The role of molecular biology in veterinary parasitology

Veterinary Parasitology 98 (2001) 169–194

The role of molecular biology inveterinary parasitology

Roger Prichard a,∗, Andy Tait b

a Institute of Parasitology, McGill University, 21, 111 Lakeshore Road,Ste Anne-de-Bellevue, Pointe-Claire, Que., Canada H9S 5G5

b Wellcome Centre for Molecular Parasitology, University of Glasgow, 56 Dumbarton Road, Glasgow, UK

Abstract

The tools of molecular biology are increasingly relevant to veterinary parasitology. The sequencingof the complete genomes of Caenorhabditis elegans and other helminths and protozoa is allowinggreat advances in studying the biology, and improving diagnosis and control of parasites. UniqueDNA sequences provide very high levels of specificity for the diagnosis and identification of parasitespecies and strains, and PCR allows extremely high levels of sensitivity. New techniques, such asthe use of uniquely designed molecular beacons and DNA microarrays will eventually allow rapidscreening for specific parasite genotypes and assist in diagnostic and epidemiological studies of vet-erinary parasites. The ability to use genome data to clone and sequence genes which when expressedwill provide antigens for vaccine screening and receptors and enzymes for mechanism-basedchemotherapy screening will increase our options for parasite control. In addition, DNA vaccines canhave desirable characteristics, such as sustained stimulation of the host immune system comparedwith protein based vaccines. One of the greatest threats to parasite control has been the developmentof drug resistance in parasites. Our knowledge of the basis of drug resistance and our ability to mon-itor its development with highly sensitive and specific DNA-based assays for ‘resistance’-alleleswill help maintain the effectiveness of existing antiparasitic drugs and provide hope that we canmaintain control of parasitic disease outbreaks. © 2001 Published by Elsevier Science B.V.

Keywords: Veterinary parasitology; Molecular biology; Genome; Diagnosis; Vaccines; Chemotherapy; Drugresistance; Nematode; Helminth; Protozoa

1. Introduction

This topic was reviewed at the 16th International Conference of the World Association forthe Advancement of Veterinary Parasitology (WAAVP) (Prichard, 1997). Since then great

∗ Corresponding author.E-mail address: [email protected] (R. Prichard).

0304-4017/01/$ – see front matter © 2001 Published by Elsevier Science B.V.PII: S0 3 0 4 -4 0 17 (01 )00429 -0

170 R. Prichard, A. Tait / Veterinary Parasitology 98 (2001) 169–194

advances have taken place in understanding the molecular biology of veterinary parasitesand their hosts, and molecular biology has become a routine tool in the search for improvedmethods of diagnosis and control of veterinary parasites and the epidemiology of parasiticdisease. This review will focus on developments in relevant genome projects, diagnosis,vaccine development, the search for new chemotherapeutic agents and understanding drugresistance in veterinary parasites.

Improvements in the prevention of parasitic disease have occurred through develop-ments in diagnostic reagents, antiparasitic drugs, understanding resistance to antiparasiticdrugs, and vaccines. Improvements have been slow. Understanding the molecular biologyof host–parasite interactions and using the tools of genomics, proteomics and bioinfor-matics offer the prospect of improving our success in preventing and controlling parasiticdiseases.

Knowledge of the molecular biology of parasites and their hosts and the use of moleculartools are becoming increasingly important to the field of veterinary parasitology. This trendneeds to be encouraged and given a high level of exposure by associations like WAAVP,and become an integral part of the teaching of veterinary parasitology. It is incumbent uponus to develop our understanding of the molecular biology of host–parasite interactions, andto apply this knowledge to the advancement of veterinary parasitology, if our field is tobe competitive for research funding, and the interest of students, veterinary practitioners,the biotechnology/pharmaceutical and agricultural industries, and fellow scientists in thebiomedical field.

2. Veterinary parasite genomics

Traditional immunological studies and empirical drug screening have served veterinaryparasitology in the past in the search for diagnostic tools, vaccines and antiparasitic drugs.However, to many, progress has been disappointing. We have virtually no defined antipara-sitic vaccines, significant breakthroughs in the discovery of new classes of broad spectrumantiparasitic drugs has occurred less frequently than once per decade over the last 40 years.Drug resistance is threatening livestock production in some parts of the world and is gettingworse, and it has been recently argued that veterinary parasitology is being marginalized(Zajac et al., 2000).

In the last few years, it has become possible to sequence the whole genome of key parasitesand related organisms, such as Caenorhabditis elegans (C. elegans Sequencing Consortium;Anon., 1998). In fact, the genome of this nematode was the first completed genome forany multicellular organism. It represents a tremendous resource for research on helminths.Degenerate primers, based on C. elegans gene sequences, have already been used to identifyand study related genes in parasitic helminths (see, e.g., Blackhall et al., 1998a–c; Forresteret al., 1999). Veterinary parasitology can also benefit from whole genome projects beingconducted on human parasites. For example, the Filarial Genome Project being conductedwith Brugia malayi (Williams et al., 2000) will be a valuable molecular tool for researchon Dirofilaria immitis and other nematodes of interest to veterinary parasitology. Similarly,the Schistosome genome project (Williams and Johnston, 1999) will provide informationuseful for research on veterinary parasites such as Fasciola hepatica, and the Leishmania,

R. Prichard, A. Tait / Veterinary Parasitology 98 (2001) 169–194 171

Table 1Ongoing genome sequencing projects on parasite pathogens of animals or zoonotic parasites (Anon., 2000)

Organism Sequencing center

ProtozoaAnaplasma marginale USDA-ARS/Washington State University/Amplicon ExpressCryptosporidium parvum University of MinnesotaNeospora caninum EST and GST Washington UniversityTheileria parva TIGRToxoplasma gondii Sanger Centre; Washington University

HelminthsAscaris suum ESTs University of Edinburgh/Sanger CentreHaemonchus contortus ESTs University of Edinburgh/Sanger CentreTrichinella spiralis University of Edinburgh/ Sanger Centre

Plasmodium and Entamoeba genome projects, although oriented to human parasites willbenefit research on protozoa important to veterinary parasitology.

It is interesting that the genome of the bacterial endosymbiont, Wolbachia spp. is alsobeing unraveled as part of the B. malayi genome project. It has been postulated that manip-ulation of the biology of Wolbachia, for example with antibiotics effective against theserickettsia-like microorganisms, may open up new methods to control filarial parasites(Williams et al., 2000).

