1

Captain’s Room: Marina Village Center: San Diego, CA- Feb 9-12

Gold Sponsors:

Silver Sponsors:

2

Oral Papers

Peter J. Roy 1,2,3, Andrew R. Burns 1, Genna M. Luciani 1,2, Gabriel Musso 4, 5, Luckshika Rajendran 1, Andy G. Fraser 1, Calum A. MacRae 4,5, John Gilleard 6

1. The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, M5S 3E1, Canada. 2. Department of Molecular Genetics, University of Toronto, Toronto, ON, M5S 1A8, Canada. 3. Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, M5S 1A8, Canada. 4. Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, and Harvard Stem Cell Institute, Boston, MA 02115, USA. 5. Department of Medicine, Harvard Medical School, Boston, MA, 02115, USA. Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, T2N 4Z6, Canada

1 THE NEMATODE CAENORHABDITIS ELEGANS AS A MODEL FOR ANTHELMINTIC LEAD DISCOVERY

Primary screens for novel anthelmintic lead molecules that rely on parasitic worms are costly and labor intensive. Here, we explore the utility of the non-parasitic nematode Caenorhabditis elegans as a primary model system to identify potential anthelmintic leads. We screened 67,012 compounds and identified 275 that reproducibly kill C. elegans. We rescreened these hits in two parasitic nematode species and two vertebrate models, and identified 30 structurally distinct potential anthelmintic leads. Genetic screens of over 27 million C. elegans mutants reveal those nematicides for which the generation of resistance may or may not be likely. Through these genetic screens, we discovered the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for the discovery of molecules with anthelmintic potential.

Mostafa Zamanian, Nick Irons, Sam Rosenberg, Robyn Tanny, and Erik Andersen

Department of Molecular Biosciences, Northwestern University, Evanston, IL

2 CAENORHABDITIS ELEGANS QUANTITATIVE GENETICS PIPELINE UNCOVERS COMPLEX DETERMINANTS OF NEMATODE

AVERMECTIN RESISTANCE.

Parasitic nematodes infect over 1.5 billion people worldwide. The morbidity and mortality inflicted by these devastating

pathogens are partly curtailed by well-organized mass drug administration programs that depend on the continued

efficacy of a limited portfolio of anthelmintic drugs. Avermectins are a class of broad-spectrum anthelmintics that are an

indispensable component of this limited arsenal. Parasite resistance to avermectin chemotherapy is widespread in

veterinary medicine and constitutes an emerging threat in human medicine. We have mapped quantitative trait loci

(QTL) associated with avermectin resistance using a statistical and quantitative genetics pipeline that exploits natural

variation in the model nematode Caenorhabditis elegans, including a population of 598 unique recombinant inbred lines

created from an advanced intercross of the laboratory strain (N2) with a wild strain from Hawaii (CB4856). These two

strains are genetically distant and differ in avermectin response. We have resolved single-nucleotide and insertion-

deletion variants distinguishing the parental strains and have defined complete genotypes for the entire recombinant

panel. Avermectin exposure phenotypes were amassed for all recombinant lines using a robotic platform that captures

a range of animal fitness and behavioral traits. Data resulting from mapping experiments recapitulate a QTL containing

a known avermectin target (glc-1) and led to the exciting discovery of five other independent QTL that significantly

contribute to variation in avermectin susceptibility. We built nearly isogenic lines (NILs) to help narrow these QTL and to

identify individual resistance genes and variants. These data present a promising pathway towards the identification of

robust genetic markers for avermectin resistance conserved in medically important parasites.

3

George von Samson-Himmelstjerna, Jürgen Krücken

Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Berlin, Germany

3 ANTHELMINTIC RESISTANCE – RECENT FINDINGS AND FUTURE THREATS

Worm control measures in humans and animals still fundamentally rely on application of a limited number of

anthelmintic drug classes and for most indications only very few choices are available. Following the longstanding and

often highly frequent use of drugs against several parasite species, nowadays certain drug classes cannot be considered

as sufficiently effective due to widespread occurrence of anthelmintic resistance (AR). This is a particular problem for the

benzimidazoles (BZs) and gastro-intestinal nematodes of small ruminants and horses. However, acetylcholine receptor

and glutamate-gated chloride channel agonists such as imidazothiazoles and macrocyclic lactones, respectively, are

increasingly affected by AR. Also monepantel, the only available compound of the recently developed amino-acetonitrile-

derivatives, faced AR in field studies. In humans, only very few studies have reported suspected AR e.g. of BZs against

hookworms and reports of poor ivermectin efficacy against Onchocerca volvolus. However, the increasing treatment

frequencies due to mass drug administration programs raise concern about future efficacy of these drugs against human

gastro-intestinal nematodes (GIN) and filaria. In veterinary helminthology, significant efforts aimed to understand

mechanisms and evolution of AR, to optimize AR detection and to evaluate strategies for prevention or reversal of AR.

Mechanisms of AR can be drug-target associated or related to drug efflux or metabolism. Only for very few drug/parasite

combinations, target associated resistance mechanisms have been described molecularly and employed as AR markers.

Novel functional and genomic tools have recently provided first insight into anthelmintic – transmembrane efflux pump

interactions. However, there is still a great paucity of detailed knowledge about the AR mechanisms for most parasite

species already displaying AR and much less is known regarding parasites for which AR is a future concern such as human

GINs. This presentation will highlight recent AR-related discoveries and provide suggestions for a One Health orientated

application of AR research findings.

Malina A. Bakowski 1; Kerstin Gagaring 1; Pamela M. White 2; Alain Debec 2; Frédéric Landmann 3; William Sullivan

2; Peter G. Schultz 1; Case W. McNamara 1;

1 California Institute for Biomedical Research, La Jolla, CA; 2 UC Santa Cruz, Santa Cruz, CA; 3 CRBM, Montpellier, France.

4 IDENTIFICATION OF ANTI-WOLBACHIAL COMPOUNDS TO CURE PARASITIC WORM DISEASE

Filarial nematode infections, namely onchocerciasis and lymphatic filariasis, infect over 200 million people worldwide

and cause significant morbidity, yet no drugs exist that kill their long-lived (average 15 years) adult stages (macrofilariae).

To identify safe and effective macrofilaricidal compounds we screened 180,000 proprietary small molecules to discover

inhibitors of Wolbachia, a bacterial endosymbiont required for adult worm fertility and viability. We carried out high-

throughput screening in 1,536-well format by adapting and miniaturizing a high-content imaging assay using Drosophila

cells infected with Wolbachia wMel. Mainly, we developed a Wolbachia 16S rRNA-specific fluorescent in situ

hybridization (FISH) staining protocol with a corresponding imaging algorithm to quantify the amount of Wolbachia in

infected cells. Using this approach, we identified 97 high-quality hit

selectivity. To validate our primary hits, we developed a complementary FISH-based ex vivo assay, wherein the amount

of Wolbachia wBp in ovaries dissected from drug-treated Brugia pahangi worms is quantified. This assay allowed us to

determine which of our candidate compounds were ineffective against filarial Wolbachia because they: 1) target a

Drosophila cell-specific host factor, 2) target a Wolbachia wMel strain-specific factor, or 3) cannot achieve a sufficient

concentration in the worm due to limited permeability or compound efflux. Invaluably, this workflow allowed us to

prioritize five hit-to-lead candidates with significant activity against filarial Wolbachia for exploratory medicinal chemistry

and pharmacokinetic studies, prior to an extended assessment in an in vivo efficacy model.

Funding for this research was provided by the Bill and Melinda Gates Foundation (Grant # OPP1107194).

4

David B Sattelle 1; Freddie A Partridge 1; Emma A Murphy 2; Nicky J Willis 3; Bruno Marinic 3; Daniel Sowood 3;

Graham M Wynne 3; Katherine J Else 2; Angela Russell 4,5.

1UCL Respiratory, Division of Medicine, University College London, The Rayne Building, 5 University Street, London WC1E

6JF, UK; 2Faculty of Life Sciences, University of Manchester, AV Hill Building, Oxford Road, Manchester M13 9PT, UK;

3Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, OX1 3TA, UK; 4Department of Pharmacology,

University of Oxford, Mansfield Road, OX1 3QT, UK.

5 DISCOVERING NEW CHEMICAL LEADS FOR THE CONTROL OF WHIPWORM, A NEGLECTED HUMAN PATHOGEN FOR

WHICH CURRENT TREATMENTS ARE INADEQUATE

The parasitic nematode Trichuris trichiura (whipworm) affects ~500 million people, resulting in disability and poor child

development. The World Health Organisation currently identifies 112 countries in need of preventative chemotherapy,

primarily in South and Central America, Asia and Africa

(http://apps.who.int/iris/bitstream/10665/44804/1/9789241503129_eng.pdf). Current anthelmintic treatments show

poor efficacy with low cure rates, thereby restricting the success of Mass Drug Administration programmes. The limited

anthelmintic drug pipeline, the growth of resistance to existing chemicals and the absence of a vaccine means that new

drugs are urgently needed. We have screened a focused library of novel drug-like molecules in order to identify new hit

classes of compound which demonstrate activity on the ex vivo model mouse parasite Trichuris muris. T. muris and T.

trichiura are similar genetically, morphologically, antigenically and physiologically and provoke similar immune responses

in their respective host species. T. muris is easily maintained in the laboratory mouse and thus provides us with a highly

relevant model for our screening pipeline. The work has involved high-throughput chemical screens using Wormwatcher,

a device developed for automated nematode phenotyping. Using this approach we have identified two novel chemical

classes which show exciting levels of activity, and importantly have no compromising toxicity in initial cell-based toxicity

and host tolerance tests. A programme of medicinal chemistry for hit-to-lead optimisation of both sets of molecule is

currently being pursued. The screening process for compound optimisation will utilise both in vitro assays on ex vivo T.

muris worms and in vivo assays to demonstrate effective parasite clearance from the host, coupled with a clear safety

profile and good host tolerance. Our goals are to develop new chemical leads, new biological tools and to identify new

drug targets, all of which are essential in developing new drug candidates for the unmet clinical need of an effective

treatment for Trichuriasis.

Angela Mousley, Louise Atkinson, Paul McVeigh, Ciaran McCoy, Neil Warnock, Johnathan Dalzell, Nikki Marks, Aaron

Maule

Parasitology Research Group, Institute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, Belfast, UK

6 NEMATODE NEUROPEPTIDES: FROM SEQUENCE TO BIOLOGY AND BACK We have maintained a long-standing interest in helminth neurobiology at Queen’s University Belfast that stemmed from

the early work of Professor D.W. Halton, and continued through research activity in the Maule and Marks laboratories. In

the early days, localisation of neuropeptides through immunocytochemical techniques in association with classical

biochemical characterisation methods, PCR-based gene detection tools, and muscle-based functional assay systems.

provided data on the importance of neuropeptides to helminth biology, and flagged the candidature of the

neuropeptidergic system as an anthelmintic target. Despite this, our understanding of the biology of neuropeptides and

their signalling systems, especially in key therapeutically relevant pathogens, is limited. The recent growth in genomic

and transcriptomic datasets for parasitic helminths, and positive shift in the cost and accessibility of gene sequencing

technologies, provides a welcome gateway to the identification of neuropeptides, their receptors and pathway

components. In addition, progress in the development of parasite-focused reverse genetics tools offers the ability to

manipulate target function in key pathogens and opportunities for novel anthelmintic discovery. This presentation will

provide an overview of the efforts to progress the identification and validation of neuropeptidergic system-derived drug

target candidates in nematode parasites, and highlight recent advances in our understanding of neuropeptide biology

across the phylum Nematoda.

5

Daniel Kulke

Bayer Health Care, Leverkusen, Germany

7 DEVELOPMENT PROGRAM OF EMODEPSIDE FOR THE TREATMENT OF HUMAN ONCHOCERCOSIS - A CONSEQUENCE OF

FRUITFUL COLLABORATIONS BETWEEN INDUSTRY AND ACADEMIA

Current mass drug administration programs (MDAs) for the treatment of human river blindness (Onchocercosis) solely

rely on the utilisation of macrocyclic lactones (ivermectin). These treatments are directed against the migrating

microfilariae of Onchocerca volvulus. Despite the outstanding effects against microfilariae, ivermectin is not effective

against adult O. volvulus, resulting in the imperative necessity of treatments at regular intervals for multiple years.

Introduction of a macrofilaricide could drastically reduce the duration of these MDAs. Emodepside is a broad-spectrum

anthelmintic used for the treatment of parasitic nematodes in cats and dogs (Profender®; Procox®). More than two

decades of intensive collaborative research in academia and pharmaceutical industry led to our current, complex

understanding of this anthelmintic compound: In 1992, the anthelmintic efficacy of PF1022A was described. By

increasing solubility and improving bioavailability, emodepside let to superior efficacy against extra-intestinal nematode

stages. It showed full efficacy against several nematode isolates being resistant to other anthelmintics, suggesting a novel

mode of action. Besides the involvement of LAT-1 in emodepside-associated inhibition of pharyngeal pumping, the

large-conductance calcium-activated and voltage-gated potassium channel SLO-1 was identified as essential for

emodepside susceptibility of nematodes. Several electrophysiological experiments demonstrated amongst others, that

emodepside directly opens nematode SLO-1 and that this modulation is significantly different from the modulation of

insect and human SLO-1 channels. Emodepside was also found to be effective against L3, L4 as well as preadult and adult

filariae in vitro (including adult Onchocerca gutturosa) and in vivo. In addition, levels of microfilaraemia were significantly

reduced in vivo (including Onchocerca lienalis in mice). Therefore, emodepside is frequently considered to be one of the

most promising candidates for evaluation as an adulticide treatment of human Onchocercosis. As a logical consequence

Bayer HealthCare and the Drugs for Neglected Diseases initiative (DNDi) entered into a collaboration to develop

emodepside for the treatment of patients suffering from Onchocercosis.

Christopher Hopkins 1; Trisha Brock 1; Thomas Marshall 1; Gongping He 1; Adrian Wolstenholme 2;

1AxumBio; 2University of Georgia

8 A DRUG DISCOVERY PLATFORM FOR FINDING COMPOUNDS ALTERING PARASITE MOLTING BEHAVIOR

Finding drugs against new anthelmintic targets will broaden the diversity of treatment options. Like the ecdysone

receptor in insects, a focus on steroid hormone receptors in C. elegans allows targeting of parasite developmental as

new pathway for controlling the spread of parasitic infections. DAF-12 is a tractable NHR target because a natural ligand,

dafachronic acid (DA), is known to be responsible for both 1) controlling entry into and out of dauer larvae in C elegans

and 2) controlling formation of il3 infective-stage larvae in Strongyloides stercoralis and Ancylostoma caninum (Wang et

al. 2009, doi: 10.1073/pnas.0904064106). Expression profiling of DA activity reveals multiple genes are induced in C.

elegans. The result is multiple daf-12 target genes are available for reporting a nematode’s molt progression into either

dauer or vegetative-growth lifestates. To create a high-throughput drug discovery platform, the promoters of DA

response genes are fused to the coding sequences of fluorescent reporter proteins. Introduction of a reporter into the

C. elegans genome creates a stable biosensor that is capable of reporting activity in a plate-reader format. Using

commercially sourced ligands, a lead compound, 5H0915 was identified with activity similar to the native DAF-12 ligand.

Structure-Activity-Relationship analysis on the lead is being used as a scaffold for the discovery novel entities with higher

ligand-binding activity. Biosensor platform customization with co-transgenic expression of parasite daf-12 homologs

allows rapid discovery of compounds with activity to a wide variety of parasites. In summary, transgenic expression of

parasite genes in C elegans creates a powerful platform for discovery of novel anthelmintic with broad-spectrum activity.

6

C. Scott Knauer; Debra J. Woods;

Zoetis Kalamazoo, MI

9 ANTHELMINTIC DRUG DISCOVERY: TURNING HITS INTO DRUGS

Parasitic nematode infections lead to large economic losses in livestock production every year. With the emergence of

resistance against all commonly used drugs, it has become increasingly difficult to control infections with the available

anthelmintics (e.g. macrocyclic lactones). Therefore, it has become vitally important to identify drugs with distinct

chemotypes and novel modes of action to combat the current resistance problem. We have previously discussed our

approach toward discovering novel anthelmintic “hits” using recombinantly expressed targets and high-throughput

quantitative motility assays. While these tools serve a critical role in discovering novel chemotypes, the identification of

hit compounds is only the first step in a very long drug discovery and development process. Progressing beyond in vitro

nematode activity to drug candidates involves building a larger understanding of in vivo safety, in vivo

pharmacokinetic/pharmacodynamics, formulation, manufacturing, commercial considerations, etc. Specific examples

will be shown to illustrate the importance of developing our scientific understanding while considering the commercial

attractiveness, in order to progress hits into novel anthelmintic drugs.

Liang Dong 1; Richard J. Martin 2;

1 Department of Electrical and Computer Engineering, Iowa State University; 2 College of Veterinary Medicine, Iowa State

University

10 LAB-ON-A-CHIP TECHNOLOGY FOR RAPID, DEEP, AND ACCURATE SCREENING OF DRUG RESISTANCE OF NEMATODES

The development of drug resistance in various nematodes has spurred a great demand for high-throughput techniques

for screening multiple drug compounds against targeted parasitic nematodes. Existing molecular mechanisms for

detecting altered genotype associated with drug resistance are time-consuming and do not easily translate to observable

changes in phenotype. Microfluidics is an attractive technology with the potential to streamline workflows and processes

in the biomedical and health sciences. Due to the small size of microscopic nematodes, there is growing interest in

studying nematodes in the areas of microfluidics and lab on a chip. This presentation will describe the efforts of a

multidisciplinary team based at Iowa State University to develop on-chip assays for screening of drug resistance of

nematode pathogens. The developed microfluidic devices are capable of measuring different parameters (motility,

electrophysiology, and biomechanics), allowing for deep and accurate phenotyping of parasitic nematodes. First, I will

present a microscale droplet-based testing ground to study how nematode parasites respond to different drug

conditions. The device is advantageous as it provides a large number of data sets in each dose response curve by

automated generation of different drug environments and exposure of worms to these environments. Second, I will

present a lens-less and image-sensor-less approach for real-time monitoring of the locomotion of microscopic

nematodes. The technology has shown promise for overcoming the constraint of the limited field of view of conventional

optical microscopy, with low cost, good spatial resolution, and high portability. Third, I will present an opto-fluidic sensor

for measurement of nematode's muscular force in response to different drug environments. The ability of the sensor to

quantify the muscular forces provides a new angle to screen different mutants at single animal resolution. Finally, I will

present a biochip device capable of immobilizing microscopic worms for detecting electropharyngeogram of nematodes.

7

Janis C. Weeks 1,2; William M. Roberts 1,2; Kristin J. Robinson 1; Melissa Keaney 3; Jon J. Vermeire 4; Joseph F.

Urban 5; Shawn R. Lockery 1,2; John Hawdon 3.

1. Institute of Neuroscience, University of Oregon, Eugene, OR; 2. NemaMetrix Inc., Eugene OR; 3. Research Center for

Neglected Diseases of Poverty, The George Washington University, Washington DC; 4. UC San Francisco, San Francisco,

CA; 5. US Dept. of Agriculture, Agricultural Research Service, Beltsville MD.

11 A MICROFLUIDIC SCREENING PLATFORM USING ELECTROPHYSIOLOGICAL RECORDINGS FROM PARASITIC LARVAL

STAGES OF HOOKWORM (ANCYLOSTOMA CEYLANICUM) AND ROUNDWORM (ASCARIS SUUM).

