodiversity Research Chair, University of Limpopo.

Abstracts

BASIC EPIDEMIOLOGICAL DATA OF ANISAKID NEMATODE LARVAE OF GOBIID FISHES (GOBIIDAE, ACTINOPTERYGII) FROM NORTHERN ADRIATIC SEA

L. RAISINGEROVÁ1, Š. MAŠOVÁ1, A. VETEŠNÍKOVÁ ŠIMKOVÁ1, R. ŠANDA2, J. VUKIĆ3, M. GELNAR1

1Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno 2Department of Zoology, National Museum, Prague 3Department of Ecology, Faculty of Science, Charles University, Prague

The aim of the presented study was to report and compare infections caused by anisakid nematode larvae at six gobiid fishes (Actinopterygii: Gobiidae) in the northern part of the Adriatic Sea in the Kvarner region of Croatia, in the vicinity of Selce village and Krk Island. Host species were collected in two periods of 2014, May and October. Basic epidemiological data are reported for 364 specimens of the following six gobiid species from the two seasons: Buenia affinis, Gobius auratus, G. niger, G. roulei, G. vittatus and Pomatoschistus marmoratus. Studied material included a total of 341 anisakid nematode larvae (116 in spring and 225 in autumn season). 35 % of all examined fish were infected with anisakids. The intensity of infection varied from one to twelve larvae per fish. Larvae were found mainly within the body cavity, liver and intestine. After morphological examination, parasites were classified into six categories according to morphotypes: Anisakidae spp., Contracaecum sp., Hysterothylacium spp., Hysterothylacium sp. 1, Hysterothylacium sp. 2 and Hysterothylacium sp. 3. Quantitative information on the prevalence, abundance and intensity of infection of anisakid nematodes were counted. Differences in anisakid fauna among fish species in two seasons were detected, while significant difference was observed in Gobius roulei. Further, DNA analyses will be performed to elucidate the exact species determination.

This study was supported by the Czech Science Foundation (project No. P505/12/G112), by the BIDA (Biodiversity - the analyses of biological systems of different levels and on different scales of environment), by the Department of Botany and Zoology, Faculty of Science, Masaryk University and the Department of Ecology, Faculty of Science, Charles University in Prague. We would like to thank to our colleagues Petra Zahradníčková, Nikol Kmentová, Markéta Pravdová, and Marcelo Kovačić for the cooperation during the fieldwork.

Abstracts

NOVEL MOLECULAR DIAGNOSTIC APPROACH FOR DETECTION OF PATHOGENS IN MEAT

N. RESLOVÁ1, 2, 3, M. KAŠNÝ1, 4, M. SLANÝ2, B. KOUDELA3, P. KRÁLÍK2

1Department of Botany and Zoology, Faculty of Science, Masaryk University Brno, Kamenice 753/5, 625 00 Brno Bohunice, Czech Republic 2Department of Food and Feed Safety, Veterinary Research Institute, Hudcova 296/70, 621 00 Brno, Czech Republic 3CEITEC – Central European Institute of Technology, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho tr. 1/3, 612 42 Brno, Czech Republic 4Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 00 Prague, Czech Republic

The changes in farming practices towards bio-production, globalization of food market with increasing animal transportation and also global climate change facilitate the risks of widespread disseminations of food-borne diseases, including the pathogens, such as zoonotic multicellular parasites. These facts consequently induce the need of improvement of the diagnostic tools also for food-borne agents in the final products such as meat. In relation to this problematics, our work is focused on development of reliable comprehensive molecular diagnostic method useful for rapid control of final meat products on the market. For this purpose, we adopted high sensitive multiplex oligonucleotide ligation- PCR assay - MOL-PCR, representing the novel diagnostic platform based on magnetic microspheres and visualization realized via MAGPIX instrument for qualitative and quantitative readout of signal. This modern approach enables simultaneous direct screen of complex samples potentially containing the DNA originating from number of different parasitic organisms. Up to this date, the specific molecular probes allowing the detection and capturing of targeted DNA from two parasitic worms - Trichinella spiralis (partial sequence of 18S rRNA gene) and Taenia saginata (partial sequence of mitochondrial COX1 gene) were designed. The calibration of the system and optimization of method is in process.

Acknowledgements: This research was supported by grant of Ministry of Agriculture MASO (Grant n. QJ1210113), grants of the Masaryk University (MUNI/A/1484/2014), CEITEC (CZ. 1.05/1.1.00/02.0068) and grants of the Charles University in Prague (UNCE 204017, PRVOUK P41, SVV).

Abstracts

ENDOPARASITES OF WILD ANIMALS OF VINEYARD AND AGRICULTURAL LANDSCAPE IN THE SOUTH MORAVIA

J. SEDLÁKOVÁ, M. BORKOVCOVÁ

Department of Zoology, Fisheries, Hydrobiology and Apiculture, Mendel University of Agriculture and Forestry Brno, Zemědělská 1, 61300 Brno, Czech Republic