Genome sequences will serve as the basis for future functional analyses of the newlydiscovered genes. In turn, proteins that are responsible for virulence, pathogenesis, hostspecificity, the induction of protective immune responses and which are essential compo-nents of metabolic processes required for the homeostasis of the parasite will be identified.This information will provide the basis for designing novel vaccines, antiparasitic drugsand diagnostic reagents.

A review of public information databases reveals that there are about eight or moregenomics projects on veterinary parasites (Table 1) (Anon., 2000). Recently, at the USDAMicrobial Genomics Workshop (Anon., 2000) a priority short list of 15 animal and foodpathogenic organisms was identified by a panel of scientists and stakeholders. Amongstthis list were the parasites Eimeria spp. and T. gondii. The latter parasite was considered amodel for apicomplexans such as Eimeria, Sarcocystis, Neospora, etc., which are importantpathogens and/or cause zoonoses. In addition, a list of other pathogens considered important,included the parasites listed in Table 2.

3. Diagnosis

3.1. Helminths

Great advances have been made in improving the sensitivity and specificity of diagnosisof veterinary parasites. Examples of the use of molecular techniques up to 1998 are givenin the reviews of Prichard (1997) and McKeand (1998). Typically, DNA is extracted from


Table 2List of parasitic organisms considered important for whole genome sequencing. Parasites are categorized on thebasis of animal host species with which the parasite is most commonly associated (Anon., 2000)

Animal host Parasite

Cattle Babesia spp., T. parva, Ostertagia ostertagiSheep H. contortusPigs A. suum, T. spiralisPoultry Eimeria spp.Horses Theileria equi, Strongylus vulgarisCats and other animals T. gondii

the sample of interest which can then be probed by DNA hybridization and analyzed byrestriction fragment length polymorphism (RFLP). More commonly, DNA is amplified bythe polymerase chain reaction (PCR) using specific primers for diagnostic sequences. Thismay be followed by RFLP, PCR linked to hybridization with specific oligoprobes; andPCR-linked single strand conformation polymorphism (SSCP). Or, non-specific primersused to produce random amplified polymorphic DNA (RAPD). These methods have beendiscussed in the previously mentioned reviews.

Another interesting approach (reviewed by Nutman et al., 1994) has been to conductthe PCR reaction having one of the primers biotinylated. The resultant double strandedDNA, with biotin attached to one strand, can then be bound to streptavidin-coated wells inmicrotitre plates. The DNA attached to the well by the biotin–streptavidin is then denaturedwith sodium hydroxide to remove the non-biotinylated single strand. A fluorescein-labeledspecific probe is introduced into the well under stringent conditions for hybridization. Afterwashing an anti-fluorescein antibody, conjugated to alkaline phosphatase is introduced, theplate incubated and washed again and then an alkaline phosphatase substrate introducedand the well read in an ELISA reader. This method avoids the use of radioisotopes and usesan ELISA approach common in immuno-diagnostic laboratories.

A number of workers have shown that the internal transcribed spacers (ITS) of ribosomal(r)DNA are species specific and provide a DNA region which allows for high sensitivity andspecificity for DNA-based assays for parasitic helminths (Gasser et al., 1997; Jacobs et al.,1997). Using this approach with the second ITS spacer (ITS2), Schnieder et al. (1999) wereable to detect as little as a single egg and differentiate between the Cooperia, Haemonchus,Trichostrongylus, Nematodirus and Ostertagia genera.

Eysker and Ploeger (2000) reviewed different methods that are available for determiningthe level of infection of ruminant animals with gastrointestinal nematodes and concludedthat DNA-based tests on faces, for DNA in nematode eggs, could be specific and show whichgastrointestinal species may be infecting an animal. However, the egg output of nematodeparasites such as O. ostertagi is low and not well correlated to worm number, rendering suchDNA-based tests not quantitative. Quantifying the level of parasite infection is importantfor optimizing parasite control strategies.

Nevertheless, Zarlenga et al. (1998) were able to use the first ITS spacer (ITS1) ofnematode rDNA to accurately estimate the proportion of O. ostertagi eggs in a mixed fecalpopulation of O. ostertagi, H. contortus, C. oncophora and Oesophogostomum radiatum.Sensitivity was equivalent to 0.05 eggs.


DNA-based assays have also been successfully employed to differentiate between dif-ferent strains within a parasitic species (Joachim et al., 1997; Zarlenga et al., 1999) and tostudy biological differences between strains. For example, Talvik et al. (1997) were ableto study differences in prepatent periods between different isolates of Oesophogostomumspp. in pigs using ITS–PCR.

3.2. Protozoa

The main groups of protozoan parasites of veterinary significance are largely diagnosedusing microscopy coupled either with conventional stains or immunofluorescence/immuno-cytochemistry. These methods involve sampling blood or lymph nodes using smears(Trypanosoma, Babesia, Theileria, etc.), fecal (Eimeria, Cryptosporidium, Giardia) ortissue smears or sections (Toxoplasma, Neospora, Eimeria). The definition of particu-lar species within a genus is dependent on morphological criteria coupled with clinicaldata and/or the detection of high antibody titres to the parasite concerned using ELISA orimmunofluorescence/immunochemical based methods. Most of these methods have stoodthe test of time and are still the main methods used in many diagnostic laboratories. How-ever, there are now a range of PCR based methods that have been devised, albeit primarilyin a research based context. In general, formats for routine diagnosis using PCR have notbeen developed although the situation is rapidly changing and the availability of microarrayformats makes the latter an attractive possibility for the future.

As with helminth diagnosis, the rDNA genes have been a well-used target for developingPCR based diagnostics. This is due to the ready availability of sequence data coupled withthe ability to design primers to the variable region of these genes, providing species speci-ficity. An additional advantage in the use of these genes is that they are invariably multicopyand thus provide an increase in sensitivity over assays using single copy sequences. PCRbased systems using the rDNA genes have been devised for Babesia species (Bashiruddinet al., 1999; Calder et al., 1996; Figueroa et al., 1993), Cryptosporidium (Morgan andThompson, 1998; Awad-el-Kariem et al., 1994; Smith, 1998) and Theileria (d’Oliveiraet al., 1995; Bishop et al., 1995). In addition, PCR based systems have been developedusing single copy sequences. Examples of these include the COWP gene of Cryptosporid-ium (Spano et al., 1997), the 14-3-3 gene and an anonymous gene fragment of Neospora(Yamage et al., 1996; Lally et al., 1996) and the major merozoite antigen gene of Theile-ria where reasonable sensitivities have been obtained (d’Oliveira et al., 1995). Multicopysequences have an obvious advantage in terms of sensitivity given that each organism willcontribute many target sequences to the PCR reaction. Such target sequences have been usedto develop methods for diagnosing tsetse transmitted African trypanosomes and not onlyprovide species identification but also sensitive detection of the parasites (Reifenberg et al.,1997). There are primers designed to detect T.b. brucei (McNamara et al., 1994), T. vivax(Masake et al., 1997) and the different ‘types’ of T. congolense (Masiga et al., 1992; Majiwaet al., 1993). With Toxoplasma, a repetitive sequence has also been identified that allows thesensitive detection of the parasite in infected tissue (Burg et al., 1989; Johnson et al., 1993).