The screening of candidate compounds and natural products for anthelmintic activity is a key component of discovering

new drugs against human and animal parasites. We previously validated in Caenorhabditis elegans a microfluidic device

(‘chip’) that records non-invasively the tiny electrophysiological signals generated by rhythmic contraction (pumping) of

the worm’s pharynx. These recordings, called electropharyngeograms (EPGs), are made from 8 worms simultaneously

and provide an automated, medium-throughput quantification of muscular and neural activity at millisecond resolution.

C. elegans is useful for high-content screening and molecular genetics, but has significant physiological differences from

parasitic nematodes. The current study tested the suitability of Ancylostoma ceylanicum (hookworm) and Ascaris suum

(roundworm) nematodes for use with the EPG platform; A. ceylanicum is a human parasite and A. suum is a zoonotic

model for the human parasite, A. lumbricoides. Microchannel dimensions in the EPG chips and culture medium

components were customized for parasitic species and chips were maintained near mammalian body temperature during

recordings. Fourth-stage larvae (L4s) of A. ceylanicum removed from the intestines of hamster hosts exhibited robust,

persistent EPG activity suitable for compound screening. This activity did not require the presence of serotonin (5-

hydroxytryptamine; 5HT). The anthelmintic drug ivermectin (IVM) inhibited EPG activity, as did aqueous extracts of bitter

melon leaves, Momordica charantia, a traditional anthelmintic in Haiti. We also recorded robust EPG activity in A. suum

L3s removed from pig lung and treated with 5HT, which was inhibited by IVM. These experiments validate the use of A.

ceylanicum L4s and A. suum L3s with the microfluidic EPG platform, providing a new tool for anthelmintic drug screening

or studies of parasitic nematode feeding behavior. A commercial microfluidic EPG platform customized for parasitic

nematodes is under development at NemaMetrix Inc. (www.nemametrix.com). Support: Bill & Melinda Gates

Foundation, Grand Challenges Explorations award to JCW.

Rahul Singh

Department of Computer Science, San Francisco State University

12 TOWARDS QUANTITATIVE, AUTOMATED, AND DEEP PHENOTYPING

One of the key challenges in drug discovery against helmintic diseases lies in the development of methods that allow

analysis of complex phenotypes elicited through genetic or chemical perturbations of the parasites. Ultimately, such

perturbations disrupt the coordinated expression and interaction of various molecules that underpin parasite biology.

Consequently, the elicited phenotypic response reflects changes in the complex network of molecular interactions within

the parasite. Therefore, the ability to quantify, analyze and reason with such phenotypes can be crucial in a number of

contexts including analysis of data from phenotypic screens for lead identification, QSAR modeling based on phenotypic

responses, phenotypic-similarity-based drug repositioning, and system modeling based on genotype-to-phenotype

relationships. This talk will focus on the computational aspects of an interdisciplinary research effort to this problem.

Specifically, we will look at technological solutions developed by us that enable quantitative, automated and highly

sensitive detection, quantification, and analysis of phenotypes exhibited by the etiological agents of various helmintic

diseases. Using schistosomiasis as the focus, I will present a number of algorithmic results and software packages created

for public use that can be employed by researchers to quantify and analyze complex parasitic phenotypes and facilitate

both drug discovery as well as fundamental biology-research.

8

Conor R. Caffrey1; Steven Chen2; Brian M. Suzuki1; Rahul Singh3; Michelle R. Arkin2

1Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences,

University of California San Diego, La Jolla, CA; 2Small Molecule Discovery Center, Department of Pharmaceutical

Chemistry, University of California San Francisco, San Francisco, CA; 3Department of Computer Science, San Francisco

State University, San Francisco, CA

13 “SHAKE, RATTLE AND ROLL:” LEARNING HOW THE SCHISTOSOME RESPONDS TO DRUGS

Automated high-throughput and/or high-content drug screening (HTS/HCS) platforms for protozoan parasites are now

well-established. In contrast, such platforms for large worm parasites are lacking. Using the schistosome flatworm as a

particularly challenging example, we developed an HTS/HCS platform to quantify the chemically induced responses of

Schistosoma mansoni. I will describe the hurdles overcome to standardize the preparation and handling of the parasite.

These include interfacing it with an automated instrumentation environment, incl. image acquisition, object

segmentation and tracking, and feature extraction. We tested the platform with seven anti-schistosomal drugs to

measure a range of static and dynamic parasite responses as a function of time and concentration. We developed a user

interface to visualize and interrogate the data, which are maintained in a customized database. We combined the high-

dimensional data into a single metric output suitable for primary first-pass library screening: data from screening

campaigns of high-value small molecule collections will be presented. The platform increases throughput, improves rigor

and will support the identification of drugs, drug targets and mechanisms of action. Supported by NIH R01AI089896 and

R21AI107390.

Marion David 1, 2; François André 3; Stéphane Orlowski 3; Roger K. Prichard 2; Anne Lespine 1

1 INRA, UMR 1331, Toxalim, Research Centre in Food Toxicology, F-31027 Toulouse, France; 2 Institute of Parasitology,

McGill University, Montreal, Canada; 3 CEA, iBiTecS/SB2SM and CNRS UMR9198, I2BC, CEA-Saclay, 91191, Gif-sur-Yvette,

France.

14 IN SILICO ANALYSIS OF MACROCYCLIC LACTONES BINDING TO CEANORHABDITIS ELEGANS P-GLYCOPROTEIN 1

Anthelmintics (AHs) remain the most effective means to treat nematode infections in animals and humans. However, AH

resistance has arisen. P-glycoproteins (Pgp) belonging to ATP-binding cassette family transporters are known to be

involved in resistance to anti-infectious drugs in many organisms including parasitic nematodes. A possible strategy to

overcome AH resistance is to block specifically the activity of nematode Pgps. Caenorhabditis elegans has served as a

model for parasitic nematodes and recently, the first nematode Pgp, Cel-Pgp-1, has been crystallized and its multidrug

transport function has been characterized in vitro. Using the Cel-Pgp1 crystal structure and molecular docking tools

dedicated to drug binding, we have identified possible binding sites and specific interacting amino acids for several

transport substrates. We characterized two major drug binding sites, one deep in the core of the inner pocket and a

second close to the cytoplasmic opening of the pocket, similar to what was predicted for mammalian Pgp (Garrigues et

al., 2002). Docking calculations of known transported substrates revealed high affinities poses, thereby assessing

trustworthiness of our docking methodology. This result allowed us to investigate the binding mode of macrocyclic

lactone anthelmintics (ML) on Cel-Pgp1. Strikingly, we found that all MLs bound in the deeper pocket with common well-

defined sub-sites for each of their main chemical groups: benzofurane on TM10 and TM12, spiroketal on TM10 and

TM11. Interestingly, the mono- or disaccharide moiety of the avermectins specifically interact with TM1, TM6, TM7 and

TM12. Such interaction does not occur with moxidectin and ivermectin aglycone. Ivermectin, abamectin, doramentin,

eprinomectin and selamectin showed highest binding affinity score, while moxidectin and iveremectin aglycone had

lower affinity. This study proposes the binding position of MLs on a nematode Pgp, and provides a valuable tool to predict

the role of nematode Pgps in transporting anthelmintics and their contribution to resistance.

9

James H. McKerrow1, Louise Goupil2, Karina Rebello3, Ana Gisele da chNCosta Neves Ferreira3

1Skaggs School of Pharmacy and Pharmaceutical Sciences, UCSD; 3Fiocruz, Rio de Janeiro, Brazil, 2Chemistry and

Chemical Biology Graduate Program, UCSF

15 PROTEASES WITH PARASITIC FUNCTIONS IN HELMINTHS

Proteases play key roles for parasitic functions of helminthes, and are targets for vaccines, drugs, and diagnostics.

“Target” proteases are often gut-associated and present in both free living and parasitic helminths. Metazoa have

conserved the same repertoire of proteases for gut digestion. These include cysteine proteases like cathepsins B, L, and

C and aspartyl proteases like cathepsin D. A homologous repertoire of gut proteases has been identified in all roundworm

and flatworm helminths studied to date. This repertoire also occurs in the free living roundworm C. elegans, and has now

been identified in the free living planaria flatworm (S. mediterranea).. The ubiquitous occurrence of these proteases and

in metazoans is distinct from the serine proteases functioning in digestion by vertebrates. When did the switch occur? It

may reflect adaptation to terrestrial life or exposure to oxygen and aerobic metabolism. Indeed cysteine proteases are

very sensitive to oxidative damage while serine proteases are not. Comparison of the proteases of the free living

planarian flatworm to that of the Schistosoma flatworm parasite has confirmed that digestive proteases are conserved,

whereas the cercarial serine protease, and surprisingly legumain (asparaginyl endopeptidase) are unique to the parasite.

The cercarial serine protease (aka elastase) evolved for a specific parasitic function, larval invasion of the host. Therefore

it is not surprising that it is absent in the free living flatworm. However the role of legumain remains uncertain. In the

schistosome parasite it might activate other gut-associated enzymes or be responsible for degradation of host

hemoglobin. Of note is the fact that a substantial quantity of inactive legumain is also produced by the schistosome.

While the free living roundworm, C. elegans, has a legumain gene, the C.elegans inhibitor, cystatin, lacks the legumain

inhibition domain found in parasitic roundworms. Taken together these observations suggest legumain has an as yet

unspecified parasitic function.

Nic Wheeler 1; Mostafa Zamanian 2; Michael Kimber 1; Matthew Berriman 3; Tim Day 1 1. Iowa State University,,2. Northwestern University, 3. Wellcome Trust Sanger Institute 16 GPCRS: UNEXPLOITED ANTHELMINTHIC TARGETS G-protein Coupled Receptors (GPCRs) are widely exploited drug targets in a substantial number of diseases, yet remain to be tapped as targets for anthelminthics. Their vast diversity, developmental ubiquity, and essentiality to parasitism maintains them as one of the foremost classes of potentially exploitable proteins. With these observations as a foundation, we sought to expand and further describe a recently discovered subfamily of GPCRs that are conserved only to flatworms – the Platyhelminth-Specific Rhodopsin-Like Orphan Family (PROF). Using a variety of in silico approaches, we expanded this family to 25 different flatworms, the majority being parasitic, including a total of 238 genes. We inferred the phylogeny of this family, showing its evolutionary distinction from the rest of the rhodopsin-like (Class A) GPCRs, and classified it into 3 subfamilies, each containing at least one representative from each flatworm analyzed. One of these subfamilies, enriched in trematode receptors, is the second largest phylum-specific family, second only to a tegumental-allergen-like protein family. We show that these genes are robustly expressed across the life cycle, particularly in the juvenile stages that are not currently targeted by marketed anthelmintics. Finally, in situ hybridization in a free-living flatworm suggests that these receptors are utilized in the neuromuscular system, raising questions about their possible role as neuropeptide receptors, which have been shown to be crucial in proper worm motor control and motility. However, this family maintains its orphan status, failing to cluster or significantly align with any other metazoan GPCRs. Conglomerating these data tells a striking tale of a massive, phylum-specific family that may be involved in neuromuscular coordination in juvenile and adult worms, the main target of the next generation of efficacious, broad spectrum anthelminthics.

10

Wenjin Law, Bruce Bamber and Rick Komuniecki,

Department of Biology. University of Toledo

17 A SCREENING PLATFORM TO IDENTIFY SELECTIVE AGONISTS FOR NEMATODE MONOAMINE RECEPTORS AS

POTENTIAL ANTHELMINTICS

Monoamines, such as 5-HT, paralyze both free-living and parasitic nematodes through a pathway requiring the Gαo-

coupled 5-HT1-like receptor, SER-4, in a limited number of interneurons (Hapiak et al., 2009). Since nematode cell lines

are not available and animal screening options are limited, we have developed a screening platform to identify 5-HT1

receptor agonists as potential anthelmintics, by expressing nematode 5-HT1-like receptors in chimeric, genetically-

engineered Caenorhabditis elegans, at sites, such as the cholinergic motorneurons, yielding robust locomotory

phenotypes upon agonist stimulation, under incubation conditions designed to dramatically increase cuticular

permeability. Using this approach, we robustly expressed 5-HT1-like receptors from a wide range of organisms, including

parasitic nematodes, Drosophila and humans and, more importantly, identified compounds with high selectively for

either nematode or human 5-HT1 receptors. Previously a 5-HT1 receptor agonist, PAPP, was used to clear Haemonchus

contortus infections from sheep (White et al., 2007). Using our screening approach, we confirmed that PAPP functioned

as a 5-HT1 receptor agonist but, more importantly, that PAPP also was active against additional nematode targets,

including the dopamine receptor, DOP-3. These studies are continuing to identify additional 1) selective high-affinity

agonists for nematode 5-HT1-like receptors and 2) molecular targets for PAPP, using a genetic screening approach to

identify mutant ser-4;dop-3 animals insensitive to PAPP-dependent paralysis.

Yan Hu 1, David Koch 1, Thanh Nguyen 1, Zeynep Mirza 1, Mostafa Elfawal, Gary Ostroff 1, Joe Urban 2’

Raffi Aroian 1

1 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA’ 2 United States

Department of Agriculture/ARS, Beltsville, MD

18 BACILLUS THURINGIENSIS CRYSTAL PROTEIN ANTHELMINTICS AND BEYOND

No new classes of anthelmintics for human therapy against soil-transmitted helminths have been approved in decades.

Cry5B made by Bacillus thuringiensis has been shown to be effective against intestinal roundworm infections in vitro and

in vivo in laboratory animals. Our current goal is to get as quickly as possible to a superior, safe, approvable, and mass-

scale deployable anthelmintic. Here, we show updated data on the ability of Cry5B to cure or impact hookworm

infections, Ascaris infections, and whipworm infections. Significantly, we are developing a novel five-step program

towards achieving a practical and affordable delivery system for Cry5B that meets the all above criteria. We have

achieved proof-of-concept data for each of the steps and have demonstrated we can successfully join several key steps

together. In the course of these studies, we are making discoveries more generally applicable to the deployment of

anthelmintics and are developing technologies towards automated moderate-high-throughput discovery of additional

novel anthelmintics.

11

Cedric Neveu1,2, Elise Courtot1,2, Abdallah Harmache1,2, Alexandra Blanchard1,2, Fabrice Guegnard1,2, Robin N.

Beech3, Nicolas Peineau4, Adrian J. Wolstenholme5, Philippe Castagnone6, Lindy Holden-Dye7, Vincent O’Connor7,

Debra J. Woods8 and Claude L. Charvet1,2

1 INRA, UMR1282 Infectiologie et Santé Publique, F-37380, Nouzilly, France, 2 Université de François Rabelais de Tours,

UMR1282 Infectiologie et Santé Publique, F-37000, Tours, France, 3 Institute of Parasitology, McGill University, Ste. Anne

de Bellevue, Québec H9X 3V9, Canada, 4 Dept. of Infectious Disease & Center for Tropical and Emerging Global Disease,

University of Georgia, Athens, GA 30602, USA, 5 School of Biological Sciences, University of Southampton, Southampton,

SO17 1BJ, UK, 6 Université François Rabelais de Tours, Département de physiologie animale, F-37000, Tours, France, 7

Veterinary Medicine Research and Development, Zoetis LLC, Kalamazoo, MI 49007, USA

19 FUNCTIONAL INVESTIGATION OF MUSCLE ACETYLCHOLINE RECEPTORS FROM PARASITIC NEMATODES

Acetylcholine receptors (AChRs) represent major targets for anti-nematodal drugs including widely used anthelmintics

such as levamisole as well as more recent molecules such as monepantel and derquantel. AChRs are made of 5 subunits

that are designated as α or non-α based on the presence of a cysteine doublet in their amino-acid sequence. Identical α

subunits have the ability to associate together to create functional receptors (homopentamers) whereas non-α subunits

have to associate with α subunits in order to generate functional receptors (heteropentamers). Even though nematodes

possess a large diversity of AChR subunits, a very small number of AChRs have been characterized so far.

In the present study, we have focused our attention on muscle AChR subtypes from animal and plant parasitic

nematodes. Using complementary functional approaches including expression in heterologous systems (Xenopus

oocytes and C. elegans) and RNAi, we highlighted subunit composition as well as pharmacological properties specific to

parasitic nematode species.

Sasa Trailovic3, Djorje Marianovic3, James G. Tipton1, Melanie Abongwa1, Fudan Zheng2, Shivani Choudhary1, Richard J. Martin1 and Alan P. Robertson1*

1Department of Biomedical Sciences, 2Department of Chemistry, Iowa State University, Ames, IA. 3Department of Pharmacology and Toxicology, College of Veterinary Medicine, University of Belgrade, Belgrade, Serbia. 20 EFFECTS OF MONEPANTEL ON NICOTINIC ACETYLCHOLINE RECEPTORS FROM ASCARIS SUUM AND OESOPHAGPSTOMUM DENTATUM. ZolvixR is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel has been demonstrated to act on deg-3/des-2 containing nAChRs in several nematodes as a positive allosteric modulator. The drug produces hypercontraction of nematode muscle. We investigated the effects of monepantel on muscle strips isolated form adult Ascaris suum. The drug did not significantly increase baseline tension when applied on its own. Contractions induced by acetylcholine and several cholininomimetic anthelmintics where antagonized by monepantel. Further investigation revealed that the antagonism was non-competitive in nature. We also investigated the effect of monepantel on nAChRs from O. dentatum heterologously expressed in Xenopus laevis oocytes. We investigated the effects of monepantel on OD3, a levamisole preferring nAChR subtype, OD4, a pyrantel preferring subtype and ACR-16 a nicotine preferring subtype. For each subtype tested monepantel applied in isolation produced no measureable currents, nor did it produce currents in the presence of choline. When monepantel was continuously applied it reduced the amplitude of acetylcholine induced currents in a concentration dependent manner for the three nAChR subtypes tested. In all three cases monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 was particularly sensitive to monepantel inhibition. Our findings suggest that the mode of action of monepantel is more complex than previously described. Research grants: NIH RO1 A1047194 to RJM & APR, Schlumberger Faculty for the Future Fellowship to MA & APR. JT Support: NIH T35 OD 012199-13.

12

Lindy Holden-Dye[1], Robert Walker[1], U Ebbinghaus-Kintscher[2], Caroline Rivers[1], Peter Urwin[3], Anna

Crisford[1], Vincent O’Connor[1]

[1]University of Southampton, Southampton, UK, SO17 1BJ; [2] Bayer CropScience Aktiengesellschaft, BCS AG-R&D-SMR-

PC-PCB-PB, Monheim, 6220, Germany; [3]School of Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK,

LS2 9JT.

21 A CROSS-SPECIES, CROSS-PHYLA CHARACTERISATION OF THE EMODEPSIDE RECEPTOR SLO-1

The emergence of resistance to widely used anthelmintic drugs is driving the need for new compounds that have

resistance breaking properties. Moreover, compounds with macrofilaricidal efficacy are needed for the treatment of

filarial disease. Emodepside, a cyclo-octadepsidepeptide, is such a compound with proven anthelmintic resistance

breaking properties and toxicity against adult filarial nematodes. The molecular basis of the efficacy of emodepside has

been extensively investigated in the model genetic nematode Caenorhabditis elegans and it signals through two

pathways, one involving latrophilin and the second the calcium-activated K+ channel, SLO-1 with the latter being the

major mechanism underpinning the anthelmintic action. In this talk the action of emodepside at SLO-1 from different

nematode species, including plant parasitic nematodes, and different phyla, encompassing human and Drosophila, will

be reviewed with a view to understanding the interaction of emodepside with the SLO-1 channel.

Caroline Rivers is a BBSRC Case student with Bayer CropScience.