The aim of the study was to determine the interrelationships between spectrum of endoparasites and species biodiversity in reporting landscape with focus on selected omnivore, carnivore and herbivore representatives. Abundance and prevalence of parasites was observed in 6 localities in the South Moravia Region, the flotation method was used to identify parasite eggs in faeces. Together 139 excrement samples of deer, pheasant, hare and marten were collected. Monitoring was carried out on a total of 6 locations, 3 locations were in vineyards (site A). These are locations Nosislav, Moravian Krumlov and Kobylí. And 3 locations in intensive agriculturally use (site B). These are locations Olbramovice, Moutnice and Zaječí. In Conclusion parasitic infestation of animals of vineyards and fields was compared. The important role was played by the heterogeneity of the surrounding landscape and the agricultural use. The total number of species of endoparasites on site A detected in animals was 30; at locations B it was 24 species. Prevalence of endoparasites was highest in deer - in both locations identically, then in pheasant in area B, the lowest prevalence was detected in hares in area A. Using ecological indices index for biodiversity was calculated: site A H = 1.1 and for the site B H = 0.8. Index evenness for site A was E = 0.35, for the site B E = 0.21. According to the values of Species dominance index, site A is less anthropogenically influenced, but the differences were not very large in comparison with the location B.

The research was financially supported by team project of IGA MENDELU Brno No. TP7/2014.

Abstracts

IMMUNOMODULATION BY PARASITES: THE BIG COMEBACK?

I. SCHABUSSOVA

Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Vienna, Austria

During the past decade, epidemiologists, parasitologists, and immunologists have made many new thrilling discoveries in the field of parasite-mediated immune regulation. Additionally, interventional human studies, as well as experiments in animal models, strongly indicate that certain parasites or products derived from parasites can confer protection from immune-mediated disease, such as allergy, autoimmunity and colitis, opening the door to novel exciting therapeutic applications of certain parasite-derived modulators to control specific immunopathological conditions in humans.However, the overall down regulation of the immune system might cause impaired responsiveness to vaccines. Here, I will discuss the immunological pathways that parasites utilise to downregulate immune responses against bystander antigens such as allergens or vaccines. In particular, our research is focused on immunomodulation induced by protozoan parasite Toxoplasma gondii and by parasitic nematode Oesophagostomum dentatum. Altogether, understanding of the mechanisms of immunomodulation by helminth parasites could be useful on one hand to utilize/modulate immune suppression induced by parasites/products/synthetic analogues to control immunopathologyand on the other hand toantagonize immune suppression and thus improve vaccine efficacy in humans.

Abstracts

HAEMOCYTE EXTRACELLULAR TRAPS IN THE SNAIL LYMNAEA STAGNALIS (LYMNAEIDAE): DO THEY OCCUR AND ARE THEY USED AS A DEFENCE RESPONSE AGAINST TREMATODES?

V. SKÁLA AND P. HORÁK

Charles University in Prague, Faculty of Science, Department of Parasitology, Viničná 7, 128 43, Prague 2, Czech Republic

One decade ago, it was discovered that activated mammalian neutrophils release granule proteins and chromatin that together form extracellular fibers (neutrophil extracellular traps; NETs) capable to degrade virulence factors and kill bacteria. This discovery was rapidly followed by similar findings in other vertebrates, and also the ability of NETs to entrap or kill eukaryotic uni/multicellular parasites was later proved. More recently, formation of extracellular trap-like fibers (ET-like fibers) that resemble the NETs of vertebrates was described as a defence response of haemocytes of several invertebrate species including one mollusc species, the blue mussel Mytilus edulis. However, it remains unknown whether ET- like fiber formation also occurs in haemocytes of snails and participates in defence response during infection of snails with trematodes. In our experiments, we isolated haemocytes of the snail species Lymnaea stagnalis and stimulated them to produce ET-like fibers with a range of compounds (e.g. PMA, lipopolysaccharide (LPS), E. coli bacteria) for various periods (2-24h). Using Sytox green to visualize nucleic acid, a few fibers protruding from cell nuclei were observed upon stimulation of the cells with LPS. To evaluate these fibers as ET-like fibers, presence of other components typical for these structures (e.g. histones) is currently investigated using specific antibodies. Next, production of ET-like fibers will be studied in L. stagnalis experimentally infected with the bird schistosome Trichobilharzia regenti which is an incompatible parasite that does not survive and develop in the snail; in vitro experiments using haemocytes and trematode larvae will also be performed. Taken together, this study provides the first insight into the ET-like fiber production in snails and aims to elucidate whether the fibers are used as a defence response of snails against invading non-compatible trematode parasites.

Acknowledgements: Charles University in Prague (research programmes PRVOUK - P41/PrF and SVV) and The Malacological Society of London.

Abstracts

ANTICOAGULATION FACTORS AND BLOOD UPTAKE BY MONOGENEANS OF THE FAMILY DIPLOZOIDAE

K. SKIPALOVÁ1, L. JEDLIČKOVÁ1, H. DVOŘÁKOVÁ1, P. BROŽ2, M. KAŠNÝ1,3 AND L.MIKEŠ1

1Department of Parasitology, Faculty of Science, Charles University in Prague. Viničná 7, 128 44 Prague 2, Czech Republic 2Bioinformatician Institute of Applied Biotechnologies a.s., Služeb 4, 108 52 Prague 10, Czech Republic 3Department of Botany and Zoology, Faculty of Science, Masaryk University Brno, Kamenice 753/5, 625 00 Brno-Bohunice, Czech Republic