Each of the range of different sources of parasite material (blood, tissues, faces, vectorsand environmental samples such as water) create different problems for PCR based diag-nosis, including inhibitors, sensitivity and the bulk of tissue in the case of tissue infective


parasites. Many of the systems that have been published are low-throughput involving PCRof purified parasites and detection of amplicons using agarose gel electrophoresis. Thus theprocessing of samples and the format for the detection of PCR products are critical issuesthat need to be addressed if high-throughput systems are to be developed. A further questionis quantitation; infection by a number of protozoan parasites leads to a carrier state afteranimals have recovered from clinical disease and such animals give a positive diagnosisusing PCR. While in some situations, such as the movement of animals into non-endemicregions, this sensitivity is important, however, in terms of diagnosing the cause of clinicaldisease such positives could be confusing. From a research point of view, the ability todetect multiple species, some of which may occur at low levels may lead to the elucidationof subtle inter-species interactions that affect clinical disease or susceptibility to infection.

These issues and questions have been addressed in some of the diagnostic tests and arecritical to achieving appropriate sensitivity and quantitation. In the case of blood borneparasites such as Trypanosoma, Theileria and Babesia, the problem of PCR inhibition hasbeen addressed and results in high levels of sensitivity. Theileria or Babesia infected bloodcan be lysed in dilute detergent so as to release the contents of the red blood cell butretain the parasites within the resulting red cell ghosts. The parasite material can then becentrifuged to concentrate the parasites before full lysis and PCR analysis (d’Oliveira et al.,1995). In the case of Trypanosoma infected blood, a system originally devised for malaria(Kyes et al., 1993) has been used in which blood spots are placed on filter papers, driedand then processed in the presence of an ion exchange resin to remove blood inhibitors(Almeida et al., 1997; Katakura et al., 1997). With these systems, as little as 3000 parasitesper milliliter can be detected. For the other groups of protozoan parasites the source ofmaterial is likely to be faeces or filtered water in the case of Cryptosporidium and Giardia,or tissue in the case of Toxoplasma or Neospora. These sources of material raise potentialproblems if infected material is used directly either in terms of inhibitors or sensitivityif parasite levels are low. However, a number of these problems have been overcome;for example with Cryptosporidium direct fecal extraction and ion exchange absorption ofnucleic acid followed by PCR leads to the detection of <1000 oocysts/g (McLauchlin et al.,1999; Smith, 1999) or as little as 1–10 oocysts per 10 000 from filters of municipal water(Chung et al., 1999). With Neospora and Toxoplasma infected tissue PCR based methodshave been devised to detect these parasites specifically (Lally et al., 1996; Muller et al.,1996), and shown to detect as few as one parasite per milligram of brain or muscle tissue(Yamage et al., 1996). It is clear that a simple extraction system, without the need to purifyparasites is an essential element to provide wide applicability. The development of realtime PCR as a general methodology for quantitating the level of original target sequencein the reaction offers the opportunity of allowing the number of parasites to be estimatedin a given sample by comparison with standards. Such a system has been developed forToxoplasma using either purified tachyzoites or extracts from paraffin embedded tissue (Linet al., 2000). Using the amplification of the B1 gene, 0.05 tachyzoites can be detected or0.34 parasite equivalents in a small tissue section. This approach could readily be appliedto other parasite species although it is quite costly in terms of equipment and reagents.

The ability to amplify parasite sequences relatively directly from samples of blood,tissue or faeces raises the issue of designing simple methods for detecting these ampli-cons from large number of samples and being able to identify specific species of parasite


simultaneously. While the use of microarrays or molecular beacons would be a very attrac-tive approach, these have yet to be developed. An illustration of how this type of technologycould be used is provided by the technique of reverse line blotting. This technique was orig-inally developed for the diagnosis of Mycobacterium tuberculosis (Kamerbeek et al., 1997),but has now been applied to the diagnosis of Theileria and Babesia species (Gubbels et al.,1999). Essentially, universal primers are used to amplify the V4 region of the rDNA gene ofall species from a lysed blood sample and the resulting amplicons labeled non-radioactively.The labeled rDNA amplicon is then used as a probe and hybridized to a membrane on whicha series of species specific oligonucleotides have been immobilized. This has been success-fully used to detect six species of Theileria and three species of Babesia and can detectsamples with 3000 parasites/ml of blood. The attraction of this technique is that only a singlePCR is required per sample and animals infected with more than one species of parasite canbe detected. In principle, this type of system could be applied to most protozoan parasitesand the filters could be customized for detecting specific groups of parasites potentiallywithout the need to undertake extensive purification of the sample. The sensitivity reportedis an improvement on microscopy and could almost certainly be increased by using a moresensitive system for detecting the hybridization signal.

Overall, the detection of protozoan parasites using molecular methods is available andpotentially provides systems that are both very sensitive and highly species specific. Someproblems remain to be solved primarily in the areas of sample preparation, application to agreater range of species and in provision of multi-species high-throughput formats.

3.3. Molecular beacons

One of the new DNA technologies relevant to the diagnosis of parasites is that of molecularbeacons (Vet et al., 1999; http://www.molecular-beacons.org/default.htm). Once a shortDNA sequence has been identified which is unique to the species or strain of interest, itcan be detected by the hybridization of a molecular beacon to this sequence. The molecularbeacon is a DNA probe with a fluorophore on one end and a fluorescence quencher on theother end of the probe. The probe is constructed with a relatively large central sequencethat is complementary to the species/strain-specific unique DNA (or RNA) sequence, butnot to itself (the probe section), and with each end of the molecular beacon containing shortstretches of DNA that are complementary to each other. In the absence of test DNA towhich the longer central part of the molecular beacon will hybridize, the two end sectionsof the molecular beacon will hybridize with each other (see Fig. 1) to form a ‘hairpin’structure. In this state, when the fluorophore is excited by light, the fluorescence energy isimmediately trapped and quenched by the quencher which is adjacent to the fluorophore.However, when the molecular beacon hybridizes to a test sequence to which the longercentral region is complementary, the hairpin is linearized and the quencher is no longeradjacent to the fluorophore. This allows a strong fluorescent signal to be emitted when themolecular beacon hybridizes to the sequence that it recognizes and is excited by light.