Paul McCusker 1, Paul McVeigh 1, Vignesh Rathinasamy 2, Hayley Toet 2, Erin McCammick 1, Anna O’Connor 1, Nikki

J. Marks 1, Angela Mousley 1, Gerard P. Brennan 1, Terry W. Spithill 2, Aaron G. Maule 1;

1Institute for Global Food Security, School of Biological Sciences, Queen’s University Belfast, UK; 2Department of Animal,

Plant and Soil Sciences, and Centre for AgriBioscience, La Trobe University, Bundoora, Australia

22 STIMULATING GROWTH - A BOOST TO FUNCTIONAL GENOMICS APPROACHES IN LIVER FLUKE

Fasciola hepatica, the liver fluke, is recognised globally as a significant agri-food industry burden, and now increasingly

as a human health issue with around 17 million people thought to be infected. In recent years efforts to identify novel

targets for drugs / vaccines have focused on understanding gene and protein expression in infective life stages of the

liver fluke. To assess the candidature of these targets we used RNA interference to silence potential target genes in

infectious life stages of the liver fluke. Unfortunately, these efforts were hampered by a lack of available in vitro

functional biology platforms, and so we initially sought to improve these techniques. We achieved over 60% survival in

vitro of juvenile fluke over six months, whilst simultaneously triggering growth, such that juveniles grew to an average

of 38.5x their original size, with a few fluke growing to >220x their original size. Juveniles exhibited extensive gut,

tegument and reproductive tissue development, similar to that seen in vivo. This fluke maintenance platform offers the

first opportunity to apply functional genomics tools to examine growth and development in juvenile liver fluke which we

have found to be supported by proliferative cells known as neoblasts. Validation of the platform has shown that RNAi

persists much longer in non-growing compared to growing juveniles. The transcript recovery seen in growing worms was

reduced via repeated, transient dsRNA exposure which subsequently resulted in increased protein knockdown compared

to that seen in non-growing juveniles. Additionally, we report the first gene knockdown in F. hepatica adults following

microinjection-based delivery of dsRNA to trigger RNAi. These enhanced methodologies provide a growing toolbox for

the interrogation of fluke biology and the validation of new control targets. This work was supported by a Glover

Memorial Scholarship grant to PMcC and BBSRC grant BB/K009583/1.

13

Paul McVeigh1, Krystyna Cwiklinski1, Grace VT Mulcahy2, Sandra M O’Neill3, Aaron G Maule1, John P Dalton1;

1School of Biological Sciences, Queen’s University Belfast, Belfast, UK; 2School of Veterinary Medicine, University College

Dublin, Dublin, Ireland; 3Department of Biotechnology, Dublin City University, Dublin, Ireland.

23 SUGAR COATING THE PILL: EXPLOITING GLYCOSYLATION OF LIVER FLUKE VIRULENCE PROTEINS FOR PARASITE

CONTROL

Glycosylation represents a major mechanism of eukaryotic co-/post-translational modification, where specific sugars

(glycans) are attached to proteins and lipids. These modifications are essential for protein folding, assessed through

quality control mechanisms within the distal ER. Glycosylating enzymes also confer structural complexity onto proteins,

by decorating recipient molecules with complex multi-branched structures bearing diverse monosaccharides. This

structural complexity is mirrored by function, since glycosylation patterns can influence protein-protein interactions. In

helminths, such interactions will ultimately determine the function and immunogenicity of parasite proteins at the host-

parasite interface. Glycosylation is therefore of interest from a control perspective, since helminth glycans represent

appealing components of vaccine preparations, while the enzymatic glycosylation machinery represents a source of

potential anthelmintic targets. This machinery, as well as the structure and function of helminth glycans, are poorly

understood. We have identified >100 genes from the Fasciola hepatica genome with homology to glycosylation

components from model organisms (yeast and human). Developmental transcriptomics suggest that these are mostly

expressed constitutively, although some genes exhibit development regulation. Using the well-annotated yeast/human

glycosylation pathways as a template, we have reconstructed the liver fluke glycosylation pathway. These data suggest

that fluke lack many of the enzymes required to produce complex glycans; in particular, biosynthetic machinery

associated with sialylation, one of the most abundant glycan modifications in vertebrates, appear to be absent. We are

also investigating functional aspects of glycosylation using a battery of pharmacological and RNAi tools targeted to

‘chokepoints’ within the fluke glycosylation pathways. Initial experiments suggest that high mannose glycans are

structurally essential components of the tegument, since their removal causes significant damage to tegumental

structure. Our ultimate aim is to study these manipulations via in vivo infection assays, to investigate the impacts of

helminth glycans on the activation of host immune cells.

Robert M. Greenberg1; Swarna Bais1; Christopher Fang-Yen2; Matthew Churgin2;

1Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania; 2Department of

Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania

24 NOVEL PHARMACOLOGICAL SENSITIVITIES OF SCHISTOSOMA MANSONI TRP CHANNELS

Praziquantel is effectively the only drug available for treatment and control of schistosomiasis, a disease that affects

hundreds of millions. There is therefore an urgent need for new or repurposed antischistosomal agents. Ion channels are

validated targets for current anthelmintics. Transient receptor potential (TRP) channels comprise a diverse family of ion

channels critical for sensory transduction and other functions. However, the roles and pharmacological sensitivities of

these channels are essentially unexplored in schistosomes and other parasitic helminths. We are using TRP channel

modulators and RNAi to examine the pharmacological sensitivities of TRP channels in schistosomes. Though the

Schistosoma mansoni genome predicts representatives of most TRP channel subfamilies, a notable exception is TRPV,

the vanilloid receptor family. Nonetheless, despite the apparent lack of TRPV-like channels, both adult and larval

schistosomes exhibit dramatic hyperactivity in response to capsaicin and other selective activators of mammalian TRPV1.

This response to capsaicin is blocked by a TRPV1 antagonist. Surprisingly, the response is also virtually eliminated by

knockdown of SmTRPA, a schistosome TRPA1-like channel. In contrast, knockdown of SmTRPA has no significant effect

on serotonin-induced hyperactivity. Though TRPA1 and TRPV1 channels are often co-expressed and share some sensory

functions (eg, nociception) in other organisms, mammalian TRPA1 channels are not sensitive to capsaicin. These results

indicate that some TRPV1-mediated sensory functions may be fulfilled by schistosome TRP channels from other

subfamilies, and that schistosome TRP channels likely have unusual pharmacological sensitivities. We are currently

expressing SmTRPA in mammalian cells and characterizing its functional and pharmacological properties. We are also

using pharmacological and molecular genetic tools to investigate the functions of other schistosome TRP channels.

Targeted disruption of these channels may provide clues about the roles they play in the parasite and possible strategies

for the development of new antischistosomal therapeutics.

14

Raymond J. Pierce1, Tino Heimburg2, Julien Lancelot1, Alokta Chakrabarti3, Martin Marek4, Tajith B. Shaik4, Manfred

Jung3, Christophe Romier4, Wolfgang Sippl2

1Univ. Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204 - CIIL - Centre d'Infection et d'Immunité

de Lille, F-59000 Lille, France..2Institute of Pharmacy, Martin-Luther University of Halle-Wittenberg, Halle/Saale,

Germany. 3Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg, Germany. 4Département de Biologie

Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg

(UDS), CNRS, INSERM, 1 rue Laurent Fries, B.P. 10142, 67404 Illkirch Cedex, France

25 STRUCTURE-BASED DEVELOPMENT OF SELECTIVE INHIBITORS OF SCHISTOSOMA MANSONI HDAC8 AS POTENTIAL

THERAPEUTIC AGENTS.

Epigenetic mechanisms that control gene expression and, in consequence, the complex developmental processes that

schistosomes undergo during their life-cycle are promising targets for novel chemotherapies. We have identified

Schistosoma mansoni histone deacetylase 8 (SmHDAC8) as a valid therapeutic target. The crystal structure of SmHDAC8

highlighted significant differences in the catalytic pocket of the enzyme compared to its human orthologue, notably

involving a charge difference and an altered architecture, which suggested that it might be possible to develop selective

inhibitors. Virtual screening of compound libraries led to the identification of small-molecule inhibitors that exploited

these differences. Chemical optimization of the structures of these inhibitors has allowed the development of an

extensive series of benzhydroxamates that are potent inhibitors of SmHDAC8. Analysis of their binding by X-ray

crystallography identifies a schistosome-specific clamp that anchors the inhibitor in the active site pocket. These

inhibitors were screened for their inhibitory activity against SmHDAC8 and the human HDACs 1, 6 and 8. Twenty-seven

compounds were active in the nM range against SmHDAC8 and showed high selectivity compared to human HDACs 1

and 6. Moreover, some showed significant selectivity compared to human HDAC8. These compounds were also screened

for their lethality toward schistosomula and four highly active compounds were also tested for their effects on adult

worms. One such compound had an EC50 value of 16 µM against schistosomula and causes a marked separation of adult

worm pairs. An analogue lacking the hydroxamate group failed to inhibit the enzyme and had no effect on the parasite.

Taken together, our results indicate that inhibition of SmHDAC8 is a valid strategy for the development of

schistosomicidal drugs.

Jonathan S. Marchant & John D. Chan Dept Pharmacology, University of Minnesota

26 ALTERING THE PERCEPTIONS OF PRAZIQUANTEL

The parasitic infection schistosomiasis afflicts 200 million people worldwide and is clinically treated by a single drug, praziquantel (PZQ). Despite the fact that PZQ has served as a stalwart anthelmintic for decades, the molecular basis of action of this clinical agent is not well understood and new insight would be welcome. In this context, our laboratory has investigated the action of PZQ on the free living planarian Dugesia japonica. In this model, PZQ causes an unusual axis duplication during regeneration to yield viable, two-headed animals. This striking phenotype provides an obvious screen for genes medicating PZQ efficacy in vivo, highlighting pathways and drugs that are likely also relevant for PZQ action in parasitic schistosomes. Identifying this broader PZQ interactome will therefore resolve new druggable nodes for development of next generations antischistosomals. Here, we highlight a ergomimetic quality to PZQ action by resolving structural aspects of the ergot alkaloid pharmacophore that phenocopy, and those that oppose, PZQ action in vivo. Ergot alkaloids that mimic PZQ act as a serotonergic blockers, whereas serotonergic agonists block PZQ action. Expression of the most predominantly expressed planarian 5-HT receptor (S7.1R) confirms an action of bipolarizing ergots as blockers of basal S7.1R signaling activity. Compounds identified as high potency blockers of this receptor in vitro mimic the high penetrance of PZQ when tested in vivo, while lower potency S7.1R blockers show lower penetrance of drug-evoked bipolarity. These data suggest that PZQ engages bioaminergic pathways in vivo to regulate axis duplication in planarians and the orthologous phenotype of motility in schistosomes. This discovery has prioritized screening approaches to discover novel ligands that regulate 5-HT receptors in schistosomes.

15

John D. Chan1 & Jonathan S. Marchant

Department of Pharmacology University of Minnesota.

27 DIVERGENT PHARMACOLOGY OF A SCHISTOSOME G PROTEIN COUPLED RECEPTOR REVEALED BY A MINIATURIZED

RECEPTOR ACTIVITY SCREEN

Despite the fact that parasitic flatworms infect hundreds of millions of people worldwide, broad spectrum drug therapy

is largely reliant on a single, decades old treatment (praziquantel) with a poorly understood mechanism. G protein

coupled receptors (GpCRs) are logical targets to focus the search for new anti-schistosomals; these proteins are

established druggable targets, accounting for over 1/3rd of all prescribed medications. Specifically, modulation of

bioaminergic GpCRs is a promising anthelmintic strategy given the crucial role of these signals in mediating flatworm

development and neuromuscular function. Therefore, we have applied a live-cell assay to monitor flatworm GpCR

activity, enabling future large scale screening of compound libraries to identify potent and selective small molecule

modulators of schistosome GpCRs. In order to investigate schistosome GpCR function and pharmacology, we

recombinantly expressed GpCRs in mammalian cells co-expressing a luminescence coupled, cAMP biosensor to provide

a real-time readout of receptor activity. These assays are robust (Z-score > 0.7), stable for prolonged continuous readings

(> 1 hour), and can be miniaturized to 96 and 384 well formats to facilitate high throughput drug screens. Pilot screening

of a GpCR library against schistosome and human serotonergic GpCRs established the feasibility of such a screen and

revealed the divergent pharmacological profiles of orthologous flatworm and mammalian receptors. For example,

chemical probes commonly used in studies of mammalian 5-HT GpCRs were inactive against the schistosome receptor

(SB258719, SB269970). On the other hand, several heterocyclic alkaloids potently inhibited the parasite receptor (IC50

< 1µM) while displaying minimal or no activity against the human sequence, demonstrating the potential of these assays

to expand our limited knowledge of invertebrate GpCR pharmacology and identify new leads to advance the anthelmintic

pipeline.

Authors: Melissa C. Sanchez 1; Amalia Sánchez Parra 1; Christian von Cabanlong 1; Pauline M. Cupit 2; and Charles

Cunningham 1

Affiliations: 1Center for Evolutionary and Theoretical Immunology, Dept. of Biology, University of New Mexico; 2Skaggs

School of Pharmacy and Pharmaceutical Sciences, University of California San Diego

28 THE EFFECT OF PRAZIQUANTEL ON THE TEMPORAL TRANSCRIPTOMAL RESPONSE OF SCHISTOSOMA MANSONI AND

THE MURINE HOST LIVER DURING INFECTION.

Upon penetrating the skin of its mammalian host, Schistosoma mansoni migrates via the bloodstream to the liver and

mesenteric veins. Mature female and male worms pair and the females lay an egg approximately every 10 minutes. While

many of these eggs are excreted in the feces, some become lodged in the liver where they cause formation of granulomas

that are responsible for much of the pathology associated with schistosomiasis. Praziquantel (PZQ) has been the drug of

choice for the treatment of this disease for approximately 30 years. The drug kills sexually mature worms but it is

ineffective against juvenile schistosomes. We have employed next generation RNA sequencing technology (Illumina)

together with the Lumenogix data analysis platform to examine the differential expression of mouse hepatic genes during

S. mansoni infection in the presence and absence of PZQ treatment. Mice were treated with a lethal dose of PZQ (or PZQ

vehicle) over 4 consecutive days beginning on day 32 post infection (p.i.). Groups of 3 mice were sacrificed on days 32,

35, 39 and 46 (p.i.) and liver tissue containing mature S. mansoni excised and total RNA extracted. The differential

expression of immune, fibrotic, and inflammatory genes and pathways will be reported and correlated with egg

deposition and granuloma formation in the liver. In a second experiment, we treated S. mansoni infected mice with PZQ

on days 25-28 (p.i.), corresponding with juvenile worms, and livers were harvested on days 25 and 28 p.i. This allowed

for a comparison of the role of ATP-binding cassette (ABC) transporter genes in adult and juvenile S. mansoni exposed to

PZQ. These experiments will provide a better understanding of the effect of PZQ on the host response to S. mansoni

infection and the differential effect of the drug on different parasite life cycle stages in vivo.

16

Andrea M. Binnebose 1; Adam Mullis 2; Shannon L. Haughney2; Richard Martin 3; Judy Sakanari 4; Balaji Narasimhan 2; Bryan H. Bellaire1 1Veterinary Microbiology and Preventive Medicine, Iowa State University; 2Chemical and Biological Engineering, Iowa State University; 3Biomedical Sciences, Iowa State University; 4Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 29 DELIVERING ANTI-WOLBACHIA DRUGS BY AMPHIPHILIC NANOPARTICLES IMPROVES MACROFILARICIDAL ACTIVITY Lymphatic filariasis (LF), a vector-borne disease represents a significant global cost and social burden, affecting up to 120 million individuals annually. The disease is caused by an endoparasitic nematode, of which one species is Brugia malayi, which exist with their symbiotic bacteria Wolbachia. Current treatment is focused on mass drug administration. Limitations include specificity of tissues, toxicity and age restriction. Additionally, the unique composition of the outer cuticle of the worms limits the permeability of the drugs. Herein, we demonstrate the use of a biodegradable polyanhydride nanoparticle for the co-delivery of the antibiotic doxycycline and antiparasitic drug, ivermectin enhances efficacy and safety. Polyanhydrides exhibit a surface erosion mechanism and have been shown to encapsulate diverse cargos with high efficiency and to target delivery to various tissues. We hypothesize that the underlying mechanism of the dramatic enhancement of macrofilarial killing is the ability of nanoparticles to behave as a Trojan horse by penetrating the outer cuticle of the worm, effectively delivering high drug concentrations directly within the microenvironment of both the worm and Wolbachia to cause death. The delivery of multiple drugs using nanoparticles addresses many of the challenges with mass drug administration. Utilizing in vitro dose titration experiments we examined the effectiveness of worms treated with nanoparticle-encapsulated drug (blue) at the highest drug concentration (195 μM) had an average time to death (ATD) of ~one day and 100% mortality. In sharp contrast, those treated with soluble drug (red) of the same concentration, had an ATD >9 days with decreasing mortality. Our confocal microscopy studies showed extensive tissue penetration by nanoparticles that was corroborated by micro-dissection of treated worms. These results combined with preliminary in vivo studies shows great promise for utilization of polyanhydride nanoparticle in drug delivery. Polyanhydride nanoparticles provide the opportunity to increase efficacy and safety of antifilarial therapies through dose sparing. Their unique tissue penetrating capabilities, tailored release, and targeting all contribute to in an increase in worm mortality. Our work has provided strong evidence for the utilization of polyanhydride nanoparticles as a versatile drug delivery platform to combat filarial disease. Ray M. Kaplan University of Georgia College of Veterinary Medicine 30 DIAGNOSTIC TESTS FOR ANTHELMINTIC RESISTANCE: A LONG AND WINDING ROAD Accurate diagnosis of anthelmintic resistance (AR) is important for both livestock health management and for research investigating the biology and epidemiology of AR. The most common and widely available method for detecting AR is the fecal egg count reduction test (FECRT). However, FECRT are labor intensive, expensive, and are poorly sensitive, only detecting resistance once it reaches relatively high levels. Since AR is a quantitative trait with worm populations demonstrating variability in phenotype, the qualitative nature of FECRT makes it a poor test for researchers needing to measure AR on a quantitative scale. An alternative approach for diagnosing AR is in vitro bioassays. A variety of such assays have been developed and validated, including: egg hatch assay (EHA), larval development assay (LDA), larval migration inhibition assay (LMIA), and direct measurement of motility (e.g. Worminator). These assays offer several advantages over FECRT, including being less expensive and more quantitative. But in vitro assays have their own sets of problems including limited sensitivity, and only being applicable to some species and some drug classes but not others. Thus, we still lack robust diagnostics for AR for many parasites and anthelmintics of importance. Molecular-based assays were thought to offer the prospect of high sensitivity, high throughput and relatively low cost. However, after several decades of effort few tests exist, and except for the benzimidazoles, we still lack the knowledge necessary to develop such tests. Despite this poor record of success, the recent rapid improvements in next-generation sequencing technologies and bioinformatics have increased our hopes of uncovering the molecular and genetic mysteries of AR. As