The aim of this study was to find molecules with potential anticoagulant activity in excretory/secretory products and in transcriptome of the monogenean Eudiplozoon nipponicum (Heteronchoinea: Diplozoidae), a blood-feeding ectoparasite occupying the gills of the common carp Cyprinus carpio. Anticoagulants and other antihaemostatic molecules facilitate blood intake by haematophagous parasites. Although these molecules have not yet been reported from monogeneans, their presence is highly probable in sanguinivorous species. They are probably produced by specialized glands and inhibit thrombosis and clot formation. Antihaemostatic molecules often act as protease inhibitors that target one or more serine proteases comprising the coagulation cascade. We tested inhibitory activity in excretory-secretory products and homogenates of E. nipponicum and other family members, Paradiplozoon homoion and Paradiplozoon bliccae, towards coagulation factors IIa and Xa, with negative results. Employing results of two transcriptome projects on E. nipponicum, we discovered three protein families of potential anticoagulants - annexins, serpins and Kunitz-domain proteins. For further analyses we focused on the Kunitz protein family. These proteins contain one or more structurally related active domains which are able to inhibit the function of proteases. In addition, we described similarities between Kunitz sequences from E. nipponicum and other hematophagous parasites which exhibit anticoagulant activity (e.g. Ixodes scapularis or Rhipicephalus microplus). By production of selected recombinant Kunitz proteins and their domains in Escherichia coli and Pichia pastoris we intend to confirm their anticoagulation activity by inhibition tests towards coagulation factors and by using coagulations tests, e.g., prothrombin time or activated coagulation time.

Abstracts

FASCIOLOID FLUKES OF LIVESTOCK AND WILD ANIMALS IN THE CZECH REPUBLIC

T. SOSNOVÁ1 AND M. KAŠNÝ1,2

1Department of Parasitology, Faculty of Science, Charles University in Prague, Vinicna 7, 128 00 Prague, Czech Republic. 2 Department of Botany and Zoology, Faculty of Science, Masaryk University Brno, Kamenice 753/5 625 00 Brno Bohunice, Czech Republic

Fasciolid flukes Fasciola hepatica and Fascioloides magna are pathogens parasiting in liver of livestock and wild ruminants. Their adults are localized in bile ducts (F. hepatica) and liver pseudocysts (F. magna), where they cause direct destruction of tissue and induce the obstruction of bile ducts. The animals, which are infected by these parasites are usually weak, gaunt and in case of strong infection they can die. Unfortunately the data reflecting the epizootological situation of fasciolosis and fascioloidosis in the Czech Republic are rather missing. Our research is therefore focused on revealing the possibilities of systematic monitoring of these two trematodosis and prediction of their occurrence at particular regions in the Czech Republic. We are evaluating the data from several sources, such as historical records presented in publications, the records of slaughter houses, regional/national veterinary administrations, game public administration, farmers, hunters and also personal sampling. Subsequently, we are correlating these data with parameters of selected biotic/abiotic factors which are available at geographic information systems portals.

Acknowledgements: Charles University in Prague (UNCE 204017, PRVOUK P41, SVV, GAUK 502313) and Masaryk University (MUNI/A/1484/2014).

Abstracts

PATHOGENESIS OF HOOKWORM/ENTERITIS COMPLEX WITH BACTEREMIA CAUSED BY UNCINARIA SPP. IN CALIFORNIA SEA LIONS AND NORTHERN FUR SEALS

T. R. SPRAKER1, T. A. KUZMINA2, E. T. LYONS3 AND R. DELONG4

1Diagnostic Medical Center, Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA 2Institute of Zoology NAS of Ukraine; 15, Bogdan Khmelnitsky street, Kiev, 01601, Ukraine 3University of Kentucky, Department of Veterinary Science, Gluck Equine Research Center, Lexington, KY, USA 4National Marine Mammal Laboratory, Alaska Fisheries Science Center/NOAA, Seattle, WA, USA

Hookworm disease or uncinariasis caused by Uncinaria lucasi (Nematoda; Ancylostomatidae) has been known in northern fur seals (Callorhinus ursinus) (NFS) since the late 1890’s. This disease can cause high mortality not only in northern fur seals but also California sea lions (Zalophus californianus) (CSL). Pups usually die from 2 to 4 months of age. The overall percentage of mortality due to hookworms is difficult to document but does vary from year to year. Field biologists monitoring the population of CSLs and NFSs that inhabit San Miguel Island, the most western of the Channel Islands, southern California, estimates that about 10–60% pup mortality occurs each year associated with uncinariasis. To gain insight into the pathogenesis of uncinariasis and possible reasons why pups were experiencing such high mortality approximately 189 CSL and 50 NFS pups were examined on necropsy from San Miguel Island, California, between June to December 2006– 2009. We found there were several extremely detrimental deviations to the accepted life cycle for Uncinaria spp. During the months of June through September 72% of the CSL and NFS pups examined were infected to some degree by Uncinaria. Approximately 15% of these pups had adult hookworms that had penetrated into the deeper layers of mucosa and submucosa of the small intestines. Approximately 10% of the pups examined had adult nematodes that had penetrated completely through the intestinal wall and entered into the abdominal cavity carrying bacteria (Salmonella sp., Klebsiella sp. and hemolytic Escherichia coli) on their cuticle. As the females parasites travel in the abdominal cavity they deposit eggs. This activity causes a bacterial peritonitis and bacteremia which then leads to encephalitis, pneumonia, pericarditis, arthritis and hepatitis in some pups causing high mortality. The conclusion from this study was that U. lyonsi in CSLs and U. lucasi in NFSs do not follow the typical pathogenesis of hookworms described in other animals. A percentage of the adult hookworms will penetrate through the intestinal wall, enter into the peritoneal cavity carrying bacteria on their cuticle and cause bacterial peritonitis and bacteremia leading to high mortality.

