MASARYK UNIVERSITY FACULTY OF SCIENCE DEPT . OF BOTANY AND ZOOLOG Y

BIOLOGICAL LY ACTIVE COMPOUNDS WIT H

POTENTIAL ANTIPARASI TIC EFFECT AND THEIR

IMPACT ON THE COURSE OF SELECTED

PARASITOSES

Ph.D. Dissertation

Radka Pecková

Supervisor: MVDr. Ivona Foitová, Ph.D . Brno 2018

Bibliographic Entry

Author Mgr. Radka Pecková Faculty of Science, Masaryk University Department of Botany and Zoology

Biological ly active compounds with potential Title of Thesis: antiparasitic effect and their impact on the processes of selected parasitoses Degree programme: Biology

Field of Study: Parasitology

Supervisor: MVDr. Ivona Foitová, Ph.D.

Academic Year: 2017/2018

Number of Pages: 139 Keywords: Giardia intestinalis ; Cryptosporidium ; Anti - protozoal activity; Plant extracts; Drug of Choice; Natural Antiparasitics; Parasites; Archidendron fagifolium ; Diospyros sumatrana ; Piper betle ; Shorea sumatrana

Bibliografický záznam

Autor: Mgr. Radka Pecková Přírodovědecká fakulta, Masarykova univerzita Ústav botaniky a zoologie

Biologicky aktivní látky s potencionálním Název práce: antiparazitárním účinkem a jejich působení na průběh vybraných parazitóz

Studijní program: Biologie

Studijní obor: Parazitologie

Vedoucí práce: MVDr. Ivona Foitová, Ph.D.

Akademický rok: 2017/2018

Počet stran: 139 Klíčová slova: Giardia intestinalis ; Cryptosporidium ; Antiprotozoární aktivita; Rostlinné extrakty; Alternativní léčiva; Přírodní antiparazitika; Paraziti; Archidendron fagifolium ; Diospyros sumatrana ; Piper betle ; Shorea sumatrana

ABSTRAK T

ABSTRACT

This thesis deals with the study of the influence of extracts of selected plants from Indonesia on parasites Giardia intestinalis (Lambl) Alexeieff, 1914 and Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Jesudoss Chelladural, Prantlová & McEvoy, 2016 . Tested plants were selected based on behavioural data and the ability to reduce the intensity of parasitic infection in Sumatran orangutans. Subsequently, they were extracted in three types of solvent s: water, methanol and methanol - tetrahydrofuran (1:1). Testing was performed using both the in vitro and in vivo models. In vitro experiments on G. intestinalis , during which four plant extracts were tested, the antiparasitic activity was reported in two of them, namely Archidendron fagifolium and Piper betle . Piper betle was also tested in vivo on Mongolian gerbils , where this activity was confirmed. The effect of extracts on C. proliferans was investigated using an in vivo model (southern multimammate mouse ( Mastomys coucha ) ) . Although Diospyros sumatrana extracts slightly decreased the number of shed oocyst s , evaluation of histological sections did not confirm the decrease in the intensity of parasitisation of the gastric tissue. The thesis also contains two methodological studies; the first concerns the comparison of the in vitro excystation protocols of Cryptosporidium parvum Tyzzer, 1912 oocyst, while the second one deals w ith the study of the course of experimental giardiasis in the gerbils. Both of these studies can be used for the future planning of experiments to test the antiparasitic activity of plant extracts.

ABSTRAK T

ABSTRAKT

Předložená práce se zabývá studiem vlivu extraktů vybraných rostlin z Indonésie na parazity Giardia intestinalis (Lambl) Alexeieff, 1914 a Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Jesudoss Chelladural, Prantlová & McEvoy, 2016 . Testované rostliny byly vybrány na základě behaviorálních dat a schopnosti snížit intenzitu parazitární infekce u sumaterských orangutanů. Následně byly extrahovány ve třech druzích rozpouštěd el: ve vodě, metanolu a metanol - tetrahydrofuranu (1:1). Te stování bylo provedeno s použitím in vitro i in vivo modelů. Při in vitro pokusech na G. intestinalis , kdy byly testovány čtyři rostliny, byla prokázána antiparazitární aktivita u dvou z nich, a to u Archidendron fagifolium a Piper betle . Piper betle byl t estován také na pískomilech, kde byla tato aktivita potvrzena. Vliv extraktů na C. proliferans byl zkoumán v in vivo modelu (krysa malá ( Mastomys coucha ) . Extrakty z rostliny Diospyros sumatrana mírně snižovaly počty vylučovaných oocyst, vyhodnocení histologických preparátů však nepotvrdilo snížení intenzity parazitární infekce v žaludeční tkáni. Práce dále obsahuje dvě metodologické studie; první se týká porovnání excystačních protokolů u oocyst Cryptosporidium parvum Tyzzer, 1912, druhá se zabývá studiem průběhu experimentální giardiózy u pískomilů. Obě tyto studie mohou být použity pro budoucí plánování experimentů zabývajících se testováním antiparazitární aktivity rostlinných extraktů.

© Radka Pecková , Masaryk University, 2018

ACKNOWLEDGEMENTS

ACKNOWLEDGEMENTS

This thesis could be created thanks to the kind support of many people I would like to acknowledge here. First, I would like to express my very great appreciation to my supervisor, MVDr. Ivona Foitová, Ph.D., for her patient guidance, advices, enthusiastic encouragement and endless optimism during the whole time of my studies, as well as for the financial support. I would like to offer my special thanks to the Laboratory of Veterinary and Medical Protistology of the Institute of Parasitology, Biology Centre CAS in České Budějovice, namely Prof. Ing. Martin Kváč, Ph.D., RNDr. Bohumil Sak, Ph.D . and RNDr. Dana Květoňová. For the opportunity to work in this laboratory, the initiation into the experimental methods, inspiring advices and a pleasant collaboration. I would like to thank Assoc. Prof. RNDr. Milan Gelnar, CSc. for giving me the opportun ity to work in Parasitology Research Group and RNDr. Andrea Bardůnek Valigurová, Ph.D. for giving me initial training in some laboratory methods and useful advices on this thesis. Collaboration and advices concerning the use of plant extracts provided by M gr. Karel Doležal, DSc. were greatly appreciated. I wish to acknowledge the help provided by the Laboratory of Electron Microscopy, Biology Centre CAS in České Budějovice particularly their help with obtaining the data from scan electron microscopy. I am a lso grateful to all my colleagues who created a pleasant workplace environment and were always willing to help. My special thanks are extended to Mgr. Markéta Fikejzová for the drawings presented in this thesis. Last but not least, I would like to thank my family and friends for their support and encouragement throughout my studies. This thesis was implemented in the Parasitology Research group at the Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno. The studies were financiall y supported by the UMI – Saving of Pongidae Foundation project “Parasites and Natural Antiparasitics in the Orang - utan”, by the Czech Academy of Sciences, Grant No. P505/11/1163 and by Scholarship program to support creative activity - research collaborati on, No. PřF_04_27.10.2006.

CONTENTS

CONTENTS

1 INTRODUCTION ...... 11

2 AIMS OF THE THESIS ...... 12

3 LITERA TURE OVERVIEW ...... 13

3.1 Biologically active compounds ...... 13

3.1.1 History of herbal drugs ...... 14

3.1.2 Self - medicative behaviour in animals ...... 15

3.1.3 Sources of secondary metabolites ...... 17

3.1.4 Biologically active compounds from plants as a treatment of parasito ses ..... 20

3.2 Plant extracts ...... 27

3.2.1 Plant extract preparation ...... 27

3.2.2 Tested plants ...... 28

3.3 Studied parasites ...... 29

3.3.1 Giardia intestinalis ...... 30

3.3.2 Cryptosporidium spp...... 34

4 MATERIALS AND METHODS ...... 41

4.1 Plant extracts ...... 41

4.1.1 Plant collection ...... 41

4.1.2 Plant extract preparation ...... 41

4.2 Giardia intestinalis ...... 42

4.2.1 In vitro experiments ...... 42

4.2.2 In vivo experiments ...... 44

4. 3 Cryptosporidium spp...... 49

4.3.1 In vitro excystation ...... 49

4.3.2 In vivo experiments ...... 52

5 SUMMARY OF RESULTS ...... 54

9

CONTENTS

5.1 Giardia intestinalis ...... 54

5.2 Cryptosporidium spp...... 56

6 DISCUSSION ...... 58

6.1 Giardia intestinalis ...... 58

6.2 Cryptosporidium spp...... 61

6.3 Future perspectives wit h regard to BAC from plant extracts ...... 62

7 CONCLUSIONS ...... 64

7.1 Giardia intestinalis ...... 64

7.2 Cryptosporidium spp...... 65

8 REFERENCES ...... 66

9 PUBLICATIONS FORMING THE DISSERTATION ...... 85

10

INTRODUCTION

1 INTRODUCTION

Biologically active compounds have a direct physiological effect on a plants, animals or another microorganisms (Macholán 2003, Opletal 2010). Natural products, such as plant extracts (pure compounds or standardized extracts) are characterized by chemical diversity and complexity (Cos et a l. 2006). They contain a broad spectrum of substances which can be utilized to treat both chronic and infectious diseases and s o they represent an important source of drugs with medicinal potential. That is also why they provide almost unlimited opportunities for research of new bioactive products, semi - synthetic medicines or lead compounds for the synthesis of medicines (Amaral e t al . 2006, Cos et al . 2006, Duraipandiyan et al . 2006). Using plants for healing has a long tradition dating back to prehistory (Cowan 1999). Nowadays, attention is being paid to extract derivatives from plants used in traditional medicine (Calzada et al . 2006, Moo - Puc et al . 2007, Li et al . 2012). According to the World Health Organization (WHO), more than 80 % of the world's population relies on traditional medicine for their primary healthcare needs and nearly 20,000 medicinal plants exist in 91 countri es (Sasidharan et al . 2011). Both Giardia intestinalis (Lambl) Alexeieff, 1914 and Cryptosporidium spp. Tyzzer, 1907 are parasites causing a significant diarrhoeal disease in humans, livestock and other animals all over the world (Thompson et al . 2005, Luj án & Svärd 2011) . Several commercial drugs have giardicidal effect, but they can produce side effects and resistance to some of these drugs has been described (Upcroft et al . 1990, Xiao et al . 1996). In case of Cryptosporidium , there is no drug that achiev es the complete removal of this parasite from the host (Zintl et al. 2009). For these reasons, the search for new, safe and effective therapeutic alternatives to treat these diseases has become necessary. One possible way is to investigate anti - parasitic effect of plants that are commonly used in traditional medicine or in the self - medication of wild animals.

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AIMS OF THE THESIS

2 AIMS OF THE THESIS

 cultivation of selected species of parasites

 development of in vitro and in vivo systems for testing anti - parasitological activity of plant extracts against selected parasites

 testing anti - p ro tozoa l activity of selected plant extracts

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LITERATURE OVERVIEW

3 LITERATURE OVERVIEW

3.1 Biologically active compounds A biologically active compound is defined as one that has a direct physiological effect on a plant, animal or another microorganism. These compounds are produced by primary and secondary metabolism of living organisms (Macholán 2003, Opletal 2010). The pr imary metabolism includes all of the chemical processes that occur in living organisms (glycolysis, pentose cycle or tricarboxylic acid cycle), resulting in growth, production of energy, elimination of waste material etc. Among biologically active primary metabolites belong amino acids and its derivatives, peptides, proteins, fatty acids, carbohydrates etc. (Macholán 2003, Opletal 2010). The secondary metabolism includes metabolic processes (biosynthesis and degradation) in which substances are only synthes ized in certain types of tissues or cells and are present in only a few species of organisms. Biologically active secondary metabolites are mainly produced by plants and fungi (80 %), their occurrence in animals is rare. Among thes e substances can be invol ved e. g. terpenes, steroids, glycosides, alkaloids or pigments (Macholán 2003). Natural products, such as plant extracts (pure compounds or standardized extracts) are characterized by chemical diversity and complexity (Cos et a l. 2006). They contain a broa d spectrum of substances which can be utilized to treat both chronic and infectious diseases and so they represent an important source of drugs with medicinal potential. That is also why they provide almost unlimited opportunities for research of new bioac tive products, semi - synthetic medicines or lead compounds for the synthesis of medicines (Amaral et al . 2006, Cos et al . 2006, Duraipandiyan et al . 2006). Natural products offer important complementary opportunities in drug discovery for various reasons (O gungbe & Setzer 2016). They occupy different regions of biologically relevant chemical space (Rosén et al . 2009), including abundant oxygen - containing functionalities (rarely nitrogen) and high degrees of chirality and complexity (Feher & Schmidt 2003). Al though outside the “rule - of - five”, numerous natural products have proven to be efficacious drugs (Ganesan 2008). They have been optimized for activity, including active transport, by evolution (Keller et al . 2006) and they also serve as lead structures for semisynthetic modification to improve activity, selectivity, or bioavailability (Newman & Cragg 2012).

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LITERATURE OVERVIEW

3.1.1 History of herbal drugs

Using plants for healing has very long tradition dating back to prehistory. There is evidence that Neanderthals living 60 thous and years ago used plants as hollyhock which is still used in ethnomedicine around the world ( Thomson 1978, Stockwell 1988, Cowan 1999). Egypt is considered the birthplace of phytotherapy. One of the oldest documents recording the process of drug discovery is the Ebers papyrus dating to the sixteenth century BC. It contains 900 formulas recommended by Egyptian priests to treat various diseases (Leil & Bertz 2014, Derda & Hada ś 2015). In ancient Greece, phytotherapy started to move out of temples. Hippocrates in the late 5 th century BC mentioned 300 to 400 medicinal plants. Galen in the 2 nd century described ways to prepare infusions, decoctions and tinctures from plants (Schultes 1978, Derda & Hada ś 2015). There are reports about the use of finely ground chrysanthemum ( Chrysanthemum cinerariaefolium ) flowers in fight against ectoparasites such as lice and fleas (Pavela 2016). In ancient Rome, granaries were often fumigated with some aromatic plants, e.g. rosemary, myrrh or juniper. Those plants were also hung near the entry openings of the granaries (Dubey 2011). In the 15 th century, a crucial breakthrough took place in medicine with the start of isolating pharmacologically active ingredients from medicinal plants. Since the beginning of the 19 th cen tury, a large number of biologically active secondary metabolites of plant origin (morphine, strychnine, caffeine, ephedrine etc.) have been found to have commercial application as drugs, flavours or pesticides (Kinghorn 1987, Derda & Hada ś 2015). The appr oach to drug discovery based on empirical evidence from the natural world is known as pharmacognosy (de Pasquale 1984) which was the principal means of drug discovery until the middle of the twentieth century. However, the difficulty of large - scale product ion of natural products made pharmaceutical industries to abandon this source of natural medicinal compounds (Trosset & Carbonell 2015). During the middle of the twentieth century, advances in biochemistry, molecular and cellular biology, and medicinal ch emistry resulted in a different approach to drug discovery (Drews 2000). This approach includes investigation of data from human epidemiology studies, combined with in vitro and in vivo experiments to illustrate the validity of a therapeutic target. The ad dition of high throughput chemical synthesis and screening permits identification of target selective, high affinity compounds. This hypothesis driven approach to pharmaceutical research and development has been a tremendous advance relative to the ancient methods of pharmacognosy, and resulted in a dramatic increase in the percentage of new drug applications approved by the Food and Drug Administration since the early 1960s (Leil & Bertz 2014). In 14

LITERATURE OVERVIEW microbiology, the discovery of antibiotics side lined the u se of plant derivatives as a treatment as well (Cowan 1999). Afterwards, the therapeutic advantages of herbal drugs (e. g. biocompatibility) were sacrificed to turn toward simpler chemistry at the risk of increased cross - reactivity with secondary therapeuti c targets and even unwanted off - targets as confirmed by recent studies in system chemical biology (Hu & Bajorath 2014, Leil & Bertz 2014) . Although therapy with pharmaceutical drugs is effective, they can produce significant undesirable side effects such a s metallic taste, vomiting, genetic damage, treatment failures, frequent relapses etc. (Katz 1982, Xiao et al . 1996). It is also known that antibiotics disturb biological balance between microorganisms of the digestive tract that have important functions i n the digestive process (Derda & Hada ś 2015). Moreover, resistance to the treatment of a broad range of parasites (protozoa, helminths and arthropods) has been described (Upcroft et al . 1990, Sangster et al. 2002). For these reasons, the search for new, sa fe and effective therapeutic alternatives to treat various diseases has become necessary. Since the late 1990s, the use of plant extracts, as well as other alternative forms of medicinal treatments, has become very popular (Cowan 1999). Nowadays, attentio n is being paid to extract derivatives from plants used in traditional medicine (Calzada et al . 2006, Moo - Puc et al . 2007, Li et al . 2012). According to the World Health Organization (WHO), more than 80 % of the world's population relies on traditional medicine for their primary healthcare needs and nearly 20,000 medicinal plants exist in 91 countries (Sasidharan et al . 2011). Plants used in t raditional medicine are characterized by good assimilability and the possibility of long term use without the danger of side effects (Derda & Hada ś 2015).

3.1.2 Self - medicative behaviour in animals

Plants produce a variety of toxic secondary compounds as a def ence against predation by animals (Howe & Westley 1988). The utilization of the chemical defences of plants by animal species to protect themselves from their own predators and parasites dates back to the beginning of the co - evolution of plant - animal relat ionships (Blum 1981). There are two forms of self - medication. First of them is prophylaxis, the prevention or reduction of the likelihood of infection (Christe et al. 2003). The second one is therapeutic self - medication, a curative use of anti - parasitic su bstances by already infected individuals (Lozano 1998, Lefèvre et al . 2010).

Honeybees, while collecting pollen and nectar from plants, gather not only nutritionally valuable food, but also highly active antimicrobial and antifungal compounds. Those, toget her

15

LITERATURE OVERVIEW with compounds secreted in the various glandular systems of honeybees facilitate their medication and sanitation (Erler & Moritz 2016). The monarch butterfly is known to feed on plants of the genus Asclepia that contain cardiac glucosides accustoming b ird predators not to feed on them because they make birds sick (Brower 1969).

In the higher vertebrates, a more sophisticated level of self - medication has evolved (Huffman 2003). Sparrows and wood rats use antiparasitic leaf material to line nests or dens (Sengputa 1981, Huffman 2003). Direct application of aromatic substances to re pel parasites infesting fur and feather has been reported in sparrows, starlings, brown bears, coatis and capuchin monkeys (Clark 1991, Grisanzio 1992, Gompper & Holyman 1993, Baker 1996). Probable self - medication behaviour has been observed in parasitized domestic animals. Sheep infected with gastrointestinal nematodes reduced their parasite burden by increasing their consumption of tannins with anthelmintic activity (Lisonbee et al. 2009). Goats in Uganda were observed to browse anti - parasitic plant Acaci a anthelmintica causing the excretion of worms in the faeces and reduction of parasite burden (Gradé et al . 2009).

Nowadays, attention is being paid to the self - medication of the great apes. The great ape diet is often rich in plants containing secondary c ompounds of non - nutritional, sometimes even toxic, value that suggests medicinal profit from their ingestion (Huffman 2003). The first documented putative self - medicative behaviour in the great apes is known as leaf - swallowing. It was observed in chimpanze es, bonobos and gorillas which swallow whole leaves and defecate them in an intact form. This behaviour with physical mode of action serves for expulsion of intestinal nematodes (Huffman 1996). Another way for the control of intestinal nematode infection b y chimpanzees is the ingestion of the bitter pith of Vernonia amygdalina (Huffman 1989, 1993).

Recently, a secondary self - medication (the external application of medicinal substances) was reported in Bornean orang - utans (Morrogh - Bernard et al. 2017). Resul ts of this study validate the anti - inflammatory activity of compounds of Dracaena cantleyi and its use to muscles and joints by orang - utans. These findings may be considered the first evidence for intentional external application of potentially bioactive s ubstances in the great apes.

It is likely that human herbal medicine have its origin within the animal kingdom. From prehistoric times man has looked to wild and domestic animals for sources of herbal medicines and narcotic stimulation. Both folklore and l iving examples provide accounts of how medicinal plants were obtained by observing the behaviour of animals (Huffman 2003).

16

LITERATURE OVERVIEW

3.1.3 Sources of secondary metabolites

3.1.3.1 Microorganisms

The discovery of penicillin from the filamentous fungus, Penicillium notatum , by Fl eming in 1929 and the observation of its broad therapeutic use in the 1940s led to a new era of medicine and promoted the intensive investigation of novel bioactive agents from natural sources ( Scriabine 1999). Microorganisms are a productive source of str ucturally diverse bioactive metabolites and have gained some of the most important products of the pharmaceutical industry. Among these belong e. g. antibacterial agents (penicillins from Penicillium species, cephalosporins from Cephalosporium acremonium , a minoglycosides, tetracyclines and other polyketides of many structural types from the Actinomycetales ), immunosuppressive agents (cyclosporins from Trichoderma and Tolypocladium species, and rapamycin from Streptomyces species), cholesterol lowering agents (mevastatin from Penicillium species and lovastatin from Aspergillus species) and antihelmintics and antiparasitic drugs (ivermectins from Streptomycetes species) (Buss & Waigh 1995).

3.1.3.2 Plants

Secondary metabolites in plants are organic compounds that are not directly involved in its normal growth, development or reproduction. The absence of these metabolites does not lead to immediate death but rather to long - term disability of surviving and reproductive potential of the plant and sometimes it does not cau se any significant change at all. Secondary metabolites are often restricted to a narrow group of phylogenetically related species (Hrckova & Velebny 2013). Climatic conditions and locality affect the chemical composition of various plant compounds (Muthau ra et al. 2011).

Any part of the plant can contain active components. Almost all of the identified plant compounds against microorganisms are aromatic or saturated organic compounds (Cowan 1999). Plants have almost unlimited possibilities for synthesis of aromatic substances. Most of them are phenols and their oxygen - substituted derivatives (Geissman 1963). Those substances are used by plants in various ways. Some of them serve as plant defence mechanisms against predators (microorganisms, insects or herbi vores), some give plants their odours and flavours, some are responsible for plant pigmentation (Cowan 1999). Humans use secondary metabolites as medicines, flavourings and drugs (Hrckova & Velebny 2013).

17

LITERATURE OVERVIEW

Secondary metabolites can be classified according t o their chemical structure (e.g. presence of rings or sugar), composition (e.g. containing nitrogen or not), solubility in different solvents or in which pathway are they synthesized (Hrckova & Velebny 2012). Major classes of useful antimicrobial phytochem icals are summarized in Tab. 1.

3.1.3.3 Animals

Concerning biological activity of compounds of animal species, we can single out marine organisms from the others. The systematic investigation of marine environments as sources for bioactive agents began in 1970s. Since this time, thousands of new metabolites were reported from a variety of marine organisms and many of them belong to totally novel chemical classes which are not found in terrestrial sources (Cragg & Newmann 2013). Natural products were proven in spon ges, coelenterates, bryozoans, marine molluscs, tunicates, echinoderms, marine worms and others (Faulkner 1988). The first marine derived product to gain approval as a drug was Ziconotide, a non - narcotic analgesic isolated as a constituent of the venom of the cone snail genus Conus , used to stun their pray prior to capture (Bulaj et al . 2003, Wallace 2006). Anticancer agents were proven at alkaloid ecteinascidin 743 isolated from the colonial tunicate Ecteinascidia turbinata , a complex polyether halichondrin B isolated from several sponge species, or bryostatin 1, a complex macrolide originally isolated from the bryozoan Bugula neritina (Cragg & Newmann 2013). Many animal species, both invertebrates and vertebrates, use seco ndary metabolites for various kinds of communication such as sexual, marking, aggregation and alarm pheromones, defensive compounds or bioluminescent compounds (luciferins) (Macholán 2003). Teprotide, isolated from the venom of the pit viper ( Bothrops jara caca ) serves for synthesis of the ACE inhibitors (captopril and enalapril) used in the treatment of cardiovascular disease (Buss & Waigh 1995). Epibatidine isolated from the skin of the poisonous frog Epipedobates tricolor has led to the development of a n ovel class of potential painkillers (Daly et al . 2005). Exendin - 4 from the venom of the Gila monster ( Heloderma suspectum ) was used for the development of an extenatide polypeptide used to improve glucose control in adults with type 2 diabetes (Eng et al . 1992).

18

LITERATURE OVERVIEW

Tab. 1: Major classes of antimicrobial compounds from plants (Cowan 1999). Class Subclass Example Mechanism Phenolics Simple phenols Catechol Substrate deprivation Epicatechin Membrane disruption Phenol acids Cinnamic acid Quinones Hypericin Bind to adhesins, complex with cell wall, inactive enzymes Flavonoids Chrysin Bind to adhesins Flavones Complex with cell wall Abyssinone Inactive enzymes Inhibit HIV reverse transcriptase Flavonols Totarol ? Tannins Ellagitannin Bind to proteins Bind to adhesins Enzyme inhibition Substrate deprivation Complex with cell wall Membrane disruption Metal ion complexation Coumarins Warfarin Interaction with eukaryotic DNA (antiviral activity) Terpenoids, Capsaicin Membrane disruption essential oils Alkaloids Berberine Intercalate into cell wall and/or DNA Piperine Lectin and Mannose - specific agglutinin Block viral fusion or polypeptides Fabatin adsorption Form disulphide bridges Polyacetylenes 8S - Heptadeca - 2(Z),9(Z) - ? diene - 4,6 - diyne - 1,8 - diol

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LITERATURE OVERVIEW

3.1.4 Biologically active compounds from plants as a treatment of parasitoses

Parasites are eukaryotes and that's why they share most molecular and biochemical characteristics with their eukaryotic hosts. This f act makes often difficult to find antiparasitic drugs being both effective and non - toxic for the host. Major targets which can mediate cytotoxicity in parasites include DNA and/or RNA, proteins of the cytoskeleton and biomembranes (Wink 2012). Traditionall y, there are two approaches that have been developed for the purpose to verify, validate and quantify antiparasitic activity of plants. The first one is provided by offering plants or plant parts to naturally or experimentally infected animals and quantify ing the consequences of their co nsumption. The second approach involves testing of plant extracts derived from medicinal plants using in vitro and in vivo systems (Athanasiadou et al . 2007). In vitro testing offers the opportunity to screen large numbers o f plant extracts at low cost, nevertheless, it should be complemented and verified by in vivo experimentation (Athanasiadou & Kyriazakis 2004). In the following chapters, the anti - parasitic activity of plant extracts or biologically active compounds obtained from them are reviewed. Because of the specialization of this thesis, the main emphasis is put on the activity against parasitic protozoans.

3.1.4.1 Protozoans

This chapter deals with the anti - parasitic activity of plant extracts or biologically active compounds against protozoal parasites of our interest, as well as against major human protozoal diseases.

Anti - giardial activity Detailed information on the biology and cultivation of Giardia intestinalis are described in Chapter 3.3.1. Scientific reports concerning biological activity of plant constituents against trophozoites and/or cysts of G. intestinalis are published from almost all parts of the world. Plants used in traditional medicine present a good source for anti - giardial assays. Barbosa et al. (2006, 2007) studied giardicidal activity of methanolic extracts and flavonoids isolated from medicinal plants used in Mexican traditional medicine. Experiment performed in suckling mice CD - 1 proved this activity in Helianthemum glomeratum (Cistaceae), Rub us coriifolius (Rosacea), Geranium mexicanum (Geraniaceae) and Cuphea pinetorum (Litraceae). Pérez - Arriaga et al. (2006) observed in in vitro conditions that curcumin, derived from the rhizome 20

LITERATURE OVERVIEW of Curcuma longa (Zingiberaceae), exhibited a cytotoxic effect in G. intestinalis trophozoites inhibiting the parasite growth and adherent capacity, induced morphological alterations and provoked apoptosis - like changes. Methanolic extracts of 21 plant species used as gastrointestinal remedies by the Luo people of Keny a and Tanzania were found to be lethal or growth inhibiting to G. intestinalis trophozoites in vitro (Johns et al. 1995). Machado et al. (2010) tested anti - giardial activity of essential oils of Thymbra capitata (Lamiaceae), Origanum virens (Lamiaceae), Th ymus zygis (Lamiaceae) and Lippia graveolens (Verbenaceae) in vitro . The oils inhibited parasite growth and adherence and promoted ultrastructural alterations like deformation in typical trophozoite appearance, round shape, irregular dorsal and ventral sur face, presence of membrane blebs, electrodense precipitates in cytoplasm and nuclei or internalization of flagella and ventral disc.