17

we look to the future with hope for new and better diagnostic tests for AR, we still face the big hurdle of understanding the relationship between genotype and phenotype, which seems to grow more complex the more we learn. Roger Prichard 1, Thangadurai Mani 1, Catherine Bourguinat 1 1 Institute of Parasitology, McGill University, Montreal, Canada 31 DIVERSITY IN ION CHANNEL RECEPTOR GENES IN DIROFILARIA IMMITIS Macrocyclic lactones (MLs) have been successfully used as heartworm preventatives against Dirofilaria immitis. Unfortunately, ML resistance in D. immitis is now confirmed and there is a need to find new anthelmintics to control this infection. Most anthelmintics target ion channel receptors. Targeting ion channels D. immitis has obvious advantages. These receptors control movement, feeding, mating and response to environmental cues. New drugs, that targets these receptors, are likely to have motility phenotypes and this facilitates screening. Knowledge of the full range of ion channel receptor genes in D. immitis, and of their genetic variability is useful information for drug design as receptor polymorphism may affect responses to a drug and anticipate possible resistance development. A total of 272 ion channels genes were identified in D. immitis. Whole genome sequencing data from 8 different populations, and geographic locations; 4 ML susceptible and 4 ML-loss of efficacy populations were used for polymorphism analysis. We have identified 42 cys-loop ligand-gated ion channels (LGICs), 35 G protein-coupled receptors (GPCRs), 43 K+ and 6 Ca2+ voltage-gated channels, 9 other Cl- channels, 7 DEG/ENaC/ASIC channels and 7 TRP-type channels. We found 2155 polymorphic loci (1844 intronic, 311 exonic) in these 272 ion channels genes/subunits. Of the polymorphic loci in exons, 165 caused non-synonymous changes. Eighteen of the exonic single nucleotide polymorphisms (SNPs) caused a change in predicted secondary structure. Other SNPs may affect gene expression, protein function or possible resistance selection. Within these genes, we found low genetic diversity, with an overall polymorphic rate of 0.19%. The effect of each SNP needs to be further evaluated in functional studies. This study has used genomic information from a large number of samples and so demonstrates for the first time the heterogeneity in these genes. John S. Gilleard1, Susan Stasiuk1, Andrew Rezansoff1, Elizabeth Redman1,Axel Martinelli2, Stephen Doyle2, Roz Laing3, Eileen Devaney3, Dae-Qyun Ro4, James A. Cotton2. 1. Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, T2N 4Z6, Canada.2. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK. 3. Institute of Biodiversity, Animal Health and Comparative Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, 464 Bearsden Road, Glasgow, Scotland, G61 1QH, UK. 4. Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 4Z6, Canada

32 INVESTIGATING MECHANISMS OF ANTHELMINTIC RESISTANCE AND DETOXIFICATION IN HAEMONCHUS CONTORTUS AND CAENORHABDITIS ELEGANS We are using Haemonchus contortus and Caenorhabditis elegans as model systems to investigate the molecular mechanisms underlying anthelmintic resistance. In this presentation I will discuss two different approaches from our current research program. Firstly, an update on the progress that we have made to genetically map ivermectin resistance loci in Haemonchus contortus using a serial backcrossing approach. This has involved introgressing resistance alleles from two different ivermectin resistant strains – MHco4(WRS) and MHco10(CAVR) – into the genetic background of the susceptible genome reference strain MHco3(ISE). I will present the latest results on mapping the introgessed region(s) using genome-wide SNP markers mapped against the latest H. contortus genome assembly. Secondly, I will present some of our work on the metabolism of benzimidazole anthelmintics in both C. elegans and H. contortus. Understanding the processes by which parasites metabolize and potentially detoxify anthelmintics is a neglected area of research that is relevant to both drug discovery and anthelmintic resistance. We have shown that members of the benzimidazole drug class (albendazole, mebendazole, thiabendazole, fenbendazole and oxfenbendazole) are heavily glucosidated by both C. elegans and H. contortus. This biotransformation reaction, which is be common in nematodes but rare in mammals, leads to detoxification and excretion of the drug from worms. We have also examined the transcriptomic response of C. elegans and H. contortus to each of the benzimidazole drugs to identify a core set of xenobiotic response genes to this drug class. We are currently examining the role of some of the genes in the glucose conjugation and detoxification of benzimidazole drugs.

18

Umer Chaudhry1, 5, E. M. Redman 1, Kamran Ashraf 3, Muhammad Zubair Shabbir 2, Muhammad Imran Rashid 3, Shoaib Ashraf 4, John S. Gilleard 1 * 1 Dept of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary Alberta, Canada, 2,3& 4 Quality Operations Laboratory, University of Veterinary and Animal Sciences, Dept of Parasitology, University of Veterinary and Animal sciences Dept of Pharmacology, University of Veterinary and Animal sciences Lahore, Punjab, Pakistan, 4 Roslin Institute, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Scotland, UK, Molecular genetic evidence for soft selective sweeps and multiple origins of benzimidazole drug resistance in Haemonchus contortus from government farms in the Punjab, Pakistan 33 MOLECULAR GENETIC EVIDENCE FOR SOFT SELECTIVE SWEEPS AND MULTIPLE ORIGINS OF BENZIMIDAZOLE DRUG RESISTANCE IN HAEMONCHUS CONTORTUS FROM GOVERNMENT FARMS IN THE PUNJAB, PAKISTAN Background: How drug selection pressure leads to the emergence of anthelmintic resistance mutations is poorly understood. We have investigated this for the small ruminant parasite Haemonchus contortus in the Punjab, Pakistan. Studying the parasite in this location has allowed us to compare parasite populations that have been intensively selected by the benzimidazole drug treatments on closed government farms with local pastoral populations exposed to minimal drug selection. Methods: Adult Haemonchus worms were collected from the abomasa of small ruminants from three government farms frequently using benzimidazole drugs and closed to animal movement for 30 years. Also from eighteen pastoral locations to investigate the standing genetic variation present in parasite populations not subject to intense drug selection. The frequency of three known benzimidazole resistance mutations was determined in each parasite population. For the seven parasite populations in which resistance mutations were found, the diversity, geographical distribution and phylogenetic relationships of isotype-1 β-tubulin resistance alleles were determined and the population genetic structure investigated using microsatellite markers. Results: The F200Y (TAC) resistance mutation was present at high frequency in H. contortus populations from government farms but not from pastoral farms consistent with their respective drug selection histories. Population genetic analysis revealed extremely low genetic differentiation between parasite populations suggesting minimal genetic drift has occurred since the founding of the government herds. Molecular genetic analysis suggested that the F200Y (TAC) mutation has arisen multiple independent times resulting in soft selective sweeps in each government herd, even in the absence of contemporary parasite migration. Two resistance haplotypes present on government farms were not detected in the local pastoral parasite populations suggesting they were absent, or present at very low frequency, in the standing genetic variation prior to drug selection. In contrast, two other resistance haplotypes present on government farms were also present in the local pastoral populations suggesting they could have pre-existed the period of drug selection. Conclusions: Overall, this work provides further support for the hypothesis that the F200Y (TAC) mutation has multiple origins in a region with both recurrent mutation and selection on the standing genetic variation likely to play a role. Richard J. Martin1, Fudan Zheng1&2, Alan P. Robertson1, Melanie Abongwa1c and Edward W. Yu2 ,

1 Department of Biomedical Sciences, Iowa State University, 2 Department of Chemistry and Astronomy, Iowa State

University

34 THE ASCARIS SUUM NICOTINIC RECEPTOR, ACR-16, AS A DRUG TARGET: FOUR NOVEL NEGATIVE ALLOSTERIC MODULATORS FROM VIRTUAL SCREENING Soil-transmitted helminths infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 µM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 µM; fmoc-1, IC50,

19

10.00 ± 1.38 µM; fmoc-2, IC50, 16.67 ± 1.95 µM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically in vivo for further characterization. Eugenio L. de Hostos 1; Jennifer Keiser 2; Xiao-Nong Zhou 3; Shu-Hua Xiao 3; Robert K. Choy 1; David A. Shoultz 1; 1PATH, San Francisco, California, United States; 2Swiss Tropical and Public Health Institute, Basel, Switzerland; 3National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People's Republic of China

35 TRIBENDIMIDINE: A POTENTIAL NEW TOOL TO COMBAT HELMINTH INFECTIONS

Tribendimidine (TrBD) was discovered in 1987 at the Chinese National Institute of Parasitic Diseases, developed by Shandong Xinhua Pharmaceutical Company, and approved by the Chinese Food and Drug Administration in 2004. Clinical studies with more than 3,400 subjects, including children as young as four years old, have shown that TrBD is well tolerated and has good efficacy against hookworms and roundworms. Similar to albendazole, TrBD has only modest efficacy against whipworms. It is also efficacious against Asian liver flukes, Opisthorchis and Clonorchis, which cause incurable cholangiocarcinoma and are a significant regional public health problem in East and Southeast Asia. Furthermore, TrBD treatment results in fewer adverse events than the standard-of-care for Asian liver flukes, praziquantel. TrBD is a nicotinic acetylcholine receptor agonist, but has a subtype selectivity that is distinct from levamisole and pyrantel. TrBD is available in an enteric coated tablet and decomposes into the active component deacylated amidantel and terephthalaldehyde. TrBD was found to have a low risk of teratogenicity in a series of reproductive and developmental toxicology studies in rats and rabbits, unlike albendazole which is known to be teratogenic and is not recommended for pregnant women. While benzimidazoles are threatened by the development of resistance and oncogenic Asian liver fluke infections remain largely untreated, TrBD is still unavailable outside of China because of lack of approval by a stringent regulatory authority. PATH intends to obtain US Food and Drug Administration approval and World Health Organization prequalification to establish an affordable and accessible supply of high-quality TrBD so that it can be added to the arsenal of drugs used for mass drug administration campaigns targeting helminth infections worldwide.

Verma Saurabh; Sudhanva Kashyap; Robertson Alan P; and Martin Richard J

Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.

36 RNAI AND PATCH-CLAMP REVEAL SUBTYPES OF ACETYLCHOLINE RECEPTORS IN BRUGIA MALAYI. Lymphatic filariasis is a debilitating disease of tropical and subtropical locations caused by parasitic nematodes such as Brugia malayi, Brugia timori & Wuchereria bancrofti. These parasites are responsible for diseases that have a high morbidity and that produce socioeconomic instability in some 120 million people across 81 endemic countries. The Global Elimination of Lymphatic Filariasis (GELF) Program using mass drug administration has reduced the prevalence of these diseases but the available drugs are not effective against adult parasites. We have been studying the nicotinic acetylcholine receptors (nAChRs) on B. malayi muscle to determine if some of the newer cholinergic anthelmintics of gastro-intestinal worms might be effective on adult filaria. We have performed single muscle-cell PCR and whole muscle-cell patch-clamp recordings to identify & characterize the nAChRs present in adult B. malayi. Our patch-clamp studies show that there are different pharmacological subtypes of nAChRs present on Brugia muscle including nicotine-sensitive and levamisole-sensitive receptors. We have also used RNA interference (dsRNA) as a reverse genetic tool for pharmacological and phenotypic characterization of the nAChRs. We were able to disrupt nAChR formation using dsRNA directed against unc-29 & unc-38. The resulting phenotype showed more than a 50% reduction in motility. qPCR confirmed that there was a mean reduction of 91% of unc-29 and 88% reduction of unc-38 transcription. Our patch-clamp recordings from the dsRNA treated worms revealed that levamisole responses were reduced to a greater extent than the nicotine suggesting the presence, again, of different nAChR subtypes: differing in sensitivity to levamisole and nicotine respectively. The combination of RNAi, patch-clamp and pharmacology is allowing us to study the properties of these nAChRs and determine the viability of them as drug targets. These receptors may be exploited for the treatment using single cholinergic anthelmintics or combinations of anthelmintics to treat the adult worms. Research grants: NIH RO1 A1047194 to RJM & APR

20

Mary J. Maclean; Ashley M. Rogers; Sally M. Williamson; Michael T. Dzimianski; Bob S. Storey ; Adrian J. Wolstenholme University of Georgia, Athens, GA, United States 37 IN VIVO EFFECTS OF DRUGS USED IN LYMPHATIC FILARIASIS MDA PROGRAMS ON BRUGIA MALAYI IN GERBILS

Lymphatic filariasis (LF) threatens nearly 20% of the world’s population and has already handicapped one-third of the 120 million people currently infected. Current control and eventual elimination of LF rely on mass drug administration (MDA) programs with three drugs: ivermectin (IVM), albendazole (ALB), and diethylcarbamazine (DEC). Only the mechanism of action of albendazole is well-understood. Our lack of understanding regarding the mechanism of action of IVM and DEC is illustrated by the disparity between results obtained in vitro versus in vivo. For Brugia malayi, the IC50 for microfilariae (Mf) was 6.1 ± 1.1 µM, 120 times the drug concentration that clears Mf in human patients. These findings suggest that the rapid clearance of Mf observed after treatment with IVM or DEC is aided by the host’s immune system. In order to gain a better insight into antifilarial drug action, we used an in vivo model with B. malayi in gerbils. Gerbils with patent infections were treated with 6 mg/kg DEC, 1 mg/kg ALB, or 0.15 mg/kg IVM to mirror human MDA dosages. These treatments had no effect on the numbers of worms present in the peritoneal cavity of infected animals. Adults and Mf were collected 1 and 7 days post-treatment and RNA was isolated for transcriptomic analysis. Preliminary data analyzing the effects of IVM on adult females and Mf revealed changes in transcripts related to muscle regulation and locomotion as well as those encoding muscle protein and collagen expression. Forthcoming analysis of the effects of these drugs in vivo will provide a better understanding of how they clear filarial parasites.

Ciaran J. McCoy 1,2; Barbara J. Reaves 1; Steeve Giguère 3; Balazs Rada 1; Adrian J. Wolstenholme 1,2; 1Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA; 2Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA; 3Department of Large Animal Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA 38 PERIPHERAL BLOOD NEUTROPHILS AND MONONUCLEAR CELLS COLLABORATE TO IMMOBILIZE AND KILL BRUGIA MALAYI MICROFILARIAE IN VITRO Wuchereria bancrofti, Brugia malayi and Brugia timori infect over 100 million people worldwide and are the causative agents of lymphatic filariasis. Anthelmintic treatment rapidly removes blood-circulating microfilariae (Mf) to impede mosquito-based disease transmission. Intriguingly the effective serum drug concentrations are much lower than those required to kill or immobilize Mf in vitro. Here we investigate the complex interaction between parasite, drug treatment and the host’s innate immune system. Importantly, we show for the first time that human polymorphonuclear neutrophils (PMN) and peripheral blood mononuclear cells (PBMC) can collaborate to kill B. malayi parasites. Blinded video analyses of PMN-worm attachment and Mf survival demonstrate that: (i) DNase treatment eliminates PMN attachment in the absence of serum, (ii) autologous serum bolsters both PMN attachment and PMN+PBMC mediated Mf killing, and (iii) serum heat inactivation (55 oC for 30 minutes) inhibits both PMN attachment and Mf killing. Despite the effects of heat inactivation, the complement specific inhibitor compstatin did not impede PMN attachment or Mf killing. The effect of DNase treatment highlights that DNA-based neutrophil extracellular traps (NETs) can facilitate PMN-Mf attachment. A fluorescent, extracellular DNA release assay was used to quantify and visualize Mf-induced NETosis over time. We show that PMN expel large amounts of NETs that readily capture but do not kill B. malayi Mf in vitro. NET morphology was confirmed by fluorescence imaging of worm-NET aggregates labelled with DAPI and antibodies to neutrophil elastase, myeloperoxidase and citrullinated histone H4. Recent observations suggest that monocytes represent the key microfilaricidal cells present within the PBMC population. Finally, we monitor Mf survival post-anthelmintic treatment (ivermectin and diethylcarbamizine) both in the presence and absence of innate immune cells. The survival assay established here could be used to investigate the mode of action of anthelmintics that promote immune cell mediated parasite killing.

21

T.G. Geary1, L. Tritten2, C. Ballasteros1, M. O'Neill1, S.A. Williams3, W. Zeaky3, E. Burkman4, A. Moorhead4 1 McGill University Montreal, Canada; 4University of Georgia, GA 39 TRANSCRIPTOMIC EFFECTS OF IVERMECTIN AND FLUBENDAZOLE ON ADULT BRUGIA MALAYI IN CULTURE Defining exposure-efficacy profiles for antiparasitic drugs in culture is of great value in developing an understanding of their pharmacodynamics in hosts. This goal is difficult to achieve when in vitro drug effects are slow and difficult to measure. This is the case for the effects of ivermectin on adult filariae; this drug cause s long-term sterilization of adult parasites in the host, but the basis for this effect remains obscure. Similarly, flubendazole is a highly potent and efficacious macrofilaricide, but has few overt effects on adult filariae in culture. We therefore investigated the effects of pharmacologically-relevant concentrations of these two drugs on adult Brugia malayi. We used RNAseq analysis initially to show that these parasites tolerate culture for up to 5 days following removal from the host; few genes show evidence of significant dysregulation at this point. We then analyzed the drug effects against this background in replicated sets of parasites obtained from multiple infected jirds. Key findings include the observation that the treatments caused dysregulation of relatively small numbers of genes, consistent with their lack of effect on viability under these conditions. Ivermectin predominately affected the expression of genes involved in reproduction and larval development, while flubendazole also altered the expression genes involved in development, as well as several cuticle collagen genes. The functional significance of these changes remains to be determined, but the fingerprints of changes in gene expression induced by these drugs can permit the comparative analysis of drug effects in vitro and in vivo.

Ali Raza 1,2; Steven R. Kopp 2; Neil H. Bagnalla 1; Abdul Jabbar 3 and Andrew C. Kotze 1 1 CSIRO Agriculture, Queensland Bioscience Precinct, University of Queensland, Brisbane, Australia 2 School of Veterinary Science, University of Queensland, Gatton, Australia 3 Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Werribee, Victoria, Australia

40 EFFECTS OF IN VITRO EXPOSURE TO IVERMECTIN AND LEVAMISOLE ON THE TRANSCRIPTION PATTERNS OF ABC TRANSPORTERS IN HAEMONCHUS CONTORTUS LARVAE

This study investigated the interaction of ATP binding cassette (ABC) transport proteins with ivermectin (IVM) and levamisole (LEV) in third-stage larvae (L3) of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the ability of L3 to tolerate subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L3 were pgp-3, pgp-9.3, mrp-5, abcf-1 and abcf-2. The resistant isolate showed significantly higher levels of transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold) after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold) following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all ABC transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both resistant and susceptible isolates showed an increase in R-123 efflux following exposure to both drugs, suggesting that these drugs also stimulated activity of existing transporter proteins in both isolates. Exposure of the resistant L3 to IVM or LEV resulted in increased tolerance towards IVM in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV only in the resistant isolate, and the subsequent increased drug tolerance, suggest that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the drug resistance displayed by this isolate.

22

Samuel Armoo1, Shannon M Hedtke1, Stephen Doyle1, Warwick Grant1 1Nematode Functional Genomics Laboratory, Department of Animal, Plant, and Soil Sciences, La Trobe University, Bundoora, Victoria, Australia 41 INTRA- AND INTER-HOST DIVERSITY OF WOLBACHIA SP. THAT IS POTENTIALLY PROBLEMATIC FOR DRUG DEVELOPMENT

Bacteria in the genus Wolbachia are intracellular microbes that infect a wide variety of arthropods and some filarial

nematodes. As opposed to arthropods, filarial nematodes are proposed to live in a symbiotic relationship with the

Wolbachia. The symbiotic nature of the relationship is supported by the observation that antibiotic treatment of the

nematode, which directly kills the Wolbachia and subsequently but indirectly kills the worm. This makes Wolbachia an

attractive target for macrofilaricidal drug development. However, resistance to antibiotics frequently arises in bacterial

populations, dependent in part on the existing genetic variation and potential for genetic exchange. We have investigated

genetic varation in wOv from two perspectives. First, we estimated the wOv:nuclear genome ratio by qPCR to assess

copy number variation. Second, we amplified total a 20-kb of the Onchocerca volvulus Wolbachia (wOv) genome, with

coverage across 13 predicted genes and 8 intergenic regions, and used deep-sequencing to estimate Wolbachia

haplotype variation within individual worms. Using a variety of population genetic and phylogenetic tools, and find that

(1) individual nematodes contain multiple strains of Wolbachia that are genetically diverse at both synonymous and

nonsynonymous sites, (2) genetic diversity is primarily structured within and between worms, rather than among

communities, and (3) bottlenecks during transmission followed by rapid population expansion play a strong role in

shaping genetic diversity of these bacteria. Preliminary analysis of the whole Wolbachia genome sequenced at lesser

depth from additional individual worms supports the conclusion that individual worms carry multiple, genetically distinct

Wolbachia genomes at variable copy number and that much of this genetic variation encodes non-conservative,

potentially high impact changes in protein sequences. The implications of this extensive quantitative and qualitative

genetic variation for Wolbachia-nematode biology and for the sustainability of antibiotic treatment as a macrofilaricidal

strategy will be discussed.