Molecular beacons can be constructed to recognize any DNA sequence of interest.Typically the test sample DNA is amplified by PCR before detection with a molecularbeacon. The chief constraint at present is the cost of having the molecular beacon synthe-sized. This and related fluorescent probe techniques will prove valuable for epidemiological


Fig. 1. Molecular beacon to detect specific DNA sequence. (A) Hairpin shape. Emission from fluorophorequenched. (B) Molecular beacon unfolded and hybridized to DNA sample. Excited fluorophore emits strongfluorescent signal.

and other studies where large numbers of samples need to be analyzed and the initial cost ofpreparing the molecular beacon and purchase of a fluorescent plate reader or real-time flu-orescent PCR machine can be amortized over many samples. Its development for diagnosisof veterinary parasites will be assisted by parasite genome projects and by other efforts toidentify species/strain unique DNA sequences.

3.4. DNA arrays

DNA arrays (see, for example, Supplement to Nature Genetics, Vol. 21, January 1999,“The Chipping Forecast”) consist of oligonucleotides tethered to a glass or silica chip. Tensof thousands of oligonucleotides can be tethered in an array on a single chip. Standard arrays(e.g. human genome, mouse genome, etc.) can be purchased. However, it is also possible tomake custom arrays, and it will be possible to assemble small arrays for the diagnosis of arange of parasites and parasite strains. There are a number of ways of using DNA microarraysfor the detection of unique DNA (or RNA) sequences. One method is to fluorescently labelall the DNA sequences in the test sample. The sample DNA that hybridizes to a specificlocation on the microarray can be detected by fluorescent array detection and the dataanalyzed by computer programs. Often more practical is to use competitive hybridizationin which the test sample competes for hybridization to the tethered oligonucleotide, on thechip, with a fluorescent labeled competitor oligonucleotide. When the test DNA is perfectlycomplimentary to the tethered oligonucleotide, it will hybridize to the chip. When the testDNA is not perfectly complementary to the tethered oligonucleotide, the fluorescent labeledcompetitor oligonucleotide will bind to the tethered oligonucleotide on the chip and displacethe test DNA. A fluorescent microarray detector and computer program can then analyze thefluorescent array for the presence or absence of the species/strain specific DNA sequence.

Set up cost for the use of DNA microarrays is high. However, once the equipment isavailable and microarrays have been prepared, cost per unit of sample analyzed will be low.


Furthermore, analysis time is extremely rapid. This development depends on more routineuse of molecular tools in veterinary parasitology. DNA microarray technology will be usedin the future for diagnosis and other purposes in veterinary parasitology. There is greatpotential for the use of DNA microarrays in regional veterinary diagnostic laboratories.

4. Vaccine development

4.1. Helminths

Progress towards the development of vaccines against helminth parasites has beenreviewed (Klei, 1997; Munn, 1997; Prichard, 1997; Newton and Munn, 1999; Smith, 1999),while Lightowlers et al. (2000) have reviewed progress towards vaccination against cys-ticercosis and hydatid disease and Ramaswamy (1999) has discussed the use of naked DNAvaccines against parasites. Considerable progress has been made in recent years in identi-fying candidate antigens for vaccines against parasitic helminths using natural preparationsfrom parasites to immunize host animals (mainly sheep) and then using the antibodies soproduced to screen expression libraries, followed by cloning and expression of the gene foreach antigen felt to have potential for vaccine development. Whilst this approach has ledto successes, it is a lengthy process, limited by the first step of preparing natural extractsfrom worms for immunization of animals. The availability of the full C. elegans genome andprogress on other helminth genome projects, as discussed above, should shorten this processby allowing specific helminth genes to be more readily identified, cloned and expressed forevaluation as vaccine candidates as proteins, peptides or DNA.

Several ‘natural’ antigens from tapeworms have been identified as vaccine candidates andconsiderable success achieved using recombinant antigens. Three oncospere antigens fromTaenia ovis (To 45W, To 16.17 and To 18) have been identified and produced in recombinantexpression systems and produced 73–99% protection in sheep against challenge with T. ovis(Johnson et al., 1989; Harrison et al., 1996). The oil adjuvants, saponin and DEAE-dextrangave the highest antibody responses and greatest degree of protection against challengeinfection with T. ovis eggs. A dialysed saponin based vaccine showed highest significantprotection up to 6 months after immunization (Harrison et al., 1999). Recent work showedthat the N-terminus of To 45W was more immunogenic than the C-terminus (Dadley-Mooreet al., 1999). Rothel et al. (1997) undertook DNA vaccination work with the To 45W andfound that initial vaccination with a plasmid containing the To 45W cDNA, pcDNA3-45Wfollowed by subsequent vaccination with the recombinant To 45W antigen resulted inantibody levels significantly higher (P < 0.05) than those obtained in sheep which hadonly received the recombinant To 45W vaccine.

Similar recombinant onchosphere antigens have shown high levels of protection againstT. saginata (Tsa 9 and Tsa 18) in cattle (99%) (Lightowlers et al., 1996a,b); T. solium(To 45W, To 16.17 and To 18 derived from T. ovis) in pigs (93%) (Plancarte et al., 1999);and E. granulosus (EG 95) in sheep (97%) (Lightowlers et al., 1996a,b).

Cathepsin L1 and L2, trematode hemoglobin, glutathione-S-transferase and fatty acidbinding protein have been used in vaccination studies against F. hepatica and F. giganticain sheep and cattle (see Smith, 1999 for summary). However, protection against worm


numbers has only been moderate. Smooker et al. (1999) showed that mice vaccinated withDNA constructs encoding F. hepatica GST had a humoral response to the DNA vaccine. Itis interesting that Fasciola cathepsin L1 and L2 specifically degrade IgG, suggesting thatthis may be a mechanism by which the parasite evades the immune system (Berasain et al.,2000).