Anti - cryptosporidial activity Detailed information on the biology and cultivation of Cryptosporidium spp. are described in Chapter 3.3.2. Experimental studies regarding the anti - cryptosporidial activity of plant compounds are realized using various species of Cryptosporidium both in vitro and in vivo . Shahiduzzaman et al. (2009) studied effect of curcumin on C. parvum maintai ned in in vitro . It was found to inhibit the parasite growth and the invasion of host cells by sporozoites. Study of Wu et al. (2011) revealed that nitazoxanide, ginkgolic acid isolated from Ginkgo biloba , inhibited the number of C. andersoni in cell cultures. The effect of matrine, the main active alkaloid extract from the traditional Chinese medicinal plant Sophora flavescens was tested on BALB/c mice and MDBK cells both experimentally inoculated with C. parvum . Results showed that matrine si gnificantly reduces the number of shed oocysts and the number of affected cells and thus inhibit C. parvum parasitisation progress (Chen & Huang 2012). Different method for the evaluation of the efficacy of blueberry extracts on C. parvum was provided by A nthony et al. (2007). This effect was based on the increase of spontaneous excystation, which is excystation at elevated temperature (37 °C) in the absence of other major host triggers. If this happens in abnormal sites, it can prevent excysted sporozoites from invading intestinal epithelium. In this study, blueberry extracts were found to increase the spontaneous excystation.

Activity against Entamoeba histolytica Entamoeba histolytica is a parasite of the large intestine. It is distributed worldwide, but is more common in developing countries and areas with poor sanitation, particularly in the tropics.

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LITERATURE OVERVIEW

Transmission occurs via the faecal - oral route. The clinical symptoms range from asymptomatic infection with the excretion of cysts, diarrh o ea and dysentery to fulminant colitis and peritonitis as well as extraintestinal amoebiasis (affecting liver, pleura, pericardium and brain) ( Derda & Hada ś 2015, http://www.who.int/ith/diseases/amoebiasis/en/) . Anti - amoebic activity of plant compounds was reported in many scientific articles. Calzada et al. (2006) tested methanolic extracts of plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. Three plants showed inhibiting effect on E. histolytica in vitro : Chiranthodendron pentadactylon (Malvaceae), Annona cherimola (Annonaceae) and Punica granatum ( Lythraceae ). In vitro testing of medicinal plants used by AIDS patient in southern Thailand proved the anti - amoebic activity of seven plants. The most active were the chloroform extracts of Boesenbergia pandurata (Zingiberaceae), Alpinia galanga (Zingiberaceae) and the methanolic extract of B. pandurata (Sawangjaroen et al. 2006). Ghoshal et al. (1996) reported amoebicidal effect of the ethanolic extract from the fruits of Piper l ongum (Piperaceae) and piperine, a pure compound of this plant, in a rat model.

Anti - plasmodial activity

Five species of Plasmodium , the cause of malaria, infect humans: P. falciparum , P. vivax , P. ovale, P. malariae and P. knowlesi . They are transmitted by female Anopheles mosquitoes. The greatest threat pose P. falciparum and P. vivax (http://www.who.int/en/news - room/fact - sheets/detail/malaria). Antimalarial drug screening used to be provided only by in vivo avian and rodent models, using non - human Plas modium species until 1976, when the asexual cycle of P. falciparum was successfully cultured in human erythrocytes. Since this year, P. falciparum has been used extensively in in vitro experiments; techniques to culture the other three human Plasmodium species have not had such a success (Kayser et al. 2002).

Medicines of plant origin have been used to treat malaria for a long time. Willcox & Bodeker (2004) state there are over 1200 plant species from 160 families used to treat malaria in traditional me dicine. The first effective antimalarial drug was quinine, an alkaloid of bitter taste present in the bark of South American tree Cinchona officinalis (Rubiaceae) (Derda & Hada ś 2015). Another effective treatment of malaria is artemisinin, obtained from Ar temisia annua (Asteraceae). It has been used as a Chinese herbal remedy for malaria since 341 BC (Li & Wu 1998). The use of artemisinin provides good therapeutic effects against diseases caused by Plasmodium vivax and P. falciparum . Furthermore, this treat ment does not cause side effects (Arnold et al ., 1990). There are many other studies concerning the efficacy of plant extracts on

22

LITERAT URE OVERVIEW plasmodia, particularly those from areas endemic for malaria (Benoit - Vical et al . 2003, Boyom et al . 2003, Venkatesalu et al . 2012) suggesting traditional phytomedicine can be as effective as pharmaceutical formulations and should be tested as sources in the search for novel drugs (Anthony et al . 2005).

Anti - leishmanial activity

Leishmaniasis is caused by different Leishmania sp ecies that are obligate intracellular parasites of mononuclear phagocytes. There are three types of the disease: cutaneous (self - healing ulcers), mucocutaneous (nasopharyngeal infections) and disseminating visceral leishmaniasis. It occurs from tropical to Mediterranean regions. The parasite is transmitted by female sandflies of the genus Phlebotomus in the Old World and Lutzomyia in the New World (Berman 1998). Before testing compounds with potential anti - leishmanial activity, it is important to take into account that there are two forms: amastigotes (major stage in mammalian host) and promastigotes (occurring only during a few hours after infection of a mammalian host) with different biochemical and molecular properties (Kayser et al. 2002).

Berberine, an alkaloid present in many plant species, showed significant in vivo activity against L. panamensis (Vennerstrom et al. 1990). Essential oil of Croton cajucara (Euphorbiaceae) used in in vitro studies caused a reduction in L. amazonensis promastigotes and a mastigotes with little or no observed toxicity in murine macrophages (Rosa et al. 2003). Water - alcohol extracts of Tanacetum parthenium (Asteraceae) inhibit the development of L . amazonensis promastigotes and reduce the survival of amastigotes in macrophag es by 10 % (da Silva et al. 2010). Ethanol extracts of Baccharis uncinella (Asteraceae) have similar effect on both promastigotes and amastigotes of L. amazonensis and L. braziliensis (Passero et al. 2011).

Anti - trypanosomal activity African trypanosomiasis This infectious disease is caused by Trypanosoma brucei gambiense and T. brucei rhodesiense in humans and by T. b. brucei , T. congolense , T. evansi , T. equiperdum and T. vivax in domestic animals. These trypanosomes are transmitted by a tse tse fly ( Glossina spp.) (Kayser et al. 2002, Anthony et al. 2005). T. brucei ssp. are extracellular parasites living in blood and lymph as elongated trypomastigotes. They can be axenically cultured in supplemented standard media (Kayser et al. 2002).

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LITERATURE OVERVIEW

Miku s et al. (2000) observed trypanocidal effect of essential oils of Melaleuca alternifolia (Myrtaceae), Melissa officinalis (Lamiaceae) and Thymus vulgaris (Lamiaceae). Abiodun et al. (2012) studied the activity of some medicinal plants from Nigerian ethnome dicine against T. brucei rhodesiense . He found out trypanocidal activity of t he ethyl acetate leaf extract of Ocimum gratissimum (Lamiaceae), the hexane, ethyl acetate, or methanol extracts of Trema orientalis (Ulmaceae), Pericopsis laxiflora (Fabaceae), J atropha curcas (Euphorbiaceae), Terminalia catappa (Combretaceae) and Vitex doniana (Verbenaceae).

South American trypanosomiasis American trypanosomiasis, also called Chagas disease, is caused by Trypanosoma cruzi and transmitted by hemophagous triatomine insects. In mammals, the parasite exists in two forms: the extracellular trypomastigote (non - dividing invasive haemolymphatic form) and the amastigote (dividing cytoplasmic form). The dividing amastigotes are cult ured in vitro most commonly in macrophages, fibroblasts and myoblasts. Trypomastigotes and epimastigotes (dividing form in a vector) can be cultured in vitro as well (Kayser et al. 2002). In the treatment of this disease, several plant species have been t ested. Leaf extracts from Serjania yucatanensis (Sapindaceae), a plant used in traditional medicine, and Bourreria pulchra (Boraginaceae) were proven to have an inhibiting effect on the development of trypomastigotes and epimastigotes in vitro and trypanoc idal activity of S. yucatanensis was also confirmed in vivo (Polanco - Hernández et al. 2012). Guaianolide, isolated from Tanacetum parthenium (Asteraceae), was shown to be effective in vitro against all forms of T. cruzi (Cogo et al. 2012). Similar results were observed by Campos et al. (2010), who tested methanolic extracts of Croton cajucara (Euphorbiaceae).

3.1.4.2 Helminths

Helminths have plagued humans since before the era of our earliest recorded history. The most common helminthiases are those caused by infe ction with intestinal helminths, ascariasis, trichuriasis and hookworm infections, followed by schistosomiasis and lymphatic filariasis (Hotez et al . 2007). According to CDC (Centres for Disease Control and Prevention), a large part of the w orld ' s populati on is infected with one or more of soil - transmitted helminths (807 - 1,121 million with ascariasis, 604 - 795 million with whipworm, 576 - 740 million with hookworm), more than 200 million people is infected with schistosomiasis and lymphatic filariasis affects over 120 million people.

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LITERATURE OVERVIEW

Most of the screenings recording anti - helminthic activity of medicinal plants are provided by in vitro methods. These studies use different model organisms: either direct parasitic species we are interested in, or species showing a natomical and physiological similarity with target helminths, or related species th at are easily available (Mali et al. 2007).

Anti - nematodial activity Haemonchus contortus (Nematoda), a highly pathogenic parasite of small ruminants, is a popular species for in vitro testing of anthelmintic properties of plants. Pomegranate ( Punica granatum , Lythraceae) inhibits transformation of eggs to filariform larvae (Prakash et al . 198 0). Crude aqueous and methanol extracts of tobacco ( Nicotiana tabacum , Solanaceae) show dose - dependent anthelmintic effect (Iqbal et al . 2006). Hydroalcoholic extract of Chenopodium ambrosioides (Amaranthaceae) and aqueous extract of Jatropha curcas (Eupho rbiaceae) inhibit the hatching of eggs, and aqueous extract of C. ambrosioides has a negative effect on the survival of adult parasites (Eguale & Giday 2009). Some plants show anthelmintic activity against Caenorhabditis elegans (Nematoda), for example the essential oil from holy basil ( Ocimum sanctum , Lamiaceae) (Asha et al . 2001) or papaya ( Carica papaya , Caricaceae) seed oil (Kermanshai et al . 2001). Indian earthworm ( Pheretima posthuma , Annelida) is often used as a test worm in in vitro anthelmintic screenings because of its anatomical and physiological resemblance with the intestinal roundworm p arasites of humans (Mali et al. 2007). The effect of many plants was proved against this organism, e.g. betel ( Piper betle , Piperaceae) (Ali & M ehta 1970), Melia azedarach (Meliaceae) (Szewezuk et al . 2003), Mimusops elengi (Sapotaceae) (Mali et al . 2007) and others.

Anti - cestodial activity Anthelmintic properties of plants were also evaluated against various species of cestodes. Ethanol extract of Melia azedarach (Meliaceae) and methanol extract of Gynandropsis gynandra (Capparidaceae) exhibited considerable activity against pork tapeworm, Taenia solium (Ajaiyeoba et al . 2001, Szewezuk et al . 2003). Tangpu et al . (2004, 2006) studied the effect o f two plants used in ethnobotanical medicine in India on rat tapeworm, Hymenolepis diminuta . He found both plants ( Trifolium repens (Fabaceae) and Strobilanthes discolor (Acanthaceae)) highly effective; both reduced the number of eggs in faeces and the rec overy of surviving worms at autopsy. Ethanol end ether extracts of Centratherum anthelminticum (Asteraceae) are active against dwarf tapeworm, Hymenolepis nana (Singh et al . 1985).

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LITERATURE OVERVIEW

3.1.4.3 Arthropods

Acaricidal activity Infestation of ticks (particularly Amblyomma variegatum , Rhipicephalus appendiculatus and Rhipicephalus microplus ) can cause severe problems in livestock, especially in developing countries (Bram 1983). They transmit protozoal, bacterial, rickettsial and viral diseases and in addition, heavy infesta tions can result in a reduction in live weight, anaemia, losses in milk production and damage to hides (Jongejan & Uilenberg 2004, Rajput et al . 2006). Years of use of chemical ectoparasiticides have resulted in the development of resistance in these paras ites as well as in negative impacts to the environment (Adenubi et al . 2016). Plant - based treatment presents an alternative approach. Phytochemicals such as cis - Jasmone, 1,8 - cineole or 1 - menthone act in different ways, for example as counteraction of growt h regulatory hormones, inhibition of egg development, disruption of mating and sexual communication or inhibition of chitin formation (Katoch et al . 2007, Chagas et al . 2012). Some compounds of plant origin are already used in commercial repellents: e.g. p yrethrum derived from Chrysanthemum cinerariifolium and C. coccineum or 2 - undecanone, originally derived from wild tomato ( Lycopersicon hirsutum ) (Witting - Bissinger et al . 2008, Adenubi et al . 2016). According to Adenubi et al . (2016), more than 200 plant species have tick - repellent or acaricidal properties using in vitro assays.

Insecticidal activity Insecticides have a wide range of application: they are used in agriculture to eliminate damage caused by phytophagous insects both in fields directly during the process of growing crops and in granaries and storehouses afterwards (Stevenson et al . 2014). They are also used in human and veterinary medicine to reduce the population density of vectors (for example mosquito species transmitting diseases such as m alaria, dengue fever, leishmaniasis, filariasis or Chagas disease) and other annoying insects (fleas, lice, ants etc.) (Pavela 2016). Because of their negative side effects (resistance in some pests, contamination of the environment, changes in natural bio logical balances etc.), alternative modes of protection are needed. The use of plant secondary metabolites synthesised by some plant species as a part of their natural self - defence against pathogens and pests seems to be a good alte rnative (Miresmailli & I sman 201 4, Pavela 2016). There are several commercially produced botanical insecticides, for example Neem products based on oil from the seeds of Azadirachta indica containing active components limonids or products based on the use of pyrethrins derived fr om Chrysanthemum cinerariifolium which was mentioned earlier. Another group of commercial botanical 26

LITERATURE OVERVIEW insecticides includes products based on essential oils whose advantage is their rapid action. The most widely used essential oils are obtained from Rosmarin us officinalis ( Lamiaceae), Mentha spp . ( Lamiaceae) , Cymbopogon schoenanthus (Poaceae), Thymus vulgaris ( Lamiaceae), Syzygium aromaticum (Myrtaceae) and Citrus spp . (Rutaceae) (Pavela 2016).

3.2 Plant extracts Plant extracts are concentrated preparations of several possible compositions and consistencies, obtained from plant materials by steam distillation, cold pressing or solvent extraction that can be precede d by pre - treatments. The most common processes used for extraction include maceration, decoction, infusion, percolation, distillation or digestion (dos Santos Gouvea et al. 2017).

3.2.1 Plant extract preparation

The processing of plant material is a critical step because during the preparation of the extract, potential active constituents can be lost, altered or destroyed (Cos et al. 2006). The role of the extraction processes is to transfer the substances of inte rest to the extraction agent so that there is no degradation of the substances (enzymatic, thermal, light - induced or oxidative) and that the extraction yield is acceptably high for the desired metabolite spectrum (Tarkowski et al . 2004).

Plant extracts are usually prepared by maceration or percolation of fresh green plants or dried powdered plant material in water and/or organic solvents. For extraction of hydrophilic compounds, polar solvents (e.g. methanol, ethanol or ethyl - acetate are used). For more lip ophilic compounds, dichloromethane or a mixture of dichloromethane/methanol 1:1 are used (Cos et al. 2006). Plant compounds active against microorganisms are most often obtained through initial ethanol or methanol extraction because the majority of the ide ntified plant compounds active against microorganisms are aromatic or saturated organic compounds (Cowan 1999). Examples of extraction solvents and the resultant active fractions are listed in Tab. 2.

One of the most frequent solvent used to make up test c ompound solutions is dimethyl sulfoxide (DMSO), methanol and ethanol. However, the disadvantage of the latter two is rapid evaporation. Other advantages of stock solutions in 100 % DMSO are elimination of microbial contamination and good compatibility with test automation and integrated screening platforms (providing e.g. good solubility during the serial dilution procedures). Because of potential 27

LITERATURE OVERVIEW toxicity of DMSO for cells and many microorganisms, the final concentration of DMSO in in vitro tests should no t exceed 1 % (Cos et al. 2006) and 2 % in animal models (Barbosa et al. 2006).

Tab. 2: Solvents used for active component extraction (Cowan 1999). Compounds commonly obtained only in one solvent are indicated in boldface type. Dichloro - Water Ethanol Methanol Chloro form Ether Acetone methanol

Anthocyanins Tannins Anthocyanins Terpenoids Terpenoids Alkaloids Flavonols

Starches Polyphenols Terpenoids Flavonoids Terpenoids

Tannins Polyacetylenes Saponins Coumarins

Saponins Flavonol Tannins Fatty acids

Terpenoids Terpenoids Xanthoxyllines

Polypeptides Steroids Totarol

Lectins Alkaloids Quassinoids

Propolis Lactones

Flavines

Phenones

Polyphenols

3.2.2 Tested plants

Four Indonesian plants were used in studies forming this dissertation: Piper betle , Archidendron fagifolium , Diospyros sumatrana and Shorea multiflora .

3.2.2.1 Piper betle

Piper betle L. (Piperaceae, betel leaf) is a plant commonly used in traditional medicine in South and Southeast Asia. It is an evergreen climbing shrub producing woody stems 5 - 20 meters lo ng. The stems produce adventitious roots used for adherence to other plants, e.g. for support (http://tropical.theferns.info/viewtropical.php?id=Piper+betle.). It is extensively grown in India, Sri Lanka, Malaysia, Thailand, Taiwan and other Southeast Asia n countries (Guha 2006). Experimental studies revealed its wide and diverse biological and pharmacological effects, for example, but not limited to, antibacterial, antifungal, antiprotozoal, antioxidant, anti - inflammatory and gastroprotective (Fazal et al. 2013, Pradhan et al. 2013). This plant 28

LITERATURE OVERVIEW contains a broad range of chemical compounds including alkaloids, carbohydrates, amino acids, tannins and steroids (Sugumaran et al. 2011). In spite of these facts, there is poor evidence of the impact of Piper betle on Giardia intestinalis (Sawangjaroen et al. 2005 ).

3.2.2.2 Archidendron fagifolium

Archidendron fagifolium (Miq.) I. C. Nielsen (Fabaceae) is a shrub or a tree growing up to a height of 16 metres. It grows in Southeast Asia (Indonesia, Philippines) and local people use the edible seeds from wild plants as a food flavouring (http://tropical.theferns.info/viewtropical.php?id=Shorea+multiflora). This plant has not been tested yet for its biological activity.

3.2.2.3 Diospyros sumatrana

Diospyros sumatrana Miq. (Ebenaceae) is a tree growing up to 1500 metres high in Indonesia, Thailand and Malaysia in lowland rainforest. Malaysians drink a decoction of roots as a protective remedy. There is a poor evidence of its pharmacological potential (Wiart 2006). Sak et al. (2017) found methanolic extracts of this plant to be effective against Encephalitozoon cinuculi infection in experimentally infected mice. According to Kanzaki et al . (1997), who compared flavonoid composition of 24 Diospyros species, D. sumatrana h as a unique combination of flavonoids which are not shared by other species.

3.2.2.4 Shorea multiflora

Shorea multiflora (Burck) Symington (Dipterocarpaceae) is a small to m edium - sized, occasionally large tree with a diffuse, hemispherical crown comprised of many ascending branches. It grows in Southeast Asia (Malaysia, Indonesia) usually up to a height of 40 metres. The tree is harvested from the wild as a commercial source of “yellow meranti” timber ( http://tropical.theferns.info/viewtropical.php?id=Archidendron +fagifolium). Its biological activity has not been tested so far.

3.3 Studied parasites In studies forming this dissertation, gastric or intestinal unicellular parasites Giardia intestinalis , Cryptosporidium parvum Tyzzer, 1912 and C. proliferans Kváč, Havrdová,

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LITERATURE OVERVIEW

Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Jesudoss Chelladural, Prantlová & McEvoy, 2016 were used.

3.3.1 Giardia intestinalis

3.3.1.1 Biology, pathogenesis and treatment

Giardia intestinalis is an anaerobic flagellated pro tozoan parasite of mammalian species, including humans, which is considered the most common cause of protozoan diarrhoea. It was observed already in 1681 by Anthony van Leeuwenhoek (Luján & Svärd 2011). Giardia belongs to the Phylum Metamonada, Class Trepo monadea, order Diplomonadida and family Hexamitidae (Thompson & Monis 2011). Members of this family are characterized by paired organelles (including two similar, transcriptionally active diploid nuclei), the absence of mitochondria and peroxisomes and a u nique attachment organelle – the ventral (adhesive) disc (Kabnick & Peattie 1990, Thompson & Monis 2011). The ventral disc is composed of a variety of cytoskeletal proteins, principally tubulin and closely related proteins called giardins (Ankarklev et al . 2010). Giardia intestinalis has a global distribution and symptomatic infections occur in developing and developed countries ( Cacciò & Sprong 2011) . It is also commonly found in domestic animals such as livestock, dogs and cats ( Thompson et al . 1993) . In 2004, Giardia was included in the World Health Organisation's Neglected Diseases Initiative (Savioli et a l. 2006). Results of molecular studies indicate the existence of seven genetic groups (assemblages). Two of them are found in both humans and animals, whereas the remaining five (C - G) are considered host specific ( Cacciò & Sprong 2011) . Transmission of this parasite is mediated through the ingestion of cysts, excreted in faeces (Fig. 1). Cysts are released intermittently (Belosevic et al . 1983) and are r emarkably stable surviving weeks to months in the environment. They can also contaminate drinking water, food, recreational water or areas, such as playgrounds and sandpits ( Cacciò & Sprong 2005). In humans, the infective dose for a symptomatic infection i s around 10 - 100 cysts. Excysted trophozoites colonize the lumen of the small intestine, without invading host tissues or entering bloodstream (Ortega & Adam 1997). The highest cases of giardiasis are reported among children (1 - 4 and 5 - 9 years old) and adul ts aged 35 - 39 years (usually parents of infected children) (Cacciò & Sprong 2011). In some immunocompromised patients, giardiasis occurs in a refractory form (Nash et al . 2001).

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LITERATURE OVERVIEW

The pathogenesis of giardiasis is a combination of parasite and host factors and therefore the subsequent clinical signs vary considerably between species (Geurden & Olson 2011). In humans, the disease can be asymptomatic, but it is often manifested by a broad spectrum of symptoms during acute or chronic phase of the infection. The acute phase is usually short - lived, characterized by flatulence and abdominal distension with cramps. Diarrhoea is initially frequent and watery but later becomes bulky, greasy and typically offensive. In chronic giardiasis, malaise, weight loss and other features of malabsorption become prominent and stools are usually pale or yellow, frequent and of small volume. Other gastrointestinal symptoms of giardiasis include bloating, abdominal pain, nausea and vomiting (Walzer et al. 1971, Nash et al . 1987, Faubert 2000, Buret & Cotton 2011). In some patients, symptoms can persist even after several treatment courses with metronidazole (Hanevik et al . 2007). In animals, a variety of clinical symptoms have been described, including diarrhoea and malabs orption (O 'Handley et al . 1999, Geurden & Olson 2011). In case of livestock, these symptoms reduce growth rate, feeding efficiency and profitability of livestock production (Olson et al. 1995). In giardiasis, the acute pathophysiology occurs without invasi on of the small intestinal tissues by the trophozoites (Gillon et al. 1982). These pathophysiological stages can involve increased rates of enterocyte apoptosis, small intestinal barrier dysfunction, activation of host lymphocytes, shortening of brush bord er microvilli, malabsorption, anion hypersecretion and increased intestinal transit rates (Cotton et al . 2011). Several compounds are known to be effective against Giardia ; in human patients, the most frequent treatment against giardiasis are nitro - imidazo les such as metronidazole or tinidazole, or benzimidazole compounds (albendazole, febendazole). Although therapy with these compounds is effective, they can produce significant undesirable side - effects such as metallic taste, vomiting, genetic damage, trea tment failures, frequent relapses and selection and evolution of resistant strains (Xiao et al . 1996, Harris et al . 2001, Upcroft & Upcroft 2001, Sangster et al . 2002). Moreover, resistance to nitro - imidazoles has been described (Upcroft et al . 1990). For these reasons, the search for new, safe and effective therapeutic alternatives to treat giardiasis has become necessary.

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LITERATURE OVERVIEW

Fig. 1: Life - cycle of Giardia intestinalis (Drawing made by Markéta Fikejzová and Radka Pecková)

3.3.1.2 In vitro cultivation

The first axe nic cultures of Giardia were grown by Meyer (1970) in a medium that was quite complicated to make and required human serum and chick embryo extracts. In 1978, Diamond developed medium to culture Entamoeba and trichomonad species (TYI - S - 33) and later he dis covered that this medium can be also used for axenic growth of Giardia trophozoites (Visvesvara 1980). In 1983, Keister modified Diamond's medium to specifically enhance giardial growth. The modifications included for example addition of bile, higher conce ntrations of cysteine, pH change to 7 - 7.2 and sterilization by filtration, rather than autoclaving. This

32

LITERATURE OVERVIEW medium was called "complete modified TYI - S - 33" and to this day, it is the most commonly used medium for trophozoite culture (Davids & Gillin 2011).

Tra ditional way to measure susceptibility of G. intestinalis to anti - giardial agents in vitro includes counting of viable cells in a cell counting chamber under a light microscope. The results are calculated as the percentage of growth inhibition when compare d with the controls grown without anti - giardial agents. The basic measure of the potency of inhibitory agents is

IC 50 (the half maximal inhibitory concentratio n), which is defined as the concentration of the drug that inhibits growth by 50 % (Cedillo - River a & Muñoz 1992).

Assessment of the efficacy of giardicidal compounds can be also evaluated by colorimetric method. Shortly, after the end of drug incubation, the remaining viable cells are fixed in methanol and stained by methylene blue. The inclusion of methylene blue by trophozoites is measured spectrophotometrically to deter mine anti - giardial activity (Bu sat t i & Gomes 2007).

Different method for assessment of susceptibility of G. intestinalis trophozoites to anti - giardial agents is measurement of oxygen uptake. This method assumes that some drugs like metronidazole inhibit the O 2 uptake of trophozoites which is used as an index of viability (Céu Sousa & Poiares - da - Silva 1 999).

3.3.1.3 In vivo experim ents

There is a variety of animals that can be used for anti - giardial activity assays. Rodents (rats, suckling mice), rabbits and mongrel dogs can be infected by cysts, Mongolian gerbils and CD - 1 mice by both cysts and trophozoites (Meyer et al . 1984, Barb osa et al . 2007).

The evaluation of the impact of giardicidal agents can be provided by two basic methods. First of them is based on faecal examination and cyst counting. This method assumes that the number of released cysts is positively related to the nu mber of trophozoites located in small intestine (Belosevic et al. 1983).

Second method is based on direct counting of trophozoites from the intestine. After animal dissection, the small intestine (the whole or just a part of it) is removed, opened longitud inally and placed to PBS or 10 % PAF fixer. Consequently, trophozoites are detached by vortexing and counted in a cell counting chamber (O'Handley et al . 2001 , Barbosa et al . 2007 ).