Ujwal Sheth 1; Roopa Ramamoorthi 1; Jennifer Dent 1 1 BIO Ventures for Global Health (BVGH)

42 PARTNERING AND REPURPOSING: ACCELERATING ANTHELMINTIC DISCOVERY AND DEVELOPMENT Half of the 17 diseases considered “neglected” by the World Health Organization are caused by helminths. These nematodes, cestodes, and trematodes combined have infected more than two billion people worldwide – most notably affecting those individuals living well below the poverty line. Current treatments for these diseases are limited in effectiveness and are often highly toxic. However, in spite of this, commercial interest in developing new and improved anthelmintics is minimal. Twenty-six drugs targeting neglected diseases were approved between 2000 and 2009, of which none were for helminthiases. Growing concern over resistance to current anthelmintics, coupled with these drugs’ incomplete efficacies and potentially life-threatening side effects, highlights the urgent need for new drugs. As eukaryotes, helminths have many molecular signaling pathways homologous to those found in humans. Therefore, drugs developed to target these conserved pathways in humans could be repurposed to treat helminth infections. Public-private partnerships, such as the WIPO Re:Search consortium, have been established to accelerate the development of new drugs, vaccines, and diagnostics for neglected infectious diseases by repurposing the pharmaceutical industry’s assets – including compound libraries and discontinued drugs – through collaborative research projects. This presentation will review biopharmaceutical companies and their compounds with anthelmintic repurposing potential, and describe several research groups that have obtained pharmaceutical company compounds, through WIPO Re:Search, to examine their anthelmintic effects.

23

Melanie Abongwa 1; Fidelis Cho-Ngwa 2; Godfred Ayimele 3; Moses Samje 2; Smith B. Babiaka 3; Ann Perera 4; Eman Mostafa 5; Adrian J. Wolstenholme 5; Richard J. Martin 1; Alan P. Robertson 1 1Department of Biomedical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA; 2Department of Biochemistry and Molecular Biology, Faculty of Science, University of Buea, P. O Box 63, Buea, South West Region, Cameroon; 3Department of Chemistry, Faculty of Science, University of Buea, P. O Box 63, Buea, South West Region, Cameroon; 4W. M. Keck Metabolomics Research Laboratory, Iowa State University, Ames, IA 50011, USA; 5Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA

43 FILARICIDAL ACTIVITIES OF EXTRACTS OF DANIELLIA OLIVERI AND PSOROSPERMUM FEBRIFUGUM The demand for new and efficacious anthelmintic drugs is on the rise, especially as resistance is likely to compromise every drug with time. There also exist other factors such as cost, poor compliance, and poor safety, which limit the use of existing drugs in resource-poor settings. Consequently, new drugs that will circumvent these problems are needed. Medicinal plants have so far been identified and shown to play a role in the remedy of various diseases, and serve as an underexplored source of lead compounds for the development of the much needed anthelmintic drugs. There is therefore the need to screen and isolate biologically active compounds from plant species for activity against various helminth species. Daniellia oliveri and Psorospermum febrifugum are among the plants that are used in NW Cameroon for the traditional treatment of onchocerciasis (caused by Onchocerca volvulus), lymphatic filariasis (caused by Brugia malayi), among other worm infections. We showed using the MTT/formazan assay and the Worminator system that extracts of D. oliveri and P. febrifugum are effective in killing the filariid parasites, Onchocerca ochengi (a good laboratory model of O. volvulus) and Brugia malayi. Remarkably, the bioactive plant extracts had IC50s as low as 4μg/mL and the majority of them showed little or no cytotoxicity on N27 rat neuronal cells. Hence, D. oliveri and P. febrifugum are potential sources of natural products with filaricidal properties. Preliminary metabolite profiling revealed identifiable peaks for compounds which will be further purified and isolated to yield compounds with enhanced activities. Supported by: - Schlumberger Foundation “Faculty for the Future” Program - NIH R01AI047194 to RJM and R21AI092185 to APR Sakong B.M., Adamu M., Adenubi O.T., Naidoo V., Eloff J.N., Mcgaw L.J Department of Paraclincal Sciences, University of Pretoria,Onderstepoort 44 RELATIONSHIP OF PLANT EXTRACTS AGAINST EGG HATCH INHIBITION, CYTOTOXICITY AND ANTIFUNGAL ACTIVITIES Internal parasites are becoming an increasing problem for livestock farmers. Inadequate access to appropriate anthelmintic drugs and development of larvae battle to anthelmintics are substantial concerns around the world. Nematode infection is therefore of economic importance as a major cause of disease and results in decreased productivity. However, small stock is the most vulnerable to these infections (Tsotetsi and Mbati, 2003). Some species of nematodes such as the blood sucking parasites whose activity usually results in anaemia. Results showed that Peddiea africana, followed by Schotra brachypetala had good relationship against antifungal, egg hatch and cytotoxicity. Similarly the water extract of Cassipourea gummiflua is the most active amongst all the water extracts. Hence, Schotra brachpetala inhibited the microorganisms with 0.04-0.08mg/ml and the EC50 ranged from 1.413-1.672, with selectivity index (SI) ranged from 0.59-13.43. It appears that acetone was the best extraction solvent of the active components of the plants against the EHA of Haemonchus contortus. It appears that acetone is the best extraction solvent of the active components of the plants. Moreover, cytotoxicity results revealed that, water extract of Cassiporea gummiflua showed good cell viability (309mg/ml) against the vero cells, followed by Senna pertesia extract with 251mg/ml. However, water extracts were relatively toxic as compared to acetone crude extracts. Therefore, the aim of the study was to check the relationship between the fungal microorganisms, cytotoxicity and EHA. Keywords: Plant extracts, fungal organisms, cytotoxicity, anthelmintic activity.

24

Makedonka Mitreva McDonnell Genome Institute, Washington University in St. Louis, St. Louis, MO, USA Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA 44 WORKSHOP: HELMINTH GENOMICS - LOOKING FOR BIOLOGY IN TERABASES OF SEQUENCE DATA Genomic approaches are revolutionizing parasitology, including helminthology, by taking full advantage of a large amount of sequences generated on the Next-Generation Sequencing platforms. The value of parasitic nematode sequence data is enhanced by applying bioinformatic tools designed to look for biology in large amount of sequence data. Appropriate high throughput analyses will accelerate progress in the parasitic nematode research by enabling researchers to formulate and test hypothesis through accessing and analyzing the available and upcoming sequence data. However, a significant gap exists between data generation and data analysis, which goes beyond summarizing the statistics from large sequencing projects. Mining the data in a way that will allow analysis of biology within the terabases of sequence data is the obvious next phase after data generation; this is where the first gap resides and where a roadmap with a goal to enable transition to post-genomic era is needed. Hence, via specific examples I will present advanced approaches to analyze helminthes at genome, transcriptome and variome level. In particular, the focus will be on: i) genome assembly and annotation, including improvements using third generation sequencing platforms, ii) genome based transcript analysis, de novo transcriptome assemblies, and differential gene expression analysis, and iii) re-sequencing genomes and variant calling/annotation and interpretation. During this process I will provide information on how to: i) navigate appropriate databases, ii) become familiar with available bioinformatics tools relevant to exploration of different ‘omics’ data while iii) gaining knowledge/ability to ask appropriate answerable questions from the sequence data. Erik C. Andersen; Mostafa Zamanian Northwestern University Evanston, IL 46 HERITABLE SMALL RNAS ARE IMPLICATED IN NEMATODE BENZIMIDAZOLE RESISTANCE Many neglected tropical diseases (NTD) are caused by parasitic nematodes. Within the last twenty years, a massive campaign to administer anthelmintic drugs was performed. Unfortunately, few anthelmintics exist and resistance is growing rapidly, prompting an urgent need to identify resistance genes. In addition, nematode-borne diseases of livestock and plants are major agricultural problems, resulting in severe economic losses. Effective future treatments of parasitic nematode infections require knowledge of which genetic variants cause resistance to a particular drug. In order to identify conserved nematode drug responses, we use two model roundworms, Caenorhabditis elegans and Caenorhabditis briggsae, and a new massively scaled quantitative phenotyping pipeline to measure population variation and perform statistical mapping procedures. Our platform utilizes automated robotic devices to rapidly and accurately measure fitness traits, such as offspring production, growth rate, and muscle behaviors for hundreds of individuals in parallel. We mapped drug resistance to ten of the most prescribed anthelmintic compounds. Surprisingly, resistance to the benzimidazoles mapped to regions of the genome with few protein-coding genes in both Caenorhabditis species. We re-sequenced and re-analyzed the genomes of the two species to facilitate an interspecies comparison that identified an abundant class of small RNAs encoded by genes in the conserved regions. These small RNAs could lead to heritable trans-generational resistance to anthelmintic compounds in parasitic roundworms. Our current efforts are focused on identified the endogenous genes targeted by these small RNAs and integrating our results with parasitic nematode sequencing projects. Bruce A. Rosa 1; Joseph F. Urban, Jr. 2; Dolores Hill 3; Valsin Fournet 3; Yue Xie 2; Allyson Dailey 4; Robin Couch4; Dante S. Zarlenga3; Makedonka Mitreva1; 1McDonnell Genome Institute at Washington University, St. Louis, MO 63108; 2USDA, Agricultural Research Service, Beltsville Human Nutrition Research Center Diet, Genomics, and Immunology Laboratory, Beltsville, MD; 3USDA, Agricultural Research Service, Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, Beltsville, MD; 4Department of Chemistry and Biochemistry, George Mason University, Manassas, VA

25

47 GENOMICS-BASED APPROACH FOR THE IDENTIFICATION OF PAN-PHYLUM CONSERVED PARASITIC NEMATODE PROTEINS THAT ACTIVATE IMMUNITY Infections from parasitic nematodes cause extensive suffering in humans and animals. They also cause major production losses in livestock due to disease, lost feed conversion and follow-up treatment. Current control relies heavily on anthelmintics. However, due to the rise in drug resistance coupled with occupational exposure, as well as drug residues in feed and less than optimal pasture management, novel approaches are required to attenuate parasitic infections. To date, years of research utilizing random cloning and immunological screening techniques have resulted in no commercially available nematode vaccines. Here, we use genomics and transcriptomics (followed by experimental testing) to develop, explore and test nematode databases to identify proteins capable of inducing innate and acquired immune responses. In particular, we will target proteins in the pan-nematode secretome in the first round of database screening. Once identified, candidate proteins with allergic and immune modulating potential are prioritized for experimental screening, synthesized, and then tested in vitro for functional activity. Those exhibiting the highest in vitro activity will be further prioritized for in vivo screening in mice. Successful candidates will ultimately be analyzed in parasite-challenged pigs and lambs/kids to confirm therapeutic potential and unilateral application. The knowledge gained herein provides both parasite vaccine targets, and validates a “proof-of-principle” demonstrating the use of vast databases to solve real problems in attenuating human and animal diseases (particularly human and animal parasitic nematodes resistant to chemical control). Young-Jun Choi 1; Stewart A. Bisset 2; Stephen R. Doyle 3; Kymberlie Hallsworth-Pepin 1; John Martin 1; Warwick N. Grant 3; Makedonka Mitreva 1; 1 McDonnell Genome Institute at Washington University, Washington University School of Medicine, 4444 Forest Park Avenue, Saint Louis, Missouri 63108, USA; 2 AgResearch, Hopkirk Research Institute, Private Bag 11008, Palmerston North 4442, New Zealand; 3 Department of Animal, Plant and Soil Sciences, La Trobe University, Victoria 3086, Australia 48 GENOMIC INTROGRESSION MAPPING OF FIELD-DERIVED MULTIPLE-ANTHELMINTIC RESISTANCE IN THE NEMATODE PARASITE TELADORSAGIA CIRCUMCINCTA Anthelmintic resistance is an increasingly serious problem, but the genetic basis of resistance remains inadequately understood, hindering our efforts to develop sustainable intervention strategies. Teladorsagia circumcincta is a parasitic nematode of major agricultural importance and a species in which the problem of anthelmintic resistance is particularly severe. To conduct a comprehensive genome-wide survey of field-derived resistance-associated alleles, we developed, through repeated backcrossing and drug selection, a pair of T. circumcincta strains that differ in their susceptibility to three major classes of broad-spectrum anthelmintics (benzimidazoles, nicotinic acetylcholine receptor agonists and macrocyclic lactones) but largely share a common genetic background. A draft genome sequence of 701 Mb (81,730 supercontigs; 25,532 protein-encoding genes; 95% CEGMA completeness) was de novo assembled based on the recurrent parental strain, and used as a reference against which variants segregating in the susceptible and the multiple-anthelmintic resistant populations were identified. Analysis of genomic regions of elevated inter-strain divergence revealed putative target sites, such as beta-tubulin isotype-1 and members of cys-loop ligand-gated ion channel superfamily. In addition, a copy number increase was observed in an ATP-binding cassette transporter in the multiple-anthelmintic resistant population in support of the hypothesis that enhanced drug efflux likely contributes to the resistance phenotype. The study provides a genomic foundation for understanding the molecular basis of anthelmintic resistance, and a starting point for future mechanistic studies and identification of marker(s) for monitoring and managing resistance.

26

Rahul Tyagi, Xin Gao, Vince Magrini, Amy Ly, Douglas P. Jasmer, Makedonka Mitreva1,2 1 McDonnell Genome Institute, Washington University in St. Louis, St. Louis, MO, USA 2 Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA 49 UNDERSTANDING NEMATODE INTESTINE COMPARTMENTALIZATION USING MRNA AND MIRNA DEEP SEQUENCING The intestine of parasitic nematodes has proven an important target for anthelmintics aimed at prevention and treatment of diseases caused by these pathogens in humans, animals and plants. The size of the swine round worm parasite Ascaris suum presents it as a particularly convenient model for studying nematode intestine. To expand utility of this model we identified and quantitatively compared expression of 15,382 coding RNAs and 277 noncoding, micro RNAs (miRNAs) among three contiguous regions of the adult A. suum intestine, presenting it at anatomical resolution not done before for any nematode species. We identified differentially expressed transcripts among regions, with the largest number of differentially expressed coding transcripts at significantly higher levels detected in the anterior region, identifying this region as the most functionally unique compared to middle and posterior regions. We further, delineated gene transcripts expression, to include 64 exon splice variants (47 genes) that are differentially expressed among these regions. A total of 2,063 mRNA transcripts were predicted to be targeted by intestinal miRNA, and correlation coefficients for miRNA:mRNA abundances (negative correlations) predicted 22 likely influential miRNAs (LIMs) and 503 likely associated miRNA:mRNA pairs (LAMPs), both reflecting attempts to delineate functional intestinal miRNA:mRNA interactions. We identified miRNAs that are conserved among intestinal miRNAs from A. suum and Caenorhabditis elegans (10 mature sequences and 13 seed sequences conserved), and prospective intestinal miRNAs from the murine gastrointestinal nematode, Heligmosomoides polygyrus (5 mature and 11 seeds). The expression levels of the inter-species conserved intestinal miRNAs were higher compared to the non-conserved miRNAs. Herewith we present the most comprehensive compilation of constitutively and differentially expressed genes along the length of the intestine for any nematode species. The information is expected to guide prospective development of many hypotheses on nematode intestinal functions encoded by mRNAs, miRNAs and interactions between these RNA populations, facilitating development of new therapeutics targeting essential functions of nematode intestinal cells.

James Wasmuth1; Jeff Wintersinger1 1Department of Ecosystem & Public Health, Faculty of Veterinary Medicine, University of Calgary 50 INTRA-SPECIFIC VARIATION AND ERRORS IN HELMINTH GENOMES: A CASE STUDY WITH HAEMONCHUS CONTORTUS. The well-publicised advances in DNA sequencing technology are enabling many groups to sequence their favourite organism. Software to perform the assembly and annotation is also becoming more user-friendly. Once published, the community assumes a high level of accuracy. Our work in helminth and protozoan genomes has revealed cases where assembly and annotation errors can have a large and negative impact on analysis and biological interpretation. Two genome assemblies for the parasitic nematode Haemonchus contortus are published. They were sequenced, assembled and annotated independently from different strains. We investigated the variation between the genomes with the aim of discriminating between legitimate biological differences and technical errors in one or both assemblies. We found important discrepancies. The genomes differed in their gene content. Specifically in genes that are known to be highly conserved across all eukaryotes, there was clear evidence for misassembly. Strikingly, only 45% of genes in one genome were orthologous in the other. One genome included many more paralogues than the other. Using the free-living and model organism Caenorhabditis elegans as a reference, we used gene location conservation to identify the likely better assembly and annotation. The differences between the two assemblies far exceeded those expected as a consequence of biological differences between two H. contortus isolates. Read depth analysis showed that assumptions on genome size, heterogeneity and the structure are likely to have significantly affected both assemblies. The findings of this work go beyond H. contortus genomics. The availability of two independent projects has allowed the first in depth comparison. For many species, one genome is available and we urge that researchers exercise caution when using such data.

27

Shannon M Hedtke 1; Katie Crawford 1; Samuel Armoo 1; Stephen Doyle 1; Warwick Grant 1

Animal, Plant & Soil Sciences Department | Agribio Building | La Trobe University | Bundoora 3086

Australia

51 META-POPULATION DYNAMICS AND RESPONSE TO IVERMECTIN IN ONCHOCERCA VOLVULUS

The probability that drug resistance arises in a population depends not only on the probability that mutations in genes that mechanistically alter phenotypic response to a drug are present (or arise) in the parasite population, but also on the probability that genes related to resistance are introduced into a naïve population due to migration of individuals across a landscape. We use population genetic tools to explore how helminth parasite populations are structured across several spatial scales, with the aim to determine how such population structure affects the predictive probability of resistance to evolve and the risk of post elimination recrudescence of disease. We have analyzed whole mitochondrial and nuclear genome data of the filarial nematode, Onchocerca volvulus, to: (1) identify interbreeding populations of the parasite, and (2) explore genetic markers associated with poor response to the drug ivermectin, a microfilaricide that is used to interrupt transmission of the parasite by mass drug administration throughout Africa and the Americas. We find that while there are genetically distinct and identifiable populations in West Africa, there is also evidence for migration among communities, as might be expected under a metapopulation model. Furthermore, we find no evidence for geographically widespread mutations associated with the strength of an individual parasite's response to drug treatment.

Pauline Fontaine; Keith Choe University of Florida, Gainesville, FL 52 THE DRUG DETOXIFICATION GENE MASTER REGULATOR SKN-1 CONFERS RESISTANCE TO ALBENDAZOLE IN C. ELEGANS Parasitic nematodes infect a fourth of the human population worldwide and have a major economic impact on livestock and crop production. Control of these parasites relies on the use of anthelmintics, but resistance is now threatening sustainable control in agriculture and community-wide human treatment programs. Resistance can arise through mutations in the drug target and increased drug detoxification. While mutations in drug targets confer resistance, how nematodes metabolize/detoxify drugs and the role of these processes in anthelmintic resistance has been little studied. The C. elegans transcription factor SKN-1 promotes resistance to a diverse array of reactive small molecules by regulating >65 drug detoxification genes including many encoding phase II metabolizing enzymes. As a consequence, we hypothesize that SKN-1 may play a role in anthelmintic resistance in C. elegans. There are three major classes of anthelmintics including benzimidazoles (BZ), for which resistance is the most widespread. The BZ albendazole targets beta-tubulin affecting reproduction and locomotion. Although a key mechanism of resistance involves mutations in the beta-tubulin drug target, differing drug resistance phenotypes suggest additional mechanisms. Using motility and egg production assays, we show here that skn-1(k1023) gain-of-function (gof) worms are resistant to albendazole whereas skn-1(zj015) loss-of-function (lof) and skn-1(RNAi)-treated worms are sensitive, demonstrating that SKN-1 activation promotes albendazole resistance and that basal SKN-1 is required for wildtype levels of resistance. We also demonstrate that ugt-22, a SKN-1 dependent detoxification gene up-regulated under albendazole and in skn-1(k1023) worms, mediates albendazole resistance of skn-1(k1023) worms. Interestingly, SKN-1 did not confer resistance to ivermectin and levamisole, suggesting specificity of the xenobiotic response in C. elegans. In summary, we report that SKN-1 promotes resistance to albendazole in C. elegans at least partly through up-regulation of ugt-22. Ongoing work includes identifying other downstream molecular mechanisms involved in albendazole resistance and investigating whether SKN-1 modulates albendazole metabolism.