Abstracts

STUDY OF THE NEMATODE FAUNA IN WATERFOWLS ON THE UKRAINIAN POLESIE

Y. YU. SYROTA, V. A. KHARCHENKO, T. A. KUZMINA

I.I. Schmalhausen Institute of Zoology of National Academy of Sciences of Ukraine

There are approximately 105 species of wild hydrophilic birds in Ukraine. The most of researchers dealt with helminth fauna of waterfowls, and only a few studies were dedicated to the parasitic nematodes. We have analyzed literature of the current knowledge on the parasitic nematode fauna in waterfowls in Ukrainian Polesie. The first publications describing of the waterfowl nematodes was published in 1874 (Shtandel, 1874). However, only a few publications in this field are known up to the XX century. K. I. Skrjabin initiated detailed studies of this subject during his helminthological expeditions on the territory of Ukraine in 1920s (Skrjabin, 1916, 1923, 1926). Next period of the studies began after the War World II. The most famous and important studies on were performed by L.A. Smogorzewka (1954, 1976), V.A. Leonov (1956), N.I. Srebradolska (1964, 1969), E.O. Saakova (1952) and M.I. Sergienko (1968). After the 1980s, the investigations on waterfowl nematodes decreased; only few publications are known after 1990. During this period scientists accumulated information not only about fauna, but about morphology, life cycles and ecology of nematodes from this region. Our literature analysis of the biodiversity of nematode fauna in waterfowls revealed that from 81 species (Smogorzewka 1976, 1990) of parasitic nematodes registered in Ukraine. Totally, 36 nematode species were registered in the Polesie region: 23 species – in Belarus (Merkusheva, 1981). 15 – in Poland (Okulewicz, 1997) and 18 – in Ukraine). Six nematode species: Eucoleus contortus, Amidostomum anseris, Echinuria uncinata, Porrocaecum crassum, Porrocaecum semiteres and Tetrameres fissispina were found to be common in all three countries. Further studies are necessary to increase our knowledge on biodiversity of the parasitic nematodes in waterfowls and clear up their taxonomic status.

Abstracts

THE EXPRESSION OF CATHEPSIN L IN DEVELOPMENTAL STAGES OF FASCIOLOIDES MAGNA

R. ŠAŠKOVÁ1, R. LEONTOVYČ1, L. ULRYCHOVÁ1,3, J. DVOŘÁK3,4, P. HORÁK1, M. KAŠNÝ1,2

1Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 00 Prague, Czech Republic. 2Department of Botany and Zoology, Faculty of Science, Masaryk University Brno, Kamenice 753/5 625 00 Brno Bohunice, Czech Republic. 3Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Flemingovo nám. 2.166 10 Praha 6 Czech Republic 4Institute of Molecular Genetics of the ASCR, v. v. i., Vídeňská 1083 142 20 Prague 4, Czech Republic

Our experimental organism Fascioloides magna is a digenetic liver fluke, which was originally introduced to Czech Republic with North American White-tailed deer. This highly pathogenic worm causes a severe liver damage to free living and also domestic ruminants in North America and Europe. Our research is focused on functionally important molecules produced by this parasite, especially those which were predominantly identified as the most abundant in the excretory- secretory products isolated from adults, e.g. cysteine peptidase cathepsin L (FmCL). FmCL is supposed to play various key roles in biological processes of all the life stages during a life cycle. Up to now the transcripts of FmCL gene in eggs, miracidia, rediae, cercariae, metacercariae and adults have been identified by using PCR and cDNA as a template. Therefore we decided to evaluate also the stage specific FmCL expression level by using qPCR and localize the transcription in adult by adoption of non radioactive in situ RNA hybridization. qPCR experiment was designed according to MIQE guidelines. Six reference genes were selected (GAPDH, ACTB, COX, ATP5B, TBP, TUBB), specific primers designed and qPCR data analyzed by using Biogazelle software. RNA in situ hybridization experiment was realized according to Schistosoma mansoni in situ hybridization protocol developed in the lab of Prof. Grevelding; The adult worms were fixed in Bouin’s solution, embedded into paraffin and histological slides were prepared. After hybridization procedure the digoxigenin labeled probes were detected by specific antibodies and reaction was observed under confocal microscope. Some primary results supporting our hypothesis about expression level variation and the transcripts localization were obtained but not the major ones. Both qPCR and RNA in situ hybridization experiments are still in process.

Acknowledgements: Charles University in Prague (UNCE 204017, PRVOUK P41, SVV, GAUK 502313) and Masaryk University (MUNI/A/1484/2014).

Abstracts

MOLECULAR CHARACTERIZATION OF CYSTATINS OF TRICHINELLA SPP.