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LITERATURE OV ERVIEW

3.3.2 Cryptosporidium spp.

3.3.2.1 Biology, pathogenesis and treatment

Cryptosporidium is a significant agent of diarrhoeal disease in humans, livestock and other animals throughout the world, and a major economic burden to the water industry . It is also a cause of respiratory cryptosporidiosis (Thompson et al . 2005). The parasite was first described in a laboratory mouse by Tyzzer in 1907 but the medical and veterinary significance of this parasite was not fully appreciated for another 70 years (Tzipori & Ward 2002). In humans, C. parvum and C. hominis are responsible for more than 90 % of c ases of cryptosporidiosis (Xiao & Ryan 2004). Furthermore, C. canis , C. meleagridis , C. felis , C. andersoni , C. muris and C. suis have also been isolated from immunocompetent humans (Rossle & Latif 2013). Cryptosporidium was originally considered as one of the coccidian protists based on its coccidian - like features of the life cycle (Levine 1988). However, this genus demonstrates special characteristics that separate it from true coccidia. These involve 1) the attachment of the parasite to the host cell where a feeder organelle is formed at the base of the parasitophorous sac , 2 ) the presence of two types of oocysts: thick - and thin - walled (the latter is responsible for autoinfection, their existence confirmed in several s pecies), 3 ) the small size of the oocyst with lack of morphological structures such as sporocyst, micropyle and polar granules, 4) insensitivity of Cryptosporidium to almost all anticoccidial agents (Thompson et al . 2005). Phylogenetic analysis revealed cr yptosporidia are more closely related to the gregarines than to coccidia (Carreno et al. 1999). Except of their phylogenetic relationship, there are other common characters in their biology, e.g. monoxenous life cycle, possibility of autoinfection (Valigur ová & Koudela 2006) or similar attachment strategy (Valigurová et al. 2007b). Because of few morphological characteristics for species discrimination, Cryptosporidium species used to be described based on host occurrence. This resulted in the description o f a large number of species and taxonomic confusion and controversy (Thompson 2002). Today, we recognise that Cryptosporidium is a phenotypically and genotypically heterogeneous assemblage of largely morphologically identical genotypes and species (Morgan et al . 1999). The application of molecular tools has had an enormous impact on the understanding of the nature of variation in Cryptosporidium (Thompson et al . 2005). Generally, the life cycle of Cryptosporidium spp. (Fig. 2) begins with the ingestion of o ocyst by the host. After excystation, four sporozoites are released in the gut (or stomach in gastric species). Subsequently, they invade epithelial cells and initiate asexual development. After

34

LITERATURE OVERVIEW

Fig. 2: Life - cycle of Cryptosporidium sp. (Drawing m ade by Markéta Fikejzová and Radka Pecková) 35

LITERATURE OVERVIEW internalization , they undergo two successive generations of merogony, releasing eight and four merozoites, respectively. The parasite might undergo multiple rounds of merogony I to produce high numbers of invasiv e merozoites. Type II merozoites give rise to the sexual developmental stages: the micro - and macrogamonts. The release of microgametes and their fusion with macrogametes gives rise to the zygote, which undergoes two asexual divisions and then forms the en vironmentally resistant oocyst containing four sporozoites (Tzipori & Ward 2002). The ability of the parasite to persist inside a single host is assigned to repeated type I merogony and the production of sporulated thin - walled oocysts. These are considered to be the source of autoinfection (Tzipori & Ward 2002). Oocysts are transmitted either directly through contact with infected humans or animals, or indirectly by drinking or eating contaminated water or food. Infection can be induced by ingestion of only 10 oocysts (Okhuysen et al. 1999). In immunocompetent individuals, the infection is usually manifested by an acute, short - term and self - limiting diarrhoea, and its course depends on a combination of host and parasite factors. Parasite forms displace the microvillus border and can lead to the loss of the mature surface epithelium. The rapid loss of surface epithelium causes marked shortening and fusion of the villi and lengthening of the crypts due to acceleration of cell division to compensate for the los s of cells. Consequently, the absorptive intestinal surface is diminished and the uptake of fluids, electrolytes and nutrients from the gut lumen is reduced. The loss of membrane - bound digestive enzymes can also occur, which, particularly in children, cont ributes to marked maldigestion in addition to malabsorption. Diarrhoea lasting 7 - 10 days results in serious dehydration and body weight loss (Tzipori & Ward 2002). Cryptosporidiosis is considered one of the most serious opportunistic infections of AIDS pat ients. Although the prevalence of these patients is not high (5 - 15 % in developed countries), the lack of effective treatment makes this infection the most troublesome among the opportunistic infections associated with AIDS (Blanshard et al. 1992). The onl y antiparasitic drug treatment with proven efficacy for intestinal cryptosporidiosis in immunocompetent individuals approved by the US Food and Drug Administration is nitazoxanide (trade name Alinia) (Carey et al. 2004). However, this treatment is not effe ctive against cryptosporidiosis in immunocompromised patients. Recovery and survival rates in these patients have been dramatically improved with the use of highly active antiretroviral therapy (causing increased CD4+ T - lymphocyte counts), together with th e introduction of protease inhibitors, which seem to directly interfere with the life cycle of the parasite. Also paromomycin, an antibiotic isolated from Streptomyces , can be effective against intestinal

36

LITERATURE OVERVIEW cryptosporidiosis when combined with protease inhib itors (Carey et al. 2004, Hommer et al. 2003). Since there is no drug that achieves the complete removal of Cryptosporidium from the host, supportive therapy is preferred in both human and animals. This consist of replacement of fluid and electrolytes, nut ritional support and anti - diarrhoeal drugs (Zintl et al. 2009).

3.3.2.2 In vitro cultivation

The lack of knowledge regarding the developmental biology of Cryptosporidium and the difficulties in treating patients and animals suffering from cryptosporidiosis have f ocused research towards the development of a reliable in vitro culture model for this parasite. The existence of a standardized and highly reproducible culture system for Cryptosporidium is necessary in several research fields. For example, a standardized culture system is considered to be the best approach to differentiate potentially infectious from non - infectious oocysts isolated from water samples. It also enables detailed studies of the life cycle to determine its affinity to other Apicomplexan parasit es and may also be used as a model for drug screening and disinfection studies (Hijjawi 2010).

The first successful in vitro culture of Cryptosporidium asexual life - cycle stages was described by Woodmansee & Pohlenz (1983) using human rectal tumour (HRT) cells maintained in RPMI 1640 supplemented with 10 % foetal bovine serum (FTS) at 37 °C and

5 % CO 2 . Since then, several laboratories have succeeded in life - cycle development of Cryptosporidium spp. in vitro that led to the formation of oocysts (Current & Long 1983, Datry et al . 1989, Gut et al . 1991, Arrowood et al . 1994). Although partial - to - complete oocyst wall formation was evident in these cultures, significant development of mature oocysts was not observ ed (Arrowood 2008). In vitro cultivation in the absence of host cells has been reported (Hijjawi et al . 2004), but it still remains controversial because of unsuccessful replication of this method by other laboratories (Arrowood 2008).

Life cycle of Crypto sporidium species is relatively complicated and so the in vitro cultivation involves several successive steps making it more difficult. Cryptosporidium oocysts for in vitro cultivation studies primarily origin from clinical human or veterinary stool sample s or from experimentally infected laboratory animals (rodents) or livestock (bovine calves, goat kids). Stool specimens may be collected unpreserved or diluted in tap water, saline, or suspended in a storage medium (2.5 % aqueous potassium dichromate (K 2 Cr 2 O 7 )). Clinically or experimentally derived oocysts are preferentially stored at 4 °C in 2.5 % K 2 Cr 2 O 7 as it

37

LITERATURE OVERVIEW inactivates other microbial contaminants and allows oocysts t o survive for long period (6 - 12 months) (Arrowood 2008).

Stool specimens with excessiv e fat content are often defatted with organic solvents (especially diethyl ether or ethyl acetate) before further processing in order to enhance oocyst recovery and purity (Current 1990). Stool samples are sieved through 20 - and 60 - mesh sieves to remove la rge debris that interferes with subsequent processing techniques. The sieved stool specimens are applied to discontinuous sucrose gradients (Arrowood & Sterling 1987) followed by final oocysts purification using a simple, micro - scale cesium chloride (CsCl) gradient technique (Arrowood & Donaldson 199 6). Alternative oocyst purification methods involve salt flotation techniques, Pecroll® or Ficoll® gradients or cellulose filtration methods (Arrowood 2008).

Successful infection of cell cultures requires excyst ation of vital sporozoites from oocysts. In its simplest form, this can be accomplished by inoculating purified, intact oocysts into host cell cultures because excystation will proceed as oocysts respond to temperature and chemical triggers naturally occur ring in culture media (Arrowood 2008). However, some researchers prefer to use excysted sporozoites for infecting cell cultures ( Mead et al . 1990, Rasmussen et al . 1993, Petry & Harris 1999) . In vitro excystation protocols of C. parvum oocysts imitate host - derived signals: exposure to acid followed by incubation in bile salts, reducing agents and proteases in 37 °C mimic transit through the acidic stomach to the alkaline small intestine (Smith et al. 2005). Results of Melicherová et al. (2016) suggest that adding the suitable concentration (5 - 10 %) of bovine serum albumin to the incubation medium increases the motility of sporozoites of C. muris which seems to be essential for the invasion of host cells. For estimating of sporo zoite viab ility, vital dyes (e. g. fluorogenic dyes such as DAPI) are usually used (Arrowood 2008).

Numerous host cell lines have been tested and advocated for cultivating Cryptosporidium species. In C. parvum , t he most commonly used cell lines are human ileocaecal a denocarcinoma (HCT - 8 ), Madin Darby canine kidney (M D C K) cells and human colonic adenocarcinoma

(Caco - 2) cells. A simple 5 % CO 2 / 95 % air mixture incubator at 37 °C conveniently supports C. parvum growth in these cell lines (Arrowood 2008).

Assessment of the efficacy of any compound against Cryptosporidium in vitro can be achieved by several ways. Traditional way involves fixation of s lides in methanol, staining (e. g. with Giemsa) and counting the cryptosporidial stages using a microscope. This method is t ime - consuming and introduces errors (Armson et al . 2003, Chen & Huang 2012). Other methods

38

LITERATURE OVERVIEW used include an enzyme - linked immunosorbent assay (ELISA) (Woods et al . 1995). The most obvious advantages of this method are increased ac curacy and faster determina tion representing a cost saving. A disadvantage is that it does not necessarily differentiate between living and dead stages (Armson et al . 2003). A similar technique to the ELISA method is the use of fluorescent dyes. This uses fluorescein diacetate and p ropidium iodide which results in red (nonviable) or green (viable) fluorescence (Arrowood et al . 1991). The latest method used for assessment of the efficacy of anti - cryptosporidial agents is a quantitative PCR method (MacDonald et al . 2002). It has the ad vantage of speed and accuracy; a disadvantage is increased number of steps required for isolation of the DNA where some amount of the DNA can be lost. However, when it is performed correctly, this method is more precise than others (Armson et al . 2003).

3.3.2.3 I n vivo experiments

Most of the animal models of various species of Cryptosporidium are used for the evaluation of compounds for inhibitory activities against this parasite. Although several sensitive rodent models have been developed, the majority of resea rchers continue to use either the neonatal mouse or immunosuppressed adult mouse, probably for reasons of availability and cost (Tzipori & Widmer 2008). Cryptosporidiosis has different symptoms in different animal species, and that is the reason why differ ent animal models are often required for different types of scientific studies. For example, new - born calves are used for studying pathogenesis and pathophysiology of the gut, mice for trials concerning specific aspects of the immune response, macaques for studying the chronic disease in the immunodeficient host (Tzipori 1983, O'Donoghue 1995). Because the search for an effective therapy against persistent cryptosporidiosis remains the major objective of some drug development programs, the use of immunocomp romised host is needed. The ideal immunodeficient animal model should require a low - to - moderate infectious dose, allow rapid and consistent establishment of persistent infection in adult animals and generate typical symptoms for cryptosporidiosis. There ar e several reported rodent models used for screening drug efficacy against infection caused by C. parvum but none of them meet all the required criteria. These models include normal neonatal mouse or neonatal SCID mouse, dexamethasone (DEX) immunosuppressed adult mouse, immunosuppressed rat, adult SCID mouse or anti - I FN γ conditioned weaned SCID mouse ( Tzipori & Widmer 2008).

Other animal models include gnotobiotic piglets, immunosuppressed gerbils or SIV - infected rhesus monkeys (Tzipori & Widmer 2008). An advantage of the two former models is that they 39

LITERATURE OVERVIEW can be used for studying infections caused by C. hominis (Akiyoshi et al . 2002, Baishanbo et al. 2005).

Assessment of the efficacy of any compound against cryptosporidiosis in vivo can be pr ovided by oocyst isolation from faecal samples by the sucrose floatation and their counting in a cell counting chamber (Chen & Huang 2012) or by real - time PCR (Higgins et al . 2001).

40

MATERIALS AND METHODS

4 MATERIALS AND METHODS

4.1 Plant extracts

4.1.1 Plant collection

Leaves of tested plants were collected in Sumatra and air dried under shade and then dehydrated by lyophilisation (freeze - drying). The research was compiled in compliance with the legal requirements for research of Indonesia. Research permit was issued by RISTEK Kementarian Riset dan Teknologi . The permission to collect plant samples was obtained from LIPI – Lembaga Ilmu Pengetahuan Indonesia (Indonesian Institute of Sciences) and KKH - Kementerian Kehutanan Direktorat Jenderal Perlindungan Hutan dan Konservasi Alam . Plant identification was done at Botany Department, LIPI – Bogor, Indonesian Academy of Sciences. Tested plants with p romising anti - parasitic activity were selected on the basis of collected behavioural data and ability to decrease parasite load in Sumatran orangutans. As humans and orangutans exhibit phylogenetic similarities (Grehan & Schwartz 2009), we focused on feedi ng behaviour with an emphasis on specific plants consumed that would lead to a reduction in parasite infections. There are only few plant species with potential medicinal value, and analyses show a positive correlation between the prevalence of these plant species in orangutan diets and the presence of parasites (based on the Jaccard index of known frequency in nature) that cannot be explained by their prevalence in the environment (Foitová et al. 2010). Voucher specimens of tested plants have been deposite d at the Department of Parasitology, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta , Indonesia under individual numbers: PL18/02 ( A. fagifolium ), PL18/03 ( D. sumatrana ), PL18/04 ( P. betle ) and PL18/05 ( S. multiflora ) .

4.1.2 Plant extract pre paration

Dried plant material was then homogenized into a fine powder in liquid nitrogen and portions of the ground material (0.33 g) were extracted separately in 10 mL of water, methanol or methanol - tetrahydrofuran (MeOH:THF; 1:1). After 16 - hours of extra ction at - 20 ˚C, the resulting homogenates were centrifuged (26,000 × g, 4 ˚C, 20 min). The sediments were then re - extracted for 1 h in the same way and centrifuged as well. Subsequently , two supernatants

41

MATERIALS AND METHODS were combined and dried in vacuum at 35 ˚C. Then af ter the plant extracts were dissolved in pure DMSO (dimethyl sulfoxide) and diluted with culture medium to obtain required final concentration of DMSO (0.5 - 2 %).

4.2 Giardia intestinalis

4.2.1 In vitro experiments

4.2.1.1 Giardia intestinalis trophozoites

The trophozoites of G. intestinalis WB isolate (ATCC® 30957™) were originally obtained from duodenal aspirate of a 30 year old human male with prolonged symptomatic giardiasis, probably acquired in Afghanistan (Smith et al . 1982), axenized and propagated i n vitro in TYI - S - 33 medium under anaerobic condition at 37 °C (Keister 1983).

4.2.1.2 Modified TYI - S - 33 medium protocol

Stock solution A:

1 l

Casein digest (hydrolysate) 22.3 g

Yeast extract (autolysate) 11.1 g

Glucose 11.1 g

Bile bovine 0.55 g

NaCl 2.22 g

Ascorbic acid 0.22 g

K 2 HPO 4 1.11 g

KH 2 PO 4 0.67 g

Ammonium ferric citrate 0.025 g

Distilled water 890 ml

Solution was filtered through filtration paper, sterilize with membrane filter and then stored at - 20 °C until use d .

42

MATERIALS AND METHODS

Stock solution B:

L - cystein HCl 2 g

Distilled water 100 ml

Solution was sterilized with 0.22 µm syringe filter and stored at - 20 °C until use d .

Stock solution C:

2N NaOH

NaOH 8 g

Distilled water 100 ml

Solution was sterilized with 0.22 µm syringe filter and stored at - 20 °C until use d .

Complete medium:

100 ml

Stock solution A 80 ml

Foetal Bovine Serum 10 ml

Stock solution B 10 ml

Stock solution C 20 drops (300 µl)

Gentamicin 0.3 ml

Antibiotics Antimycotics 1 ml

Solution was prepared under sterile conditions in small volumes and stored at 4 °C.

4.2.1.3 Antigiardial activity assay

In vitro testing against G. intestinalis was performed on each of the target extracts dissolved in DMSO following modified protocols of various laboratories ( Calzada et al . 2006 , Sawangjaroen e t al . 2005). A 12 - well culture plate containing 2 ml of medium (TYI - S - 33) was

43

MATERIALS AND METHODS inoculated with G. intestinalis trophozoites to achieve an inoculum of 25 × 10 3 trophozoites/ml. Afterwards, the extracts were added to the wells to obtain the required range of concentration: 6.25 - 400 µg/ml. The extract of Arabidopsis thaliana , the Eurasian plant routinely used as a laboratory model in research, was used as a negative control. Each test included 100 µg/ml metronidazole (a standard giardicidal drug), a control (cu lture medium plus trophozoites and DMSO) and a blank (culture medium with trophozoites). After incubation for 72 h at 37 ° C in anaerobic conditions, trophozoites were detached by chilling and counted with a cell counting chamber. The criterion used for viab ility of trophozoites was their motility. The results were calculated as the percentage of live trophozoites compared to the controls grown without plant extracts. Subsequently, the half maximal inhibitory concentration (IC 50 ) was calculated. Each concentr ation was tested in triplicate in at least two experiments.

For determining the degree of the antigiardial activity, individual extracts were divided into categories according to Sawangjaroen et al. (2005) as follows: IC 50 < 20 µg/ml = highly active,

20 < IC 50 ≤ 100 µg/ml = active, 100 < IC 50 ≤ 250 µg/ml = moderately active, 250 < IC 50 ≤

500 µg/ml = weekly active, IC 50 ≥ 500 µg/ml = inactive.

4.2.2 In vivo experiments

4.2.2.1 Giardia intestinalis cysts and trophozoites

Giardia intestinalis cysts (human isolate H - 3, pas saged through gerbils) were obtained from Waterborne™, Inc. (New Orleans, LA, USA). Acquiring of trophozoites is described in chapter 4.2.1.1.

4.2.2.2 Laboratory animals

The Mongolian gerbils ( Meriones unguiculatus ) (Institute of Parasitology, Biology Centre, CAS , v.v.i. in České Budějovice), were used in this study. All animals were supplied sterilized diet (TOP - VELAZ Praha, Czech Republic) and sterilized water ad libitum . They were kept separately in plastic cages supplemented with raised bottom grids to avoid r e - infection. The breeding of test animals was regulated by Czech legislation (Act No. 246/1992 Coll., on protection of animals against cruelty). These documents correspond with the legislation of the European Commission. The experimental procedures were ar ranged according to protocols approved by the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences and Institute and National Committees (Protocols No. 52/2014). 44

MATERIALS AND METHODS

4.2.2.3 Parasitation and examination of cyst shedding

Mongolian gerbils ( Merion es unguiculatus ) were inoculated orally using an oesophagus tube either with 0. 5 × 10 6 cysts or 10× 10 6 trophozoites of G. intestinalis per animal . The pattern of cyst shedding was monitored daily in all of the animals. Their faeces were taken three times a day (because of a possibility of intermittent shedding) directly from the bottom of the cages and their consistence was noted. Each day, after the last collection, specimens were weighed. To maintain consistency, the same amount of faeces from these indivi dual three samples/animal/day was placed in 15 ml glass tube. Then 10 ml of distilled water was added to each tube. Faeces were examined the next morning by Sheather's sugar flotation method (Garcia et al . 1983). The number of shed cysts was counted using a cell counting chamber (number of cysts per 1 gram of faeces).

4.2.2.4 Antigiardial activity assay

In vivo antigiardial activity of the extract was tested using a method previously described by Barbosa et al. (2006) with modifications.

Twenty - four Mongolian gerbils (six weeks old) were used for this study. After starting cyst shedding (9 DPI), gerbils were divided into six groups of three and treated with Piper betle extracted in water, methanol or methanol - tetrahydrofuran (1:1), 2 % DMS O as a control, Metrozol and pure PBS. There were also six control groups of one non - infected gerbil treated with the same treatment as the former groups.

Gerbils were treated intragastrically twice a day for nine days with the dose of 40 mg of the extract per 100 g of body weight dissolved in 200 µL of water or PBS. The dose of 3.75 mg of Metrozol (commercial treatment, containing 0.75 mg of metronidazole) used as a positive control was diluted in 200 µL of water and administered once a day. The control gr oup received 2 % DMSO solution in PBS twice a day as well as the group without treatment that received just PBS solution.

Tenth day after the start of treatment, gerbils were sacrificed by cervical dislocation and dissected. Their duodena were then process ed for examination using histological sectioning and scanning with electron micro scopy. These methods were perform ed to see the visual aspect of the tissues as well as to get an approximate picture about the number of trophozoites in the

45

MATERIALS AND METHODS intestine. For thi s purpose, five millimetres of duodenal tissue were taken for SEM, the rest for histology. The histology sections were made for each five millimetres of the tissue.

The amount of released cysts is positively related to the number of trophozoites located in small intestine (Belosevic et al . 1983). Because of this fact, the evaluation of the impact of plant extracts was based on the course of cyst shedding.

4.2.2.5 Time course of parasitation

In this study, nineteen Mongolian gerbils were used (15 inocula ted with gi ardia trophozoites, four as a negative control). Six gerbils were used to examine the course of the infection during the experiment; two animals (male and female) were sacrificed by cervical dislocation at four to five - day intervals (9, 14, 18 DPI) and the ir small intestines were processed for next examination using histological sectioning. The remaining nine gerbils were euthanized and dissected at the end of the experiment, i. e. 23 DPI. Their small intestines were examined for the presence of G. intestin alis trophozoites by three different methods: three were processed for histological sectioning, three for scan electron microscopy and in last three, the numbers of trophozoites in the small intestine were counted following the protocol of O'Handley et al . (2001). In short, intestinal segments were removed from the duodenum, proximal and distal jejunum and ileum. They were split longitudinally and placed in a tube containing 5 ml of PBS. Segments were incubated for 30 min at 37 °C and then the numbers of tr ophozoites were counted in a cell counting chamber at 400 × magnification on a n Olympus BX51 light microscope. After euthanasia, gerbils' blood was taken and sent to a specialized laboratory for complete blood count examination. Intestinal tissues determine d for histological procedure and scanning electron microscopy were rinsed by PBS and the numbers of trophozoites present in these fluids were counted using a cell counting chamber . Histological sections were made from each 0.2 cm of small intestine . The bl ocks were cut using Microm HM360 automated rotary microtome and sections were examined using an Olympus CX41 light microscope at 400 × magnification. The number of trophozoites was divided to semiquantitative categories ranging from 0 to 5 (0 – no trophozoi tes, 5 – high abundance of trophozoites).

46

MATERIALS AND METHODS

4.2.2.6 Histological procedure

Histological procedure was made according to a protocol described in Valigurová (2007 a ).

1. Fixation

Pieces of host tissues (intestines) were removed, rinsed by PBS and stretched by pins to a cardboard (to avoid rolling up of the tissue). Tissues were straightened by prefixation and then put to a vial with AFA (Alcohol - Formalin - Acetic Acid) fixative:

 Formalin (40 % aqueous formaldehyde): 60 ml  95 % ethanol: 500 ml  Glacial acetic acid: 40 ml  Distilled water: 400 ml

The volume of fixative should be at least 10 times the volume of the tissue, fixation should take at least overnight.

2. Dehydration

The tissues were moved to embedding cassettes and dehydrated through a graded alcohol series:

 70 % ethanol: 60 min  80 % ethanol: 2 × 45 min  90 % ethanol: 2 × 45 min  100 % ethanol: 3 × 30 min

3. Clearing in xylene

 100 % ethanol : xylene (1:1) : 2 × 10 min  Xylene 1: 10 min  Xylene 2: 40 min

4. Paraffin infiltration

In a thermost at at 56.7 °C:

 Paraffin bath 1: 1 h  Paraffin bath 2: overnight  Paraffin bath 3: overnight

47

MATERIALS AND METHODS

5. Embedding in fresh paraffin

The tissues were embedded into containers and poured with fresh paraffin using Leica EG1150 Modular Tissue Embedding Center. Consequently, containers were cooled rapidly. After paraffin hardening, blocks were trimmed and mounted onto a wooden holder.

6. Sectioning

Sections were cut on a motorized rotatory microtome to get 7 µm thick serial sections. Microscope slides with sectio ns were dried in the incubator at 40 °C for one week.

7. Staining with haematoxylin - eosin

a. Deparaffinization and rehydration:  Xylene 1: 5 min  Xylene 2: 5 min  100 % ethanol: 5 min  96 % ethanol: 5 min  distilled water: 5 min b. Staining in Ehrlich´s haematoxylin for 10 min. c. Differentiation in acid alcohol (5 drops of hydrochloric acid in 100 ml of 96 % ethanol). d. Washing in running tap water for 30 min. e. Staining with 0.1 % aqueous eosin for 1 - 5 min. f. Dehydration:  96 % ethanol: 3 min  100 % ethanol: 5 m in  Carbol - xylene (1:3): 5 min g. Clearing in xylene : 2 × 5 min. h. Mounting in Canada or Damar balsam and drying at 40 °C.

4.2.2.7 Statistical evaluation

Antigiardial activity assay

Trends in course of cyst shedding during the entire treatment trials were computed as linear regression within each group. There were considerable differences in numbers of shed cysts in single gerbils during the treatment period and therefore these values were standardized.

48

MATERIALS AND METHODS

Standardization was performed as observed value divided by obse rved value on day 0. The statistical evaluation was carried out in statistical package R (version 3.2.3).

Time course of parasitation

For comparison of male and female cyst shedding and weight, Mann - Whitney U test was used. Correlations between other vari ables were counted using Pearson correlations.

4.3 Cryptosporidium spp.

4.3.1 In vitro excystation

4.3.1.1 Cryptosporidium parvum oocysts

C. parvum oocysts were obtained from the faeces of naturally infected bovine calves. All of the experimental procedures were conducted in accordance with applicable laws of the Czech Republic on the use of experimental animals and the safe use of pathogenic agents. The study was conducted under protocol approved by the Institute of Parasitology, Biology Centre of the Czech Academy of Scie nces and Central Commission for Animal Welfare, Czech Republic (protocol no. 073/2010).

4.3.1.2 Stool collection and purification of oocysts prior to excystation test

After collection, faeces were stored unpreserved at 4 °C before use. The stool sample was defatt ed with organic solvent diethyl ether before further processing for a higher quality of oocyst purity (ratio of stool to solvent 2:1). This stool was passed through a sieve (standard tea sieve) and processed using the microscale caesium chloride (CsCl) gra dient technique (Arrowood & Donaldson 1996) as follows: the sieved stool sample was diluted with phosphate - buffered saline (PBS ) and passed through a finer, nylon sieve (40 µm mesh size). The sieved sample was centrifuged (800× g for 20 min at 4 °C), super natant discarded, pellet resuspended in PBS repeatedly, until the supernatant was clear. The sediment was resuspended in a small amount of PBS to get a liquid suspension. RTA tubes (2.0 ml) were filled with 1 ml of caesium chloride suspension (21.07 g of C sCl in 100 ml of deionized H 2 O) and 500 µl of liquid sieved stool solution was carefully layered over the CsCl solution. Tubes were centrifuged at 12 ,100 × g for 3 min at 20 °C. Oocysts were collected from the middle layer, transferred to microcentrifuge tubes, dilute d with PBS and centrifuged (750 × g for 3 min at 20 °C) three times

49

MATERIALS AND METHODS so that CsCl was washed out. Pellets from all tubes were pooled , centrifuged again and stored at 4°C.

4.3.1.3 Test of viability

Viability of the oocysts was tested with propidium iodi de staining: 10 µl of the oocyst suspension was diluted with 100 µl of PBS and 30 µl of stock solution of propidium iodide was added. This suspension was incubated for 3 min at room temperature in the dark and then examined using a n Olympus IX70 fluorescen ce microscope with an excitation wavelength 535 nm.

4.3.1.4 Excystation methods

In each of the following six methods, three 1.5 ml microcentrifuge tubes were used; each contained 0.5 × 10 6 oocysts in 1.5 ml of PBS .

1. Method by Rasmussen et al . (1993) Oocysts we re washed three times in PBS (centrifugation at 12 ,100 × g for 3 min). McCoy´s medium, used in the original paper, was replaced with PBS. Oocysts were then suspended in 2 % sodium hypochlorite and incubated for 10 min at room temperature . After that, oocyst s were washed five times in PBS, suspended in an excystation medium consisting of 0.75 % taurocholic acid (sodium taurocholate, NaT) and 0.25 % trypsin in PBS and incubated at 37 °C for 45 min.

2. Method by Petry & Harris (1999) Oocysts were centrifuged (12 ,100 × g for 3 min), supernatant discarded and the pellet with oocysts was re - suspended in 1:20 diluted commercial bleach (0.25 % sodium hypochlorite) and incubated for 10 min on ice. Afterwards, oocysts were washed three times in water (12 ,100 × g for 3 min), suspended in excystation solution (0.75 % sodium taurocholate in RPMI 1640 medium) and incubated at 37 °C for 2 h.

3. Method by Mead et al . (1990) Oocysts were centrifuged (12 , 100× g for 3 min) in order to remove PBS, then suspended in 1 ml of 0.5 % sodium hypochlorite, incubated for 5 min at 4 °C and centrifuged (6 , 800× g for 1 min). The oocyst sediment was neutralized by suspending in 0.1 % sodium thiosulfate (dilu ted 50

MATERIALS AND METHODS in PBS), centrifuged (2,450 × g for 3 min) and washed twice with PBS. Subsequently , oocyst suspensions were suspended in excystation solution (consisting of 0.75 % sodium taurocholate and 0.25 % trypsin diluted in PBS) and incubated for 1 h at 37 °C. Afterwards, the oocyst/sporozoite suspensions were washed twice in PBS.