28

Ronald E. Baynes Center for Chemical Toxicology Research and Pharmacokinetics, Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University Raleigh, NC 53 PHARMACOKINETICS OF ANTHELMINTICS IN SEVERAL ANIMAL MODELS. This presentation will focus on the pharmacokinetics of several anthelmintics used in human and veterinary medicine with the ultimate aim of understanding how these drugs (1) are absorbed and distributed to targeted sites (e.g., effector compartment) at therapeutic or sub-therapeutic drug concentrations and (2) persist at these target sites or eliminated rapidly. The benzimidazoles and avermectins will be discussed amongst several other anthelmintic drug classes to describe the pharmacokinetics across several animal species as these drugs are more widely used in treatment and control of nematodes in veterinary species and there is substantial pharmacokinetic plasma and tissue data for these species. As to be expected there are pharmacokinetic differences between monogastric and ruminant species. There are also pharmacogenomic differences across breeds within species as has been demonstrated in our recent work with four swine breeds. In brief, there were significant breed and gender differences for oxfendazole for area under the curve (AUC) and time to maximum concentration (Tmax). There were differences in change in gene expression across breeds for sulfotransferase (SULT1A1) and gender for cytochrome P450 (e.g., CYP3A29) following fenbendazole exposure. Future work will help determine whether these differences may be associated with under-dosing or over-dosing in a swine herd and whether this could be translated to improved therapeutics in human populations. This presentation will also compare the drug absorption and disposition following different routes of administration with special focus on transdermal delivery. Species and anatomical differences and formulation effects amongst other factors will be described along with the several successes and challenges associated with transdermal drug delivery of anthelmintics (e.g., avermectins) in veterinary species. As pig skin is similar anatomically and biochemically to human skin, this model allows for the development of novel therapeutic delivery systems.

Melissa Miller 1; Ray Kaplan 1; 1Department of Infectious Diseases, The University of Georgia, Athens, Georgia, United States of America 1Department of Infectious Diseases, The University of Georgia, Athens, Georgia, United States of America; 2Department of Comparitive Biology and Experimental Medicine, The University of Calgary, Calgary, Alberta, Canada

54 CHARACTERIZATION AND MAINTENANCE OF RESEARCH ISOLATES OF PARASITIC NEMATODES Access to laboratory isolates is a fundamental component of a parasitology research program, however, maintenance

and characterization of isolates are costly and challenging. While numerous assays have been developed to characterize

the genotype and anthelmintic susceptibility of isolates, they are often limited to use within a single species, suffer from

reproducibility across laboratories, or may be too costly and labor intensive to implement on a consistent basis. Our

laboratory has established and maintained avermectin susceptible and resistant isolates of Haemonchus contortus,

Cooperia spp., and Dirofilaria immitis. We have evaluated several in vitro and molecular assays to differentiate these

isolates, finding some to be robust and others to lack the ability to differentiate isolates. In Haemonchus contortus, the

larval development assay yielded a resistance ratio of 60.0 and has been used consistently to monitor the resistance

status of our isolates. However, both worminator (larval motility) and larval migration inhibition assays yielded resistance

ratios of less than 2.0, suggesting motility of third-stage larvae is a poor indicator of resistance to avermectins. In Cooperia

spp., worminator assays conducted with abamectin, doramectin, eprinomectin, ivermectin, and moxidectin also

determined that motility of third-stage larvae is a poor indicator of macrocyclic lactone resistance. We employed

microsatellite genotyping with a panel of neutral polymorphic loci to develop molecular markers for our isolates of H.

contortus and Dirofilaria immitis. Pooled third-stage larvae or microfilariae can be easily obtained, genotyped, and

compared to the reference background haplotype for each isolate. These markers can also be used to evaluate the

genetic distance between field and laboratory isolates. Utilization of a combination of in vitro and molecular assays to

characterize research isolates can help to maintain isolate purity. These tools are valuable to research laboratories cycling

multiple isolates of parasitic nematodes with various levels of anthelmintic susceptibility.

29

Sarah Preston 1; Abdul Jabbar 1; Robin Gasser 1. 1. Faculty of Veterinary and Agricultural sciences, The University of Melbourne, Parkville, Victoria, 3010, Australia 55 WORKING TOWARD NEW DRUGS AGAINST PARASITIC WORMS IN PUBLIC-PRIVATE PARTNERSHIP Diseases caused by parasitic worms have a devastating and chronic impact on plant animal, and human health and productivity globally. For most of these diseases, there are no vaccines, current treatments are frequently ineffective or excessive, and drug resistance in some parasites is emerging against what is a very limited number of drug classes. Therefore, there is a need to work toward improved treatments. In collaboration with academic, philanthropic and industry partners, we are employing a cross-disciplinary approach to find new classes of effective anthelmintics and their targets. Complemented by the use of informatic tools and genomic/transcriptomic resources, we are predicting and prioritizing essential molecules as targets, and are now employing a newly established whole-organism, chemical screening assay to test and then repurpose prioritized compound groups to parasitic worms. Our low-cost assay records larval motility microscopically by video-capture, and achieves high levels of reproducibility, and low levels of intra- and inter-assay variabilities. This assay has major advantages over conventional methods (such as larval development, motility and migration inhibition methods), particularly in terms of set-up costs, ease of use, accuracy of results, throughput, time and cost, and can screening thousands of chemicals per week. In public-private partnerships, we have screened various libraries well-curated chemicals for anti-nematode activity, and identified at least six effectors, some of which are currently undergoing hit-to-lead optimization. For example, we have identified two chemicals in the classes biphenyl amides and pyrazolo[1,5-α]pyridines (originally designed as anti-inflammatory drugs), which reproducibly and significantly inhibit worm motility and development. These inhibitors fit the Lipinski rule-of-five and show considerable promise for optimization. Together with medicinal chemists, we are now synthesizing series of analogues for subsequence assessment of activity in vitro and in vivo in animals, and absorption, distribution, metabolism, excretion and toxicity (ADMET) testing.

Deirdre A. Joy

Program Officer, Parasite Genomics, National Institute of Allergy and Infectious Diseases, Fishers Ln MD

56 NIH FUNDING

I will be presenting on current NIH and NIAID-specific parasite genomics priorities, initiatives and funding opportunities.

30

Poster Pitch and Presentation

Christopher cFarland1, A. G. Maule1, A. Mousley1, N. Marks1

1Molecular Bioscience-Parasitology, Institute for Global Food Security, School of Biological Sciences, Queen’s University

Belfast, Belfast, BT9 7BL

1P: THE IMPACT OF LIVER FLUKE AND RUMEN FLUKE CO-INFECTION ON FUTURE AGRICULTURAL OUTPUTS: PREVENTING

WIDESPREAD ANTHELMINTIC RESISTANCE

Over 55% of farm animal diseases in Europe result from helminth parasites, costing billions of dollars per year through produce losses and expenditure on anthelmintic drugs. Liver Fluke disease (fasciolosis) affects over 700 million domesticated livestock worldwide. Economic losses in the agricultural sector attributed to fasciolosis are estimated globally at a conservative $3.2 billion per annum due to: livestock mortality; reduced productivity; decreased fertility; and, increased susceptibility to other diseases. Comparatively, the economic costs and worldwide prevalence of rumen fluke disease (paramphistomosis) remains relatively unknown. Until recently rumen fluke prevalence and pathogenicity was considered low in comparison to fasciolosis. However, chronic ulcerative rumenitis (presence of adult rumen fluke) and acute parasitic gastroenteritis (caused by internal migration of immature fluke) are now often attributed to morbidity and mortality, particularly in young animals and are emerging as important causes of productivity loss in livestock. Rumen fluke exhibit a heteroxenous life cycle similar to Fasciola hepatica, increasing the likelihood of prevalence expansion and a geographical distribution comparable to that observed in liver fluke. Most of the treatments employed to treat liver fluke infections show minimal efficacy in the control of rumen fluke numbers. However, oxcylozanide and more recently closantel, commonly employed as fasciolides, have been shown to reduce rumen fluke numbers. The overarching aim of the following project is to examine the presence/prevalence of liver fluke triclabendazole resistance in Northern Ireland, employing FECRTs and egg hatch assays. The impact of co-infection with rumen fluke infections will be investigated and the efficacy of closantel and oxyclozanide treatments to treat both trematodes examined.

Susan J. Stasiuk 1; Gillian MacNevin 2; Dae-Qyun Ro 2; and John S. Gilleard 1. 1. University of Calgary, Faculty of Veterinary Medicine, Alberta, Canada, 2. University of Calgary, Faculty of Biological Sciences, Alberta, Canada

2P: BIOTRANSFORMATION OF BENZIMIDAZOLE ANTHELMINTICS IN CAENORHABDITIS ELEGANS AND HAEMONCHUS

CONTORTUS.

Understanding the processes by which parasites metabolize and remove anthelmintics is an important goal both for drug discovery and anthelmintic resistance research. We are investigating metabolism of benzimidazole drugs in order to explore these processes. HPLC and LC/MS/MS analysis of the metabolites produced by C. elegans and H. contortus, has shown that each of five different members of the benzimidazole (BZ) family were modified by the addition of a glucose (+other) moiety, and that C. elegans and H. contortus produce similar metabolites, suggesting they may share similar detoxification pathways, and that C. elegans may be a valid model in which to study these processes for strongylid parasites. We have shown that BZs are metabolized to a much greater extent than the other important anthelmintics: Levamisole, Ivermectin or Moxidectin. Biotransformation of drugs by the addition of glucose appears to be nematode specific as it is rare in mammals. Glucosidation is catalyzed by the mammalian UDP-glucuronosyl/glucosyl transferase (UGT) enzymes which may also be involved in the biotransformation of benzimidazoles in nematodes as we show that Chrysin, a pharmacological inhibitor of UGT, decreases the amount of metabolite produced. We have used a bespoke Larval Motility Digital Analysis (LaMDA) system to measure the effects of drug exposure on worm motility in C. elegans and found that metabolism of ABZ reduces the potency of the drug, suggesting that metabolism is a protective response and a possible mechanism of resistance. Using RNA-Seq to examine the global transcriptomic response to drug exposure has identified candidate enzymes that may be responsible for the metabolism of benzimidazoles in C. elegans and H. contortus, and we are now undertaking functional studies to explore this further. These results provide some fundamental insights into how nematodes metabolize an important class of anthelmintic drugs.

31

Natalie A. Hawryluk1, Simon Townson2, Suzanne Gokool2, Marc Hubner3, Achim Hoerauf3, Coralie Martin4, Agnieszka Chojnowski5, Tamara Kreiss5, Monika Prorok5, John Siekierka5, Vikram Khetani6, Jerome B. Zeldis6, Stacie S. Canan1, Ivan Scandal7

1Celgene Global Health, San Diego, CA; 2Northwick Park Institute for Medical Research, London, UK; 3Institute for Medical Microbiology, Immunology & Parasitology, University Hospital of Bonn, Germany; 4Biodiversité et Adaptation des Microorganismes Eucaryotes à leur Environnement, Muséum National d’Histoire Naturelle France; 5Sokol Institute of Pharmaceutical Life Sciences, Montclair State University, Montclair, NJ; 6Celgene Global Health, Summit, NJ; 7Drugs for Neglected Diseases initiative, Geneva Switzerland.

3P: DISCOVERY PROGRAM FOR IDENTIFICATION MACROFILARICIDE AGENTS FOR TREATMENT OF ONCHOCERCIASIS

Lymphatic filariasis and onchocerciasis are helminthic diseases caused by parasitic nematodes infecting over 150 million people worldwide. Following the discovery of diethylcarbamazine and ivermectin, two drugs targeting the larval stage of the parasites, transmission and burden of these diseases has been significantly reduced. However, despite these advancements, modern treatments targeting the adult stage of the parasites are still needed. Recently, a pipeline of drug candidates in development has been identified through collaborations between PDPs, academic groups and pharmaceutical companies. However loss of candidates through attrition for these diseases is common, and the evaluation of new chemical series is needed. DNDi and Celgene Global Health have assessed focused compound libraries in an in vitro phenotypic screening effort targeting O. linealis, O. gutturosa, and L. sigmodontis. A sub-set of active molecules, with EC50 in the low micromolar range and with good pharmacokinetic profiles, were prioritized for in vivo proof of principle efficacy studies in L. sigmodontis murine models.

Harischandra H., Zamanian M., Day T.A. and Kimber M.J

Iowa State University, Ames, IA

4P: MECHANISMS OF HOST MANIPULATION BY EXOSOME-LIKE-VESICLES (ELVS) RELEASED BY THE FILARIAL NEMATODE

PARASITE, BRUGIA MALAYI

Lymphatic Filariasis (LF) is a mosquito borne disease caused by filarial nematodes including Brugia malayi and is the second leading cause of permanent disability worldwide. There is a recognized need for novel therapeutics to control LF and other parasitic nematode diseases but progress toward this goal is frustrated by significant gaps in our understanding of general parasite biology. The interaction between the host and parasite is critical for establishing and maintaining infection. Manipulating this interface to the detriment of the parasite opens up potential avenues for disrupting infection and transmission of LF. Our efforts are to deepen the understanding of the mechanisms through which B. malayi manipulate the host to their benefit. Previously, we have shown that exosomes-like-vesicles (ELVs), a specific type of extracellular vesicle, secreted by these parasites contain small RNA and protein effector molecules with the potential of manipulating the host immune system. Here we explore the mechanistic basis for ELV bioactivity across the B. malayi life cycle. ELVs were purified from B. malayi culture media using standard protocols. Nanosight analysis of these purifications revealed that ELVs are secreted by all parasite life stages. Confocal microscopy shows these ELVs are internalized by RAW and J774A.1 murine macrophage cell lines in a stage specific manner; preliminary data indicate uptake via phagocytosis. qPCR analysis suggest that exposure to these parasitic ELV[s drives a more classical than alternative activation phenotype in these macrophages, deviating from what is widely seen in the literature. Understanding the way the parasite functions is the first step towards developing drugs that can effectively target them. This work opens up a potential avenue for novel drugs or strategies manipulating the parasite-host interface and introduces the possibility of eliminating parasites resistant to current drugs, accelerating the eradication of the disease.

32

Melissa C. Sanchez 1; Amalia Sánchez Parra 1; Christian von Cabanlong 1; Pauline M. Cupit 2; and Charles Cunningham 1 1Center for Evolutionary and Theoretical Immunology, Dept. of Biology, University of New Mexico; 2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego

5P: THE EFFECT OF PRAZIQUANTEL ON THE TEMPORAL TRANSCRIPTOMAL RESPONSE OF SCHISTOSOMA MANSONI AND

THE MURINE HOST LIVER DURING INFECTION

Upon penetrating the skin of its mammalian host, Schistosoma mansoni migrates via the bloodstream to the liver and mesenteric veins. Mature female and male worms pair and the females lay an egg approximately every 10 minutes. While many of these eggs are excreted in the feces, some become lodged in the liver where they cause formation of granulomas that are responsible for much of the pathology associated with schistosomiasis. Praziquantel (PZQ) has been the drug of choice for the treatment of this disease for approximately 30 years. The drug kills sexually mature worms but it is ineffective against juvenile schistosomes. We have employed next generation RNA sequencing technology (Illumina) together with the Lumenogix data analysis platform to examine the differential expression of mouse hepatic genes during S. mansoni infection in the presence and absence of PZQ treatment. Mice were treated with a lethal dose of PZQ (or PZQ vehicle) over 4 consecutive days beginning on day 32 post infection (p.i.). Groups of 3 mice were sacrificed on days 32, 35, 39 and 46 (p.i.) and liver tissue containing mature S. mansoni excised and total RNA extracted. The differential expression of immune, fibrotic, and inflammatory genes and pathways will be reported and correlated with egg deposition and granuloma formation in the liver. In a second experiment, we treated S. mansoni infected mice with PZQ on days 25-28 (p.i.), corresponding with juvenile worms, and livers were harvested on days 25 and 28 p.i. This allowed for a comparison of the role of ATP-binding cassette (ABC) transporter genes in adult and juvenile S. mansoni exposed to PZQ. These experiments will provide a better understanding of the effect of PZQ on the host response to S. mansoni infection and the differential effect of the drug on different parasite life cycle stages in vivo.

Naiara Cristina Clemente Santos Tavares De Paula 1; Fernanda Sales Coelho 1; Pedro Henrique Nascimento Aguiar 1; Policarpo Ademar Sales Junior 2; Juliano Michel Araújo 1; Luiza Freire Andrade 1; Wolfgang Sippl 3; Manfred Jung 4; Antonello Mai 5; Raymond J. Pierce 6; Guilherme Oliveira 7; Marina de Moraes Mourão 1 1- Medical Helmintology and Malacology Group, CPqRR/FIOCRUZ, Brazil , 2- Anti-Trypanosoma Cruzi Drug Screening Platform (PlaBio), CPqRR/FIOCRUZ, Brazil, 3- Martin Luther Universität Halle, Wittenberg, Germany, 4- Albert Ludwigs Universität, Freiburg; Germany, 5- Università degli Studi di Roma La Sapienz, Italy, 6- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 – UMR 8204 – CIIL – Centre d’Infection et d’Immunité de Lille, F59000 Lille, France, 7- Vale Technology Institute, Brazil 6P: HISTONE MODIFYING ENZYMES ARE ESSENTIAL TO VIABILITY AND REPRODUCTION OF SCHISTOSOMA MANSONI AND ARE POTENTIAL THERAPEUTIC TARGETS

Schistosomiasis is the second most prevalent parasitic disease in the world. Treatment relies on a single drug, Praziquantel, and due to the identification of drug resistant parasites, the development of new chemotherapy against schistosomiasis is needed. Histone modifying enzymes (HMEs) play a central role in regulating chromatin epigenetic modifications and are implicated as therapeutic targets in various diseases. In this work, we employed RNA interference to validate 1 histone deacetylase (SmHDAC8), 5 demethylases (HDM) and 10 methyltranferases (HMT) as drug targets in Schistosoma mansoni; from which 4 were chosen for experimental validation. Additionally, specific inhibitors developed by the A-PARADDISE consortium were used to interrogate HMEs as drug targets against S. mansoni. To elucidate the roles of HMEs, schistosomula were exposed to dsRNAs, injected in mice and evidenced that SmHDAC8 is important to parasite development and survival. Additionally, HDAC8, PRMT3, and KDM1/KDM2 seem to be associated in egg production since infected mice presented significantly lower egg burdens and female worms presented underdeveloped ovaries. For inhibitor screening, schistosomula were exposed to 252 inhibitors and parasite viability was assessed by measurement of lactate produced in the medium and by propidium iodide staining. These inhibitors were also tested on adult worms, in which parasites mobility was evaluated using the WormAssay software. In the lactate and propidium iodide assays, 43 and 66 active inhibitors were identified, respectively. Using the WormAssay, 86 compounds were active in male worms, 109 were active in female worms, and of those, 57 were active in both sexes. IC50 was also measured for the active compounds and cytotoxicity was tested in mouse fibroblast cells. These results indicate that HMEs are

33

essential to parasite viability, oviposition and/or development of the reproductive system, confirming their potential as drug targets. In addition, some inhibitors seem to be potential candidates for drugs against schistosomiasis.