L. ŠKORPÍKOVÁ1, B. KOUDELA3, J. ILGOVÁ 1, M. GELNAR1 AND M. KAŠNÝ1,2

1Department of Botany and Zoology, Faculty of Science, Masaryk University, Kamenice 753/5 625 00 Brno Bohunice, Czech Republic 2Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 00 Prague, Czech Republic 3Department of Pathological Morphology and Parasitology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Science Brno, Palackého 1/3, 612 42 Brno, Czech Republic

Nematodes of the genus Trichinella are intracellular parasites which invade the cells of small intestine and skeletal muscles. Their global distribution is corresponding to the wide range of their hosts involving mammals, birds and reptiles. Six (T1, T2, T3, T4, T5 and T7) of twelve genotypes (T1-T12) of Trichinella are causative agents of human trichinellosis, a serious disease which has been documented in 55 countries. Our work is focused on cystatins - inhibitors of C1 cysteine peptidases. Their ancestor gene have undergone a complex and dynamic evolution implicating the current high variability of the cystatin group. Despite this fact, three amino acid conserved motifs of cystatin active site remain preserved. Cystatins are involved in many essential biological processes of helminths, including Trichinella spp., like development, growth, migration, digestion, reproduction and also in manipulation of the host immunne system. The presence of some cystatins was confirmed in excretory-secretory products of Trichinella muscle larvae and “multi cystatin-like domain” protein was recognized as a one of the most dominant components.

Acknowledgements: This research was supported by the Ministry of Education, Youth and Sports of the Czech Republic (Grant CONTACT II n. LH12096), by Charles University in Prague (UNCE 204017, PRVOUK P41, SVV) and by project of Masaryk University (MUNI/A/0888/2013)

Abstracts

ENOLASE: A SUITABLE ANTIGEN FOR THE IMMUNODIAGNOSTICS OF TRICHOBILHARZIA REGENTI INFECTIONS IN DUCKS

L. TURJANICOVÁ AND L. MIKEŠ

Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Prague 2, Czech Republic

Bird schistosomes of the genus Trichobilharzia are well known as the causative agent of cercarial dermatitis in bathers. Cercarie penetrate mammalian skin on the basis of biochemical signals similar to those of their natural definitive hosts - waterfowl. In our study, we focused on the search for a sensitive immunodiagnostic method of infections caused by the neurotropic nasal schistosome - Trichobilharzia regenti in ducks. The diagnosis of infections by T. regenti can be performed by direct methods such as the detection of parasite eggs in the nasal cavity of ducks or finding of schistosomula or adult worms at autopsy. The observed life span of T. regenti in the definitive host is 23-25 days. Neverteless, we can prove a past infection by indirect techniques based on the detection of Trichobilharzia-specific antibodies. The aim of our work was to find and to characterize antigens of T. regenti with possible diagnostic potential. We examined by Western blotting samples of sera obtained from experimentally infected ducks. Specific IgY antibodies recognized several antigens, most commonly those of molecular sizes of 47 and 50 kDa, both in cercarial and schistosomula homogenates. Following 2D eletrophoresis, corresponding protein spots were identified by mass spectrometry as enolases employing transcriptomic and genomic reference data of T. regenti and Schistosoma mansoni, respectively. Preparation of recombinant antigens for evaluation of their immunodiagnostic potential is under way.

Acknowledgements: This work was supported by grants of the Czech Science Foundation (13- 29577S) and the Charles University in Prague (GAUK 243-259305, PRVOUK P41, SVV260074/2014).

Abstracts

INTERACTIONS OF THE EGGS AND MIRACIDIA OF TRICHOBILHARZIA REGENTI WITH THE DUCK NASAL MUCOSA

L. VLČKOVÁ AND P. HORÁK

Department of Parasitology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44 Prague 2, Czech Republic

Trichobilharzia regenti as a member of bird schistosomes is a dixenous trematode bound to aquatic environment. In ducks, cercariae penetrate the skin; then the flukes migrate through the nervous system and mature in blood vessels of the nasal mucosa where they lay the eggs. Miracidia hatch directly in the nasal mucosa, and then they leave the duck to the aquatic environment. The parasite causes an inflammation of the bird nasal mucosa. In addition, due to the migration of too many schistosomula through the spinal cord, a paralysis of ducks was observed. Cercariae can also penetrate the skin of mammals including humans (in humans they cause itchy rash known as swimmers’ itch); mainly in sensitized hosts they are eliminated soon by the immune system and die in the skin, whereas in non-sensitized hosts (and some other cases) the parasites migrate via peripheral nerves and spinal cord to the brain, but they cannot complete their development. Similarly to the research performed on the genus Schistosoma, we will analyze different phases of miracidia development within the eggs. Immunohistochemistry will be used to show the presence of host immune cells in eggs vicinity, detection of the antigenic products secreted by miracidia using antibodies and the pathogenic changes of the duck nasal mucosa.

Abstracts

POSSIBLE DEFENCE MECHANISMS AGAINST TREATMENT IN TAPEWORMS

I. VOKŘÁL1, Z. SEIFERTOVÁ1, M. VEVERKOVÁ1, L. STUCHLÍKOVÁ2, L. PRCHAL2, B. SZOTÁKOVÁ2 AND L. SKÁLOVÁ2

1Dept. of Pharmacology and Toxicology, 2Dept. of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Hradec Králové, Czech Republic