4. Method by B lack et al . (1996) Oocysts were suspended in 500 µl of PBS, added to 500 µl of excystation medium (150 mg of sodium taurocholate and 50 mg of trypsin dissolved in 5 ml PBS) and vortexed. Oocysts were then incubated at 37 °C for 2 h and afterwards at room t emperature for 30 min. Suspensions were centrifuged (9 ,700 × g for 5 min) and re - suspended in PBS.

5. Method by Gut & Nelson (1999) Oocysts were centrifuged (12 ,100 × g for 3 min), resuspended in 10mM HCl and incubated for 10 min at 37 °C. Afterwards, oocys ts were centrifuged again (12,100 × g for 3 min), resuspended in RPMI 1640 medium (pre - warmed to 37 °C) with 0.8 % sodium taurocholate and incubated for 10 min at 37 °C. Subsequently, s uspensions were centrifuged (12,100 × g for 3 min) and resuspended in PBS .

6. Method by Rennecker et al . (1999) Oocysts were added to centrifuge tubes containing 2 ml of excystation medium (1.5 % sodium taurocholate and 0.5 % trypsin in PBS; pre - warmed to 37 °C). The tubes were then vortexed and incubated at 37 °C for 1 h. Af ter that, the tubes we re centrifuged (6 800 × g for 3 min) and resuspended in PBS.

4.3.1.5 Calculation of released sporozoites and statistical evaluation

All the tubes were then centrifuged (12 ,100 × g for 3 min) and the supernatant volume was reduced to 0.5 ml. T he samples were assessed immediately after the excystation treatments. A cell counting chamber was used to determine the excystation rates by counting the percentage of empty oocysts. Oocysts which had not lost their contents and appeared intact were evalu ated as not - excysted, while those that had released at least one sporozoite were considered to be excysted. The percentage of non - viable oocysts, determined in an earlier viability test, was excluded. The numbers of empty oocysts were counted using an Olym pus BX51 light

51

MATERIALS AND METHODS microscope at 400 × magnification. The viability of released sporozoites was assessed morphologically using light microscopy: motile sporozoites were considered viable. One - way ANOVA was used for statistical analysis (STATISTICA 12 software).

4.3.2 In vivo experiments

4.3.2.1 Cryptosporidium proliferans oocysts

Oocysts of the gastric species C. proliferans originated from naturally infected East African mole rat ( Tachyoryctes splendens ) and was kept in SCID mice and southern multimammate mice ( Mastomys coucha ) under laboratory conditions.

4.3.2.2 Laboratory animals

Eight - week old southern multimammate mice ( Institute of Parasitology, Biology Centre, CAS, v.v.i. in České Budějovice), were used in this study. All animals were supplied with sterilized diet (TOP - VE LAZ Praha, Czech Republic) and sterilized water ad libitum . Each group was kept separately in plastic cages. The breeding of test animals was regulated by Czech legislation (Act No. 246/1992 Coll., on protection of animals against cruelty). These documents correspond with the legislation of the European Commission. The experimental procedures were arranged according to protocols approved by the Institute of Parasitology, Biology Centre of the Czech Academy of Sciences and Institute an d National Committees ( Protocol No. 52/2014).

4.3.2.3 Parasiti sation and examination of oocyst shedding

Oocysts used for experimental inoculation were collected from faeces and purified by Sheater 's sugar flotation method (Garcia et al. 1983) and caesium chloride gradient centrifugation ( Arrowood & Donaldson 1996 ). Mice were inoculated orally using an oesophagus tube with 10 6 viable oocysts of C. proliferans . Their faeces were taken each morning and examined microscopically for the presence of ooc ysts using staining method according to Miláček & Vítovec (1985). The intensity of oocyst shedding was evaluated as the number of oocyst per gram of faeces (OPG).

52

MATERIALS AND METHODS

4.3.2.4 Treatment of cryptosporidiosis using plant extracts

Experiment I

M ice were treated daily for 14 days, initiating two months post inoculation, with the dose of 12.5 mg of the extract per 100 g of body weight administered per os . Extracts used for this trial were either Piper betle , Diospyros sumatrana or Arabidopsis thaliana (negative control) extracted in methanol, dissolved in DMSO and diluted in sterile water to obtain a final concentration of 0.5 % DMSO. Halocur (commercial drug used for treatment of cryptosporidiosis in calves) and 0.5 % DMSO were used as controls.

Experiment II

Second exp eriment was performed after the evaluation of th e first one. Mice were treated twice a day for 21 days, initiating three months post inoculation, with the dose of 40 mg of the extract per 100 g of body weight. Extracts used for this experiment were either Piper betle , Diospyros sumatrana or Arabidopsis thaliana extracted in three extra ction media: methanol, methanol - tetrahydrofuran and sterile water. Extracts were then dissolved in DMSO and diluted in sterile water to obtain a final concentration of 0.5 % DMSO. Halocur and 0.5 % DMSO were used as controls.

At the end of both experiments, mice were sacrificed by cervical dislocation and dissected. Their stomachs were then processed for examination using histological sectioning and scanning electron microsco py.

53

SUMMARY OF RESULTS

5 SUMMARY OF RESULTS

This chapter is divided into two main parts according to the studied parasite; papers in subchapters are sorted chronologically. Complete results of the studies are presented in attac hed Papers I - V.

5.1 Giardia intestinalis

Paper I

Pecková, R., Doležal, K., Sak, B., Květoňová, D., Kváč, M., Nurcahyo, W. & Foitová, I. (2016). Effect of Piper betle on Giardia intestinalis infection in vivo . Experimental Parasitology 184:39 - 45.

Data of cyst shedding from 18 gerbils divided into six groups of three was evaluated by linear regression. Gerbils treated with the extract dissolved in MeOH or MeOH:THF showed a slight decline in the number of shed cysts but this result was not statistically sign ificant (there was no decrease in the number of cysts excreted from day 1 to day 9). Significant decline in cyst shedding was found in gerbils treated with the extract dissolved in water. Surprisingly, there was also a decline in cyst shedding in gerbils t reated with pure DMSO but it was not statistically significant. On some of the histological sections, there were apparent villus atrophies characteristic for giardiasis, especially in case of negative controls with high number of present trophozoites. Howe ver, the number of trophozoites was not correlated with the presence of villus atrophy.

54

SUMMARY OF RESULTS

Paper II

Pecková, R., Sak, B., Květoňová, D., Kváč, M., Koriťáková, E. & Foitová, I. (2018). The course of experimental giardiasis in Mongolian gerbil. Parasitology Research 117(8):2437 - 2443.

Gerbils started to shed cysts on 7 or 8 DPI. Number of shed cysts varied considerably among days and single individuals from 0 to 18 × 10 6 cysts per gram of faeces. There were differences in cyst shedding in males comparing to f emales. Levels of consistency of faeces were divided into three groups (1 – normal, 2 – soft, 3 – diarrhoeal). In three male gerbils, diarrhoea occurred in a few days during infection, in others the consistency of faeces was normal. Statistical comparison of these values with the number of shed cysts showed no significant correlation between these variables. Infected gerbils had lower body weight gain in comparison with control group. Most values of complete blood count fell into standard range although the re were some exceptions. First of them was values of mean corpuscular haemoglobin concentration (MCHC) which should be in the range of 30.6 - 33.3 %. In our case, the range of values was 25.9 - 30.9 %. The range of eosinophils in blood is standardly 0 - 4 %, how ever in one gerbil this value reached 9 %. In both gerbils euthanized on 14 DPI, the whole small intestine was filled with mucus. According to histological screening, the distribution of trophozoites in the small intestine varied in different periods of th e infection. In general, the highest abundance of trophozoites in all periods was found in the duodenum whilst the lowest one was found in the ileum. The highest number of trophozoites at the total was observed in the duodenum in gerbils sacrificed on 14 D PI. After this day, number of trophozoites decreased in all the parts of the intestine. Number of shed cysts was positively correlated with number of trophozoites rinsed from the intestine but there was no correlation between number of shed cysts and troph ozoites on histological sections. Also, with an increasing number of shed cysts, mean corpuscular haemoglobin concentration (MCHC) significantly decreased. Values of other physiological parameters (complete blood count, sex, body weight, consistency of fae ces) did not correlate with the intensity of infection (expressed by number of trophozoites in the small intestine or number of shed cysts).

55

SUMMARY OF RESULTS

Paper III

Pecková, R., Doležal, K., Sak, B., Květoňová, D., Kváč, M., Nurcahyo, W. & Foitová, I. (2018 ). Effect o f Indonesian plants on Giardia intestinalis infection in vitro . Submitted to Experimental Parasitology .

Four Indonesian plant extracts in three different solvents were used in this study. The most effective plant extract against G. intestinalis trophozoites was the aqueous extract of

A. fagifolium (IC 50 = 5.63 µg/ml), followed by aqueous extract of P. betle (IC 50 = 8.63 µg/ml).

In addition, the methanolic extract of A. fagifolium showed a strong effect on trophozoites (IC 50 = 40.14 µg/ml). The e xtract of A. thaliana , used as a negative control, had no effect on trophozoites as we expected, whilst metronidazole killed almost all of them.

5.2 Cryptosporidium spp.

Paper IV

Pecková, R., Stuart, P. D., Sak, B., Květoňová, D., Kváč, M. & Foitová, I. (20 16). Statistical comparison of excystation methods in Cryptosporidium parvum oocysts. Veterinary Parasitology 230:1 - 5.

In this study, six excystation methods were compared. All of the released sporozoites were motile, so they were considered to be viable. The most efficient excystation method evaluated was that described by Rasmussen et al . (1993) (92.5 %) followed by Petry & Harris (1999) (85.7 %) and Mead et al . (1990) (83.7 %). Lower excystation rates were obtained by using excystation method described by Black et al . (1996) (70.7 %) and the lowest ones by Gut & Nelson (1999) (58.5 %) an d Rennecker et al . (1999) (55.1 %). There were significant differences in percentage of excysted oocysts among groups excysted by different methods. Analysis of contrasts in planned comparisons of least squares means showed significant differences between methods using pre - incubation with sodium hypochlorite and those without this pre - incubation (the former were significantly more efficient). Significantly higher excystation rates were observed in methods using sodium hypochlorite pre - incubation compared to pre - incubation with hydrochloric acid ( HCl ) . HCl pre - incubation did not differ from no pre - incubation with HCl. Among the other variables tested (the use of trypsin, 56

SUMMARY OF RESULTS excystation medium, and incubation time), none showed statistically significant differenc es between groups.

Paper V Valigurová, A., Pecková, R., Doležal, K., Sak, B., Květoňová, D., Kváč, M., Nurcahyo, W. & Foitová, I. (2018). Limitations in the screening of potentially anti - cryptosporidial agents using laboratory rodents with gastric cryptos poridiosis. Folia Parasitologica , in press.

The variations in oocyst shedding were generally comparable in all experimental groups treated with either plant extracts or Halocur. A decline in oocyst number in Experiment I can be observed in groups treated for 14 days with D. sumatrana extracted in methanol and Halocur. In contrast, non - treated controls, controls treated with pure DMSO and animals treated with P. betle and A. thaliana extracted in methanol exhibited increased oocyst shedding. In case of Expe riment II, a decline in oocyst shedding was observed in groups treated for 21 days with A. thaliana extracted in MeOH:THF, D. sumatrana in MeOH:THF and Halocur. An increase occurred in non - treated controls and mice treated with pure DMSO, P. betle extracte d in all three solvent media, A. thaliana extracted in methanol and water and D. sumatrana in water. Despite an obvious decline in oocyst shedding in some animals, histological examination revealed heavy cryptosporidiosis in all non - treated as well as trea ted animals. Comparison of results in these two groups did not reveal significant difference in parasitation intensity or in associated pathological changes to the gastric mucosa in animals treated with Indonesian plant extract. Surprisingly, the control a pplication of A. thaliana with expected neutral expected effect, seemed to have a positive impact on gastric mucosa (exhibiting less intense p athological folding) , particularly when extracted in MeOH:THF . Halocur was shown to be ineffective treatment of gastric cryptosporidiosis in mice caused by C. proliferans , except for better preservation of epithelial cells lining the glands in treated mice.

57

DISCUSSION

6 DISCUSSION

Us ing plants as a treatment against various parasitic diseases has very long tradition dating back to prehistory (Cowan 1999). Until the middle of the twentieth century, the principal approach to drug discovery was based on empirical evidence from the natura l world (Trosset & Carbonell 2015). After that, pharmacological research gave preference to simpler chemistry and herbal drugs were sidelined (Hu & Bajorath 2014). Although therapy with pharmaceutical drugs is effective, they can produce significant undesi rable side effects (Xiao et al. 1996) and resistance to the treatment of a broad range of parasites (protozoa, helminths and arthropods) has been described (Upcroft et al . 1990, Sangster et al. 2002). Since the late 1990s, the use of plant extracts, as wel l as other alternative forms of medicinal treatments, has become popular again (Cowan 1999). The aim of this thesis was to test leaf extracts of selected plants from Indonesia. Tested plants with promising anti - parasitic activity were selected on the basis of collected behavioural data and ability to decrease parasite load in Sumatran orangutans. The thesis also contains two methodological studies. The first one deals with the comparison of excystation methods in oocysts of Cryptosporidium parvum , the second one concerns the course of experimental giardiasis in Mongolian gerbils.

6.1 Giardia intestinalis Possible anti - giardial activity was tested using both in vitro and in vivo models. In in vitro experiment, extracts of four plants ( Archidendron fagifoliu m , Shorea multiflora , Piper betle and Diospyros sumatrana ) in three solvents (water, methanol and methanol - tetrahydrofuran) were used. The most effective plant against G. intestinalis trophozoites was A. fagifolium . Its aqueous extract was determined as hi ghly active and the methanolic extract as active. This plant has not yet been tested for its biological activity. Nevertheless, another species of this genus, A. clypearia was found to contain xanthine oxidase inhibitors (Nguyen et al. 2017) and it is also used as a treatment for various inflammatory diseases in traditional medicine (Woo et al. 2013). Another studies, particularly on animal hosts, are highly recommendable.

This experiment also revealed high activity of the aqueous extract of P. betle agains t G. intestinalis trophozoites. The aqueous extract of P. betle was also shown to have anti - giardial activity in our study on Mongolian gerbils. In this study, we evaluated the impact of P. betle in

58

DISCUSSION three solvents on the course of giardiasis in gerbils bas ed on the intensity of cyst shedding. A significant decline of cyst shedding was observed just in the group of gerbils treated with the aqueous extract. This match in in vitro and in vivo assays represents an important outcome because in general, most of t he antiparasitic properties of extracts and isolated natural products have been tested in vitro only and confirmation of the in vitro research results in animal models is indispensable (Cos et al. 2006, Wink 2012) since the results of in vitro studies are not always verified by in vivo experimentation (Athanasiadou & Kyriazakis 2004).

Piper betle has been already studied for its biological and pharmacological effects. Its aqueous extracts have shown to have anti - adherence effects on early plaque settlers (R azak & Rahim 2003), vasodilatory activity (Runnie et al. 2004) and the hepatoprotective and antioxidant effects (Saravanan et al. 2002, 2003). Nevertheless, there is poor evidence of the impact of this plant on G. intestinalis . A study by Sawangjaroen et al. (20 05 ) found giardicidal activity from the chloroform extract of P. betle . On the other hand, antigiardial activity from aqueous extract was not observed. This might be a result of a different method of plant extraction or different origin of plant (se ason and/or region). The fact that the chemical composition of various plant compounds is affected by region and climatic conditions under which plant species were growing was already reported (Gessler et al. 1995). Betel leaf contains a broad range of che mical compounds including alkaloids, carbohydrates, amino acids, tannins and steroids (Sugumaran et al. 2011). However, the active compounds responsible for activity against intestinal microorganisms remain unclear and need to be investigated (Sawangjaroen et al. 2005 ).

In future planning of in vivo assays concerning testing of giardicidal effect of plant extracts, results obtained in our study dealing with the course of experimental infection in Mongolian gerbils could be very helpful. This study was intended to find out the precise distribution of G. intestinalis trophozoites in small intestine in the course of infection and to compare these values with other physiological parameters (complete blood count , sex, body weight, consistency of faeces, trophozoite number in small intestine or number of shed cysts).

According to histological screening, the highest number of Giardia trophozoites in all periods of the infection was found in the duodenum followed by the proximal jejunum, whilst t he lowest one was found in the ileum. This finding is consistent with observation of Koudela & Vítovec (1998) who made similar experiment in goat kids. On the contrary, O'Handley et al . (2001) found the proximal and distal jejunum to be the site of maximum trophozoite numbers

59

DISCUSSION in dairy calves (third week post infection). These controversial results could be caused by different host used in these experiments.

Evaluation of histological sections revealed that the highest abundance of trophozoites in the small intestine was observed on 14 DPI, followed by 9 DPI; the abundance of trophozoites on days 18 and 23 post infection was very low. On the other hand, the number of trophozoites rinsed from the lumen of the intestine was gradually increasing with the peak on 18 DPI. Together with findings of high amount of mucus in the small intestine on 14 DPI these results indicate relocation of trophozoites from the surface of villi and crypts to the lumen of the intestine as a result of increased mucus secretion (Buret et al . 1990).

There was a different pattern in cyst shedding in males comparing to females. At the beginning, females excreted more cysts than males, while at the end of the experiment, this ratio was the opposite. The course of giardiasis in males and femal es may vary in different animal species. Oberhuber & Stolte (1990) found it equally distributed among male and female human patients. Experimental studies in mice by Roberts - Thomson et al. (1980) revealed lower levels of infection in female than in male mi ce. This effect can be related to sex hormone levels or reflect the influence of loci associated with sex chromosomes (Eidinger & Garrett 1972, Roberts - Thomson et al. 1980).

In three male gerbils, diarrhoea occurred in a few days during infection, in other s the consistency of faeces was normal. Comparable results were obtained by Koudela & Vítovec (1998) who observed three of eight infected goat kids to have diarrhoea. Similarly, no association between bouts of diarrhoea and peaks of giardia cysts shedding was found in dogs (Horejs & Koudela 1994).

The assessment of blood picture values revealed that values of MCHC were decreasing with an increasing number of shed cysts and these values themselves were lower than standard ones. This may suggest iron deficien cy which is often presented in giardiasis (De Vizia et al. 1985, Monajemzadeh & Monajemzadeh 2008, Weiss 2010). Parasite infections are associated with an increased number of eosinophils in blood (Young & Meadows 2010). In our study, only one male gerbil h ad an increased value of eosinophils.

To conclude, for future planning of experiments, I would recommend to use both males and females and evaluate them separately, as they have different pattern of cyst shedding and show different clinical signs (diarrhoe a and increased number of eosinophils was found only in males, although not in all of them). Comparison of these findings could bring very interesting results. Concerning the optimal length of the experiments focussing on plant extract testing, I find the

60

DISCUSSION most appropriate to finish the experiment in a period between 14 and 18 days post inoculation. This is a time of the peak in the infection (assessed by histological screening and counting of trophozoites from the lumen of the small intestine) after which t he infection is naturally eliminated by the host's immune system.

6.2 Cryptosporidium spp. Primary intention for testing potential anti - cryptosporidial activit y of selected plant extracts was to use in vitro methods (preliminary screening) followed by in vivo ones as cell culture infectivity experiments have several advantages over mouse infectivity experiments. The former ones are considered to be more precise, faster, easier to perform and less genetically variable. Moreover, they are cheaper, less material demanding and not ethically controversial (Rasmussen et al. 1993, O'Donoghue 1995, Shin et al . 2001, Smith et al ., 2005). Inoculation of cell cultures is preceded by several successive steps including oocyst excystation and release of motile sporozoites, l ocalization of potential host cell , attachment to and invasion (Smith et al. 2005). Considering that excystation of sporozoites from oocysts is essential for successful in vitro assays and also for further research and we decided to perform an experiment to compare various protocols for in vitro excystation of Cryptosporidium parvum oocysts to find the most effective one. Those protocols differed in pre - incubation steps, excystat ion media or incubation time. The most efficient excystation protocols investigated in this study were those described by Rasmussen et al. (1993), Petry & Harris (1999) and Mead et al. (1990). Common feature of all these methods is that sodium hypochlorite is used as a pre - incubation step unlike the remaining, less efficient methods. This confirms results of Woodmansee (1987), that bleaching increases the excystation rate. This is probably because sodium hypochlorite treatment results in thinning and perfor ation of the outer and inner layers of the oocyst wall. At the same time it possibly exposes receptors located in different levels in the oocyst wall, which require acid and bile stimulation before they become exposed in their host (Smith et al. 2005). On the other hand, we did not find any influence of the type of excystation medium, the incubation time or the presence of trypsin on the excystation ratio.

Next steps after the oocyst excystation included inoculation of HCT - 8 cells in RPMI - 1640 medium (suppl emented with foetal bovine serum , antibiotics, antimycotics , Gentamicin and non - essential amino acids) by released sporozoites and incubation at 37 °C and 5 % CO 2 . Although some laboratories find this method efficient for testing of potential anti - cryptosp oridial agents (Shahiduzzaman et al. 2009, Wu et al. 2011), we have failed to develop

61

DISCUSSION a successful model for in vitro cultivation of C. parvum (invasion of sporozoites into cells). Because of this failure, we moved from in vitro methods to in vivo ones.

Fo r testing of potential anti - cryptosporidial activity of selected plant extracts in vivo , southern multimammate mouse ( Mastomys coucha ) experimentally inoculated with gastric species Cryptosporidium proliferans was used. Although some plant extracts caused a decline in oocyst shedding ( Diospyros sumatrana extracted in methanol in Experiment I, Arabidopsis thaliana extracted in MeOH:THF, D. sumatrana in MeOH:THF in Experiment II and Halocur in both experiments), histological examination revealed heavy gastric cryptosporidiosis in all non - treated as well as treated animals. Surprisingly, the extract of A. thaliana , which was used as a negative control, had a positive impact on the gastric mucosa pathologically altered by chronic cryptosporidiosis. This plant is used as a model organism in molecular biology research but except a study of Mynářová (2015) documenting a positive effect of A. thaliana extrac t on the reduction of spores of microsporidian Encephalotozoon cuniculi in the tissues of experimentally inoculated BALB/c mice, antimicrobial activity of secondary metabolites of A. thaliana against human pathogens has not been well examined (Romeo et al. 2018).

Even though a decline in oocyst shedding may be considered as a partial success because of a reduction of potential environmental contamination (McAllister et al. 2001), the aim of such studies is to find a drug that would be able to eliminate the parasite from the host at all. Because even Halocur which is standardly used as a treatment for intestinal cryptosporidiosis, was found to be ineffective against gastric cryptosporidiosis caused by C. proliferans , the best way for the testing of potential anti - cryptosporidial agents in vivo seems to be the use of different host - parasite model (e.g. C. parvum – SCID mouse/neonatal mouse).

6.3 Future perspectives with regard to biologically active compounds from plant extracts Plant products used in studies form ing this dissertation were in form of rough extracts. Proving of biological activity of these rough extracts is the first step for the discovery of compounds responsible for biological activity of the plant. If the biological activity is demonstrated only in extracts extracted in a certain type of extracting agent, the type of this agent may indicate the type of organic substances responsible for the biological activity of the plant. Screening of the biological activity of extracts is followed by purificati on by solid phase extraction (SPE) (Madlener et al. 2009). During this process, compounds that are dissolved or 62

DISCUSSION suspended in a liquid mixture are separated from other compounds in the mixture according to their physical and chemical properties. It is commo nly used to concentrate and purify samples for next analyses (Hennion 1999). Fractions obtained by SPE are subsequently tested for their potential biological activity. If it is proved, those fractions are further purified by a preparative high performance liquid chromatography (HPLC) and obtained fractions are tested for their potential biological activity again. Fractions with proven biological activity are then examined by mass spectrometry and/or nuclear magnetic resonance that may clarify the structure of the active compound (Madlener et al. 2009).

63

CONCLUSIONS

7 CONCLUSIONS

In this chapter, the main results of this thesis are summarized.

7.1 Gi ardia intestinalis  The aqueous extracts of Archidendron fagifolium and Piper betle were found to be effective against G. intestinalis trophozoites obtained by in vitro assay. They were

evaluated as highly active (IC 50 < 20 µg/ml). Methanolic extract of A. fagifolium was

determined as active (20 < IC 50 ≤ 100 µg/ml).  The aqueous extract of Piper betle caused a significant decline in cyst shedding in Mongolian gerbils experimentally inoculated with G. intestinalis .  In the study dealing with the course of experimental giardiasis in Mongolian gerbils, we have reached the following conclusion: o The highest number of trophozoites in all periods of the parasitation was found in the duodenum followed by the proximal jejunum, whilst the lowest one was found in the ileum. o The highest abundance of trophozoites in the small intesti ne was observed on the 14 th day post inoculation (DPI), followed by 9 DPI; the abundance of trophozoites on days 18 and 23 post - inoculation was very low. o The number of trophozoites rinsed from the lumen of the intestine was gradually increasing with the pe ak on 18 DPI. o There were differences in cyst shedding in males comparing to females. At the beginning, females excreted more cysts than males, while at the end of the experiment, this ratio was the opposite. o Gerbils with giardiasis had lower body weight in comparison with control groups (12.1 % decrease in males, 9.5 % in females). o Values of MCHC (mean corpuscular haemoglobin concentration) were decreasing with an increasing number of shed cysts and these values themselves were lower than standard ones, whi ch may suggest iron deficiency, often presented in giardiasis.

64

CONCLUSIONS

7.2 Cryptosporidium spp.  Comparison of different excystation methods in Cryptosporidium parvum oocysts showed that those pre - incubation steps which included sodium hypochlorite increased the excys tation ratio.  In the study concerning anti - cryptosporidial activity of selected plant extracts in vivo ( C. proliferans – southern multimammate mouse), the following results were obtained: o A decline in oocyst number in Experiment I can be observed in groups treated for 14 days with Diospyros sumatrana extracted in methanol and Halocur . o In case of Experiment II, a decline in oocyst shedding was observed in groups treated for 21 days with Arabidopsis thaliana extracted in MeOH:THF, D. sumatrana in MeOH:THF and Halocur. o Despite an obvious decline in oocyst shedding in some animals, histological examination revealed heavy cryptosporidiosis in all non - treated as well as treated animals. Comparison of results in these two groups did not reveal significant differen ce in parasitation intensity or in associated pathological changes to the gastric mucosa in animals treated with Indonesian plant extract s . o Surprisingly, the control application of A. thaliana with expected neutral expected effect, seemed to have a positi ve impact on gastric mucosa (exhibiting less intense pathological folding) , particularly when extracted in MeOH:THF. o Halocur was shown to be ineffective treatment of gastric cryptosporidiosis in mice caused by C. proliferans , except for better preservatio n of epithelial cells lining the glands in treated mice.

65

REFERENCES

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84

PUBLICATIONS FORMING THE DISSERTATION

9 PUBLICATIONS FORMING THE DISSERTATION

Paper I

Pecková Radka , Stuart D. Peter, Sak Bohumil, Květoňová Dana, Kváč Martin, Foitová Ivona (2016): Statistical comparison of excystation methods in C ryptosporidium parvum oocysts. Veterinary Parasitology 230: 1 - 5.

Paper II

Pecková Radka , Doležal Karel, Sak Bohumil, Květoňová Dana, Kváč Martin, Nurcahyo Wisnu, Foitová Ivona (2018): Effect of Piper betle on Giardia intestinalis infection in vivo . Experimental Parasitology 184: 39 - 45.

Paper III

Pecková Radka , Sak Bohumil, Květoňová Dana, K váč Martin, Koriťáková Eva, Foitová Ivona (2018): The course of experimental giardiasis in Mongolian gerbil. Parasitology Research 117(8): 2437 - 2443 .

Paper IV

Pecková Radka , Doležal Karel, Sak Bohumil, Květoňová Dana, Kváč Martin, Nurcahyo Wisnu, Foitová Ivona (2018) : Effect of Indonesian plants on Giardia intestinalis infection in vitro . Submitted to Experimental Parasitology .

Paper V

Valigurová Andrea, Pecková Radka , Doležal Karel, Sak Bohumil, Květoňová Dana, Kváč Martin, Nurcahyo Wisnu, Foitová Ivona (2018): Limitations in the screening of potentially anti - cryptosporidial agents using laboratory rodents with gastric cryptosporidiosis. Folia Parasitologica , in press .

85

PUBLICATIONS FORMING THE DISSERTATION

Paper I

Statistical comparison of excystation methods in Cryptosporidium parvum oocysts

Veterinary Parasitology 230: 1 - 5 (2016)

PECKOVÁ R. , STUART D. P., SAK B., KVĚTOŇOVÁ D., KVÁČ M., FOITOVÁ I .