Marina De Moraes Mourão ¹; Sandra Grossi Gava1,2; Lívia Das Graças Amaral Avelar4; Naiara Cristina Clemente Santos Tavares De Paula¹; Anna Christina De Matos Salim4; Flávio Marcos Gomes Araújo4; Guilherme Corrêa De Oliveira3 1- Medical Helminthology and Malacology, CPqRR/FIOCRUZ, Brazil, 2- Biological Science Institute, Federal University of Minas Gerais, Brazil, 3- Vale Technology Institute, Brazil, 4- Genomics and Computational Biology Group, CPqRR/FIOCRUZ, Brazil

7P: FUNCTIONAL ROLES OF SMP38 MAPK AND IDENTIFICATION OF SPECIFIC GENE TARGETS REGULATED BY MAPKS PATHWAYS IN SCHISTOSOMA MANSONI

Eukaryotic protein kinases (ePKs) are important in regulatory mechanisms of cellular functions. Mitogen-activated protein kinases (MAPKs) influence innumerous tissue-specific biological activities in most organisms and are widely studied as targets for drug development. Recently, we identified orthologs and demonstrated that MAPKs (SmJNK, SmERK1, SmERK2) are involved in the reproductive development and survival of Schistosoma mansoni and, thus, are potential targets for drug development. In this study, we aim to elucidate the functional roles of Smp38 and to identify essential S. mansoni specific gene targets regulated by SmJNK, SmERK1, SmERK2 and Smp38 MAPKs pathways. In order to achieve this, schistosomula were Smp38-knockdown by RNA interference and infected mice. We evidenced that Smp38 is important to the parasite viability, had significantly lower egg burdens, and female worms presented underdeveloped reproductive tract. Additionally, worms depleted for Smp38 exhibited large damages in the tegument and seems to be involved in the activation of detoxification enzymes. To elucidate genes target of regulation by the MAPKs signaling pathways in the parasite, RNAseq analysis of SmJNK, SmERK1, SmERK2, and Smp38 knockdown parasites were performed. We generated 11 paired-end libraries containing 100 bp reads, ranging from 47 to 184 million reads per library. The sequences were assembled to the genome version 5.0. Differentially expressed genes were identified when comparing each knockdown group of parasites to unspecific control. Our results help to characterize the importance of MAPK pathway in the maintenance and survival of schistosomes and suggest the MAPKs enzymes as valuable drug targets to schistosomiasis control. Keywords: Schistosoma mansoni; RNA-Seq; MAPKs Financial support: CAPES; CAPES/University of Nottingham; FAPEMIG; CNPq, CPqRR-FIOCRUZ

Ruby Coates1,2,3, Ciaran J. McCoy1,2, Mary J. Maclean1,2, Adrian J. Wolstenholme1,2 1 Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA; 2 Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA; 3 University of Bath, United Kingdom

8P: A STUDY OF DIROFILARIA IMMITIS MICROFILARIAE AND INNATE IMMUNE CELLS IN AN IN VITRO SYSTEM

Heartworm prophylaxis for dogs is dependent on macrocyclic lactones such as ivermectin (IVM). The concentration of IVM required to immobilize Dirofilaria immitis microfilariae in vitro (IC50 of 43 µM) is significantly higher than the effective microfilaricidal concentration of IVM in vivo (in vivo molarity of ~ 25 nm). This 1700 fold difference is attributed to the involvement of the host immune response in clearance of the microfilariae. We previously found that peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophils (PMN) attach to D. immitis microfilariae in vitro, and increased attachment was observed as IVM concentration was increased. Their nuclear morphology also indicated that the PMNs attached to the microfilariae were activated. However, killing of microfilariae by PBMCs and PMNs in isolation was not observed. A previous study reported that the combination of PBMCs and PMNs resulted in the killing of Strongyloides stercoralis. We aim to determine if PBMCs and PMNs can attach to and kill D. immitis microfilariae in vitro, and if their collaboration can result in increased killing. Blood plasma, PBMCs and PMNs were isolated from uninfected dog blood and incubated with D. immitis microfilariae extracted from infected dog blood. No serum controls were used to determine if the presence of serum is required for cell attachment and killing. The possible role of

34

complement in killing was established using heat inactivated serum controls and DNase treatment was used to verify the involvement of Neutrophil Extracellular Traps in PMN attachment to microfilariae. The attachment and killing were measured simultaneously using blinded video analysis, with videos taken after incubation of 18, 24 and 40 hours. The data described here supports the hypothesis that PMNs and PBMCs contribute to D. immitis related immune responses and warrant further investigation.

Kelsey L. Paras, Melissa Miller, Ray Kaplan 1University of Georgia College of Veterinary Medicine Department of Infectious Diseases 9P: COMPARISON OF FECAL EGG COUNTING METHODS IN FOUR LIVESTOCK SPECIES

Accurate quantification of strongylid eggs is necessary for evaluating anthelmintic efficacy and making anthelmintic treatment decisions. Numerous fecal egg counting (FEC) techniques exist, however, they vary in detection sensitivity and efficiency of egg recovery. In this study we evaluated the Modified Wisconsin, 3-chamber (high sensitivity) McMaster, and the relatively new Mini-FLOTAC method. These methods have 5, 8 and 5 eggs per gram (EPG) detection sensitivity, respectively, and are commonly used in labs throughout the world. The objective of this study was to determine which method has the highest sensitivity for egg recovery, and if differences existed among four common host species. We performed multiple replicate FEC using each method on fecal samples from five horses, sheep, cattle and llamas. For ruminants, Mini-FLOTAC consistently yielded the highest EPG, suggesting a higher level of egg recovery. Mini-FLOTAC recovered 55.7% and 59.4% more eggs than Wisconsin and 44.6% and 29.6% more eggs than McMaster in sheep and cattle, respectively. McMaster recovered 20.2% and 43.2% more eggs than Wisconsin. In contrast for horses and llamas, both McMaster and Mini-FLOTAC yielded similar but higher EPG than Wisconsin; Mini-FLOTAC recovered 50.0% and 30.5% and McMaster recovered 40.9% and 50.1% more eggs than Wisconsin for horses and llamas, respectively. The most likely explanation for the superior egg recovery of the Mini-FLOTAC is due to fewer steps where eggs could be lost, and having a sieve with larger diameter pores than the cheesecloth used for straining samples in the Wisconsin and McMaster methods. Based on these data, Mini-FLOTAC is the preferred method for ruminant species, but for equine and camelid species, both the McMaster and Mini-FLOTAC are preferred over the Wisconsin method. However, given the improved efficiency of sample set up, the Mini-FLOTAC can be recommended for all host species.

Christina Bulman 1; Chelsea Bidlow 1; Pamela White 2; Laura Chappell 2; Andy Yang 3; Wenbo Yang 3; William Sullivan 2; Judy Sakanari 1 1 Dept. of Pharmaceutical Chemistry, University of California, San Francisco, 2 Dept. of Molecular, Cell & Developmental Biology, University of California, Santa Cruz, 3 CSIB, Dept. of Integrative Biology, University of California, Berkeley

10P: SEARCHING FOR GOLD: EFFECTS OF AURANOFIN ON ADULT BRUGIA PAHANGI IN VITRO

Auranofin is an FDA-approved, gold-containing drug that has been shown to be effective in killing adult filarial worms (Onchocerca ochengi and Brugia spp.) in vitro and decrease worm burdens in gerbils infected with adult Brugia. In an effort to better understand the effects of auranofin on adult filarial worms, we 1) measured gold levels present in worms treated with varying concentrations of auranofin using inductively coupled plasma optical emission spectrometry, 2) measured both motility and metabolic activity to determine the effects of drug concentration and length of exposure on worms and 3) looked at the effects of auranofin on the endosymbiont, Wolbachia, to determine titers in treated worms. We found that the motility of adult female Brugia treated with 3uM and 1uM auranofin was inhibited by >90% by Day 1 and Day 4, respectively but that 86% of worms treated with 0.3 uM auranofin survived for over 10 days in vitro. Worms treated for 24 hrs with 3 uM had 10 ppb gold (50 nM auranofin) per worm whereas worms treated with 1 uM had 4 ppb (extrapolated) gold levels per worm (20 nM auranofin). Preliminary studies using quantitative image analysis of ovaries from worms treated with 1 uM auranofin for 24 hours showed that Wolbachia titers were significantly decreased compared to control worms. Metabolic activity as determined by an MTT assay indicated that despite the >90% inhibition of motility of worms treated with 0.3 uM auranofin, they were still metabolically active unlike those treated with higher concentrations of auranofin. This may suggest that even though worms are barely moving, Wolbachia may still be viable and that 0.3 uM auranofin may have a bacteriostatic effect on Wolbachia. We plan to next focus on studies using

35

auranofin in our Brugia gerbil model to determine how the in vitro worm gold concentrations and Wolbachia titers translate to an in vivo setting. This work is funded in part by the Bill & Melinda Gates Foundation

Thavy Long 1 ; R. Jeffrey Neitz 2, 3; Rachel Beasley 4; Chakrapani Kalyanaraman 3; Brian M. Suzuki 1; Matthew P. Jacobson 3; Colette Dissous 5; James H. McKerrow 1; David H. Drewry 6; William J. Zuercher 7; Rahul Singh 4; Conor R. Caffrey 1; 1 Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA; 2 Small Molecule Discovery Center, Department of Pharmaceutical Chemistry, University of California San Francisco, CA 94158, USA; 3 Department of Pharmaceutical Chemistry, University of California San Francisco, CA 94158, USA; 4 Department of Computer Science, San Francisco State University, San Francisco, CA 94040, USA; 5 Center of Infection and Immunity of Lille, Université Lille Nord de France, Inserm U1019, CNRS-UMR 8204, Institut Pasteur de Lille, 59019 Lille Cedex, France; 6 Department of Chemistry, Meryx Inc, Chapel Hill, NC 27517, USA; 7 Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA 11P: STRUCTURE-BIOACTIVITY RELATIONSHIP FOR BENZIMIDAZOLE THIOPHENE INHIBITORS OF POLO-LIKE-KINASE 1

(PLK1), A POTENTIAL DRUG TARGET IN SCHISTOSOMA MANSONI

Schistosoma flatworm parasites cause schistosomiasis, a chronic and debilitating disease of poverty in developing countries. Praziquantel is employed for treatment and disease control, however, its efficacy spectrum is incomplete (less active or inactive against immature stages of the parasite) and being the only drug available there is concern regarding the potential for drug resistance. Thus, there is a need to identify new drugs and drug targets. Here, we show that RNA interference (RNAi) of the Schistosoma mansoni ortholog of human polo-like kinase (huPLK)1 elicits marked deleterious phenotypic alterations in larvae (schistosomula). Phenotypic screening and analysis of schistosomula and adult parasites with small molecule inhibitors of huPLK1 currently in pre-clinical and clinical phase trials for various cancers identified a number of potent (low micromolar concentration) anti-schistosomals. Among these was a GlaxoSmithKline (GSK) benzimidazole thiophene inhibitor of huPLK1 that has completed Phase I clinical trials for treatment of solid tumor malignancies. Through the recent public availability of GSKs ‘Published Kinase Inhibitor Sets (PKIS)’ 1 and 2 collections, we phenotypically screened an expanded series of 38 benzimidazole thiophene PLK1 inhibitors. Computational phenotypic analysis of controls and PLK1 inhibitor-treated populations of somules demonstrated a clear and objective distinction in their phenotypes. The distribution of phenotypes exhibited by both these populations was mapped and visualized through projection to a low-dimensional spaces using principal component analysis (PCA) and had a distinct shape and topology. We also obtained a structure-activity relationship (SAR) that held for both schistosomula and adult parasites. The most potent inhibitors generated marked phenotypic alterations at 1-2 µM within 1 h of application. Among these were compounds previously characterized as potent inhibitors of huPLK1 in cell assays. We discuss the data and the apparent SAR in relation to a future program targeting schistosome PLK1 and/or developing benzimidazole thiophenes as anti-schistosomals.

36

Claude L. Charvet1,2; Elise Courtot1,2; Robin N. Beech1,3; Abdallah Harmache1,2; Adrian J. Wolstenholme4; Lindy Holden-Dye5; Vincent O’Connor5; Nicolas Peineau6; Debra J. Woods7; Cedric Neveu1,2 1 INRA, UMR1282 Infectiologie et Santé Publique, F-37380, Nouzilly, France, 2 Université de François Rabelais de Tours, UMR1282 Infectiologie et Santé Publique, F-37000, Tours, France, 3 Institute of Parasitology, McGill University, Ste. Anne de Bellevue, Québec H9X 3V9, Canada, 4 Dept. of Infectious Disease & Center for Tropical and Emerging Global Disease, University of Georgia, Athens, GA 30602, USA, 5 School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK, 6 Université François Rabelais de Tours, Département de physiologie animale, F-37000, Tours, France, 7 Veterinary Medicine Research and Development, Zoetis LLC, Kalamazoo, MI 49007, USA

12P: PARASCARIS EQUORUM ACR-26 AND ACR-27 GENES ENCODE A PYRANTEL AND MORANTEL-SENSITIVE

ACETYLCHOLINE RECEPTOR

Parascaris equorum is the only ascarid parasitic nematode in equids. In nematodes, acetylcholine receptors represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animals possess two closely related genes that are essentially absent in free-living or plant parasitic species: acr-26 encodes an alpha subunit while acr-27 encodes a non-alpha subunit. Here, we cloned acr-26 and acr-27 from P. equorum. We demonstrated that the co-expression of the Peq-ACR-26 and Peq-ACR-27 subunits in Xenopus laevis oocytes resulted in a functional acetylcholine-receptor. Interestingly, affinities of Peq-26/27 AChR for pyrantel (EC50 0.32 ±0.26 µM) and morantel (EC50 0.98 ±0.26 µM) were higher than for the natural ligand ACh (EC50 34.9 ±1.1 µM). This receptor had a similar pharmacology pattern to the AChR made of ACR-26 and ACR-27 subunits from the distantly related parasite species Haemonchus contortus. Importantly, we report that the heterologous expression of Peq-acr-26 and Peq-acr-27 as transgenes in the model nematode Caenorhabditis elegans also drastically increased morantel and pyrantel sensitivity in-vivo, thus mirroring the pharmacological properties previously observed in Xenopus oocytes. Our results describe a novel class of nematode AChR representing a pharmacological target of prime interest for drug screening and relevant anthelmintic combinations.

Umer Chaudhry1, David Jon Bartley2, Roz Laing3, Eileen Devaney4, Neil Sargison 1*

1Roslin Institute, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Scotland, United Kingdom

2 Moredun Research Institute, Pentlands Science Park, Midlothian, Scotland, United Kingdom, 3 Institute of Biodiversity

Animal Health and Comparative Medicine, College of Medicine, Veterinary Medicine and Life Sciences, University of

Glasgow, United Kingdom, 4 Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary

Medicine and Life Sciences, University of Glasgow, United Kingdom

13P: THE USE OF FORWARD GENETIC APPROACH TO INVESTIGATE THE MECHANISMS OF ANTHELMINTIC RESISTANCE IN

H. CONTORTUS

Control of parasitic nematodes are heavily dependent upon the use of anthelmintics. Resistance to the all major class of anthelmintics has been observed in livestock. Most parasitic nematodes are difficult experimental subjects for making mechanistic study of anthelmintic resistance (AR). The small ruminant parasite H. contortus is a more amenable model system in which to study mechanism and genetics of AR based on the number of aspects of parasite biology. Genetic crossing of parasitic nematodes is a difficult task because the adult parasites are concealed in the host. Nevertheless, the high fecundity of H. contortus and ability to viably transplant into the abomasum makes genetic crossing feasible. Our approach involves genetic mapping of AR loci in the progeny of a F2 mapping crosses generated between two well characterized genetically divergent isolates of H. contortus, the anthelmintic sensitive genome isolate-MHco3 (ISE) and multi-drug resistant isolate-MHco18 (UGA). In this particular study, L4 female worms from a drug susceptible strain were transplanted with L4 male worms from a drug resistant strain directly into the abomasum of a recipient ovine host. Eggs recovered in the faeces of the recipient host (F1 progeny) were cultured to the L3 stage in vitro and then used to orally infect another host to obtain F1 adult worms. These F1 adult worms were surgically recovered from the abomasum on necropsy and used in the next generation F2 progeny. The sensitivity of the F1 and F2 progeny to AR was assessed using in-vitro bioassays with free living life stages. A panel of microsatellite markers was also used to monitor the back crossing procedure of F1 and F2 progeny. We demonstrate that after few arounds of backcrossing, worms that are phenotypically resistant to AR has similar genetic background to the susceptible reference strain. This

37

work provides a novel genetic approach to study anthelmintic resistance and provides a proof a concept of the use of potentially extremely powerful forward genetic in an important model parasite. The strains generated from this study will be the valuable resources to further look for the evidence of genetic linkage to AR resistance conferring locus providing a starting point for more detailed studies of the genome wide region to identify AR mutations. Mark McHugh 1, Saurabh Verma 2, Shivani Choudhary 2, Melanie Abongwa 2, Sudhanva Kashyap 2, JoAnne Powell-Coffman 1, Alan Robertson 2, Richard Martin 2 1 Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50011, USA, 2 Department of Biomedical Sciences, Iowa State University, Ames, Iowa 50011, USA

14P: DIFFERENTIAL EXPRESSION OF FOUR NICOTINIC ACETYLCHOLINE RECEPTOR SUBUNITS (NACHRS) IN ASCARIS SUUM TISSUES

Ascaris suum is a large nematode parasite of the pig which appears genetically to be the same as Ascaris lumbricoides, a common parasite of humans. In this parasite, muscle movement is driven by activation of nicotinic acetylcholine receptors (nAChRs) that are found synaptically and extra-synaptically over the surface of the muscle cells. In the absence of effective vaccines, and indiscriminate use of cholinergic anthelmintics, development of resistance in both animals and humans are of major concern. Hence, this has led to our interest in the biology of nematode parasite nAChRs. nAChRs are ion-channel comprising of 5 subunits that form a channel pore in the membrane, which opens in the presence of acetylcholine or selective agonists like pyrantel. Different pentameric arrangements of nAChR subunits produce different nAChR subtypes that vary in pharmacology. Here, in this study, different tissues of A. suum adult female worms were dissected in an attempt to detect and determine the type and amount of nAChR subunits present, namely, Asu-unc-38, Asu-unc-29, Asu-lev-8 and Asu-acr-8 by RT-PCR. Expression of each subunit was found in the head region, pharynx, gut, somatic muscle and ovijector. However, expression of nAChR subunit in the intestine was unexpected due to absence of muscle tissue. Subsequently qPCR was used quantitate expression levels in the different tissues. Again we found the presence of these subunits in the gut and muscle: the level of expression in the muscle was however greater than in the intestine. The presence of nAChR subunit message in excitable tissues was expected, but its presence in non-excitable tissue (gut) suggests that some nematode nAChRs also serve a paracrine and/or developmental role. Future experiments that investigate the role of nAChRs in non-excitable tissue of nematode parasites will enhance our understanding of the effects and mode of action of cholinergic anthelmintics. James Kearn[1], Anna Crisford[1], Elizabeth Ludlow[1], Peter E. Urwin[2], Catherine J. Lilley[2], Vincent O’Connor[1],

Lindy Holden-Dye[1]

[1]University of Southampton, Southampton, UK, SO17 1BJ; [2]School of Biology, Faculty of Biological Sciences, University

of Leeds, Leeds, UK, LS2 9JT." Fluensulfone, a novel nematicide

15P: FLUENSULFONE, A NOVEL NEMATICIDE Fluensulfone (full name: (5-chloro-2-(3,4,4-trifluorobut-3-enylsulfonyl)-1,3-thiazole) (also known as MCW-2 and Nimitz®) is a new nematicide belonging to the heterocyclic fluoroalkenyl sulfones. It is a contact nematicide that is applied via direct soil application and used for protecting a range of crop plants from plant parasitic nematodes (PPN). Studies have shown that fluensulfone has direct nematicidal activity against a number of PPNs. It has an excellent profile of selective toxicity and is well tolerated by a wide range of phyla encompassing non-target invertebrates, vertebrates and mammals. Remarkably, it only has weak efficacy against Caenorhabditis elegans constraining the use of this as a platform to define the mode of action. A systematic analysis of the effect of fluensuflone on the behaviour, physiology and biochemistry of a target species of PPN Globodera pallida indicates that fluensulfone imparts a potent, slowly developing, lipid sparing, metabolic insult which is selectively nematicidal to plant parasitic nematodes. Understanding the mode of action of fluensulfone has the potential to provide new insight into nematode metabolism and energy homeostasis. Acknowledgements; James Kearn was supported by a postgraduate studentship funded by Adama. Anna Crisford and Elizabeth Ludlow were funded by BBSRC grant no BB/J006890/1.