Much work has been done on the so-called ‘hidden’ antigens in nematode parasites sincethe commercialization of a recombinant vaccine against the Australian cattle tick, Boophilusmicroplus (Willadsen et al., 1995). These are antigens not normally seen by the immunesystem, but to which an immune reaction can be directed if the antigen is presented to theimmune system via vaccination. Usually the hidden antigen occurs in the gut membrane ofthe parasite. Moderate to high levels of protection have been reported in sheep vaccinatedby the H. contortus gut aminopeptidases, known as H11 (Smith et al., 1997). Anotherpeptidase has been implicated in immunity in naturally immune lambs, a developmentallyregulated zinc metallopeptidase, present in membrane preparations from blood feeding H.contortus. This metallopeptidase has been cloned and characterized (Redmond et al., 1997).Other proteases which are of interest for vaccine development are an extensive family ofcathepsin B-like cysteine proteases which have been cloned and characterized and shownto be targeted by the immune system in immune sheep (Rehman and Jasmer, 1999; Skuceet al., 1999). The immunogenic properties of a recombinant Cu/Zn superoxide dismutasesfrom H. contortus have also been assessed (Liddell and Knox, 1998). Another proteinimplicated in immunity to H. contortus and present in gut preparations from the worm,glutamate dehydrogenase, has recently been cloned and characterized (Skuce et al., 1999).These efforts to produce recombinant antigens and screen them as vaccine candidates isalready being greatly assisted by access to the C. elegans genome. As the whole genome ofpathogenic helminths such as H. contortus are completed, this process is likely to accelerateleading to highly effective anti-nematode vaccines.

4.2. Protozoa

Live attenuated vaccines are available for a number of protozoan parasites includingBabesia bovis (Callow et al., 1997), Theileria annulata (Pipano, 1995), Eimeria spp. (Shirleyand Bedrnik, 1997) and Toxoplasma (Buxton et al., 1991). While these have proved to behighly effective, live vaccines suffers from a number of disadvantages such as the require-ment for cold chains, the potential of contamination with other pathogens, shelf life, produc-tion, etc. The possibility of developing sub-unit vaccines is raised by advances in molecularbiology particularly in relation to being able to produce and deliver antigens. The optimismof the early days of molecular parasitology has been tempered by the realization that theidentification of relevant antigens and their delivery is complex. In considering this field,the discussion is divided into an outline of the current progress towards the development ofsub-unit vaccines followed by the consideration of the future challenges presented by theavailability of genome sequences of a number of important parasites.

The critical issues, in the context of the availability of gene libraries and a suite ofmolecular techniques, concern the identification of relevant antigens and the use of differentdelivery systems. Rather than providing a full review of current progress on all researchin this area, the focus will be on the parasites with which there is an extensive research


effort, namely Theileria parva, T. annulata, B. bovis and Babesia bigemina, and the generalresearch strategies that have been applied. In the interests of brevity the extensive work onvaccination against the ricketsial pathogens (Palmer et al., 1999; Mahan et al., 1999) willnot be considered here. Progress with vaccination against the protozoan tick borne parasiteshas recently been reviewed in a special issue of Parasitology Today (Vol. 15, pp. 253–300).To date, no protozoan sub-unit vaccine is immediately in prospect but a vaccine against thetick B. microplus is currently on the market (Tick GARD Plus in Australia and GAVACPlus in Cuba). This vaccine is based on the use of a recombinant gut antigen (Bm86)and vaccination leads to reduction in the number of engorged females and the resultantreduction in the number of larvae (up to 90% reduction per generation). As the antigen isnot normally exposed to the immune response, no natural boosting of immunity occurs as aresult of field challenge (Willadsen and Jongejan, 1999; Willadsen, 1997; Willadsen et al.,1989). Preliminary results with a 90 kDa antigen from R. appendiculatus have been reported(Rutti et al., 1991) and suggest that an analogous approach could be applied to other tickgenera and species. It is not known what effect vaccination against the tick will have on thetransmission of protozoan parasites and this area needs further research. These results raisethe whole issue of whether vaccination against the vectors of disease is a viable method tocontrolling transmission. This could potentially be a very useful approach where multiplespecies of parasite are transmitted by the same vector or where vaccination strategies againstthe parasite are unlikely to be feasible.

The identification of the relevant antigens is a key step in the development of a sub-unitvaccine and depends heavily on a knowledge of protective immune mechanisms in order todevise relevant assay systems and target particular life cycle stages. However, advances havebeen made in the absence of such information using pragmatic approaches. In principal,there are five approaches to antigen identification that have been used or could be applied. Inthe first, proteins are fractionated by biochemical means, the fractions tested for protectiveresponses in the target host species and the gene(s) encoding the antigens within the activefractions cloned from an expression library for further evaluation in protection assays.In the second, a similar approach is applied but CD4+ T-cell (obtained from immuneanimals) stimulation assays are used to identify active fractions with subsequent isolationof individual antigens from expression libraries. In the third, CD8+ T-cells from immuneanimals are used as an assay for the relevant antigens. These antigens need to be presented aspeptides and so a system for transfecting the antigen presenting target cells or the availabilityof a set of candidate molecules that can be added to the assay as peptides are required. In thefourth, molecules considered to have a critical role in the infection process (for example hostcell invasion) are selected, cloned and expressed before being evaluated in immunizationtrials. In the fifth, which has only been tested in malaria (Conway et al., 2000) and notdirectly with any veterinary parasite, genetic variation is examined in sets of specific genesfor evidence of immune selection. If evidence for such selection is obtained, it suggeststhat the antigen concerned is critical for parasite survival and is also the target for theprotective immune response. The application of some of these approaches is illustratedby the research on Theileria and Babesia recombinant antigens. One of the most difficultareas in this research are the criteria in any preliminary vaccine trials for the inclusion ofa particular antigen in further work. This is compounded by the array of potential deliverysystems and adjuvants that may critically affect the outcome after challenge.


While our understanding of protective immune mechanisms involved in B. bovis andB. bigemina infections is not complete, it is clear that antibody plays a significant role(Mahoney, 1986) and there is increasing evidence for a significant role for activatedmacrophages and CD4+ T-cells (Brown and Palmer, 1999; Brown et al., 1991). Systematictesting of fractionated B. bovis merozoite proteins led to the identification of five antigensthat conferred a level of protection to vaccinated animals (Wright et al., 1992). The proteinsare not abundant, are secreted from the parasite and are not immunodominant antigens(Wright et al., 1992; Harper et al., 1996). Combinations of these antigens, tested underfield conditions, gave significant protection compared to unvaccinated controls as well asprotection levels close to those provided by the live vaccine. Using CD4+ T-cell screening,a further set of antigens have been identified (Hines et al., 1992; Jasmer et al., 1992; Brownet al., 1996) that include two of the antigens identified in the earlier experiments as wellas two major merozoite surface antigens and a set of spherical body antigens (SBPs) thatappear to be secreted by the parasite and located on the cytoplasmic face of the red blood cell(Dowling et al., 1996). The rhoptry protein RAP-1 was identified in both sets of studies andis clearly a major candidate antigen for inclusion in a sub-unit vaccine; T-cell epitopes havebeen mapped on this molecule (Brown et al., 1996, 1998). In B. bigemina the RAP-1 proteinsare encoded by a gene family making their analysis more complex, however, immunizationwith a recombinant protein derived from one family member shows a protective effect onchallenge although this involves a reduction in parasitaemia relative to controls rather thancomplete protection (McElwain et al., 1991; Brown et al., 1998). The potential for rhoptryantigens to induce a protective response is illustrated by the effect shown on immunizationof cattle with purified whole rhoptries (Machado et al., 1999) where 7/8 cattle showed noparasitaemia on challenge with 500 B. bigemina infected tick larvae. Using proteomicsand amino acid sequencing or other approaches it would seem relatively straightforward toclone and express the constituent genes and test their expression products in immunizationexperiments. Thus the current data provide a relatively optimistic prospect for a sub-unitvaccine against this group of parasites.