86

Veterinary Parasitology 230 (2016) 1–5

Contents lists available at ScienceDirect

Veterinary Parasitology

journal homepage: www.elsevier.com/locate/vetpar

Research paper

Statistical comparison of excystation methods in Cryptosporidium

parvum oocysts

a,∗ a,1 b b b

Radka Pecková , Peter D. Stuart , Bohumil Sak , Dana Kvetoˇ novᡠ, Martin Kvácˇ , a

Ivona Foitová

a

Department of Botany and Zoology, Masaryk University, Kotlárskᡠ2, 611 37, Brno, Czech Republic

b

Institute of Parasitology, Biology Centre of the Czech Academy of Sciences of the Czech Republic, v.v.i., Branisovskᡠ31, 37005, Ceskéˇ Budejovice,ˇ Czech

Republic

a r t i c l e i n f o a b s t r a c t

Article history: Excystation of sporozoites of Cryptosporidium parvum from oocysts is essential for successful in vitro

Received 26 July 2016

assays. It has also been traditionally used as a measure for oocyst viability and infectivity. Laboratories

Received in revised form

use various excystation protocols so there is a need to clarify which method is the best. In this study,

27 September 2016

six different protocols for in vitro excystation of C. parvum oocysts were compared to find the most

Accepted 7 October 2016

efficient excystation method (expressed as percentage excystation). Tested protocols differed in chemical

pre-incubation steps, excystation media or time of incubation.

Keywords:

There were significant differences in percentage of excysted oocysts among groups excysted by differ-

Cryptosporidium parvum

ent methods. There were also significant differences in percentage of excysted oocysts between methods

Excystation methods

using pre-incubation with sodium hypochlorite and those without. The other variables examined; the

In vitro cultivation

Sodium hypochlorite presence of trypsin, kind of excystation medium and the incubation time, did not show statistical differ-

Trypsin ences in percentage excystation among groups.

Pre-incubation steps which included sodium hypochlorite, enhancing the permeability of the oocysts

were found to increase the excystation ratio and methods using this step were the most effective.

© 2016 Elsevier B.V. All rights reserved.

1. Introduction tation protocols of C. parvum oocysts imitate host-derived signals:

exposure to acid followed by incubation in bile salts, reducing

◦

Cryptosporidium parvum is a protozoan parasite that infects agents and proteases in 37 C mimic transit through the acidic

epithelial cells in the microvillus border of the gastrointesti- stomach to the alkaline small intestine. The timed release of sporo-

nal tract of many mammalian species, including humans. In zoites from oocysts is likely related to the transit time of oocysts

immunocompetent individuals, cryptosporidiosis can cause a through the stomach and into the small intestine where excyst-

short-term gastrointestinal diarrheal disease. In some populations, ing sporozoites can invade epithelial cells. Released sporozoites are

like immunocompromised patients, children and the elderly, it considered “short-lived” and have a limited time to successfully

can result in significant morbidity and mortality (Thompson et al., invade and proliferate in host cells, thus assuring parasite survival.

2005; Fayer, 2008). Although a variety of treatments have been Cell culture infectivity assays have several advantages over

tested, no reliably effective and worldwide accessible therapy for mouse infectivity assays. We consider them more precise, faster

cryptosporidiosis is available (Jenkins, 2004; Smith and Corcoran, (days instead of weeks), easier to carry out (mice have to be

2004). The use of an in vitro system that enables complete devel- acquired, housed, fed, handled, sacrificed, necropsied etc.) and

opment of C. parvum, could support efforts to identify efficacious less genetically variable. Furthermore, they are less expensive and

anticryptosporidial agents prior to testing in animals. In vitro excys- materially demanding and not ethically controversial in compar-

ison with in vivo assays (O’Donoghue, 1995; Shin et al., 2001;

Rasmussen et al., 1993; Smith et al., 2005). Inoculation of cell

cultures is preceded by several successive steps including oocyst ∗

Corresponding author.

excystation, cell localization, attachment and invasion (Smith et al.,

E-mail addresses: [email protected] (R. Pecková), [email protected]

2005). It follows that excystation of sporozoites from oocysts is

(P.D. Stuart), [email protected] (B. Sak), [email protected] (D. Kvetoˇ nová),ˇ

essential for successful in vitro assays and also for further research.

[email protected] (M. Kvác),ˇ ivona [email protected] (I. Foitová).

1

Present address: Zoology Department, Trinity College Dublin, Ireland.

http://dx.doi.org/10.1016/j.vetpar.2016.10.007

0304-4017/© 2016 Elsevier B.V. All rights reserved.

2 R. Pecková et al. / Veterinary Parasitology 230 (2016) 1–5

Table 1

Summary of excystation methods used.

Method by Rasmussen et al. Petry and Harris Mead et al. (1990) Black et al. (1996) Gut and Nelson Rennecker et al.

(1993) (1999) (1999) (1999)

Pre-incubation 2% sodium 0,25% sodium 0,5% sodium 10 mM HCl

hypochlorite hypochlorite hypochlorite, 0,1%

sodium thiosulfate

Excystation 0,75% NaT and 0,75% NaT in RPMI 0,75% NaT and 1,5% NaT and 0,5% 0,8% NaT in RPMI 1,5% NaT and 0,5%

medium 0,25% trypsin in medium 0,25% trypsin in trypsin in PBS medium trypsin in PBS PBS PBS

◦ ◦ ◦ ◦ ◦ ◦

Incubation 45 min (37 C) 2 h (37 C) 1 h (37 C) 2 h (37 C) 10 min (37 C) 1 h (37 C)

time 30 min (room temperature)

◦

Excystation has been traditionally used as a measure for oocyst for 3 min at 20 C) three times so that cesium chloride was washed

viability and infectivity, although, according to some studies, it out. Pellets from all tubes were pooled, centrifuged again and stored

◦

is not so precise. Neumann et al. (2000) discovered that intact at 4 C.

C. parvum oocysts that fail to excyst in vitro remain infectious to

neonatal mice. In spite of this fact, excystation provides a conve- 2.3. Test of viability

nient measure of oocyst and sporozoite health and if excystation

yields sporozoites at rates higher than 60–70%, the preparation is Viability of the oocysts was tested with propidium iodide stain-

considered to be suitable for culture (Arrowood, 2008). There are ing: 10 ␮l of the oocyst suspension was diluted with 100 ␮l of PBS

various excystation protocols that are used in different laboratories, and 30 ␮l of stock solution of propidium iodide was added. This

so there is a need to clarify which of these methods is the best. suspension was incubated for 3 min at room temperature in the

In this study, we compared six of the most well known dark and then examined using a fluorescence microscope with an

in vitro excystation protocols differing in pre-incubation steps excitation wavelength 535 nm.

(pre-treatment with sodium hypochlorite/HCl/no pre-treatment),

excystation media (PBS/RPMI and trypsin/no trypsin) or incubation

2.4. Excystation methods

time. All of these variables were tested in the past (Woodmansee,

1987; Kar et al., 2011) but their combination can bring different

In each of the following six methods, three 1.5 ml microcen-

results in the excystation success rate. 6

trifuge tubes were used; each contained 0.5 × 10 oocysts in 1.5 ml

of PBS (Table 1).

2. Materials and methods

2.4.1. Method by Rasmussen et al. (1993)

2.1. Cryptosporidium parvum oocysts Oocysts were washed three times in PBS (centrifugation at

12,100 × g for 3 min). McCoy’s medium, used in the original paper,

Cryptosporidium parvum oocysts were obtained from the faeces was replaced with PBS. Oocysts were then suspended in 2% sodium

of naturally infected bovine calves. All of the experimental proce- hypochlorite and incubated for 10 min. After that, oocysts were

dures were conducted in accordance with applicable laws of the washed five times in PBS, suspended in an excystation medium

Czech Republic on the use of experimental animals and the safe consisting of 0.75% taurocholic acid (sodium taurocholate, NaT) and

◦

use of pathogenic agents. The study was conducted under proto- 0.25% trypsin in PBS and incubated at 37 C for 45 min.

col approved by the Institute of Parasitology, Biology Centre of the

Czech Academy of Sciences and Central Commission for Animal 2.4.2. Method by Petry and Harris (1999)

Welfare, Czech Republic (protocol no. 073/2010). Oocysts were centrifuged (12,100 × g for 3 min), supernatant

discarded and the pellet with oocysts was re-suspended in 1:20

diluted commercial bleach (0.25% sodium hypochlorite) and incu-

2.2. Stool collection and purification of oocysts prior to

bated for 10 min on ice. Afterwards, oocysts were washed three

excystation test

times in water (12100 × g for 3 min), suspended in excystation

◦

solution (0.75% sodium taurocholate in RPMI 1640 medium) and

After collection, faeces were stored unpreserved at 4 C for one

◦

incubated at 37 C for 2 h.

week before use. The stool sample was defatted with organic sol-

vent diethyl ether before further processing for a higher quality

of oocyst purity (ratio of stool to solvent 2:1). This stool was 2.4.3. Method by Mead et al. (1990)

×

passed through a sieve (standard tea sieve) and processed using the Oocysts were centrifuged (12,100 g for 3 min) in order to

microscale cesium chloride (CsCl) gradient technique (Arrowood remove PBS, then suspended in 1 ml of 0.5% sodium hypochlorite,

◦

×

and Donaldson, 1996) as follows: the sieved stool sample was incubated for 5 min in 4 C and centrifuged (6800 g for 1 min). The

diluted with phosphate-buffered saline (PBS) and passed through oocyst sediment was neutralized by suspending in 0.1% sodium

×

a finer, nylon sieve (40 ␮m mesh size). The sieved sample was cen- thiosulfate (diluted in PBS), centrifuged (2450 g for 3 min) and

◦

trifuged (800 × g for 20 min at 4 C), supernatant discarded, pellet washed twice with PBS. Subsequently, oocyst suspensions were

resuspended in PBS repeatedly, until the supernatant was clear. The suspended in excystation solution (consisting of 0.75% sodium tau-

sediment was resuspended in a small amount of PBS to get a liq- rocholate and 0.25% trypsin diluted in PBS) and incubated for 1 h at

◦

uid suspension. RTA tubes (2.0 ml) were filled with 1 ml of cesium 37 C. Afterwards, the oocyst/sporozoite suspensions were washed

chloride suspension (21.07 g of CsCl in 100 ml of deionized H2O) twice in PBS.

and 500 ␮l of liquid sieved stool solution was carefully layered over

the CsCl solution. Tubes were centrifuged at 12100 × g for 3 min at 2.4.4. Method by Black et al. (1996)

◦

20 C. Oocysts were collected from the middle layer, transferred to Oocysts were suspended in 500 ␮l of PBS, added to 500 ␮l of

microcentrifuge tubes, diluted with PBS and centrifuged (750 × g excystation medium (150 mg of sodium taurocholate and 50 mg

R. Pecková et al. / Veterinary Parasitology 230 (2016) 1–5 3

100

90

80

70

60 Percentage of empty oocysts 50

40 Rasmussen Mead Gut & Nelson Petry & Harris Black Rennecker

Methods

Fig. 1. Average percentage of successful excystation from different methods tested. Oocysts were excysted using six excystation methods which differed in chemical pre-

incubation steps (sodium hypochlorite, sodium thiosulfate, HCl, non-pre-incubation), excystation media (RPMI or PBS with different proportion of NaT and trypsin) or time

of incubation.

◦

of trypsin dissolved in 5 ml PBS) and vortexed. Oocysts were then incubated at 37 C for 1 h. After that, the tubes were centrifuged

◦

incubated at 37 C for 2 h and afterwards at room temperature for (6800 × g for 3 min) and resuspended in PBS.

30 min. Suspensions were centrifuged (9700 × g for 5 min) and re-

suspended in PBS.

2.5. Calculation of percentage of released sporozoites and

statistical analysis

2.4.5. Method by Gut and Nelson (1999)

×

Oocysts were centrifuged (12,100 g for 3 min), resuspended All the tubes were then centrifuged (12,100 × g for 3 min) and

◦

in 10 mM HCl and incubated for 10 min at 37 C. Afterwards, the supernatant volume was reduced to 0.5 ml. The samples were

×

oocysts were centrifuged again (12,100 g for 3 min), resuspended assessed immediately after the excystation treatments. A haemo-

◦

in RPMI 1640 medium (pre-warmed to 37 C) with 0.8% sodium cytometer was used to determine the excystation rates by counting

◦

taurocholate and incubated for 10 min at 37 C. Subsequently, sus- the percentage of empty oocysts. Oocysts which had not lost their

×

pensions were centrifuged (12,100 g for 3 min) and resuspended contents and appeared intact were evaluated as not-excysted,

in PBS. while those that had released at least one sporozoite were con-

sidered to be excysted. The percentage of non-viable oocysts,

determined in an earlier viability test, was excluded. The numbers

2.4.5. Method by Rennecker et al. (1999) of empty oocysts were counted using an Olympus light microscope

×

Oocysts were added to centrifuge tubes containing 2 ml of at 400 magnification. The viability of released sporozoites was

excystation medium (1.5% sodium taurocholate and 0.5% trypsin assessed morphologically using light microscopy: motile sporo-

◦

in PBS; pre-warmed to 37 C). The tubes were then vortexed and zoites were considered viable.

Table 2

Comparisons among single groups found out by least significance difference (LSD) test in post-hoc comparisons.

Method Rasmussen et al. (1993) Petry and Harris (1999) Mead et al. (1990) Black et al. (1996) Gut and Nelson (1999) Rennecker et al. (1999)

** ** **

Rasmussen 0.274228 0.161001 0.003221 0.000094 0.000039

* ** **

Petry and Harris 0.274228 0.733705 0.026820 0.000623 0.000239

* **

Mead 0.161001 0.733705 0.050470 0.001147 0.000428

** * *

Black 0.003221 0.026820 0.050470 0.061025 0.021808

** ** **

Gut and Nelson 0.000094 0.000623 0.001147 0.061025 0.580990

** ** **

Rennecker 0.000039 0.000239 0.000428 0.021808* 0.580990

LSD test for variable excystation rate.

Probabilities for post-hoc tests.

Error: Between MS = 52.834, df = 12.000.

*

p < 0.05.

**

p < 0.01.

4 R. Pecková et al. / Veterinary Parasitology 230 (2016) 1–5

One-way ANOVA was used for statistical analysis (STATISTICA ences between the present results and those by Gold et al. (2001)

12 software). were probably caused by the fact that all of the oocysts used in this

study were bleached during the purification process. Consequently,

even the authors later call the oocysts “non-bleached”, they were

3. Results

actually bleached in the first step of the experiment.

Trypsin, a serine protease found in the duodenum, is commonly

3.1. Test of viability

used by many investigators in in vitro excystation protocols to imi-

tate the duodenal conditions (Mead et al., 1990; Black et al., 1996;

According to the propidium iodide viability test, 98% of all

Rennecker et al., 1999; Rasmussen et al., 1993). We did not find

oocysts used for testing were viable.

any influence of the presence of trypsin on the excystation ratio

which is in agreement with results of Woodmansee (1987), who

3.2. Excystation methods

found trypsin unnecessary for excystation and even inhibitory in

higher concentrations. Robertson et al. (1993) also discovered that

In this study, six excystation methods were compared. All of

the inclusion of trypsin does not increase the number of excysted

the released sporozoites were motile, so they were considered to

sporozoites, but it has clear effects both on sporozoites and oocysts:

be viable. The most efficient excystation method evaluated was

it increases the translucency of intact oocysts as well as the motility

that described by Rasmussen et al. (1993) (92.5%) followed by

of both excysted and unexcysted sporozoites.

Petry and Harris (1999) (85.7%) and Mead et al. (1990) (83.7%)

We also did not find any influence of the type of excystation

(Fig. 1). Lower excystation rates were obtained by using excysta-

medium or the incubation time on the excystation ratio, but the aim

tion method described by Black et al. (1996) (70.7%) and the lowest

of this study was to compare protocols used by different research

ones by Gut and Nelson (1999) (58.5%) and Rennecker et al. (1999)

groups to find the most effective one. Studies concerning determi-

(55.1 %).

nation of optimal concentrations of chemical triggers, media or the

There were significant differences in percentage of excysted

incubation time have already been published (Woodmansee, 1987;

oocysts among groups excysted by different methods (p = 0.00015;

Robertson et al., 1993; Gold et al., 2001).

one-way ANOVA) (data not shown). Analysis of contrasts in

According to previous findings, variation in excystation rates

planned comparisons of least squares means showed significant

and infectivity of C. parvum oocysts can be dependent on age or

differences between methods using pre-incubation with sodium

strain both in vitro and in vivo (Hijjawi, 2003; Kar et al., 2011). This

hypochlorite and those without this pre-incubation (the former

suggests that working with oocysts of another strain or age could

were significantly more efficient (p < 0.001)) (Table 2). Signifi-

bring different results and this should be investigated. However, in

cantly higher excystation rates were observed in methods using

this study, we used fresh oocysts from naturally infected animals.

sodium hypochlorite pre-incubation compared to pre-incubation

This way of investigation corresponds to reality the most and it

with HCl (p < 0.001). HCL pre-incubation did not differ from no pre-

should be the end target of such studies. Based on the present study,

incubation with HCL. Among the other variables tested: the use of

we can conclude that sodium hypochlorite enhances the excysta-

trypsin, excystation medium, and incubation time, none showed

tion rate. In contrast the presence of trypsin, type of excystation

statistically significant differences between groups.

medium used or the incubation time does not affect the excystation ratio.

4. Discussion

5. Conclusions

The results from this study give much needed clarity as to

which methods of excystation for Cryptosporidium parvum work

The objectives of our study were to compare different excysta-

best from those identified in previous studies (Fayer and Leek,

tion protocols of the oocysts of C. parvum to find the most effective

1984; Woodmansee, 1987; Campbell et al., 1992). These six meth-

one. Excystation is an essential process for successful in vitro assays

ods have not been compared before and therefore this study could

and using the right method can save time and material to a consid-

be a help for other researchers.

erable extent. The protocols that we tested differed in chemical

From the results of the present study, we can conclude that

pre-incubation steps, excystation media or time of incubation.

the most efficient excystation protocols investigated were those

Those pre-incubation steps which included sodium hypochlorite

described by Rasmussen et al. (1993), Petry and Harris (1999) and

were found to increase the excystation ratio and methods using

Mead et al. (1990). In all these methods, sodium hypochlorite is

this step are therefore the most effective.

used as a pre-incubation step in comparison with the remain-

ing, less efficient protocols. This confirms results of Woodmansee

Conflict of interest statement

(1987), that bleaching increases the excystation rate. This is prob-

ably because sodium hypochlorite treatment results in thinning

The authors declare that they have no competing interests.

and perforation of the outer and inner layers of the oocyst wall.

At the same time it possibly exposes receptors located in different

levels in the oocyst wall, which require acid and bile stimulation Acknowledgements

before they become exposed in their host (Smith et al., 2005). The

bleach can also provide additional sanitization of the oocyst sam- This study was financially supported by Czech Academy of Sci-

ple in order to prevent undesirable microbial contamination of the ences, Grant No. P505/11/1163 and Scholarship program to support

cell culture (Arrowood, 2002). Also results of the study of Kar et al. creative activity – research collaboration, No. PrFˇ 04 27.10.2006.

(2011) come to the conclusion that in young oocysts (3 months

old), pre-treatment with NaClO increases the excystation rate. Con- References

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Paper I I

Effect of Piper betle on Giardia intestinalis infection in vivo

Experimental Parasitology 184: 39 - 45 (2018)

PECKOVÁ R. , DOLEŽAL K., SAK B., KVĚTOŇOVÁ D., KVÁČ M. , NURCAHYO W. , FOITOVÁ I .

92

Experimental Parasitology 184 (2018) 39e45

Contents lists available at ScienceDirect

Experimental Parasitology

journal homepage: www.elsevier.com/locate/yexpr

Effect of Piper betle on Giardia intestinalis infection in vivo

** Radka Peckova a, , Karel Dolezal b, Bohumil Sak c, Dana Kveto nov a c, Martin Kvac c, * Wisnu Nurcahyo d, Ivona Foitova a, a Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlarska 2, 611 37, Brno, Czech Republic b Department of Chemical Biology and Genetics & Laboratory of Growth Regulators, Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacký University and Institute of Experimental Botany, Academy of Sciences of Czech Republic, 78371, Olomouc-Holice, Czech Republic c Institute of Parasitology, Biology Centre of the Czech Academy of Sciences of the Czech Republic, v.v.i., Branisovska 31, 37005, Ceske Budejovice, Czech Republic d Department of Parasitology, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta, Indonesia highlights graphical abstract

 Antigiardial activity of the extracts of the betel leaf was tested in vivo.  Plants were extracted in water, methanol and methanol:tetrahydrofuran.  The antigiardial activity was evalu- ated by the course of cyst shedding.  A significant decline in cyst shedding was found in group treated with water extract.

article info abstract

Article history: Piper betle has been used as a medicinal plant in traditional medical systems throughout South and South Received 15 May 2017 East Asia. Experimental studies have revealed its wide and diverse biological and pharmacological ef- Received in revised form fects. In this study, antigiardial activity of Piper betle was tested using experimental infections of Giardia 8 November 2017 intestinalis, the most common cause of protozoal diarrhoea worldwide, in Mongolian gerbils. Plants were Accepted 12 November 2017 extracted in water, methanol and methanol:tetrahydrofuran. Gerbils were treated for ten days intra- Available online 14 November 2017 gastrically twice a day, with the dose of 40 mg of the extract per 100 g of body weight. Drug metroni- dazole was used as a negative control. Gerbils' faeces were taken every day and examined by flotation Keywords: fi Antigiardial activity method, the number of shed cysts were counted using a haemocytometer. After gerbils' sacri ce and Drug of choice dissection, their duodena were then processed for examination using histological sectioning and scan- Giardia intestinalis ning electron microscopy. The antigiardial activity was evaluated by the course of cyst shedding Natural antiparasitics throughout the entire experiment. A significant decline in cyst shedding, evaluated by linear regression Parasites was found in gerbils treated with the aqueous extract. Our results indicate that the aqueous extract of Piper betle P. betle shows giardicidal effects. © 2017 Elsevier Inc. All rights reserved.

1. Introduction

* Corresponding author. Giardia intestinalis is an anaerobic flagellated protozoan parasite ** Corresponding author. E-mail addresses: [email protected] (R. Peckova), [email protected] (I. Foitova). of mammalian species, including humans, which is considered the https://doi.org/10.1016/j.exppara.2017.11.005 0014-4894/© 2017 Elsevier Inc. All rights reserved. 40 R. Peckova et al. / Experimental Parasitology 184 (2018) 39e45 most common cause of protozoan diarrhoea. It has a global distri- 2. Methods and materials bution and symptomatic infections occur in developing and developed countries (Caccio and Sprong, 2011). According to WHO 2.1. Plant extract preparation (1996), about 200 million people in Asia, Africa and Latin America have symptomatic giardiasis and about 500 thousand new cases are Leaves of Piper betle L. (Piperaceae) were collected in Sumatra reported each year. It is also commonly found in domestic animals and air dried in the shade and then dehydrated by lyophilisation such as livestock, dogs and cats (Thompson et al., 1993). In 2004, (freeze-drying). The research complied with the legal requirements Giardia was included in the World Health Organisation's Neglected for research of Indonesia. Research permit was issued by RISTEK Diseases Initiative (Savioli et al., 2006). Kementarian Riset dan Teknologi. The permission to collect plant Transmission of this parasite is mediated through the ingestion samples was obtained from LIPI e Lembaga Ilmu Pengetahuan of cysts, excreted in faeces. They can contaminate drinking water, Indonesia (Indonesian Institute of Sciences) and KKH - Kementer- food, recreational water or areas (Caccio et al., 2005). Excysted ian Kehutanan Direktorat Jenderal Perlindungan Hutan dan Kon- trophozoites colonize the lumen of the small intestine (Ortega and servasi Alam. Adam, 1997). Dried plant material was homogenized into a fine powder in Several compounds are known to be effective against Giardia;in liquid nitrogen. Portions of the ground material (approx. 1 g) were human patients, the most frequent treatment against giardiasis are weighed separately into 100 ml Erlenmeyer flasks. Extraction sol- nitro-imidazoles such as metronidazole or tinidazole, or benz- vent (30 mL of water, methanol or methanol:tetrahydrofuran imidazole compounds (albendazole, febendazole). Although ther- (MeOH:THF, 1:1)) was added to each flask. The flasks were subse- apy with these compounds is effective, they can produce significant quently vortexed for 1 min, and after 16 h of extraction at À20 C, undesirable side-effects such as metallic taste, vomiting, genetic the resulting homogenates were centrifuged (26,000 Â g, 4 C, damage, treatment failures, frequent relapses and selection and 20 min). The sediments were then re-extracted for 1 h in the same evolution of resistant strains (Xiao et al., 1996; Harris et al., 2001; way and centrifuged as well. These two supernatants were then Upcroft and Upcroft, 2001; Sangster et al., 2002). Moreover, resis- combined, divided to the three aliquots corresponding to 0.33 g tance to these treatments has been described (Upcroft et al., 1990; fresh weight and dried in vacuum at 35 C. Subsequently, 0.33 g of Leitsch, 2015). For these reasons, the search for new, safe and plant extracts originally dissolved in MeOH and MeOH:THF were effective therapeutic alternatives to treat giardiasis has become dissolved in 41.25 mL of pure DMSO (dimethyl sulfoxide) and 4 mLof necessary. this solution were diluted in 196 mL PBS to get 2% concentration of Nowadays, attention is being paid to extract derivatives from DMSO. Water extracts were both dissolved and diluted in distilled plants used in traditional medicine (Calzada et al., 2006; Moo-Puc water. et al., 2007; Li et al., 2012). They contain a broad spectrum of substances which can be utilized to treat both chronic and infec- 2.2. Giardia intestinalis cysts tious diseases. That is why they provide almost unlimited oppor- tunities for research of new bioactive products, semi-synthetic Giardia intestinalis cysts (human isolate H-3, passaged through medicines or lead compounds for the synthesis of medicines gerbils) were obtained from Waterborne™, Inc. (New Orleans, LA, (Amaral et al., 2006; Cos et al., 2006; Duraipandiyan et al., 2006). USA). There are a large number of papers concerning the efficacy of plant extracts against G. intestinalis trophozoites. Several com- 2.3. Laboratory animals pounds were shown to reduce parasite growth, adhesion capacity and morphology in vitro, for example curcumin and peppermint The Mongolian gerbils, Meriones unguiculatus (Institute of (Perez-Arriaga et al., 2006; Vidal et al., 2007). In in vivo studies, Parasitology, Biology Centre, CAS, v.v.i. in Ceske Budejovice), were extracts from Mexican medicinal plants were shown to kill Giardia used in this study. Six weeks old females (approx. 80 g) were trophozoites in mice and yucca extracts in gerbils (McAllister et al., divided into treatment groups. All animals were supplied sterilized 2001; Barbosa et al., 2006). diet (TOP-VELAZ Praha, Czech Republic) and sterilized water ad The betel (Piper betle) has been used as a medicinal plant in libitum. They were kept separately in plastic cages supplemented traditional medicine throughout South and South East Asia since with raised bottom grids to avoid re-infection. The breeding of test ancient times. Experimental studies have revealed its wide and animals was regulated by Czech legislation (Act No. 246/1992 Coll., diverse biological and pharmacological effects. For example, it has on protection of animals against cruelty). These documents corre- conveyed antibacterial, antifungal, antiprotozoal, antioxidant, spond with the legislation of the European Commission. The anti-inflammatory and gastroprotective properties as well as experimental procedures were arranged according to protocols many others (Fazal et al. 2013; Pradhan et al., 2013). This plant approved by the Institute of Parasitology, Biology Centre of the contains a broad range of chemical compounds including alka- Czech Academy of Sciences and Institute and National Committees loids, carbohydrates, amino acids, tannins and steroids (Protocols No. 52/2014). (Sugumaran et al., 2011). In spite of these facts, there is a poor evidence of the impact of Piper betle on Giardia intestinalis.The 2.4. Antigiardial activity assay only study concerning this impact was realized in vitro by Sawangjaroen et al. (2005). The authors found a positive result In vivo antigiardial activity of the extract was tested using a from Piper betle extracted in chloroform; the minimum inhibitory method previously described by Barbosa et al. (2006) with modi- concentration of the extract was 250 mg/ml. fications (gerbils were used instead of mice, the treatment period In this study, in vivo antigiardial activity of Piper betle leaf ex- took ten days instead of two and the number of treated animals in tracts in three different solvents (water, methanol (MeOH) and one group was three instead of six used in the original article). methanol:tetrahydrofuran (MeOH:THF)) was tested. This plant Mongolian gerbils (Meriones unguiculatus) were inoculated has not previously been investigated in vivo as an antigiardial orally using an oesophagus tube with 0.5*106 viable cysts of Giardia agent. The results will therefore provide an important insight into intestinalis. After starting cyst shedding (9 DPI (days post infection), the problems of using this plant as a potential antigiardial examined by Sheather's sugar flotation method (Garcia et al., phytodrug. 1983)), they were divided into six groups of three and treated R. Peckova et al. / Experimental Parasitology 184 (2018) 39e45 41 with Piper betle extracted in water, methanol or methanol:te- the number of cysts excreted from day 1 to day 9) (Fig. 1). Signifi- trahydrofuran, 2% DMSO as a control, Metrozol and pure PBS. There cant decline in cyst shedding was found in gerbils treated with the were also six control groups of one non-infected gerbil treated with extract dissolved in water. Surprisingly, there was also a decline in the same treatment as the former groups. cyst shedding in gerbils treated with pure DMSO but it was not Gerbils were treated intragastrically twice a day for nine days statistically significant (Fig. 1). with the dose of 40 mg of the extract per 100 g of body weight There was also a significant decline in number of shed cysts in dissolved in 200 mL of water or PBS. The dose of 3.75 mg of Metrozol the group of gerbils treated with metronidazole (Fig. 1). (commercial treatment, containing 0.75 mg of metronidazole) used Histological sections and superficial views from scanning elec- as a positive control was diluted in 200 mL of water and adminis- tron microscope of gerbils' small intestinal tissues are shown on tered once a day. The control group received 2% DMSO solution in Figs. 2 and 3, respectively. We can see healthy, non-infected tissue PBS twice a day as well as the group without treatment that (Figs. 2a and 3a), tissue of a gerbil infected with G. intestinalis, non- received just PBS solution. treated (Figs. 2b and 3b, c). Figs. 2c and 3d show tissues of a gerbil Faeces were taken three times a day (because of a possibility of infected with G. intestinalis, treated with aqueous P. betle extract. intermittent shedding) directly from the bottom of the cages. All Giardia trophozoites are present rarely. Tissues of a gerbil infected the specimens from individual animals were stored separately in with G. intestinalis and treated with Metrozol are represented on eppendorf tubes. Each day, after the last collection, specimens were Figs. 2d and 3e. Almost no trophozoites of G. intestinalis are present weighted and the same amount of faeces from these individual there. On some of the histological sections, there are apparent villus three samples/animal/day was placed in 15 ml glass tube. Then atrophies characteristic for giardiasis, especially in case of negative 10 ml of distilled water was added to each tube. Faeces were controls with high number of present trophozoites (Fig. 2b). examined next morning by Sheather's sugar flotation method However, the number of trophozoites was not correlated with the (Garcia et al., 1983). The number of shed cysts were counted using a presence of villus atrophy. haemocytometer (number of cysts per 1 g of faeces). Tenth day after the start of treatment, gerbils were sacrificed by cervical dislocation 4. Discussion and dissected. Their duodena were then processed for examination using histological sectioning and scanning with electron micro- Giardiasis is considered the most common protozoal diarrheal scopy. These methods were provided to see the visual aspect of the disease occurring in humans all over the world. Scientists have tissues as well as to get an approximate picture about the number been trying to find new, plant-based drugs useful in the treatment of trophozoites in the intestine. For this purpose, 5 mm of duodenal of giardiasis that could substitute the commercial drugs with un- tissue were taken for SEM, the rest for histology. The histology pleasant potential side effects (Barbosa et al., 2006). sections were made for each 5 mm of the tissue. In this study, we tested plant extracts from Piper betle dissolved The amount of released cysts is positively related to the number in methanol, methanol:tetrahydrofuran and water to potential of trophozoites located in small intestine (Belosevic et al., 1983). antigiardial activity in vivo. This plant has already been studied for Because of this fact, the evaluation of the impact of plant extracts its biological and pharmacological effects but there is poor evi- was based on the course of cyst shedding. dence existing of the impact that P. betle has on Giardia intestinalis. In studies concerning this impact, different solvents for extract preparations were used. We observed a significant decline of cyst 2.5. Statistical analysis shedding just in the group of gerbils treated with the aqueous extract which corresponds to the most realistic conditions. Trends in course of cyst shedding during the entire treatment Aqueous extracts of P. betle have already demonstrated anti- trials were computed as linear regression within each group. There adherence effects on early plaque settlers (Razak and Rahim, were considerable differences in numbers of shed cysts in single 2003), vasodilatory activity (Runnie et al., 2004)andthehep- gerbils during the treatment period and therefore these values atoprotective and antioxidant effects (Saravanan et al., 2002, were standardized. Standardization was performed as observed 2003). value divided by observed value on day 0. The statistical evaluation We did not find any significant influence of the extracts dis- was carried out in statistical package R (version 3.2.3). solved in methanol or methanol:tetrahydrofuran on the course of cyst shedding. This is in accordance with the observation of 3. Results Sawangjaroen et al. (2005) who did not find giardicidal activity of the methanol extract of Piper betle. On the other hand, they did not In this study, data of cyst shedding from 18 gerbils divided into observe the influence of the aqueous extract on G. intestinalis as we six groups of three was evaluated. The statistical evaluation of the did, but this could be due to the different nature of their study as course of cyst shedding obtained by linear regression is shown in they carried out just in vitro studies. Table 1. Gerbils treated with the extract dissolved in MeOH or We used a concentration of DMSO (2%) in according with MeOH:THF showed a slight decline in the number of shed cysts but Barbosa et al. (2006), therefore we did not expect any influence of this result was not statistically significant (there was no decrease in the course of infection in gerbils. However, there was a decline in cyst shedding in control DMSO group. We suppose that this un- expected decline could be due to the combination of several factors. Table 1 In in vivo models, we must take into account genetic variability of Linear regression. Marked correlations (*) are significant at corresponding P value. each animal as well as homeostatic mechanisms and pathways of Treatment R2 SE P value the host including the immune system. Animals used in laboratory research are regularly exposed to stress by routine maintenance or P. betle (MeOH) 0.096 0.248 0.702 P. betle (MeOH:THF) À0.114 0.399 0.777 monitoring procedures which can also affect the experiment at fi P. betle (H2O) À0.104* 0.057 0.088 various levels. Nevertheless, the signi cant decline in cyst shedding Negative control À0.120 0.076 0.137 was observed in case of aqueous extract which was not diluted in Metrozol À0.577* 0.162 0.002 DMSO. DMSO À0.645 0.443 0.168 Metronidazole also caused a decline in numbers of cyst 42 R. Peckova et al. / Experimental Parasitology 184 (2018) 39e45