Tapeworms are probably the least studied parasites in terms of metabolism and transport of xenobiotics compared to flukes and roundworms. On the other hand, knowledge about all detoxification mechanisms in tapeworms can be important for the future drug research and also can contribute to understanding of drug resistance development. Our first aim was to study metabolism of selected anthelmintics (albendazole, flubendazole, mebendazole and praziquantel) in two tapeworms, Hymenolepis diminuta and Moniezia expansa. Second aim was to answer if active efflux can participate in defence against treatment. For study of drug metabolism, tapeworms were incubated for 24 hours with selected anthelmintics. After samples extraction LC/MS analysis was performed. Medium and tissue samples were compared to blank samples and potential metabolites were subjected to MS/MS analysis for structure determination. For study of efflux tapeworms were incubated in medium with albendazol for 2 hours. Subsequently, they were divided to two groups. One group inactivated and second left without any influencing. Both groups were put into the fresh drug free medium for 30 minutes. After 30 minutes, concentration of drug in medium was measured and compared. In case of metabolism, most of found metabolites were produced during Phase I. Only two found metabolites were from the Phase II. No metabolites of praziquantel were detected (studied only in H. diminuta). On the contrary flubendazole and mebendazole were extensively reduced in both tapeworms. Reduced flubendazole was further metabolized in H. diminuta and methylated metabolites (Phase II) were found. On the other hand, only in M. expansa metabolites produced by oxidative metabolism, albendazole sulfoxide and albendazole sulfone, were observed. Regarding active efflux, our results show that for albendazole, there is an evidence of active efflux.

Financial support for this project was provided by Charles University in Prague, Specific Academic Research SVV 260 186 and Research program "Research and Study of the Drugs" (PRVOUK P40)

Abstracts

IN VITRO CULTIVATION OF THE TREMATODE SPECIES FASCIOLOIDES MAGNA AND TRICHOBILHARZIA REGENTI

K. VRBOVÁ1, J. PANKRÁC1 AND M. KAŠNÝ1,2

1Department of Parasitology, Faculty of Science, Charles University of Prague, Viničná 7, 128 44 Prague 2, Czech Republic 2 Department of Botany and Zoology, Faculty of Science, Masaryk University Brno, Kamenice 753/5 625 00 Brno Bohunice, Czech Republic

Many species of the class Trematoda are severe human and animal pathogens. Therefore they are frequently subjects of intensive research, where the setting of standardized conditions is one of the essential premises of relevant results. The long term in vitro cultivation of trematodes could help to solve many complications which are arising during experimental infections of animals or during the cultivation of worms from naturally infected hosts. The in vitro approach also enables e.g. detailed investigation of parasite biology or evaluation of the effect of antitrematode drugs. The first cultivation experiments were realized ca 100 years ago. Since that time significant advances have been achieved, especially on model organisms such as Schistosoma mansoni and Fasciola hepatica. However, until now no trematode species has been successfully in vitro cultivated continuously from egg to fully developed adult producing viable eggs. The main aims of our research are to elaborate a procedure of in vitro excystment of metacercariae of fasciolid flukes, transformation of cercariae of schistosomatid trematodes and subsequent cultivation of larval stages, juveniles and adults of Fascioloides magna and Trichobilharzia regenti. The suitability of proposed in vitro cultivation methods will be verified by characterization of the development of selected organ systems of cultivated worms. Recently the composition of SCM medium (Schistosoma culture medium, Basch) was optimized, transformation of Trichobilharzia regenti/T. szidati cercariae to schistosomula performed and their successful cultivation documented.

Acknowledgements: Czech Science Foundation (Grant No.GA13-29577S), Charles University in Prague (UNCE 204017, PRVOUK P41, SVV, GAUK 502313) and Masaryk University (MUNI/A/1484/2014).

Abstracts

AN EVALUATION OF DIFFERENT DISCRIMINATIVE APPROACHES USED FOR DISCRIMINATION OF CRYPTIC SPECIES

P. ZAHRADNÍČKOVÁ1, MAXWELL BARSON2, WILMIEN J. LUUS-POWELL3AND IVA PŘIKRYLOVÁ1,3

1Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic. 2Department of Biological Sciences, University of Zimbabwe, PO Box MP167, Mt. Pleasant Harare, Zimbabwe. 3Department of Biodiversity, University of Limpopo, Turfloop Campus, Private Bag X1106, Sovenga, 0727 South Africa.

Present study tackles with various discriminative approaches tested on selected species of highly diverse genus Gyrodactylus von Nordmann, 1832 (Gyrodactylidae) what is presumably group of viviparous ectoparasitic monogeneans of fish. Parasites were collected on cichlids from several distinct localities in Africa but the majority from Lake Chivero, Lake Kariba and River Zambezi in Zimbabwe during 2011 and 2012. Studied individuals form a group of species with same morphological type of main attachment organ hard parts that represent main morphological determination feature within the genus Gyrodactylus. However, morphometric differences within species are small, molecular distances are sufficient for species distinguishing. Exhaustive analyses of minor shape differences facilitated to detect cryptic species differentiated prior by genetic sequences. Thus the suitability and accuracy of varied techniques use-full for species discernment were compared. Various methods such as geometric morphometry, elliptic Fourier, principal component analyses and discriminative cluster analyses were applied on shapes and metrical characters of the hamuli and marginal hooks. Evaluation of the suitability of methods for the detection/distinguishing cryptic species was based on the presumption that species discrimination according to molecular approaches of ribosomal DNA ITS segment is valid. Additionally, phylogenetic relationship between studied species and other Gyrodactylus species collected from cichlids in Africa were estimated using methods Maximum likelihood and Bayesian inference. The results clearly show the significance of the use in molecular confirmation of species identity in standard taxonomy and also the potential of statistical techniques in preliminary species discrimination.