38

Mark Kaji1; Robin Beech1; Tim Geary1;

1 Institute of Parasitology, McGill University. 21,111 Lakeshore Road, Ste Anne de Bellevue QC, H9X 3V9.

16P : HOOKWORM GLUTAMATERGIC ION CHANNELS AND THEIR SENSITIVITY TO IVERMECTIN

Hookworms infect hundreds of millions of humans around the world, residing in the host small intestine and inducing anemia from taking blood meals. Those most at risk are pregnant women and children, who can experience severe malnutrition and even death from large worm burdens without proper medical treatment. To date, the only approved therapeutic treatment is the benzimidazole class of anthelmintics. However, widespread reduction of efficacy is present in veterinary medicine and there is a very real risk of this being mirrored in human helminth infections. The lack of an approved and effective second-line treatment is a glaring deficit in our therapeutic arsenal for controlling infection and reducing transmission. The macrocyclic lactone ivermectin (IVM) has previously been investigated for treating both Necator americanus and Ancylostoma spp. hookworms with mixed results. Studies show inconsistent cure rates, with N. americanus far less sensitive to IVM than Ancylostoma spp. Because of the wide distribution of N. americanus and overlap of heterogeneous Ancylostomatidae hookworm infections, mass drug administration of IVM is not considered a viable treatment option. The molecular determinant(s) for this differential drug insensitivity, however, have not been elucidated. IVM allosterically and irreversibly activates glutamate-gated chloride channels (GluCls) exclusively expressed in invertebrates. These receptors have been studied in Caenorhabditis elegans and Haemonchus contortus with IVM treatment inducing phenotypes of flaccid paralysis and inhibition of pharyngeal pumping. Here, we present the identification of seven putative hookworm genes encoding GluCl subunits orthologous to Glc-2,3,4,5,6, Avr-14 and Avr-15 from H. contortus and C. elegans. By comparing genes between IVM-sensitive and -insensitive parasite species, we can identify functional mechanisms for IVM and structurally related macrocyclic lactone activity. With a more comprehensive functional understanding of IVM-receptor interactions, we will gain insight for more effective use of current macrocyclic lactones and rational drug development.

Cuiqin Huang1,2, Sue Howell1, Ray Kaplan1 and Adrian Wolstenholme1,3 1Department of Infectious Diseases and 3Center for Tropical & Emerging Global Diseases, University of Georgia, USA, 2College of Life Sciences, Longyan University, Fujian Province, China

17P: DETECTION OF MACROCYCLIC LACTONE RESISTANCE IN FIELD ISOLATES OF HAEMONCHUS CONTORTUS: DOES DYF-

7 GENOTYPE CORRELATE WITH THE LARVAL DEVELOPMENT ASSAY?

Current bioassays for the detection of macrocyclic lactone (ML) resistance are slow and time-consuming. A recent report that polymorphisms in the sequence of the dyf-7 gene of Haemonchus contortus were correlated with resistance suggested that a quicker molecular test might be possible. In order to assess this, we analyzed partial sequences of the dyf-7 gene amplified from archived samples of eighteen H. contortus field isolates, all of which were from the United States, and which had been submitted to the University of Georgia for resistance testing using the DrenchRite™ larval development assay (LDA). The samples included representative isolates that spanned the range from completely susceptible to highly resistant. Genomic DNA was extracted from either eggs or L3 larvae that had been stored under ethanol. The amplified dyf-7 fragments were cloned into pCR-Blunt and multiple plasmids isolated from the resultant colonies for sequencing. The sequences were aligned and the proportion of putative ‘resistant’ and ‘susceptible’ alleles compared to the resistance status as determined in the DrenchRite™ LDA. All of the sequence polymorphisms reported previously were found in these isolates. Initial comparisons of the sequences from two laboratory isolates, UGA/2004 (which is moderately ML-resistant) and UGA/S (which is susceptible), demonstrated alleles fairly consistent with those reported previously for susceptible and resistant isolates. However, in the field isolates there was little correlation between the proportion of the putative ‘resistant’ and ‘susceptible’ sequences and the ML-resistance status. ‘Resistant’ sequences were present in very susceptible isolates and ‘susceptible’ sequences present in highly resistant ones. Possible reasons for this discrepancy will be discussed, but these results suggest that a molecular diagnostic based on dyf-7 sequences will not be a reliable indicator of the ML-resistance status of US field populations of H. contortus.

39

Alexandra Grote1; Denis Voronin2; Nirvana Nursimulu3; Swapna Seshadri3; Sara Lustigman2; John Parkinson3; Elodie Ghedin1 1Department of Biology, Center for Genomics & Systems Biology, New York University; 2Molecular Parasitology, New York Blood Center; 3Molecular Genetics, University of Toronto

18P: MECHANISMS OF FILARIAL ENDOSYMBIOSIS

Brugia malayi, a causative agent of lymphatic filariasis (also called Elephantiasis), has an obligate bacterial endosymbiont, Wolbachia. Current modes of treatment are limited to microfilaricides, which must be administered on a regular basis and whose efficacy is at risk due to the potential for drug resistance; there are no macrofilaricides or vaccines currently available. Eliminating Wolbachia with antibiotics affects the ability of the parasite to develop, reproduce, and survive, thus making Wolbachia an attractive target for treatment. While it is known that the relationship between the nematode and the bacteria is co-dependent, the molecular basis remains poorly understood. To better understand the molecular interplay between these two organisms we profiled the transcriptomes of B. malayi and Wolbachia by dual-RNAseq across the life-cycle of the parasite. This helped us identify the functional pathways necessary for this essential symbiotic relationship provided by the co-expression of nematode and bacterial genes. We have identified significant stage-specific differential expression in both Wolbachia and B. malayi during development of the worm. In Wolbachia we find genes involved in ATP production and purine biosynthesis as well as genes involved in the oxidative stress response significantly upregulated during female worm development. Correspondingly, in B. malayi we find genes involved in growth and embryogenesis as well as antioxidants significantly upregulated during female worm development. We are currently using these data to also characterize the endosymbioic relationship at the metabolic level using Flux Balance Analysis and thus identify choke points that could be exploited for therapy. We are using metabolic reconstruction to create a draft metabolic network based on available enzyme annotation data for the genomes of B. malayi and Wolbachia, as well as the Wolbachia-free filarial parasite, Loa loa. Using in silico knockouts, we will determine the necessary pathways for growth and virulence of the filaria and determine how these pathways are influenced by the presence of Wolbachia. The identification of these differential pathways is the first step in novel drug target discovery. Cécile Ménez, Dalia Kansoh, Jean-François Sutra, Anne Lespine INRA UMR1331, Université de Toulouse, INP, TOXALIM, F-31027 Toulouse, France 19P: RESISTANCE TO MACROCYCLIC LACTONES: IS THER A FITNESS COST TO ACQUIRED TOLERACE TO IVM AND MOX IN C. ELEGANS The anthelmintic macrocyclic lactones (MLs) ivermectin (IVM) and moxidectin (MOX) are the most widely administrated drugs to treat nematode infections. Their widespread use has led to the emergence of resistance to these two drugs. Our objectives were to investigate the adaptation of nematodes to IVM and MOX and the potential costs of this adaptation in the model nematode Caenorhabditis elegans. Therefore, we have selected C. elegans worms under IVM or MOX selection pressure through stepwise exposure to increasing doses of each drug. We have determined their acquired tolerance to MLs, and their susceptibility to other anthelmintics or xenobiotics, in regard to unselected parental worms (N2Bristol). The possible impact of acquired tolerance to MLs on reproductive capacity, fitness and survival was then evaluated by measuring changes in life-history traits of IVM- and MOX-selected animals. Both IVM and MOX selection led to similar ML tolerance profile (order of susceptibility: MOX>IVM>eprinomectin) and a dye-filling defective phenotype linked to a defect in amphid sensory neurons structure integrity. Both ML-selected strains displayed a higher susceptibility to levamisole, triclabendazole sulfoxide and chloroquine compared with the wild-type unselected strain, while susceptibility against the albendazole sulfoxide and colchicine was unchanged. Increased tolerance to both ML was associated with decreased reproductive fitness (decrease in the total number of progeny and the number of F1 progeny becoming reproductive adults), indicating that the adaptation to MLs induced a cost in term of reproductive capacity. Finally, lifespan of ML-selected strains was evaluated. The increase in sensitivity to other anthelmintics and xenobiotics and the compromised reproductive fitness of the ML-selected strains suggest that there is some biological cost imposed by acquired tolerance to MLs. This could help to better understand mechanisms underlying failure in drug efficacy and opens possibilities for managing anthelmintic resistance in parasite nematodes in the field.

40

Melissa Miller 1; Sue Howell 1; Adriano Vatta 1; Bob Storey 1; Ciaran McCoy 1; Adrian Wolstenholme 1; Elizabeth Redman 2; John Gilleard 2; Ray Kaplan 1; 1Department of Infectious Diseases, The University of Georgia, Athens, Georgia, United States of America; 2Department of Comparitive Biology and Experimental Medicine, The University of Calgary, Calgary, Alberta, Canada

20P: EVALUATION OF CHANGES IN POPULATION GENETIC STRUCTURE AND DRUG SUSCEPTIBILITY FOLLOWING

REPLACEMENT OF A MULTIPLE-ANTHELMINTIC RESISTANT POPULATION OF HAEMONCHUS CONTORTUS

A population of Haemonchus contortus that was highly resistant to benzimidazoles and avermectin/milbemycins and borderline resistant to levamisole was replaced with a susceptible laboratory isolate of H. contortus in a flock of sheep. Anthelmintic susceptibility and population genetics of the newly established worm population were subsequently evaluated for 3.5 years using in vivo, in vitro, and molecular methods. Initially post-replacement, albendazole yielded 98.5% fecal egg count reduction; larval development assay showed reversion to susceptibility, pyrosequence genotyping of single nucleotide polymorphisms in positions 167 and 200 of the isotype-1 beta tubulin gene indicated susceptibility; and microsatellite genotyping indicated the background haplotype was similar to the susceptible laboratory isolate; confirming successful replacement. In an effort to maintain anthelmintic susceptibility, targeted selective treatment based on FAMACHA was implemented using albendazole. Surprisingly, within 1.5 years post-replacement, resistance to albendazole, ivermectin, and moxidectin was confirmed via fecal egg count reduction test, larval development assay, and pyrosequence genotyping. Levamisole was then used for all treatments using targeted selective treatment. However, within one year, resistance was detected to levamisole. Population genetic analysis with Nei’s genetic distance matrix using a panel of 10 neutral polymorphic microsatellite markers demonstrated there was a gradual change in the genetic structure of the population until the final population was similar to the initial resistant population. The isotype-1 β-tubulin haplotypes present in the population were also evaluated at 6 time points throughout the study. Phylogenetic network analysis was employed to evaluate changes that have occurred in susceptible and resistant β-tubulin haplotypes throughout the study. This work demonstrates the power of combining molecular, in vitro, and in vivo assays to study phenotypic and genotypic changes in a field population of H. contortus.

Pamela M. White 1, Adan Codina 1, Walter Bray 2, Alain Debec 3, Scott Lokey 2, Laura R. Serbus 4, William Sullivan 1 1 Molecular, Cell, and Developmental Biology, University of California Santa Cruz, Santa Cruz, California, United States of America; 2 Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, United States of America; 3 Polarity and Morphogenesis Group, Jacques Monod Institute, CNRS, University Paris Diderot,UPMC Batiment Buffon, Paris, France; 4 Department of Biological Sciences, Florida International University Modesto A. Maidique Campus, Miami,Florida, United States of America

21P: A GENOME-WIDE CELL BASED RNAI SCREEN REVEALS WOLBACHIA MAINTAINS INTRA-CELLULAR TITER THROUGH

INCREASED UBIQUINATION AND PROTEOLYSIS OF HOST PROTEINS

Wolbachia are gram-negative, obligate, intracellular bacteria carried by millions of arthropod and filarial nematode hosts worldwide. The presence of Wolbachia in the latter are the cause of African River-blindness and Lymphatic Filariasis. Here we use a Wolbachia-infected Drosophila cell line and genome wide RNAi screening to identify host factors that influence Wolbachia titer. By screening an RNAi library targeting 15,600 transcribed host genes, we identified 39 candidate genes that dramatically reduced Wolbachia titer and 45 that increased Wolbachia titer. Strikingly, among the set of gene knockdowns that reduced Wolbachia titer, 6 were associated with the ubiquitin and proteolysis pathways. To test the in vivo relevance of these results, we found that both drugs and mutants that disrupt proteolysis result in reduced levels of Wolbachia in the Drosophila oocyte. In addition, we find the presence of Wolbachia dramatically influences the distribution and abundance of ubiquitin foci in Drosophila cells. Given Wolbachia are incapable of producing their own amino acids, Wolbachia induced upregulation of proteolysis via ubiquination may be a key mechanism provisioning Wolbachia with required amino acids.

41

Fernando Calahorro, Peter Johnson, Teresa Fereiro, Hywel Morgan, Vincent O’Connor, Lindy Holden-Dye

Centre for Biological Sciences and Centre for Hybrid Biodevices. Blg 85. Highfield Campus. University of Southampton.

SO17 1BJ, UK.

22P: PHENOTYPIC SIGNATURES OF COMPOUND EFFECTS ON NEUROMUSCULAR SIGNALLING IN THE C. ELEGANS

PHARYNGEAL SYSTEM

The Caenorhabditis elegans pharyngeal system provides an information rich experimental platform to investigate the efficacy of chemicals with known and unknown mode of action. This well characterised system can provide insight into effects mediated via established and putative anthelmintic targets. This is achieved via extracellular recordings which deliver an electrophysiological waveform reporting on the neuromuscular and synaptic physiology of the circuit that supports the muscle’s rhythmic cycle of contraction and relaxation. We have previously described an integrated microfluidic and electrophysiological experimental platform that records this waveform. Here we report the application of this approach for providing phenotypic signatures for different classes of neuroactive and nematicidal chemicals in C. elegans. We have optimized medium throughput plate assays in which worms can be cultivated in chemicals of interest overnight prior to investigation in the device. This approach is particularly useful in instances where access across the cuticle is slow or mode of action implies that chronic exposure is required for biological activity. The format and ergonomics of loading are suited to investigation of both the chronic or acute effects of important classes of chemicals on wild-type, mutant or genetically modified C. elegans sculpted to express targets of experimentally intractable nematode species. We are establishing a portfolio of phenotypic signatures for effects of compounds on neuromuscular signalling and synaptic physiology in the pharyngeal circuit that will provide a route to i) assigning compounds to discrete classes for mode of action ii) validating mode of action iii) confirming effects in the intact organism and iv) rank order of potency for lead optimisation. Acknowledgements: Funded by BBSRC grant no BB/L02439X/1. 23: Wasim Hussain 1; Paul McVeigh 1; Paul McCusker 1; Angela Mousley 1; Nikki Marks 1; Aaron Maule 1 1 Molecular Biosciences: Parasitology, The Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, United Kingdom

23P: INVESTIGATING THE RNA-INTERFERENCE PATHWAY IN LIVER FLUKE

The liver fluke Fasciola hepatica is a trematode parasite that infects both humans and ruminants. It causes the disease Fascioliasis in humans and Fasciolosis in animals. It is estimated that worldwide up to 17 million people are infected with F. hepatica and that the parasite costs an estimated $3 billion to the agriculture industry. The drug Triclabendazole can be used to treat both acute and chronic F. hepatica infections however cases of resistance are being reported worldwide. Efforts to develop novel drugs are dampened by the lack of tools for drug-target validation in this species. It has been nearly 10 years since RNA-interference (RNAi) was first reported in F. hepatica. Since then, many genes have been successfully knocked down in liver fluke but the exact mechanism of RNAi is still unclear. Using a bioinformatics approach we report the putative pathway of RNAi in F. hepatica including both micro (miRNA) and short-interfering (siRNA) RNAi pathways. Identified were homologs for the nucleases Drosha and Dicer as well as the main component of the RNAi-Induced-Silencing-Complex (RISC), Argonaute. Also identified was the dsRNA transporter SID-1. In other species, SID-1 has been shown to transport dsRNA from the gut to neighbouring cells and is required for RNAi to function. Expression analysis reveals that SID-1 is expressed in the juvenile fluke stage but not in the mature adult stage. This expression profile matches our own observations on differences in the susceptibility to environmental dsRNAs of these two life stages.

42

Kirsty McCann 1, Shannon M Hedtke 1, Stephen Doyle 1, Dave Spratt 2, Warwick Grant

1Nematode Functional Genomics Laboratory, Department of Animal, Plant, and Soil Sciences, La Trobe University,

Bundoora, Victoria, Australia 2. CSIRO Ecosystems, Canberra, Australia

24 P: TOWARDS DEVELOPING AN ANIMAL MODEL FOR TESTING ANTIHELMINTHICS ON MICROFILIARIAL NEMATODES

Filarial nematodes infect and cause disease in humans and animals, and are transmitted via insect vectors. The causative

agent of onchocerciasis in humans, Onchocerca volvulus, affects over 37 million people world-wide, and causes a range

of symptoms, from mild itching to blindness. Many of these symptoms are caused by the host immune response to

parasite death. This immunological response is unpredictable, and drug treatments that kill microfilaria can illicit severe

responses in humans, including death. An animal model that recapitulates the immunopathology of disease would aid in

not only understanding the disease further but would provide a predictive screening model in which candidate drugs

could be tested for safety as well as efficacy prior to human trials. We have begun work on developing an animal model

that would be suitable for drug testing. The parasitic nematode Circopethifilaria johnstoni has a life cycle analogous to

that of Onchocerca volvulus, and, crucially, elicits in its bush rat Rattus fuscipes hosts similar immune pathology, including

eye damage. One challenge to developing this system as a model in the laboratory is that C. johnstoni is vectored by ticks

in the genus Ixodes. Ticks have long life cycles relative to other insect vectors (such as mosquitoes or black flies), and at

least one Australian tick species can cause paralysis in small lab animals, while being morphologically indistinguishable

in immature stages from non-paralytic species. We have identified genetic markers that are suitable for distinguishing

paralysis-causing ticks from other species, and have used these markers to determine the species to which ticks belong

prior to attachment to bushrats. In the course of this work, we have found evidence to support a molecular revision of

the current classification of at least some Australian ixodid ticks.