Fig. 1. Course of G. intestinalis cyst shedding (number of cysts per 1 g of faeces) by days in tested groups. R. Peckova et al. / Experimental Parasitology 184 (2018) 39e45 43

Fig. 2. Histological sections of small intestine of tested gerbils, light microscope. a. Healthy, non-infected tissue. b. Tissue of a gerbil infected with G. intestinalis, non-treated, with apparent villus atrophy. Arrows show giardia trophozoites. c. Tissue of a gerbil infected with G. intestinalis, treated with aqueous P. betle extract. No giardia trophozoites are visible. d. Tissue of a gerbil infected with G. intestinalis, treated with Metrozol. No trophozoites of G. intestinalis are present.

shedding, but the elimination was not total; at the end of the aqueous P. betle leaf extract. For a greater understanding of this experiment, cysts were still present in gerbils' faeces. This is in issue, in vivo assays should be performed, optimally with a pro- agreement with observations of McAllister et al. (2001) who did longed treatment period. similar experiments on lambs. Some histological sections of gerbils' small intestinal tissues show apparent villus atrophies, especially in case of negative con- 5. Conclusions trols with high number of present trophozoites. However, the number of trophozoites was not correlated with the presence of The aim of this study was to test the impact of Piper betle leaf villus atrophy. These pathological changes in the mucosa of the extracts on potential antigiardial activity in vivo for the possibility small intestine were described in several studies (Gillon and of using this plant as a potential antigiardial remedy that could be a Ferguson, 1984). substitute for current commercial treatment. Our results represent Some authors demonstrate typical circular marks of various an important insight into this issue indicating that the aqueous depths left on the microvillus surface by departed trophozoites extract of P. betle shows giardicidal effects. Nevertheless, being the using scan electron microscopy (Erlandsen, 1974; Owen et al.,1979). first report on the effect of P. betle extracts on G. intestinalis in vivo, Nevertheless, we did not observe these changes. more studies, optimally with a prolonged treatment period, are The results obtained in this study indicate antigiardial activity of required. 44 R. Peckova et al. / Experimental Parasitology 184 (2018) 39e45

Fig. 3. Superficial view of small intestine of tested gerbils, scanning electron microscope. a. Healthy, non-infected tissue. b, c. Tissue of a gerbil infected with G. intestinalis, non- treated, with high number of giardia trophozoites. d. Tissue of a gerbil infected with G. intestinalis, treated with aqueous P. betle extract. Giardia trophozoites are present rarely. e. Tissue of a gerbil infected with G. intestinalis, treated with Metrozol. Almost no trophozoites of G. intestinalis are present.

Funding histolytica and Giardia lamblia to plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. J. Ethnopharmacol. 108, 367e370. This work was supported by the UMI e Saving of Pongidae Cos, P., Vlietinck, A.J., Berghe, D.V., Maes, L., 2006. Anti-infective potential of natural Foundation project “Parasites and Natural Antiparasitics in the products: how to develop a stronger in vitro ‘proof-of-concept’. e Orang-utan” and by the Czech Academy of Sciences, Grant No. J. Ethnopharmacol. 106, 290 302. Duraipandiyan, V., Ayyanar, M., Ignacimuthu, S., 2006. Antimicrobial activity of P505/11/1163 and Scholarship program to support creative activity some ethnomedicinal plants used by Paliyar tribe from Tamil Nadu, India. BMC - research collaboration, No. PrF_04_27.10.2006. Complement. Altern. Med. 6, 35e41. Erlandsen, S.L., 1974. Scanning electron microscopy of intestinal giardiasis lesions of the microvillus border of the villus epithelial cells produced by trophozoites of Acknowledgements Giardia. In: Johari, O. (Ed.), Scanning Electron Microscopy. IIT Research Institute, Chicago, pp. 775e782. The authors would like to thank the State Ministry of Research Fazal, F., Prajwal, P., Mane, Manoj, P., Rai, Karadka, R., Thilakchand, Harshith, P., Bhat, Prathibha, S., Kamble, Princy, L., Palatty, Manjeshwar, Shrinath, Baliga, 2013. The and Technology (RISTEK) and the Directorate General for Nature phytochemistry, traditional uses and pharmacology of Piper betle Lin. (betel Conservation (PHKA) for their cooperation and for permission to leaf): a pan-asiatic plant. Chin. J. Integr. Med. 1e11. conduct research in the Gunung Leuser National Park. We are also Garcia, L.S., Brewer, T.C., Shimizu, R.Y., 1983. Techniques for the recovery and identification of Cryptosporidium oocysts from stool specimens. J. Clin. Micro- grateful to Stepan Mikula for assistance with statistical evaluation biol. 18, 185e190. and Hannah Kaluzynski for English corrections. Gillon, J., Ferguson, A., 1984. Changes in the small intestinal mucosa in giardiasis. In: Erlandsen, S.L., Meyer, E.A. (Eds.), Giardia and Giardiasis. Biology, Pathogenesis, and Epidemiology. Plenum Press, New York, pp. 163e183. References Harris, J.C., Plummer, S., Lloyd, D., 2001. Antigiardial druggs. Appl. Microbiol. Bio- techn. 57, 614e619. Amaral, F.M.M., Ribeiro, M.N.S., Barbosa-Filho, J.M., Reis, A.S., Nascimento, F.R.F., Leitsch, D., 2015. Drug resistance in the microaerophilic parasite Giardia lamblia. Macedo, R.O., 2006. Plants and chemical constituents with giardial activity. Rev. Curr. Trop. Med. Rep. 2, 128e135. Bras. Farmacogn. 16, 696e720. Li, L.-D., Li, W.-C., Liu, C.-W., Shi, W.-J., Gong, P.-T., Li, J.-H., Zhang, W.-J., Yang, J., Li, H., Barbosa, E., Calzada, F., Campos, R., 2006. Antigiardial activity of methanolic extracts Zhang, X.-C., 2012. Giardia intestinalis: effects of Pulsatilla chinensis extracts on from Helianthemum glomeratum Lag. and Rubus coriifolius Focke in suckling trophozoites. Parasitol. Res. 111, 1929e1935. mice CD-1. J. Ethnopharmacol. 108, 395e397. McAllister, T.A., Annett, C.B., Cockwill, C.L., Olson, M.E., Wang, M.E., Cheeke, P.R., Belosevic, M., Faubert, G.M., MacLean, J.D., Law, C., Croll, N.A., 1983. Giardia lamblia 2001. Studies on the use of Yucca schidigera to control giardiasis. Vet. Parasitol. infections in Mongolian gerbils: an animal model. J. Infect. Dis. 147 (2), 97, 85e99. 222e225. Moo-Puc, R.E., Mena-Rejon, G.J., Quijano, L., Cedillo-Rivera, R., 2007. Antiprotozoal Caccio, S.M., Thompson, R.C.A., McLauchlin, J., Smith, H., 2005. Untravelling cryp- activity of Senna racemosa. J. Ethnopharmacol. 112, 415e416. tosporidium and Giardia epidemiology. Trends Parasitol. 21, 430e437. Ortega, Y.R., Adam, R.D., 1997. Giardia: overview and update. Clin. Infect. Dis. 25 (3), Caccio, S.M., Sprong, H., 2011. Epidemiology of giardiasis in humans. In: Lujan, H.D., 545e549. € Svard, S. (Eds.), Giardia. A Model Organism. Springer Wien New York, Wien, Owen, R.L., Nemanic, P.D., Stevens, D.P., 1979. Ultrastructural observations of giar- pp. 17e28. diasis in a murine model. Gastroenterology 76, 757e769. Calzada, F., Yepez-Mulia, L., Aguilar, A., 2006. In vitro susceptibility of Entamoeba R. Peckova et al. / Experimental Parasitology 184 (2018) 39e45 45

Perez-Arriaga, L., Mendoza-Magana,~ M.L., Cortes-Z arate, R., Corona-Rivera, A., Sawangjaroen, N., Subhadhirasakul, S., Phongpaichit, S., Siripanth, C., Jamjaroen, K., Bobadilla-Morales, L., Troyo-Sanroman, R., Ramírez-Herrera, M.A., 2006. Cyto- Sawangjaroen, K., 2005. The in vitro anti-giardial activity of extracts from plants toxic effect of curcumin on Giardia lamblia trophozoites. Acta Trop. 98, 152e161. that are used for self-medication by AIDS patients in southern Thailand. Para- Pradhan, D., Suri, K.A., Pradhan, D.K., Biswasroy, P., 2013. Golden hearth of the sitol. Res. 95, 17e21. nature: Piper betle L. J. Pharmacogn. Phytochem. 1 (6), 147e167. Sugumaran, M., Poornima, M., Venkatraman, S., Lakshmi, M., Srinivasansethuvani, Razak, F.A., Rahim, Z.H., 2003. The anti-adherence effect of Piper betle and Psidium 2011. Chemical composition and antimicrobial activity of sirugamani variety of guajava extracts on the adhesion of early settlers in dental plaque to saliva- Piper betle Linn Leaf oil. J. Pharm. Res. 4 (10), 3424e3426. coated glass surfaces. J. Oral Sci. 45, 201e206. Thompson, R.C., Reynoldson, J.A., Mendis, A.H., 1993. Giardia and giardiasis. Adv. Runnie, I., Salleh, M.N., Mohamed, S., Head, R.J., Abeywardena, M.Y., 2004. Vaso- Parasitol. 32, 71e160. relaxation induced by common edible tropical plant extracts in isolatedrat aorta Upcroft, J.A., Upcroft, P., Boreham, P.F., 1990. Drug resistance in Giardia intestinalis. and mesenteric vascular bed. J. Ethnopharmacol. 92, 311e316. Int. J. Parasitol. 20, 489e496. Sangster, N., Batterham, P., Chapman, D.H., Duraisingh, M., Jambre, L.L., Shirley, M., Upcroft, P., Upcroft, J.A., 2001. Drug targets and mechanism of resistance in the Upcroft, J.A., Upcroft, P., 2002. Resistance to antiparasitic drugs: the role of anaerobic protozoa. Clin. Microbiol. Rev. 14, 150e164. molecular diagnosis. Int. J. Parasitol. 32, 637e652. Vidal, F., Vidal, J.C., Gadelha, A.P., Lopes, C.S., Coelho, M.G., Monteiro-Leal, L.H., 2007. Saravanan, R., Prakasam, A., Ramesh, B., Pugalendi, K.V., 2002. Influence of Piper Giardia lamblia: the effects of extracts and fractions from Mentha x piperita Lin. betle on hepatic marker enzymes and tissue antioxidant status in ethanol- (Lamiaceae) on trophozoites. Exp. Parasitol. 115, 25e31. treated Wistar rats. J. Med. Food 5, 197e204. WHO, The World Health report, 1996. Fighting Disease Fostering Development. Saravanan, R., Rajendra Prasad, N., Pugalendi, K.V., 2003. Effect of Piper betle leaf World Health Organization, Geneva. extract on alcoholic toxicity in the rat brain. J. Med. Food 6, 261e265. Xiao, L., Saeed, K., Herd, R.P., 1996. Efficacy of albendazole and febendazole against Savioli, L., Smith, H., Thompson, A., 2006. Giardia and cryptosporidium join the Giardia infection in cattle. Vet. Parasitol. 61, 165e170. neglected diseases initiative. Trends Parasitol. 22, 203e208. PUBLICATIONS FORMING THE DISSERTATION

Paper II I

The course of experimental giardiasis in Mongolian gerbil

Parasitology Research 117(8): 2437 - 2443 (2018)

PECKOVÁ R. , SAK B., KVĚTOŇOVÁ D., KVÁČ M., KORIŤÁKOVÁ E., FOITOVÁ I .

100

Parasitology Research (2018) 117:2437–2443 https://doi.org/10.1007/s00436-018-5932-5

ORIGINAL PAPER

The course of experimental giardiasis in Mongolian gerbil

Radka Pecková1 & Bohumil Sak2 & Dana Květoňová2 & Martin Kváč2 & Eva Koriťáková3 & Ivona Foitová1

Received: 22 March 2018 /Accepted: 15 May 2018 /Published online: 24 May 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Fifteen Mongolian gerbils were inoculated with 10 × 106 viable trophozoites of Giardia intestinalis. Their faeces were examined daily by flotation method and the number of shed cysts was counted. Two animals (male and female) were euthanised at 4- to 5- day intervals (9, 14, 18 days post-infection (DPI)). The remaining nine gerbils were sacrificed and dissected at the end of the experiment (23 DPI). Their small intestinal tissues were processed for examination using histological sectioning and scanning electron microscopy and their complete blood count (CBC) was examined. The highest number of trophozoites at the total was observed in the duodenum in gerbils sacrificed on 14 DPI. Number of shed cysts was positively correlated with number of trophozoites rinsed from the intestine. Infected gerbils had lower body weight gain in comparison with control group and in three male gerbils; diarrhoea occurred during infection. Cyst shedding was negatively correlated with values of mean corpuscular haemoglobin concentration. Females showed another pattern in cyst shedding than males. This information needs to be taken into account while planning the experiments.

Keywords Giardial intestinal distribution . Giardia intestinalis . Giardiasis . Gerbil model

Introduction This parasite is transmitted through the ingestion of cysts excreted in faeces. They are released intermittently (Belosevic Giardia intestinalis, an anaerobic flagellated protozoan para- et al. 1983) and can survive and stay infective weeks to site of mammalian hosts, is considered the most common months in the environment. They can contaminate drinking cause of protozoan diarrhoea. It is distributed worldwide and and recreational water, food and other areas (Cacciò et al. symptomatic infections occur in both developing and devel- 2005). Excysted trophozoites colonise the lumen of the small oped countries (Cacciò and Sprong 2011). It is also commonly intestine, without invading host tissues or entering blood- found in domestic animals such as livestock, dogs and cats stream (Ortega and Adam 1997). (Thompson et al. 1993). In 2004, Giardia was included in the The pathogenesis of giardiasis is a combination of parasite World Health Organisation’s Neglected Diseases Initiative and host factors, and therefore, the subsequent clinical signs (Savioli et al. 2006). vary considerably between species (Geurden and Olson

Section Editor: Panagiotis Karanis

* Radka Pecková Ivona Foitová [email protected] [email protected]

Bohumil Sak 1 [email protected] Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic Dana Květoňová [email protected] 2 Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, v.v.i., Branišovská 31, 37005 České Martin Kváč Budějovice, Czech Republic [email protected] Eva Koriťáková 3 Institute of Biostatistics and Analyses, Faculty of Medicine, Masaryk [email protected] University, Brno, Czech Republic 2438 Parasitol Res (2018) 117:2437–2443

2011). In humans, the disease can be asymptomatic, but it is bottom grids to avoid re-infection. The gerbils were purchased often manifested by a broad spectrum of symptoms during from Institute of Parasitology, Biology Centre, CAS, v.v.i. in acute or chronic phase of the infection. Most commonly, the České Budějovice. The breeding of animals was regulated by gastrointestinal symptoms of giardiasis include diarrhoea, Czech legislation (Act No. 246/1992 Coll., on protection of bloating, abdominal pain, nausea, vomiting, as well as anorex- animals against cruelty). These documents correspond with ia and failure to thrive (Walzer et al. 1971;Nashetal.1987; the legislation of the European Commission. The experimen- Buret and Cotton 2011). In some patients, symptoms can per- tal procedures were arranged according to protocols approved sist even after several treatment courses with metronidazole by the Institute of Parasitology, Biology Centre of the Czech (Hanevik et al. 2007). In animals, a variety of clinical symp- Academy of Sciences and Institute and National Committees toms have been described, including diarrhoea and malab- (Protocol No. 52/2014). sorption (O'Handley et al. 1999;GeurdenandOlson2011). In case of livestock, these symptoms reduce growth rate, feed- Giardia intestinalis trophozoites ing efficiency and profitability of livestock production (Olson et al. 1995). In giardiasis, the acute pathophysiology occurs The trophozoites of Giardia intestinalis WB isolate (ATCC® without invasion of the small intestinal tissues by the tropho- 30957™) were originally obtained from duodenal aspirate of zoites (Gillon and Ferguson 1984). These pathophysiological a 30-year-old human male with prolonged symptomatic giar- stages can involve increased rates of enterocyte apoptosis, diasis, probably acquired in Afghanistan (Smith et al. 1982), small intestinal barrier dysfunction, activation of host lympho- axenized and propagated in vitro in TYI-S-33 medium under cytes, shortening of brush border microvilli, malabsorption, anaerobic condition at 37 °C (Keister 1983). anion hypersecretion and increased intestinal transit rates (Cotton et al. 2011). Experimental protocol: time course of infection The most frequent treatment against giardiasis is nitro- in gerbils infected with G. intestinalis imidazoles such as metronidazole, tinidazole or benzimid- azole compounds (albendazole, febendazole). Although ther- Four Mongolian gerbils were used as a negative control; 15 apy with these compounds is effective, they can produce sig- gerbils were inoculated orally using the oesophagus tube with nificant undesirable side effects and resistance to nitro- 10*106 viable trophozoites of Giardia intestinalis. The pattern imidazoles has been described (Upcroft et al. 1990;Xiaoet of cyst shedding was monitored daily in all of the animals. al. 1996;Harrisetal.2001; Sangster et al. 2002). For these Their faeces were taken three times a day (because of a possi- reasons, the search for new, safe and effective therapeutic bility of intermittent shedding) directly from the bottom of the alternatives to treat giardiasis has become necessary. cages and their consistence was noted. Each day, after the last Because of the reasons described above, giardiasis belongs collection, specimens were weighed. To maintain consistency, to commonly studied diseases. There are experiments trying to the same amount of faeces from these individual three samples/ find new, alternative treatments to chemical compounds, and animal/day was placed in 15-ml glass tube. Then, 10 ml of for these assessments, animal experimental models are neces- distilled water was added to each tube. Faeces were examined sary. Mongolian gerbils are one of the most common laboratory the next morning by Sheather’s sugar flotation method (Garcia animals used for studying giardiasis (Meyer et al. 1984). For the et al. 1983). The number of shed cysts was counted using a most exact evaluation of these trials, the knowledge of precise haemocytometer (number of cysts per 1 g of faeces). distribution of Giardia trophozoites in small intestine as well as the course of the infection is essential. This study was per- Parasitological dissection and evaluation formed to find out this information, together with other param- eters like values of complete blood count, sex, clinical signs, Six gerbils were used to examine the course of the infection trophozoite number in small intestine or number of shed cysts. during the experiment; two animals (male and female) were euthanised at 4- to 5-day intervals (9, 14, 18 DPI) and their small intestines were processed for next examination using Materials and methods histological sectioning. The remaining nine gerbils were sacrificed by cervical dislocation and dissected at the end of Laboratory animals the experiment, i.e. 23 DPI. Their small intestines were exam- ined for the presence of G. intestinalis trophozoites by three Eight- to ten-week-old male and female Mongolian gerbils different methods: three were processed for histological sec- (Meriones unguiculatus) were used in this study. All animals tioning, three for scan electron microscopy and in last three, were supplied sterilised diet (TOP-VELAZ Praha, the numbers of trophozoites in the small intestine were count- Czech Republic) and sterilised water ad libitum. They were ed following the protocol of O'Handley et al. (2001). In short, kept separately in plastic cages supplemented with raised intestinal segments were removed from the duodenum, Parasitol Res (2018) 117:2437–2443 2439 proximal and distal jejunum and ileum. They were split lon- gerbils sacrificed on 14 DPI. After this day, number of tropho- gitudinally and placed in a tube containing 5 ml of PBS. zoites decreased in all the parts of the intestine. Histological Segments were incubated for 30 min at 37 °C, and then the sections of gerbils’ duodenal tissues (gerbils sacrificed on 9, numbers of trophozoites were counted in a haemocytometer at 14, 18 and 23 DPI) are shown on Fig. 2. 400× magnification on a light microscope. The total number of trophozoites rinsed from the intestine After euthanasia, samples of gerbil blood were taken and sent is marked on Fig. 3. to a specialised laboratory for complete blood count examination. We did not observe any alterations in villus architecture, Intestinal tissues determined for histological procedure and characteristic of giardiasis. scanning electron microscopy were rinsed with PBS, and the numbers of trophozoites present in these fluids were counted Cyst shedding and clinical signs using a haemocytometer. Histological sections were made from each 0.2 cm of small Gerbils started to shed cysts on 7 or 8 DPI. Number of shed intestine and examined using a light microscope at 400× mag- cysts varied considerably among days and single individuals nification. The number of trophozoites was divided to semi- from 0 to 18 × 106 cysts per gram of faeces (Table 1). quantitative categories ranging from 0 to 5 (0—no trophozo- There were differences in cyst shedding in males compar- ites; 5—high abundance of trophozoites). ing to females (Fig. 4). At the beginning (i.e. 7–11 DPI), fe- males excreted more cysts (statistically significant on 8 and Statistical analyses 11 DPI, p = 0.05 and 0.019, respectively). On days 12 to 15 DPI, the number of shed cysts in males and females was For comparison of male and female cyst shedding and weight, almost equal, and from 16 DPI, this number was higher in Mann-Whitney U test was used. Correlations between other males (statistically significant on 16 DPI, p =0.024). variables were counted using Pearson correlations. Levels of consistency of faeces were divided into three groups (1—normal; 2—soft; 3—diarrhoeal). In three male gerbils, diarrhoea occurred in a few days during infection; in Results others, the consistency of faeces was normal. Statistical com- parison of these values with the number of shed cysts showed Distribution of trophozoites in small intestine no significant correlation between these variables. and histopathology Infected gerbils had lower body weight gain in comparison with control group (median 6.3 vs. 12.5 g in males, p =0.021; According to histological screening, the distribution of tro- and 3.9 vs. 9.2 g in females, p =0.049). phozoites in the small intestine varied in different periods of Measured parameters of complete blood count were com- the infection (Fig. 1). In general, the highest abundance of pared to control values stated in Schalm’s Veterinary trophozoites in all periods was found in the duodenum whilst Haematology (Zimmerman et al. 2010). Most values fell into the lowest one was found in the ileum. The highest number of standard range although there were some exceptions. First of trophozoites at the total was observed in the duodenum in them was values of mean corpuscular haemoglobin

Fig. 1 Median value of the duodenum proximal jejunum mid jejunum distal jejunum ileum distribution of trophozoites in the 3.5 small intestine during the experiment, based on histological 3.0 screening. The number of trophozoites (y-axis) is divided to 2.5 categories ranging from 0 to 5 (0—no trophozoites; 5—high abundance of trophozoites) 2.0

1.5

1.0 NUMBER OF TROPHOZOITES

0.5

0.0 9 DPI 14 DPI 18 DPI 23 DPI 2440 Parasitol Res (2018) 117:2437–2443

Fig. 2 Histological sections of duodenum of tested gerbils infected with G. intestinalis,light microscope. a Tissue of a gerbil euthanised on 9 DPI. Arrows show giardia trophozoites. b Tissue of a gerbil euthanised on 14 DPI. Arrows show giardia trophozoites. c Tissue of a gerbil euthanised on 18 DPI. No trophozoites of G. intestinalis are present. d Tissue of a gerbil euthanised on 23 DPI. No trophozoites of G. intestinalis are present

ab

cd

concentration (MCHC) which should be in the range of 30.6– 7,000,000 33.3%. In our case, the range of values was 25.9–30.9%. The 6,000,000 range of eosinophils in blood is standardly 0–4%; however, in 5,000,000 one gerbil, this value reached 9%.