Abstracts

MOLECULAR AND SEROLOGICAL DIAGNOSIS OF INFECTIONS CAUSED BY TRICHOBILHARZIA SPP.

M. VAŠČIKOVÁ, L. TURJANICOVÁ AND P. HORÁK

Department of Parasitology, Faculty of Science, Charles University in Prague Viničná 7, 128 44 Prague 2, Czech Republic

Cercariae of the genus Trichobilharzia penetrate the skin of definitive hosts (ducks), but they are also able to penetrate the skin of accidental hosts (mammals). The penetration is frequently accompanied by an inflammatory response known as cercarial dermatitis. The goal of our experiments is to detect parasite DNA (T. regenti and T. szidati) in the serum and cerebrospinal fluid of infected ducks, and in the serum of infected mice (c57BL/6). We use commercial kits and HotSHOT method for DNA extraction. Universal primers were designed for a tandem repeated sequence that was presented by Hertel et al. (2002). We were capable of detecting 0.1 femtograms of DNA isolated from cercarie of T. regenti by using semi-nested PCR. With this method we were also able to detect parasite DNA from 6 out of 12 sera of infected ducks. DNA obtained from these positive samples were sequenced and then they were compared using BLASTN; our results showed 93-97% identity with the sequences of T. regenti. Sera of infected animals were also tested by ELISA and Western blot using the homogenate of T. regenti and T. szidati cercariae as antigen. Results from ELISA indicated progressive increase in the level of IgM antibody from the 1st infection to 10 days after the 4th infection, then the level of IgM gradually declined. The level of IgM 100 days after the 4th infection was still higher if compared to uninfected mice. The increase in the level of IgG antibody was detected from the 2nd infection. The course of the levels of IgG is similar to that of IgM. In the recent study by Lichtenbergová et al. (2008) IgG and IgE antibodies specifically recognized 34 kDa antigen that was identified in the homogenate of cercariae of T. regenti. Our results from Western blot analysis showed 21 and 22 kDa antigens which were recognized by both IgG and IgM antibodies of mice. Our intention is to separate these antigens by 2D electrophoresis and then identify protein spots by mass spectrometry. We also plan to test cross-reactions using the sera of Trichobilharzia-infected mice and S. mansoni antigen. Results of these experiments could help to develop reliable and specific diagnostic tests.

Acknowledgements: The work was supported by the Czech Science Foundation grant No. 13- 29577S and by the Charles University in Prague (GAUK 243-259305).

Addresses of Participans

Presentation in Czech language:

PARAZITI: „PRAVDU MÁ TEN, KDO VYLÉČIL!“

HANA BLÁHOVÁ

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65 Addresses of Participans

PARTICIPANT CONTACT INFORMATION

Bernardová Nicol, Ústav imunologie a mikrobiologie, 1.LF UK v Praze, Studničkova 7, 128 00, Praha 2 a Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Bláhová Hana, Celostní medicína, Jílová 29b, 639 00, Brno, Czech Republic, [email protected] Brabec Jan, Parazitologický ústav, Biologické centrum AVČR, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected] Bulantová Jana, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Cibulková Lucie, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Civáňová Kristina, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic [email protected] Čadková Zuzana, Katedra zoologie a rybářství, Fakulta agrobiologie, potravinových a přírodních zdrojů, Česká zemědělská univerzita v Praze, Kamýcká 957, Prague 6, 165 21, Czech Republic, [email protected] Dalton John, Queen's University Belfast, Medical Biology Centre, School of Biological Sciences, 97 Lisburn Road Belfast BT9 7BL, United Kingdom, [email protected] Dvořáková Hana, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Fajtová Pavla, Ústav organické chemie a biochemie AV ČR, v.v.i., Flemingovo nám. 2., 166 10, Praha 6, Czech Republic, [email protected] Gelnar Milan, Ústav botaniky a zoologie, PřF MU, Kotlářská 2, 611 37, Brno, Czech Republic, Czech Republic, [email protected] Georgieva Simona, Parazitologický ústav, Biologické centrum AVČR, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected] Haas Wilfried, Former Section Parasitology, Institute for Zoology, University Erlangen-Nuernberg, Staudtstrasse 5, 91058, Erlangen, Germany, [email protected] Hernandez Orts Jesus Servando, Departamento de Zoología, Instituto de Biología, Universidad Nacional Autónoma de México (UNAM), Avenida Universidad 3000, Ciudad Universitaria, CP 04510, Distrito Federal, México a Parazitologický ústav, Biologické centrum AVČR, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected] Horák Petr, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Chanová Marta, Ústav imunologie a mikrobiologie, 1. LF UK v Praze, Studničkova 7, 128 00, Praha 2, [email protected] Ilgová Jana, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Jedličková Lucie, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Jílková Adéla, Ústav organické chemie a biochemie AV ČR, v.v.i., Flemingovo nám. 2., 166 10, Praha 6, Czech Republic, [email protected] Jirsová Dagmar, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno a Ústav lesnické botaniky, dendrologie a geobiocenologie, Lesnická a dřevařská fakulta, Mendelova univerzita v Brně, Zemědělská 3, 61300, Brno, Czech Republic, [email protected] Kašný Martin, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Kmentová Nikol, Ústav botaniky a zoologie, PřF MU, Kotlářská 2, 611 37, Brno, Czech Republic, [email protected] Kostadinova Aneta, Parazitologický ústav, Biologické centrum AVČR, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected]