Research on the development of a sub-unit vaccine against Theileria parva and T. annulatahas focussed on the elucidation of the mechanisms of immunity and the isolation andcharacterization of surface antigens considered to be important in host cell invasion. Theprotective immune response to T. parva highlights a major role for cytotoxic T-cells (Emery,1981; McKeever et al., 1994) with parasite peptides presented on MHC Class I moleculesof infected lymphocytes acting as the target that mediates their lysis (McKeever et al.,1999; Morrison et al., 1995). In contrast, the protective response to T. annulata involves acomplex interplay between NK cells, activated macrophages and cytotoxic T-cells (Prestonet al., 1999; Boulter and Hall, 2000). The humoral response seems to play a limited rolein parasite infected animals. The major sporozoite surface antigen from both species hasbeen cloned, sequenced (p. 67, T. parva; SPAG, T. annulata) and then used as an expressedrecombinant antigen in a series of laboratory based immunization trials (Boulter et al., 1995,1998; Nene et al., 1996; Morrison and McKeever, 1998). The results of these trials showthat this molecule can confer a protective response with both species of parasite with up to70% of immunized animals being protected in the case of T. parva (Musoke et al., 1992)and significant reductions in a number of disease parameters including parasitaemia, withT. annulata (Boulter and Hall, 2000). The major merozoite surface antigen of T. annulata


(Tams-1) has also been expressed in recombinant form and used in small scale immunizationtrials (d’Oliveira et al., 1997). The results initially suggested that this molecule elicited aprotective response but this result has not proved to be reproducible. However, extensiveanalysis of the sequence variation between alleles of this gene from field isolates showthat it is under strong selective pressure (Shiels et al., 1995) supporting the view that itplays a critical role in parasite survival and is recognized by the immune system. Whilethese antigens almost certainly warrant inclusion in a sub-unit vaccine, it is clear that sucha vaccine will require the inclusion of antigens that stimulate cellular responses to theschizont infected lymphocyte. Approaches to the identification of such molecules are beingdeveloped and include (in T. parva) the elution of peptides from MHC Class I molecules toasses their ability to sensitize target cells to cytotoxic T-cell killing and the transfection ofcells expressing Bovine Class I molecules with parasite cDNAs (McKeever et al., 1999).Once active molecules are identified the issue of antigen delivery will need to be addressed.

These examples of current progress towards the development of sub-unit vaccines illus-trate the range of approaches and some of the complexities that are involved in this area ofresearch as well as demonstrating the value of understanding the mechanisms of immunity(Morrison et al., 1995). These considerations apply to any of the protozoan parasites wherea research effort is being mounted to develop a vaccine. In the next few years, the genomesequences of many of the protozoan parasites will be completed and it is important to con-sider how this information will impact on vaccine research. The optimists in the communitywould argue that this will rapidly lead to the identification of candidate vaccine antigens and,while this is possibly true, the potential numbers of candidate molecules could be daunting.It is envisaged that the initial analysis would involve a bio-informatics approach to identifymolecules containing relevant motifs such as signal peptides and those required for presen-tation by the cellular immune system as well as data on stage specificity of gene expression.At the present time it is difficult to judge how many molecules would be identified in thisway but it is likely to be at least 100’s depending on the sophistication and specificity ofthe motif searches. It is clear that such approaches will require high throughput functionalscreening systems and the design and testing of these will be critical to the full exploitationof the complete genome sequences.

5. Drug resistance

5.1. Genetics of anthelmintic resistance

Genes involved in the mechanism of action of an anthelmintic are likely to be, but are notnecessarily involved with mechanisms of anthelmintic resistance. Resistance mechanismscan broadly be considered as due to changes in the drug receptor or effector (involvingsome of the same genes involved with the drug mode of action), or with modulation ofdrug concentration at its site of action. Genes which code for drug transport or metabolism,for example, can be involved in drug resistance but have no direct role in the mechanismof action. If the mechanism of resistance is due solely to mutation(s) in genes involvedin the mode of action of a class of anthelmintics, different anthelmintics with the samemode of action are likely to share the same mechanism of resistance. However, if the


mechanism of resistance is entirely or in part due to modulation of drug concentration,different anthelmintics in the same mode of action class may not necessarily show the samelevel of resistance because of the effect of different chemical substituents on transport ormetabolism.

Levamisole resistant mutants of C. elegans lack a normal complement of physiologicallyfunctioning acetylcholine receptors (Lewis et al., 1980). The cloning and expression ofthe levamisole resistance genes, lev-1, unc-29 and unc-38 has revealed that they encodesubunits of acetylcholine-gated cation (Na+ and K+) channels in C. elegans including alevamisole/morantel binding site (Fleming et al., 1997). The lev-1 appears to be a struc-tural subunit of a five subunit acetylcholine-gated channel. A mutation, Glu237Lys in thesequence encoding a transmembrane region of lev-1 changes the ion channel from cationicto anionic and renders C. elegans insensitive to levamisole. In the parasitic nematode, O.dentatum there appear to be a number of cholinergic channels which respond to levamisolewith variable conductances (Martin et al., 1997). In H. contortus, there appear to be highand low affinity levamisole binding sites on cholinergic receptor subunits, with differencesbetween resistant and susceptible worms consistent with the low affinity receptors in resis-tant worms readily becoming unresponsive to levamisole (Sangster et al., 1998a,b). Thesestudies suggests that several genes may be involved with the effects of levamisole and possi-bly with levamisole resistance and in one isolate of H. contortus in which this question wasaddressed, levamisole resistance appeared to be multigenic (Sangster et al., 1998a,b). Otherwork suggested that it was a recessive autosomal trait in H. contortus (Dobson et al., 1996).