4,000,000 In both gerbils euthanised on 14 DPI, the whole small

3,000,000 intestine was filled with mucus.

2,000,000 Number of trophozoites Number of Scanning electron microscopy 1,000,000

0 9 141823Small intestinal tissues from three gerbils sacrificed on 23 DPI DPI were processed for SEM. The distribution of trophozoites in Fig. 3 Median value of the number of trophozoites rinsed from the small the intestine corresponded to the one from histological sec- intestine during the experiment tions which means there were almost no trophozoites there. Parasitol Res (2018) 117:2437–2443 2441

Table 1 Number of shed cysts corpuscular haemoglobin concentration (MCHC) significant- − DPI N Mean Median Min Max ly decreased (Pearson correlation r = 0.62, p = 0.032). Values of other physiological parameters (complete blood 7 15 1,939,648 250,000 0 23,200,000 count, sex, body weight, consistency of faeces) did not corre- 8 15 516,449 166,667 0 2,291,667 late with the intensity of infection (expressed by number of 9 13 216,795 120,563 0 888,889 trophozoites in the small intestine or number of shed cysts). 10 13 394,970 39,063 0 3,450,000 11 13 2,642,469 86,806 46,296 18,000,000 12 13 107,020 38,580 0 880,000 Discussion 13 13 504,105 35,714 0 3,500,000 14 13 901,438 112,847 0 6 250,000 15 11 610,859 138,889 9921 2 250,000 The aim of this study was to find out the precise distribution of 16 11 336,331 12,478 0 2,285,714 G. intestinalis trophozoites in small intestine in the course of 17 11 437,330 73,333 0 4,133,333 infection and to compare these values to other physiological parameters (complete blood count, sex, body weight, consis- 18 11 2,021,895 68,750 19,097 9,895,833 tency of faeces, trophozoite number in small intestine or num- 19 9 171,964 18,084 0 1,277,778 ber of shed cysts). 20 9 233,310 54,253 0 1,083,333 According to histological screening, the highest number of 21 8 126,110 31,440 0 366,667 Giardia trophozoites in all periods of the infection was found 22 9 1,116,279 138,889 0 5,318,182 in the duodenum followed by the proximal jejunum, whilst the DPI days post-infection lowest one was found in the ileum. This finding is consistent with observation of Koudela and Vítovec (1998) who made We did not observe any changes in villus architecture, typical similar experiment in goat kids. On the contrary, O'Handley et of giardiasis. al. (2001) found the proximal and distal jejunum to be the site of maximum trophozoite numbers in dairy calves (third week post-infection). These controversial results could be caused by Correlation analysis different host used in these experiments. Evaluation of histological sections revealed that the highest Number of shed cysts was positively correlated with number abundance of trophozoites in the small intestine was observed on of trophozoites rinsed from the intestine (Pearson correlation 14 DPI, followed by 9 DPI; the abundance of trophozoites on r =0.77,p = 0.04), but there was no correlation between num- days 18 and 23 post-infection was very low. On the other hand, ber of shed cysts and trophozoites on histological sections. the number of trophozoites rinsed from the lumen of the intestine Also, with an increasing number of shed cysts, mean was gradually increasing with the peak on 18 DPI. Together with

Fig. 4 Cyst shedding in males (blue) and females (red); *p < 0.05 2442 Parasitol Res (2018) 117:2437–2443 findings of high amount of mucus in the small intestine on The peak of the infection according to histological screening 14 DPI, these results indicate relocation of trophozoites from is in the second week both in males and females, whilst the the surface of villi and crypts to the lumen of the intestine as a peak of cyst shedding is in the third week in males and the result of increased mucus secretion (Buret et al. 1990a). second week in females. This information needs to be taken Neither histological sections nor superficial views from into account while planning the experiments. scanning electron microscope of gerbils’ small intestinal tis- sues showed any alterations in villus architecture, characteris- Acknowledgements This work was financially supported by the Czech tic of giardiasis (e.g. villus atrophy, villus blunting, crypt hy- Academy of Sciences, Grant No. P505/11/1163 and Scholarship program to support creative activity-research collaboration, No. PřF_04_ perplasia). These changes have been previously observed in 27.10.2006. infected laboratory rodents, calves and goat kids (Buret et al. 1990b; Ruest et al. 1997;KoudelaandVítovec1998;Pecková Compliance with ethical standards et al. 2018), but they do not always occur (Farthing 1993; Hardin et al. 1997). Some authors demonstrate typical circular Conflict of interest The authors declare that they have no conflict of marks of various depths left on the microvillus surface by interest. previously dislodged trophozoites using SEM (Erlandsen 1974;KoudelaandVítovec1998). Nevertheless, we did not observe these changes. References Thereweredifferencesincystsheddinginmalescomparingto females. At the beginning, females excreted more cysts than Belosevic M, Faubert GM, MacLean JD, Law C, Croll NA (1983) males, while at the end of the experiment, this ratio was the Giardia lamblia infections in Mongolian gerbils: an animal model. – opposite. The distribution of giardiasis among males and J Infect Dis 147(2):222 225 Buret AG, Cotton J (2011) Pathophysiological processes and clinical females can differ in different animal species. Oberhuber and manifestations of giardiasis. In: Luján HD, Svärd S (eds) Giardia. Stolte (1990) found it equally distributed among male and female A Model Organism. Springer, Wien, pp 301–318 human patients. Experimental studies in mice by Roberts- Buret A, Gall DG, Nation PN, Olson ME (1990a) Intestinal Protozoa and Thomsonetal.(1980)revealedlowerlevelsofinfectioninfemale epithelial cell kinetics, structure and function. Parasitol Today 6(12): – thaninmalemice.Thiseffectcanberelatedtosexhormonelevels 375 380 Buret A, Gall DG, Olson ME (1990b) Effects of murine giardiasis on or reflect the influence of loci associated with sex chromosomes growth, intestinal morphology, and disaccharidase activity. J (Eidinger and Garrett 1972; Roberts-Thomson et al. 1980). Parasitol 76:403–409 In three male gerbils, diarrhoea occurred in a few days Cacciò SM, Sprong H (2011) Epidemiology of giardiasis in humans. In: during infection; in others, the consistency of faeces was nor- Luján HD, Svärd S (eds) Giardia. A model organism. Springer, – mal. Similar results were obtained by Koudela and Vítovec Wien, pp 17 28 Cacciò SM, Thompson RCA, McLauchlin J, Smith H (2005) (1998) who observed three of eight infected goat kids to have Untravelling cryptosporidium and Giardia epidemiology. Trends diarrhoea. Also, in a study carried out by Horejs and Koudela Parasitol 21:430–437 (1994) in dogs, no association between bouts of diarrhoea and Cotton JA, Beatty JK, Buret A (2011) Host parasite interactions and peaks of giardia cysts shedding was found. In our experiment, pathophysiology in Giardia infections. Int J Parasitol 41:925–933 statistical analysis did not reveal any correlation between con- De Vizia B, Poggi V, Cucchiara S, Acampora A (1985) Iron malabsorp- tion in giardiasis. J Pediatr 107(1):75–78 sistency of faeces and other studied parameters. Eidinger D, Garrett TJ (1972) Studies of the regulatory effects of the sex Infected gerbils had lower body weight in comparison with hormones on antibody formation and stem cell differentiation. J Exp control groups (12.1% decrease in males, 9.5% in females). Med 136:1098–1116 Loss of weight and failure to thrive are common symptoms of Erlandsen SL (1974) Scanning electron microscopy of intestinal giardia- giardiasis (Nash et al. 1987; Buret and Cotton 2011). sis lesions of the microvillus border of the villus epithelial cells produced by trophozoites of Giardia. In: Johari O (ed) Scanning Most values of blood picture fell into standard range al- Electron Microscopy. IIT Research Institute, Chicago, pp 775–782 though there were some exceptions. Values of MCHC were Farthing MJ (1993) Diarrhoeal disease: current concepts and future decreasing with an increasing number of shed cysts and these chalenges. Pathogenesis of giardiasis. Trans R Soc Trop Med Hyg values themselves were lower than standard ones. This may 87(3):17–21 suggest iron deficiency which is often presented in giardiasis Garcia LS, Brewer TC, Shimizu RY (1983) Techniques for the recovery and identification of Cryptosporidium oocysts from stool speci- (De Vizia et al. 1985; Monajemzadeh and Monajemzadeh mens. J Clin Microbiol 18:185–190 2008; Weiss 2010). Parasite infections are associated with an Geurden T, Olson M (2011) Giardia in pets and farm animals, and their increased number of eosinophils in blood (Young and zoonotic potential. In: Luján HD, Svärd S (eds) Giardia. A model – Meadows 2010). In our study, only one male gerbil had an organism. Springer, Wien, pp 71 92 increased value of eosinophils. Gillon J, Ferguson A (1984) Changes in the small intestinal mucosa in giardiasis. In: Erlandsen SL, Meyer EA (eds) Giardia and giardiasis. To conclude, giardiasis in Mongolian gerbils is Biology, pathogenesis, and epidemiology. 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Paper V

Limitations in the screening of potentially anti - cryptosporidial agents using laboratory rodents with gastric cryptosporidiosis

Folia Parasitologica , in press

VALIGUROVÁ A., PECKOVÁ R. , DOLEŽAL K., SAK B., KVĚTOŇOVÁ D., KVÁČ M. , NURCAHYO W. , FOITOVÁ I .

122

Institute of Parasitology, Biology Centre CAS Folia Parasitologica 2018, 65: XXX doi: 10.14411/fp.2018.XXX http://folia.paru.cas.cz

Research Article Limitations in the screening of potentially anti-cryptosporidial agents using laboratory rodents with gastric cryptosporidiosis

Andrea Valigurová1, Radka Pecková1, Karel Doležal2, Bohumil Sak3, Dana Květoňová3, Martin Kváč3,4, Wisnu Nurcahyo5, Ivona Foitová1,5

1 Department of Botany and Zoology, Faculty of Science, Masaryk University, Brno, Czech Republic; 2 Department of Chemical Biology and Genetics & Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, and Institute of Experimental Botany, Academy of Sciences of Czech Republic, Olomouc-Holice, Czech Republic; 3 Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic; 4 Department of Animal Husbandry Sciences, Faculty of Agriculture, University of South Bohemia in České Budějovice, Czech Republic; 5 Department of Parasitology, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta, Indonesia

Abstract: The emergence of cryptosporidiosis, a zoonotic disease of the gastrointestinal and respiratory tract caused by Cryptosporid- ium Tyzzer, 1910, triggered numerous screening studies of various compounds for potential anti-cryptosporidial activity, the majority of which proved ineffective. Extracts of Indonesian plants, Piper betle and Diospyros sumatrana, were tested for potential anti-cryp- tosporidial activity using Mastomys coucha (Smith), experimentally inoculated with Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016. None of the plant extracts tested showed significant activity against cryptosporidia; however, the results indicate that the following issues should be addressed in similar experimental studies. The monitoring of oocyst shedding during the entire experimental trial, supplemented with histological examination of affected gastric tissue at the time of treatment termination, revealed that similar studies are generally unreliable if evaluations of drug efficacy are based exclusively on oocyst shedding. Moreover, the reduction of oocyst shedding did not guarantee the eradication of cryptosporidia in treated individuals. For treatment trials performed on experimentally inoculated lab- oratory rodents, only animals in the advanced phase of cryptosporidiosis should be used for the correct interpretation of pathological alterations observed in affected tissue. All the solvents used (methanol,methanol-tetrahydrofuran and dimethylsulfoxid) were shown to be suitable for these studies, i.e. they did not exhibit negative effects on the subjects. The halofuginone lactate, routinely administered in intestinal cryptosporidiosis in calves, was shown to be ineffective against gastric cryptosporidiosis in mice caused byC. proliferans. In contrast, the control application of extract Arabidopsis thaliana, from which we had expected a neutral effect, turned out to have some positive impact on affected gastric tissue. Keywords: Cryptosporidium, gastric, oocyst, pathology, treatment

The phylum Apicomplexa comprises exclusively par- course of gastric cryptosporidiosis in both immunocom- asitic protists infecting invertebrates and vertebrates, petent and immunodeficient animals is an asymptomatic including humans. One of the most significant and wide- and chronic infection (Kváč et al. 2008, 2011). In humans, spread pathogens are coccidia of the genus Cryptosporid- gastric involvement is reported to be very common in pa- ium Tyzzer, 1907, causative agents of zoonotic disease tients with cryptosporidiosis when combined with severe (cryptosporidiosis) of the gastrointestinal and respiratory immunodepression (Rivasi et al. 1999). tracts. In healthy hosts, cryptosporidiosis is self-limiting; The recently described species Cryptosporidium pro- nevertheless, in immunocompromised hosts, it represents a liferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, chronic and debilitating condition (Chen et al. 2002). Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Although gastric cryptosporidia have been reported in Prantlová et McEvoy, 2016 (previously known as strain fish, reptiles, amphibians, birds and mammals (Jirků et al. TS03 of Cryptosporidium muris Tyzzer, 1907), used in 2008, Ryan 2010, Nakamura and Meireles 2015), there is a this study, develops exclusively in the glandular part of the dearth of useful studies dealing with the treatment of gas- stomach, similar to C. muris and Cryptosporidium ander- tric cryptosporidiosis. In contrast to intestinal species, the soni Lindsay, Upton, Owens, Morgan, Mead et Blagburn,

Address for correspondence: Ivona Foitová, Department of Botany and Zoology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic Phone: +420 549 494 447; Fax: +420 549 498 331; E-mail: [email protected]

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. doi: 10.14411/fp.2018.XXX Screening of potentially anti-cryptosporidial agents

2000, with a life cycle corresponding to that of C. muris to be effective against the infection withC. parvum in mice (see Tyzzer 1910, Melicherová et al. 2014, Kváč et al. (Abu El Ezz et al. 2011). In contrast, an in vitro study using 2016). Though C. proliferans had been considered identi- HCT-8 cells inoculated with C. andersoni did not confirm cal with C. muris in previous years, their clinical courses of the significant inhibition of parasite growth when exposed parasitisation in Mastomys coucha (Smith) differ consid- to garlicin (antifungal component extracted from garlic) erably (Kváč et al. 2016). Compared to C. muris, rodents (Wu et al. 2011). The authors concluded that garlicin in- shed oocysts of C. proliferans for a much longer period hibits the growth of cryptosporidia in vivo by enhancing and at a greater intensity, and only C. proliferans induc- macrophage activity, rather than by exerting direct effects es significant clinical and pathological changes, such as on the parasite. This study also showed anti-cryptosporid- weight loss and massive proliferation of the gastric mucosa ial activity of ginkgolic acids extracted from maidenhair associated with a considerable increase in stomach weight tree (Ginkgo biloba) sarcotesta. With the exception of the (Kváč et al. 2016). The change in the ratio of glandular to prophylactic effect of garlic, however, none of these nat- non-glandular surfaces from 55 : 45 to 80 : 20, detected in ural products were able to completely eradicate crypto- M. coucha infected with C. proliferans, was not observed sporidiosis. in C. muris infection (Kváč et al. 2016). As humans and orang-utans exhibit phylogenetic simi- Cryptosporidiosis is recognised as a major medical con- larities (Grehan and Schwartz 2009), we focused on orang- cern as there is no effective treatment for either intestinal utans’ feeding behaviour with an emphasis on specific or gastric cryptosporidiosis (Fayer et al. 2000, Thompson plants consumed that would lead to a reduction in parasite et al. 2005). Numerous compounds have been screened in infections. Recently we documented a secondary self-med- vitro and in vivo for potential anti-cryptosporidial activi- ication (the external application of a medicinal substance) ty, but the majority turned out to be ineffective and only in Bornean orang-utans (Morrogh-Bernard at al. 2017). a few agents have shown promise. The most commonly These findings validate the anti-inflammatory properties of used drugs used against cryptosporidiosis include antibiot- Dracaena cantleyi and its application to muscles and joints ics (e.g. paromomycin or azithromycin) and halofuginone, by orang-utans, and may serve as the first evidence for the which are partially effective. In contrast to chemothera- deliberate external application of substances with bioactive peutics, often with considerable side effects and a certain potential for self-medication in great apes. level of toxicity, the use of natural products or dietary sup- We selected few plants including Piper betle and Dio- plements with anti-cryptosporidial activity could represent spyros sumatrana with promising antiparasitic activity on a new and safe approach to the effective pharmacological the basis of behavioural data and decreases in parasite load control of cryptosporidiosis. For example, L-arginine was (Foitová et al. 2010). Our analyses show a positive cor- shown to have a protective role during infection with Cryp- relation between the prevalence of these plant species in tosporidium parvum Tyzzer, 1912 in undernourished mice orang-utan diets and the presence of parasites (based on (Castro et al. 2012). Several studies have found probiotics the Jaccard index of known frequency in nature) that can- to be effective against cryptosporidiosis in humans and an- not be explained by their prevalence in the environment. imals, reporting prompt clinical improvement and resolu- The betel, P. betle, has been used as a medicinal plant in tion of the infection following treatment (Rotkiewicz et al. traditional medicine throughout South and South East Asia 2001, Pickerd and Tuthill 2004). Lactobacillus spp. signif- since ancient times. Experimental studies have revealed its icantly reduced the viability of oocysts of C. parvum (see wide and diverse biological and pharmacological effects Foster et al. 2003). Administration of exogenous agmatine (Pecková et al. 2018). Diospyros sumatrana has not yet seems to alter the metabolism of C. parvum enough to in- been studied for pharmacological potential, but our study terfere with its ability to colonise the mammalian intestine shows its possible potential. (Moore et al. 2001). Mangiferin, widely distributed in This study aimed to test extracts of Indonesian plants higher plants and one of the constituents of folk medicines selected by orang-utans for self-medication for potential (Yoshimi et al. 2001), has significant anti-cryptosporidial anti-cryptosporidial activity, using a rodent host that had activity comparable to the same dose (100 mg/kg/day) of been experimentally inoculated with C. proliferans. The paromomycin (Tarantino et al. 2004, Perrucci et al. 2006). extract of Arabidopsis thaliana (the Eurasian plant rou- Curcumin, which is active against a variety of diseases, tinely used as a model in research laboratories) was used was found to be effective againstC. parvum in cell cultures as a control with an expected neutral effect. Halofuginone (Shahiduzzaman et al. 2009). lactate (Halocur), an oral solution used for the treatment of Garlic (Allium sativum) appears to be a prophylactic and cryptosporidiosis in calves, was tested for its potentially a promising therapeutic agent, as it successfully eradicated positive effect. cryptosporidial oocysts from the faeces and intestines of infected immunocompetent mice that had received garlic MATERIALS AND METHODS two days before the experimental infection and contin- ued for two weeks (Gaafar 2012). The administration of Preparation of plant extracts garlic to human HIV patients with chronic diarrhoea and The dried leaves obtained from Piper betle (akar sirih), Dio- confirmed cryptosporidiosis resulted in complete or partial spyros sumatrana (kayu hitam) and Arabidopsis thaliana (thale remission (Fareed et al. 1996). Onion (Allium cepa) and cress) were homogenised to a fine powder in liquid nitrogen. cinnamon (Cinnamomum zeylanicum) oils also turned out Portions of the ground material (0.33 g) were then extracted sep-

Folia Parasitologica 2018, 65: XXX Page 2 of 17 doi: 10.14411/fp.2018.XXX Screening of potentially anti-cryptosporidial agents arately in 10 ml of water, methanol (methanol) or methanol-tet- parasitisation intensity of infected mice administered with only rahydrofurane (methanol-THF, 1 : 1). After 16 hours of extraction distilled water. The coefficient of determination (r2) was calcu- (overnight) at -20 °C, the resulting homogenates were centrifuged lated for each linear regression. All computation was carried out (26,000 g, 4 °C, 20 min); the sediments were then re-extracted with the SigmaPlot 13.0 (Systat Software Inc., San Jose, CA). for one hour in the same way and centrifuged. Afterward, these The histopathological changes of parasitised gastric mucosa were two supernatants were pooled and dried in a vacuum at 35 °C, evaluated post mortem. and then dissolved in 100 µl of pure Dimethylsulfoxid (DMSO), Trial 1. Groups of mice were treated daily for 14 days, be- except for samples dissolved in sterile water, which were further ginning two months post inoculation with C. proliferans, with diluted in sterile water. 12.5 mg per 100 g of body mass (BM) of either P. betle, D. suma- trana or A. thaliana extracted in methanol, dissolved in DMSO, The parasite used in this study and diluted with sterile water to obtain a final concentration of The gastric species Cryptosporidium proliferans used in this 0.5% DMSO. The effect of the Halocur (100 µg/kg BM) and the study and our previous studies (Kváč et al. 2008, 2011, 2016 diluent (0.5% DMSO in sterile water) was evaluated in infected Melicherová et al. 2014, 2016) originated from a naturally infect- control mice. ed East African mole rat Tachyoryctes splendens (Rüppell) and Trial 2. A second trial was conducted after the completion of was kept in severe combined immunodeficiency (SCID) mice and the first trial. The treatments began three months post inoculation southern multimammate mice (Mastomys coucha) under labora- with C. proliferans. Three extraction media were used: metha- tory conditions. nol, methanol-THF and sterile water. Extracted material was dis- solved in DMSO (except for that dissolved in sterile water) and Laboratory animals and experimental inoculations with diluted with sterile water to obtain a final concentration of 0.5% oocysts of Cryptosporidium proliferans. DMSO. A dose of 40 mg of per 100 g BM of either P. betle, D. su- Eight-week old M. coucha mice (Biology Centre, CAS, České matrana or A. thaliana extract was administered twice a day for Budějovice) were used for this study. To prevent environmental 21 days. Additionally, the effect of the Halocur (100 µg/kg BM) contamination with oocysts, each group of mice was housed in and the diluent (0.5% DMSO in sterile water) was evaluated in plastic cages with sterile wood-chip bedding and supplied with infected control mice. sterilised food and water ad libitum. The rearing of animals was regulated by Czech legislation (Act No. 246/1992 Coll., on pro- Parasitological dissection and tissue processing for micro- tection of animals against cruelty); these documents are consist- scopic evaluation ent with legislation by the European Commission. All housing, After either 14 (Trial 1) or 21 (Trial 2) days of treatment, con- feeding, and experimental procedures were conducted under pro- trol and treated animals were euthanised by cervical dislocation tocols approved by the Institute of Parasitology, Biology Cen- and dissected according to protocols described by Melicherová tre, CAS and Institute and National Committees (Protocols No. et al. (2014). For histological sectioning, gastric tissue was fixed 52/2014). in AFA (Alcohol-Formalin-Acetic Acid) solution and processed For the experimental inoculation of mice, oocysts collected according to Valigurová et al. 2008. The blocks were cut using a from faeces were purified using Sheather’s sugar flotation meth- Zeiss Hyrax M 300 rotary microtome and the 7 µm thick sections od (Arrowood and Sterling 1987) and modified caesium chloride were stained with haematoxylin-eosin. Preparations were viewed gradient centrifugation (Kilani and Sekla 1987). Each mouse was using an Olympus BX61 microscope. inoculated orally by an oesophagus tube with a dose of 106 viable For scanning electron microscopy, samples of gastric tis- oocysts of C. proliferans. Afterwards, fresh mouse faeces were sue were fixed overnight at 4 °C in freshly prepared 2.5% glut- collected daily in the morning and examined microscopically for araldehyde (v/v) in cacodylate buffer (0.1 M; pH 7.4), washed the presence of oocysts using staining according to Miláček and 3 × 15 min in the buffer, postfixed in 2% 4 OsO in cacodylate Vítovec (1985). The intensity of oocyst excretion was assessed as buffer for two hour at room temperature, and washed again the number of oocysts per gram of faeces (OPG) as previously de- 3 × 15 min in buffer. After dehydration in a graded acetone series, scribed Kváč et al. (2007). In addition, faecal consistency, faecal specimens were critical point-dried using CO2, coated with gold, colour and general health status were examined daily. and examined using a JEOL JSM-7401F – Field Emission Scan- ning Microscope. Abbreviations used in Figs. 1–9: LM – light The treatment of parasitised animals using plant extracts microscopy; SEM – scanning electron microscopy The potential antiparasitic effect of Indonesian plants P.( be- tle and D. sumatrana), was compared with the expected null ef- RESULTS fect of A. thaliana, as well as with the potentially positive effect of the Halocur oral solution (Intervet Production S.A., Rue de Histopathological observations of the gastric tissue Lyons, France). Mice infected with C. proliferans two or three of uninfected and infected rodents months before treatment with plant extracts were divided into In the stomach of healthy (control) Mastomys coucha groups (three animals per group) and treated with the following individuals, the surface of the gastric mucosa was smooth treatment doses administered per os. Treated non-infected and with a brain-like ornamentation (Fig. 1A). In histologi- untreated infected and non-infected control groups were includ- cal sections stained with haematoxylin-eosin, the gastric ed in all experiments. The effect of administered extracts/drugs/ mucosa, along with a thin layer of muscularis mucos- diluents on the course of parasitisation was evaluated as change ae and subjacent submucosa, appeared homogeneously in the parasitisation intensity expressed by OPG in comparison to pink with well-demarcated blue nuclei (Fig. 1B). It was

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D E F

Fig. 1. Healthy gastric mucosa of Mastomys coucha (Smith). A – general view of surface of the gastric mucosa exhibiting constricted gastric pits; note the remnants of mucus not washed away during rinsing (SEM); B – general view of gastric mucosa showing the longi- tudinally sectioned pits and glands (LM); C – general view of mucosa with cross-sectioned gastric glands (LM, histology); D – detailed view of constricted gastric pits in longitudinal section (LM, histology); E – detailed view of constricted pits in cross section (LM, his- tology); F – detailed view of constricted glands in cross sections (LM, histology); asterisk – gastric pit; m – mucosa; mm – muscularis mucosae; sm – submucosa; white arrow – mucus with cell debris. possible to distinguish quite easily the tubular glands thelium, especially as parasitisation progressed (most like- and necks ended by pits invaginating the luminal surface ly due to the increased space requirements). The duration of mucosa (Fig. 1B,C). The gastric pits and glands were of the prepatent period (18–21 days) and the chronology of obviously constricted and contained within a thin lamina pathological changes correspond to previously published propria (Fig. 1A–F). A tall simple columnar epithelium data (Melicherová et al. 2014). lined the mucosal surface and gastric pits (Fig. 1B). Sur- In the first trial, the use of mice in a relatively early face mucous cells lining the pits as well as mucous neck stage of cryptosporidiosis (two months post inoculation cells demarcating the necks of gastric glands appeared pale with C. proliferans) was shown to be unsuitable for the mi- (Fig. 1B), while the cells forming the base of glands were croscopic evaluation of treatment effects, as affected gas- stained darker with prominent, intensively stained nuclei tric mucosa exhibited only mild to moderate pathological (Fig. 1C,F). changes. The gastric tissue was also irregularly affected by In mice infected with Cryptosporidium proliferans, par- cryptosporidia in an island-like manner, where individu- asite endogenous stages were restricted to the epithelial al parasitised gastric pits were surrounded by regions of cells in the glandular part of the gastric mucosa. This spe- healthy epithelium. The affected gastric tissue showed no cies primarily parasitises epithelial cells lining the gastric obvious alterations and the pits appeared almost fully con- pits and glands, though some parasites could be found at- stricted when evaluated under SEM (Fig. 2A). Despite the tached to cells lining the luminal surface of the gastric epi- mild character of pathological alterations visible by SEM,

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A B

Fig. 2. Pathological alterations to gastric mucosa induced by Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016 in control Mastomys coucha (Smith) from Trial 1. A – superficial view of surface of the gastric mucosa exhibiting slightly enlarged gastric pits (SEM);B – general view of the gastric mucosa with longitudinally sectioned pits. The gastric pits and glands exhibit moderate dilation when viewed in tangential and cross sections (LM, histology); asterisk – gastric pit, black arrowhead – cryptosporidia, m – mucosa, white arrow – mucus with cell debris, white arrowhead – cryptosporidia-free pit/gland. histological sectioning revealed the moderate dilatation hibited various degrees of oedema and the infiltration of of gastric glands due to the presence of numerous crypto- the lamina propria and submucosa with neutrophils (Fig. sporidia (Fig. 2B). 3B,E). Stomach weight (due to proliferating mucosa) and Pathological changes in affected gastric tissue became epithelial height were considerably greater than in non-in- more prominent three months post inoculation with C. pro- fected animals. Interestingly, despite the chronicity of in- liferans, when the parasitisation entered a chronic phase fection, no clinical signs were observed in infected rodents. (Fig. 3A–I). Such mice, used in Trial 2, had the surface of their gastric glandular epithelium markedly deformed Treatment with plant extracts and Halocur due to intense pathological changes. At the macroscopic Trial 1 served as the primary screening of experimental level, the gastric mucosa was typified by a cauliflower-like protocols and the selected treatment doses based on related appearance. The epithelium proliferated into the luminal literature and empirical data on other unicellular parasites space, so that the stomach exhibited extensive folding that used in our research (Pecková et al. 2018). On the basis of was especially visible under SEM (Fig. 3A,C,D). This was the results obtained with daily doses of 12.5 mg/100 g BM the result of an increase in the volume of the lamina pro- applied for 14 consecutive days, we decided to increase pria, which then increased the distance between individual the treatment doses to 40 mg per 100 g BM administered gastric glands and caused the longitudinal folds to become twice daily for 21 consecutive days in Trial 2. Furthermore, twisted and obviously deformed. The progress of parasiti- in Trial 2, we tested and compared the efficacy of select- sation caused a distinctive form of diffuse mucosal hyper- ed plant extracts obtained using various solvent media – trophy typified by the presence of enlarged/giant gastric methanol, methanol-THF, and sterile water. In both trials, folds and intensive epithelial hyperplasia. an oral administration containing 100 µg/kg BM halofug- The gastric glands, packed with various developmental inone lactate (Halocur), a salt whose antiprotozoal prop- stages of C. proliferans and necrotic material, were mark- erties and efficacy against Cryptosporidium parvum have edly dilated and hypertrophied (Fig. 3B,E–I). In addition, been demonstrated under in vitro and in vivo conditions numerous cryptosporidia developed attached to the luminal (Giadinis et al. 2008, Petermann et al. 2014), was used as surface of the gastric mucosa outside the dilated pits (Fig. a control treatment. 3D). The affected glands, lined with many undifferentiated One of the parameters used to evaluate parasitisation in- cells, lost their normal architecture; the atrophic epithelial tensity was the monitoring of the number of shed oocysts cells of the affected glands exhibited cuboidal or squamous detected in faeces. Generally, the variations in oocyst shed- metaplasia (Fig. 3G–I). Besides the more intense staining ding were comparable in all experimental groups treated of affected tissue in histological sections, another feature with either plant extracts or Halocur. A decline in oocyst typical of advanced cryptosporidiosis was the thickening number in Trial 1 can be observed in groups treated for of muscularis mucosae (Fig. 3B). Parasitised tissue ex- 14 days with Diospyros sumatrana extracted by metha-

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D E F

G H I

Fig. 3. Pathological alterations to gastric mucosa induced by Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016 in control Mastomys coucha (Smith) from Trial 2. A – superficial view of the gastric mucosa exhibiting extensive folding and intense parasitisation (SEM); B – general view of the gastric mucosa and submucosa in longitudinal section (LM, histology); C, D – detailed view of dilated pits filled with numerous parasites (SEM); E – gastric mucosa showing the longitudinally sectioned pits and glands (LM, histology); F–H – detailed view of parasitised pits and glands in longitudinal section (LM, histology); I – detailed view of cross-sectioned gastric glands filled with parasites (LM, histology); asterisk – gastric pit; black arrowhead – cryptosporidia; m – mucosa; mm – muscularis mucosae; sm – submucosa; white arrow – mucus with cell debris; white arrowhead – cryptosporidia-free pit/gland.