Addresses of Participans

Krasnovyd Vadym, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Krčmářová Veronika, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Kudlai Olena, Parazitologický ústav, Biologické centrum AVČR, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected] Kuchta Roman, Parazitologický ústav, Biologické centrum AVČR, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected] Kuzmina Tetiana, Institute of Zoology NAS of Ukraine; 15, Bogdan Khmelnitsky street, Kiev, 01601, Ukraine, [email protected] Leontovyč Adrian, Ústav organické chemie a biochemie AV ČR, v.v.i., Flemingovo nám. 2., 166 10, Praha 6, Czech Republic, [email protected] Leontovyč Roman, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Leštinová Kateřina, Parazitologický ústav, Biologické centrum AVČR, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected] Macháček Tomáš, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Majer Martin, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Mašová Šárka, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Melounová Klára, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Michálková Veronika, Ústav botaniky a zoologie AV ČR, v.v.i., Květná 8, 603 65, a PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Mikeš Libor, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Novák Jan, Ústav imunologie a mikrobiologie, 1. LF UK v Praze, Studničkova 7, 128 00, Praha 2, Czech Republic, [email protected] Pakosta Tomáš, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Pankrác Jan, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Panská Lucie, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Pavlíková Veronika, Ústav imunologie a mikrobiologie, 1.LF UK v Praze, Studničkova 7, 128 00, Praha 2 a Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Perháčová Terézia, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Peštová Jitka, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, zelvi- [email protected] Pravdová Markéta, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Přikrylová Iva, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic a Department of Biodiversity, School of Molecular and Life Sciences, University of Limpopo, Sovenga, South Africa, [email protected] Rahmouni Chahrazed, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Raisingerová Ludmila, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Reslová Nikol, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno; Výzkumný ústav veterinárního lékařství, v.v.i, Hudcova 296/70,

67 Addresses of Participans

621 00, Brno a CEITEC – Středoevropský technologický institut, Veterinární a farmaceutická univerzita Brno, Palackeho tr. 1/3, 612 42 Brno, Czech Republic, [email protected] Řehulková Eva, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Sedláková Jana, Ústav zoologie, rybářství, hydrobiologie a včelařství, Mendelova univerzita v Brně, Zemědělská 1, 61300, Brno, Czech Republic, [email protected] Schabussova Irma, Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna,Vienna, Austria, [email protected] Scholz Tomáš, Parazitologický ústav, Biologické centrum AVČR, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected] Skála Vladimír, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Skipalová Karolína, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Soldánová Miroslava, Parazitologický ústav, Biologické centrum AVČR, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected] Sosnová Tereza, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Spraker Terry, Diagnostic Medical Center, Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA, [email protected] Syrota Yaroslav, I.I. Schmalhausen Institute of Zoology of National Academy of Sciences of Ukraine,Vul. B. Khmelnytskogo 15, 01601, Kyiv, [email protected] Šašková Romana, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Škorpíková Lucie, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Turjanicová Libuše, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Uhrová Lucie, Přírodovědecká fakulta, Jihočeská univerzita v Českých Budějovicích, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected] Ulrychová Lenka, Ústav organické chemie a biochemie AV ČR, v.v.i., Flemingovo nám. 2., 166 10, Praha 6, Czech Republic a Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Valdejch Jaroslav, Katedra zoologie a rybářství, Fakulta agrobiologie, potravinových a přírodních zdrojů, Česká zemědělská univerzita v Praze, Kamýcká 957, Prague 6, 165 21, Czech Republic, [email protected] Vaščiková Michaela, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Vetešníková Šimková Andrea, Ústav botaniky a zoologie, PřF MU, Univerzitní kampus Bohunice, Kamenice 5, 625 00, Brno, Czech Republic, [email protected] Vlčková Linda, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Vokřál Ivan, Katedra farmakologie a toxikologie, Farmaceutická fakulta v Hradci Králové, Univerzita Karlova v Praze, Akademika Heyrovského 1203, 500 05, Hradec Králové, Czech Republic, [email protected] Vrbová Kristýna, Katedra parazitologie, PřF UK, Viničná 7, 128 44, Praha 2, Czech Republic, [email protected] Vyhlídalová Tereza, Přírodovědecká fakulta, Jihočeská univerzita v Českých Budějovicích, Branišovská 31, 370 05, České Budějovice, Czech Republic, [email protected] Zahradníčková Petra, Ústav botaniky a zoologie, PřF MU, Kotlářská 2, 611 37, Brno, Czech Republic, [email protected] Zikmundová Jana, Přírodovědecká fakulta, Jihočeská univerzita v Českých Budějovicích, Branišovská 31, 37005, České Budějovice, Czech Republic, [email protected]

22nd Helminthological Days 2015. Programme & Abstracts Terezia Perháčová, Libuše Turjanicová & Vladimír Skála (Eds.)

Published by Charles University in Prague, Faculty of science Graphic design: T. Perháčová, L. Turjanicová, L. Jedličková & V. Skála 1st edition, 2015 Number of copies: 85 Printed by Tisk Pětka s.r.o., Horova 1631, 252 63 Roztoky u Prahy ISBN 978-80-7444-032-8.

The abstracts are published as received from authors, without any changes or corrections. The authors are fully responsible for the content, stylistic and language representation of their abstracts.