Benzimidazole resistance in H. contortus has been shown to be due to selection onisotype I (�8–9) and isotype II (�12–16) �-tubulin genes (Geary et al., 1992; Kwa et al.,1993; Lubega et al., 1994; Beech et al., 1994). A conserved mutation at amino acid 200 ofisotype I �-tubulin, from phenylalanine to tyrosine has been shown to be associated withand to contribute to the benzimidazole resistance phenotype (Kwa et al., 1994, 1995). Wehave also shown, using point directed mutagenesis, expression and benzimidazole bindingand polymerization studies that the Phe200Tyr mutation on isotype II also contributes tobenzimidazole resistance (R.K. Prichard, M. Oxberry, Y. Bounhas, S. Sharma, G. Lubega, T.Geary, unpublished) and in some benzimidazole resistant strains a null isotype II may havebeen selected (Kwa et al., 1993). In addition to the Phe200Tyr mutation, we have foundthat in field and laboratory selected benzimidazole resistant strains, reported by Beechet al. (1994) that the Phe200Tyr mutation is not present and have found that a Phe167Tyror Phe167His mutation is present. Expression and pharmacological characterization hasshown that the codon 167 mutation, as with the codon 200 mutation, results in the lossof the benzimidazole high affinity receptor (R.K. Prichard, M. Oxberry, Y. Bounhas, S.Sharma, G. Lubega, T. Geary, unpublished). The findings that both isotype I and isotype II�-tubulins are involved with benzimidazole resistance and mutations at both codons 167 and200 can confer resistance have important implications for the design of genetic markers forbenzimidazole resistance. A DNA based method for diagnosis of benzimidazole resistancehas been described (Elard et al., 1999) based on the Phe200Tyr mutation in isotype I.

Different avermectin resistant strains of H. contortus show different phenotypes (Gillet al., 1998) and it has been argued that these differences may reflect selection pressures(Gill and Lacey, 1998). These findings suggest that more than one gene may contribute toresistance to macrocyclic lactones. Selection pressure may well influence the selection on


different genes involved in avermectin resistance. However, other factors such as the geneticdiversity of different populations under selection and the macrocyclic lactone used in theselection may also influence which genes show restricted genetic diversity after macrocycliclactone selection in a given parasite population. Thus different avermectin resistant popula-tions may not show an identical genetic composition with respect to avermectin resistance.Where ivermectin resistance has been found in the field, typically there have been 5–30treatments with an avermectin anthelmintic (Shoop, 1993).

The first gene found to be associated with macrocyclic lactone resistance in H. contortuswas a P-glycoprotein gene, PGP-A (Xu et al., 1998; Blackhall et al., 1998a) found in threestrains of ivermectin (IVC and IVF17 strains) and moxidectin (MOF17) selected wormswhich were compared with their unselected parental strains (BBH in the case of IVC, andPF17 in the case of IVF17 and MOF17). These three selected strains were selected in theUSA (Rohrer et al., 1994; Wang et al., 1999) by repeated treatment with sub-LD95 doserates of ivermectin or moxidectin. Subsequently, it was independently found that there wasselection on the same PGP-A gene (referred to as A27) and another P-glycoprotein gene(A28) in two experimentally selected ivermectin resistant strains (Warren and CAVRS) inAustralia (Sangster et al., 1999). Another P-glycoprotein gene in H. contortus, hcpgp-1,has been investigated for possible association with ivermectin resistance. A limited RFLPanalysis, on a multidrug (benzimidazole/ivermectin/closantel resistant) isolate (RSA) fromSouth Africa, indicated that hcpgp-1 was not associated with resistance (Kwa et al., 1998).However, using a H. contortus/H. placei backcross method, it was subsequently found thathcpgp-1 is selected by ivermectin in the CAVRS avermectin resistant strain experimentallyproduced in Australia (Le Jambre et al., 1999), but the authors concluded that althoughthis gene is associated with ivermectin resistance it was not the major gene responsiblefor avermectin resistance in this strain. It is interesting that P-glycoprotein genes seem tobe closely linked with each other in C. elegans (Lincke et al., 1992), thus selection onone or more P-glycoprotein genes during treatment with macrocyclic lactone anthelminticsmay select certain alleles of other P-glycoprotein genes because they will segregate withthe P-glycoprotein gene(s) which contribute to avermectin resistance. Ivermectin is a potentligand for P-glycoproteins (Didier and Loor, 1996; Pouliot et al., 1997) and multidrug resis-tance (mdr) reversing agents, which inhibit the transport functions of some P-glycoproteinscan partially reverse macrocyclic lactone resistance in H. contortus (Xu et al., 1998; Molentoand Prichard, 1999). The overwhelming evidence of an association between P-glycoproteingenes and macrocyclic lactone resistance suggests that these genes will be useful markersfor this type of resistance in H. contortus and probably other parasitic nematodes.

GluCl genes are implicated in the mechanism of action of macrocyclic lactoneanthelmintics. Recent findings in C. elegans have led to the following hypothesis for iver-mectin action on the pharynx of this worm, resulting in starvation, and for ivermectinresistance in C. elegans in vitro (Dent et al., 2000). Three GluClα subunit gene, products ofthe genes avr-15 (GluClα2), avr-14 (GluClα3) and GLC-1 (GluClα1) may respond to iver-mectin. The avr-15 is located the pharyngeal muscle, allowing ivermectin to act directly onthe pharynx. In contrast, avr-14 and GLC-1 are located on extrapharyngeal neurons whichare connected to the pharyngeal cells via linking neurons in which the gap junction innexingenes unc-7 and unc-9 products, which do not bind ivermectin themselves, convey thehyperpolarization signal from the extrapharyngeal neurons to the pharynx. In this model,


the amphid dye filing Dyf gene, osm-1, and other Dyf genes may act additively to regulateivermectin uptake and access to the various GluCl ivermectin-receptors, especially AVR-14(Dent et al., 2000).

Comparison of the polymorphism in two ivermectin selected (IVC and IVF17) and amoxidectin (MOF17) selected strain of H. contortus with the two parental unselected strains(BBH and PF17, respectively) revealed that there was also selection by these macrocycliclactone anthelmintics on a GluCl gene (Blackhall et al., 1998b) which was subsequentlycharacterized as two alternatively spliced cDNAs, HcGluCla and HcGluClb (Forrester et al.,1999). The full length cDNA, HcGluCla, derived from unselected H. contortus, has beenexpressed in Cos-7 cells and shown to have separate receptors for ivermectin/moxidectinand glutamate.