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2,200,000

2,000,000

1,800,000

1,600,000

1,400,000

1,200,000

1,000,000

2,200,000

2,000,000

1,800,000

1,600,000

1,400,000

1,200,000

1,000,000

Day of treatment

Fig. 4. Infection dynamics of Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Or- tega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016 in Trial 1. Groups of eight-week-old Mastomys coucha (Smith) inoculated with a dose of 106 oocysts two months before treatment and subsequently treated daily for 14 days with 12.5 mg per 100 g of body mass of either Piper betle, Diospyros sumatrana or Arabidopsis thaliana extracted in methanol. The linear regression including regression coefficient is included for each experimental group. nol and Halocur (Fig. 4). In contrast, non-treated controls, 7A–I) neither helped to eradicate the parasites nor cured controls treated with pure DMSO and animals treated with the pathological changes induced by C. proliferans. When Piper betle and Arabidopsis thaliana extracted by meth- compared to non-treated controls (Fig. 3A–I), no signifi- anol exhibited an increase in oocyst shedding (Fig. 4). In cant difference was observed in parasitisation intensity or Trial 2, a decline in oocyst shedding occurred in groups in associated pathological alterations to the gastric mucosa treated for 21 days with A. thaliana by methanol-THF, D. in treated animals examined in SEM or histological prepa- sumatrana by methanol-THF and Halocur (Fig. 5). An in- rations. crease occurred in non-treated controls and animals treated Surprisingly, the application of a control with no expect- with pure DMSO, P. betle extracted by methanol, P. betle ed effect, the extract of A. thaliana (Fig. 8A–I), seemed by methanol-THF, P. betle by sterile water, A. thaliana by to have a positive impact on parasitised gastric mucosa, methanol A. thaliana by sterile water, D. sumatrana by especially when dissolved in methanol-THF (Fig. 8G–I). methanol, and D. sumatrana by sterile water (Fig. 5). The This was obvious especially in SEM preparations, where coefficient of determination, however, was low in all exper- the pathological folding of parasitised gastric tissue ap- imental groups, with the exception of non-treated controls peared less intense (Fig. 8G) when compared to those in and controls treated with pure DMSO in Trial 2 (Fig. 5). non-treated animals (Fig. 3A,C,D) and the effects of oth- Despite an obvious decrease in oocyst shedding in er treatments (Figs. 7A,D,G, and 8A,D). Some degree of some animals, post mortem histological examinations at this effect ofA. thaliana on parasitised gastric mucosa was the end of both trials revealed heavy cryptosporidiosis in also visible in histological sections; i.e. the architecture of all non-treated (Figs. 1–3) and treated animals (Figs. 6–9). the gastric glands was closer to the normal state and the Only histological sections from animals treated for 21 days epithelial cells lining the gastric glands were slightly less (Trial 2) are shown to demonstrate the status of parasitised atrophic (Fig. 8I). gastric tissue at the end of the trial. Histopathological data Although it slightly decreased oocyst excretion, even show that, independently of the solvent medium used, ex- prolonged treatment with Halocur was insufficiently effec- tracts from P. betle (Fig. 6A–I) and D. sumatrana (Fig. tive in treating gastric cryptosporidiosis (Fig. 9A–C). The

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3,500,000

3,000,000

2,500,000

2,000,000

1,500,000

1,000,000

3,500,000

3,000,000

2,500,000

2,000,000

1,500,000

1,000,000

3,500,000

3,000,000

2,500,000

2,000,000

1,500,000

1,000,000

3,500,000

3,000,000

2,500,000

2,000,000

1,500,000

1,000,000

Fig. 5. Infection dynamics of Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Or- tega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016 in Trial 2. Groups of eight-week-old Mastomys coucha (Smith) inoculated with a dose of 106 oocysts three months previously were treated twice a day for 21 days with a dose of 40.0 mg per 100 g of body mass of either Piper betle, Diospyros sumatrana or Arabidopsis thaliana extracted in methanol, ethanol-tetrahydrofuran (methanol-THF) or sterile water. The linear regression including regression coefficient is included for each experimental group.

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A B C

D E F

G H I

Fig. 6. The effect of Piper betle extract on Mastomys coucha (Smith) gastric mucosa parasitised with Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016 in Trial 2. A–C – P. betle in sterile water: A – superficial view of the gastric mucosa (SEM);B – general view of the gastric mucosa in longitudinal section (LM, histology); C – detailed view of cross-sectioned glands filled with parasites (LM, histology); D–F – P. betle in methanol: D – superficial view of the gastric mucosa (SEM); E – general view of the gastric mucosa in longitudinal section (LM, histology); F – detailed view of cross-sectioned glands filled with parasites (LM, histology); G–I – P. betle in methanol-THF: G – superficial view of the gastric mucosa (SEM); H – general view of the gastric mucosa and submucosa in longitudinal section (LM, histology); I – detailed view of cross-sectioned glands filled with parasites (LM, histology); asterisk – gastric pit; black arrowhead – cryptosporidia; m – mucosa; mm – muscularis mucosae; sm – submucosa; white arrow – mucus with cell debris; white arrowhead – cryptosporidia-free pit/gland.

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D E F

G H I

Fig. 7. The effect of Diospyros sumatrana extract on Mastomys coucha (Smith) gastric mucosa parasitised with Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016 in Trial 2. A–C – D. sumatrana in sterile water: A – superficial view of the gastric mucosa (SEM);B – general view of the gastric mucosa in longitudinal section (LM, histology); C – detailed view of tangentially-sectioned glands filled with parasites (LM, histology); D–F – D. sumatrana in methanol: D – superficial view of the gastric mucosa (SEM);E – general view of the gastric mucosa in longitudinal section (LM, histology); F – detailed view of cross-sectioned glands filled with parasites and neighbouring empty glands (LM, histology); G–I – D. sumatrana in methanol-THF: G – superficial view of the gastric mucosa (SEM); H – general view of the gastric mucosa in longitudinal section (LM, histology); I – detailed view of tangentially-sectioned glands filled with parasites (LM, histology); asterisk – gastric pit, black arrowhead – cryptosporidia; m – mucosa; mm – muscularis mucosae; sm – submucosa; white arrow – mucus with cell debris; white arrowhead – cryptosporidia-free pit/gland.

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D E F

G H I

Fig. 8. The effect of Arabidopsis thaliana extract on Mastomys coucha (Smith) gastric mucosa parasitised with Cryptosporidium proliferans Kváč, Havrdová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016 in Trial 2. A–C – A. thaliana in sterile water: A – superficial view of the gastric mucosa (SEM); B – general view of the gastric mucosa in longitudinal section (LM, histology); C – detailed view of cross-sectioned glands filled with parasites (LM, histology); D–F – A. thaliana in methanol: D – superficial view of the gastric mucosa (SEM); E – general view of the gastric mucosa in longitudinal section (LM, histology); F – detailed view of cross-sectioned glands filled with parasites (LM, histology); G–I – A. thaliana in methanol-THF: G – superficial view of the gastric mucosa (SEM); H – general view of the gastric mucosa in longitudinal section (LM, histology); I – detailed view of cross-sectioned glands filled with parasites (LM, histology); asterisk – gastric pit; black arrowhead – cryptosporidia; m – mucosa; mm – muscularis mucosae; sm – submucosa; white arrow – mucus with cell debris; white arrowhead – cryptosporidia-free pit/gland.

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Fig. 9. The effect of Halocur on Mastomys coucha (Smith) gastric mucosa parasitised with Cryptosporidium proliferans Kváč, Havr- dová, Hlásková, Daňková, Kanděra, Ježková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016 in Trial 2. A – superficial view of the gastric mucosa, halocur in PBS (SEM); B – general view of the gastric mucosa in longitudinal section, halocur in PBS (LM, histology); C – detailed view of cross-sectioned glands filled with parasites, halocur in PBS (LM, histology); asterisk – gastric pit, black arrowhead – cryptosporidia, m – mucosa, mm – muscularis mucosae, sm – submucosa, white arrow – mucus with cell debris, white arrowhead – cryptosporidia-free pit/gland. generally recommended dose for intestinal cryptosporidi- In addition, other pathogens might escalate the impact osis, corresponding to 100 µg/kg BM of halofuginone ad- of cryptosporidiosis on host tissue. For example, in mice ministered for seven consecutive days, was reported to be simultaneously infected with Cryptosporidium muris and effective in the past (Giadinis et al. 2008, Petermann et al. Helicobacter felis, the gastric glands were more severely 2014). In our study, the period of Halocur administration parasitised by cryptosporidia, and their stomachs showed was extended to 14 (Trial 1) and 21 days (Trial 2). Treat- more severe cellular infiltrates(Tatar et al. 1995). The fun- ed animals showed no symptoms of toxicity (no clinical dus glands of nude mice inoculated with strain RN 66 of C. signs of overdosing). However, SEM observations of the muris showed dilatation with mild epithelial changes (Tay- intensively folded surface of the gastric mucosa, with the lor et al. 1999), and only mice receiving an inoculum of presence of various developmental stages of C. prolifer- 1,000,000 oocysts showed an inflammatory reaction. ans, confirmed heavy infection (Fig. 9A). Histopathologi- A study using SCID mice reported only mild gas- cal changes (Fig. 9A–C) were very similar to those found tric cryptosporidiosis without gross pathologic findings in untreated individuals (Fig. 3A–I). The only difference, (Jalovecká et al. 2010). The maximum peak of parasitisa- noticeable at higher magnification, was the better preser- tion intensity was 21 DPI, whereas the highest numbers vation of epithelial cells lining the glands (Fig. 9C). These of immune cells occurred in the gastric epithelium at 28 cells appeared less atrophic with more preserved nuclei DPI, a period when the majority of mice had already been compared to non-treated animals or those treated with cured of the infection. Immunocompetent mice inoculated P. betle and D. sumatrana. with either C. muris CB03 or Cryptosporidium proliferans developed T cell responses leading to a clearance of the DISCUSSION primary infection and complete resistance to re-infection with the same strain (Jalovecká et al. 2010, Kváč et al. Histopathological changes related to gastric 2011). In contrast, the intensity of infection with C. pro- cryptosporidiosis liferans in experimentally infected Mastomys coucha con- Studies on animal gastric cryptosporidiosis usually re- tinued to increase throughout experiments with maximum port only mild histopathological changes represented by oocysts’ shedding at 126 DPI and animals developed life- the dilatation and epithelial metaplasia of gastric glands. long (chronic) infection (Melicherová et al. 2014, Kváč et These are generally reported without obvious alterations to al. 2016). host health status, and with no or only insignificant inflam- Despite the considerable gross pathology of gastric ep- matory responses in the lamina propria, although inflam- ithelium documented in this and previous studies (Meli- matory infiltrates are occasionally seen (e.g. Taylor et al. cherová et al. 2014, Kváč et al. 2016), neither clinical 1999, Masuno et al. 2006, Kváč et al. 2008). Nevertheless, signs of cryptosporidiosis, nor weight lost were observed there are obvious differences in histopathological changes in southern multimammate mice. In contrast to previous induced by different gastric species, load of parasite inocu- studies (Melicherová et al. 2014, Kváč et al. 2016), we ob- lum or immunological status of their host. served the inflammatory infiltration of muscularis mucosae

Folia Parasitologica 2018, 65: XXX Page 12 of 17 doi: 10.14411/fp.2018.XXX Screening of potentially anti-cryptosporidial agents and submucosa with neutrophils. Great variations in oo- been recommended to treat intestinal cryptosporidiosis in cyst shedding accompanied by heavy cryptosporidiosis re- calves with fluid therapy and the correction of acid-base vealed in histological sections of southern multimammate disturbances (Tzipori 1998). Colostrum containing an- mice stomach in this study are also rather inconsistent with ti-Cryptosporidium antibodies also appears to be benefi- the recent study by Kváč et al. (2016), in which high num- cial (O’Donoghue 1995). bers of cryptosporidian developmental stages were typical- More recent studies have reported halofuginone (Ha- ly associated with high oocyst shedding. locur) administration at the recommended dose of 100 µg/ In cattle parasitised by Cryptosporidium andersoni, de- kg for 7–10 consecutive days as very effective in stopping spite the lack of apparent clinical signs, severe infection diarrhoea and preventing deaths without side effects (Gia- was observed in the abomasum with prominent hyperpla- dinis et al. 2008, Petermann et al. 2014). At this dose, it sia of mucosa, along with a moderate degree of inflamma- appears to inhibit the reproduction of cryptosporidia with- tory infiltration of lamina propria (Masuno et al. 2006). in the host and encourages the development of immunity The number and length of gastric pits increased consider- in lambs (Causapé et al. 1999). Nitazoxanide, though not ably because of the increasing number of epithelial cells. effective in immunocompromised patients, significantly Thickening and granulation of abomasal mucosa were shortens the duration of diarrhoea and decreases mortality often reported (Anderson 1998). The epithelium of the in adults and malnourished children (Gargala 2008). stomach antrum in an immunocompetent human patient Similarly, newly synthesised nitro- or non nitro- thi- with isolated gastric cryptosporidiosis was shown to be azolide compounds, derived from nitazoxanide, have disorganised, fragile, and infiltrated by neutrophils (Ram- been shown to be effective against Cryptosporidium par- say et al. 2007). Gastric involvement in AIDS patients is vum (see Gargala 2008). Furthermore, compounds active usually considered to be secondary to retrograde spread against protein disulphide isomerases (PDI2 and PDI4), the from the small intestine (Val-Bernal et al. 2013). Relat- epidermal growth factor (EGF) receptor, pp60v-src, and ed to Cryptosporidium gastropathy, patients suffer from pp110gag-fes, as well as new isoflavone derivatives, seem vomiting and epigastric pain. The gastric wall might ex- to represent promising targets (Ortega-Pierres et al. 2009). hibit a lack of distensibility, stiffness, thickening, distor- SCID mice orally administered with the chicken egg yolk tion or erosions of the mucosal folds involving the antrum antibody against C. parvum infection demonstrated partial region (Val-Bernal et al. 2013). Regularly in the same bi- reduction in oocyst shedding (Kobayashi et al. 2004). opsy, areas with cryptosporidia were contiguous to neg- Whilst the majority of these studies have been per- ative ones. Various degrees of mucosal alterations were formed on intestinal cryptosporidia, usually C. parvum, observed, even in the same individual. Besides hyperplas- there is still a lack of experimental work dealing with the tic reactive changes, high intensity of infection correlates treatment of gastric cryptosporidiosis and most of the few with erosions and acute inflammation. Commonly, indi- published works deal with the treatment of AIDS patients. viduals with gastric cryptosporidiosis show no significant One of these papers has shown a positive effect of para- endoscopic alteration to the gastric mucosa, even though momycin on reducing inflammation of the gastric mucosa histological features are highly modified. In this and pre- and mild relief from pain and diarrhoea, despite parasite vious studies (Melicherová et al. 2014), we also observed persistence in mucosa (Ventura et al. 1997). Another study a patchy (island-like) distribution of C. proliferans. Sim- reported the eradication of AIDS-related gastric crypto- ilarly to the course of cryptosporidiosis in other homoio- sporidiosis with azithromycin and suggested long-term therm vertebrates, the rodents in our study did not show treatment (Dı́az Peromingo et al. 1999). any clinical signs of cryptosporidiosis. In our study, none of the Indonesian plant extracts were shown to be effective against gastric cryptosporidiosis, de- Trends in drug development and screening studies spite their proven activity against other protists parasitising testing potentially anti- cryptosporidial compounds the small intestine (Pecková et al. 2018). For the first trial, Cryptosporidium spp. represent a highly problematic the dosage was calculated based on behavioural observa- target for drug development. One of the self-protective tion of self-medication of wild animals (e.g. the number of strategies of cryptosporidia against the harsh conditions plant leaves consumed), but the dosage for the second trial of the host’s gastrointestinal tract is their unique epicel- was increased to 40 mg to increase the potential antipara- lular localisation within a parasitophorous sac of host cell sitic effect. This dosage was calculated based on maximum origin (Valigurová et al. 2007, 2008). The oocysts of cryp- concentrations of extracts reported in the literature (e.g. tosporidia sporulate inside the host and infective oocysts Bin-Hafeez et al. 2003, Squires et al. 2011). Although ad- are transmitted by the faecal-oral route. Besides the most ditional assays with different doses in a wide scale would commonly used antibiotics and halofuginone, numerous be of interest, such an extensive experiment would be too compounds have been screened for potential anti-cryp- demanding of time and material, especially in the number tosporidial activity, but the majority were ineffective. of laboratory rodents required. Hence, respecting the rules Although some drugs have shown promise in calves and for breeding animals, regulated by Czech legislation and lambs, they are too expensive (paramomycin) or highly the legislation of the European Commission on protection toxic at effective doses (halfuginone lactate and lasalocid) of animals against cruelty, we have designed our experi- (Tzipori 1998). Therefore, along with antibiotics admin- ments so that we do not use too many laboratory rodents istered to control secondary bacterial infections, it has unnecessarily, as do most of the world’s laboratories.

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The oral administration of Halocur – even for a pro- limitations in the screening of anti-cryptosporidial longed period – was not sufficiently effective either, though drugs. it slightly decreased the degree of oocyst shedding and During the course of our experiments, we found a num- seemed to facilitate the regeneration of epithelial cells lin- ber of unexpected difficulties and limitations. One of these ing the gastric pits. Nevertheless, in contrast to previous re- is the need for sufficient (preferably more than estimated) ports, it did not seem to inhibit the reproduction of crypto- stocks of tested plant extracts for individual trials, which sporidia, although some slight decrease might be observed can be a problem in screening studies using exotic plants in the trendline showing oocyst excretion (Figs. 4, 5). Sim- where their availability and quantity might be strictly lim- ilarly, halofuginone treatment did not produce a satisfac- ited (such as those used in our study). Primary screening tory therapeutic outcome for infection with Cryptosporid- of experimental protocols, therapeutic doses and the effec- ium serpentis Levine, 1980 affecting the gastric mucosa of tiveness of selected plant extracts in various solvent media snakes (Graczyk et al. 1996), suggesting its ineffectiveness consumes a lot of material before starting the animal ex- against gastric cryptosporidiosis. periments. Therefore, for pilot screening of the antiparasit- Of special interest, however, was the positive impact of ic effect, it is preferable to use an in vitro system, at least Arabidopsis thaliana on the gastric mucosa pathologically for parasites where cultivation is possible (e.g. C. parvum). altered by chronic cryptosporidiosis. Arabidopsis thaliana In vitro studies require smaller volumes of plant extracts is a small Eurasian annual flowering plant routinely used and this approach helps to minimise the number of animals as an important model plant in molecular biology research used and to reduce their distress during experiments. and is reported to be edible (Lindh et al. 2008, Hansson et Another issue was the variability of the course of infec- al. 2016). The extract from the green parts (leaves) of this tion in tested animals inoculated at the same doses, result- plant was used as a control and we expected it to have a ing in variations in oocyst shedding. Similarly to Sréter et neutral effect on animal health and its parasitised gastric al. (1995) relatively small numbers of animals are required tissue, as we found only a few studies reporting the posi- for the estimation of the length of the prepatent period, but tive effect ofA. thaliana seed extract. large numbers of animals are needed for the estimation of The use of plant-derived products as antimicrobial the mean of oocyst excretion. Additionally, histological agents has been investigated in depth. Isothiocyanates observations of treatment effects during early stage cryp- (ITCs) are bioactive products resulting from enzymatic tosporidiosis can be misleading, due to mild histopatho- hydrolysis of glucosinolates (GLs), the most abundant sec- logical changes and the patchy occurrence of the parasite. ondary metabolites in the plant order Brassicales. Although Promising reports from studies focusing on anti-crypto- the antimicrobial activity of ITCs against foodborne and sporidial drug development are usually based on a reduc- plant pathogens has been well documented, little is known tion in oocyst shedding. The results of this study, however, about their antimicrobial properties against human path- indicate that the evaluation of parasitisation intensity based ogens (Romeo et al. 2018). Concurrently, during the trial exclusively on the number of oocysts shed in faeces can be finalisation in this study, an unexpected positive effect of misleading. For example, despite a decline in oocyst shed- A. thaliana plant extract on the reduction of spores of mi- ding in some treatment groups (including those adminis- crosporidian Encephalitozoon cuniculi Levaditi, Nicolau tered with Halocur), all known developmental stages of et Schoen, 1923 in the tissues of experimentally inoculated C. proliferans, from early stages invading epithelial cells, BALB/c mice was documented (Mynářová 2015). Differ- or freshly attached to the epithelium surface to oocysts en- ent genetic programs, activated upon pathogen recognition veloped by a parasitophorous sac, were observed in corre- and leading to the production of inducible antimicrobial sponding SEM and histological preparations. This means compounds, have been identified in this plant (Tierens et that despite the fact that fewer oocysts were excreted in al. 2001). Using the fungus Neurospora crassa as a test host faeces, their development did not stop. Recently, it has organism, Tierens et al. (2001) analysed the antimicrobial been shown that rats infected with Cryptosporidium occul- compounds from aqueous extracts of leaves of Arabidopsis tus Kváč, Vlnatá, Ježková, Horčičková, Konečný, Hlásk- and suggested their role in the plant’s protection against ová, McEvoy et Sak, 2018 shed fewer oocysts than would some pathogens. The treatment of mice with Alloxan-in- be predicted from the massive infection of the colonic ep- duced diabetes with A. thaliana at a dose of 200 mg/kg BM ithelium (Kváč et al. 2018). This could be explained by led to a significant reduction in blood glucose levels and the presence of two types of oocysts, i.e. thick- and thin- an improvement in insulin resistance (Rashid et al. 2013, walled, in life cycle of C. proliferans and other species/ 2014; Taha et al. 2014). The consumption of A. thaliana genotypes (Current and Reese 1986, Uni et al. 1987, Meli- reversed most of the histological changes in the liver of the cherová et al. 2014). It is likely that the treatment simply diabetic mice, stimulated protein synthesis by increasing induces increased production of thin-walled oocysts, which the number of ribosomes and significantly reduced oxida- excyst once they separate from host epithelium, are not tive stress in diabetes (= antioxidant effect) (Rashid et al. usually excreted in faeces and appear to be responsible for 2014). Moreover, as A. thaliana plants have a close rela- autoinfection. The multiplication of cryptosporidia inside tionship with species of Brassica eaten by humans it is of the same host via autoinfective oocysts (= sexual stage) particular interest with respect to further investigations. appears to be beneficial for increasing parasite genetic var- iability, and thereby fitness and infectivity(Melicherová et Methods used for treatment efficacy evaluation: al. 2014). Alternatively, the drug-affected parasite could

Folia Parasitologica 2018, 65: XXX Page 14 of 17 doi: 10.14411/fp.2018.XXX Screening of potentially anti-cryptosporidial agents begin investing in asexual multiplication and undergo mul- rately assess anti-cryptosporidial treatment efficacy in lab- tiple rounds of merogony I to produce high numbers of in- oratory-housed animals, the most reliable and economic vasive merozoites. Autoinfection and the recycling of type approach seems to be the monitoring of oocyst shedding I merogony provide an explanation for persistent chronic accompanied by histological (gastric tissue) or SEM anal- infections (Bouzid et al. 2013). ysis (applicable for intestinal tissue) of the parasitised ep- A further problem in similar studies is the correct choice ithelium post mortem. Although such an approach is not of microscopic techniques for screening for parasite pres- applicable for medical purposes or for studies dealing with ence and morphopathological changes of the parasitised livestock, experimental studies on small laboratory animals tissue. The surface topology detectable by SEM is insuffi- based on the most accurate evaluation would provide im- cient for gastric cryptosporidia if not supplemented by his- portant information on the actual effect of the drug tested. tological sectioning as the mucus, a thick substance natu- rally produced by surface cells and cells of the gland necks Acknowledgements. The authors would like to acknowledge support from the Department of Botany and Zoology, Faculty to prevent self-digestion of the gastric mucosa, might of Science, Masaryk University, towards the preparation of this hamper the view inside the gastric gland. This relatively manuscript. The authors also to thank the State Ministry of Re- thick layer, forming a non-transparent film after chemical search and Technology (RISTEK) and the Directorate General of fixation, is usually almost impossible to wash -away, de Forest Protection and Nature Conservation (PHKA) for their co- spite the careful and repetitive rinsing of stomach tissue operation and for their permission to conduct research in Gunung (Melicherová et al. 2014). In addition, SEM analyses did Leuser National Park. The study was financially supported by the not prove helpful in evaluating parasitisation intensity of ʻUMI – Saving of Pongidaeʼ Foundation project ʻParasites and gastric tissue, as it did not enable close examination of Natural Antiparasitics in the Orang-utanʼ and by the Czech Sci- constricted or only slightly dilated pits. Transmission elec- ence Foundation (Grant No. P505/11/1163), by the Ministry of tron microscopy represents a powerful tool for evaluating Education, Youth, and Sports, Czech Republic (Grant LO1204 pathological aspects along with the presence of parasites, from the National Program of Sustainability I). The authors would also like to thank Mallory Abel for editing and formatting but it is too expensive and time consuming for studies not the final draft. focusing on ultrastructural aspects. Hence, to more accu-

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Received 19 September 2017 Accepted 31 May 2018 Published online xxx 2018

Cite this article as: Valigurová A., Pecková R., Doležal K., Sak B., Květoňová D., Kváč M., Nurcahyo W., Foitová I. 2018: Lim- itations in the screening of potentially anti-cryptosporidial agents using laboratory rodents with gastric cryptosporidiosis. Folia Parasitol. 65: XXX

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