Membranes from ivermectin selected strains of H. contortus had higher Bmax for glu-tamate binding than did unselected strains (Paiement et al., 1999a; Hejmadi et al., 2000)and ivermectin decreased the Bmax for glutamate binding in the susceptible strain but notin the ivermectin selected strain. Furthermore, glutamate attenuates the inhibitory effecton pharyngeal pumping of moxidectin, but not of ivermectin in susceptible H. contortusand of both moxidectin and ivermectin in ivermectin selected H. contortus (Paiement et al.,1999b). These studies suggest that an up-regulation of glutamate binding is involved inivermectin resistance, whilst in susceptible H. contortus ivermectin decreases glutamatebinding, but potentiates the action of glutamate on channel opening in C. elegans andDrosophila melanogaster (Arena et al., 1992; Cully et al., 1996).

HG1, a putative member of the amino acid gated anion channel subunit family (Laughtonet al., 1994) has also been found to be selected by ivermectin (strains IVC and IVF17) andmoxidectin (strain MOF17) compared with their parental unselected strains (BBH and PF17,respectively) (Blackhall et al., 1998c). Further work is required to characterize the functionof this gene. A number of other genes have been investigated for association with macro-cyclic lactone selection in H. contortus, including a GluCl �-subunit, a N-acetlycholinereceptor, phosphoenolpyruvate carboxykinase and phosphofructokinase (Blackhall et al.,1998b,c; Blackhall, 2000) and none of these genes were linked with macrocyclic lactoneselection. Other genes of potential interest for association with macrocyclic lactone resis-tance is the Hc-gbr2 which has high homology to C. elegans avr-14 (gbr-2) (Jagannathanet al., 1999).

5.2. Resistance to anti-protozoal drugs

Drug resistance in the parasitic protozoa is primarily a problem with Eimeria spp. inpoultry and Trypanosoma in cattle and camels. The lack of widespread resistance in othergenera and species is probably due to the lack of effective or extensively used drugs, theabsence of extensive prophylactic treatment and the availability of alternative methods ofcontrol such as vaccines or, in the case of vector borne diseases, control by the use ofinsecticides or acaricides. This has meant that there has been a limited research effort eitherto develop DNA based markers for drug resistant organisms or to investigate the mechanismsof action of the available compounds. The exception is the analysis of the mechanisms ofresistance to melarsoprol, which is used in the treatment of late stage human sleepingsickness (Pepin and Milord, 1994).


Resistance to the main commercially available coccidial drugs is widespread in poultry(Chapman, 1993) and many of the isolates are resistant to several drugs with different modesof action. In general, although data is available on the modes of action of a number of theavailable drugs, explanations of the mechanisms of resistance are at best speculative andmore often not available. The approach to dealing with resistance has been to prolong theeffective life of the available compounds either by using shuttle programs with differentdrugs or by the use of vaccination alternated with chemotherapy. In addition, because ofthe value of the poultry market, the pharmaceutical industry has produced new compoundswith different modes of action on a fairly regular basis. There is an increasing problemwith drug resistance to trypanocidal drugs in sub-Saharan Africa. In a recent analysis 140isolates of T. congolense were characterized using drug tests in mice (Geerts et al., 2001)and it was shown that 31.4% were resistant to isometamidium, 9.3% to diminazene aceturateand 11.4% to both compounds (Eisler et al., 2000). As these drugs were developed over40 years ago, this is perhaps not surprising given their extensive use, however, it seemsunlikely that new drugs will be developed as the investment is difficult to justify given thefinancial size of the market. This is clearly a cause for concern given the substantial impactthat trypanosomosis has in this region. Little is known about the mechanism of resistance orthe mode of action of the available drugs (Geerts et al., 2001). However, the mechanism ofdrug resistance has been investigated in the case of the melaminophenyl class of arsenicals(melarsoprol in humans and cymelarsen in cattle) although they are primarily of value in thetreatment of human disease (Barrett and Fairlamb, 1999). Biochemical studies of laboratoryderived drug resistant lines have shown that the arsenicals are selectively taken up by anadenosine transporter (P2) and that this transporter is altered in the resistant lines resultingin no adenosine or drug transport (Carter and Fairlamb, 1993). The gene encoding the P2transporter has been cloned and sequenced (Mäser et al., 1999); comparison of the sequenceof this gene with the homologue from a drug resistant isogenic line has shown a number ofsequence differences six of which resulted in amino acid changes. The properties conferredby the expression of this gene in a yeast system suggests that it codes for the P2 transporterand, interestingly, suggest that isometamidium is also imported by the same transporter(Mäser et al., 1999). If this is a common mechanism of resistance in the field, the sequencealterations in the resistant gene could be used as a basis for a simple assay for resistance.Thus, overall there is relatively limited research activity on drug resistance mechanismsand the development of markers for resistance with the parasitic protozoa partly reflectingthe availability of alternative strategies to dealing with resistance (Eimeria spp.) and itsoccurrence in only a few of the important parasites.

6. The development of molecular screens for antiparasitic drug discovery

6.1. Anthelmintics

Economic pressures have led to virtually all major pharmaceutical companies start-ing the search for new classes of drugs with mechanism-based screening. In the caseof anthelmintics, the role of mechanism-based screening has been reviewed by Gearyet al. (1999b). Mechanism-based screening depends on the identification of a biochemical


process in the parasite that can be used for high throughput screening in the laboratory.Typically, the gene for a particular receptor or enzyme is expressed in a suitable vector,usually a eucaryotic cell (e.g. yeast). The assay may be run with whole transfected cells orsub-cellular preparations. Typically, the gene of interest may be expressed with a reportergene (Bronstein et al., 1994) which allows for rapid automated monitoring of responsesto chemical libraries, by fluorescence or chemiluminescence detection in multiwell plates.Isolation of receptors and enzymes from parasites is not feasible for high throughput screen-ing, so the whole screening process has become dependent on the expression of parasitegenes (or model organism genes, such as C. elegans). This process tends to produce many‘hits’ and second stage screening, e.g. in whole C. elegans in vitro is used prior to in vivoassays in small animal model host/parasite systems. Most of the initial ‘hits’ do not survivethrough the initial in vivo screens due to toxicity or lack or sufficient efficacy. Candidatedrugs that have processed through these different screens from the molecular screens tothe model in vivo screens may then proceed to initial target animal screening. As can beseen the molecular, mechanism-based screens are now the foundation of the anthelminticdiscovery process. As has been emphasized by Geary et al. (1999a), our ability to discovernew anthelmintics depends on our understanding of the basic biology of parasites.

6.2. Antiprotozoal drugs

Apart from the Eimeria spp. of poultry, the large pharmaceutical companies have hadrelatively limited interest in developing new compounds due to the relatively small eco-nomic size of the markets. However, with both biochemical research and the increasingnumber of EST and genomic sequences, the number of potential targets is increasing allthe time. The strategies for both target identification and subsequent identification of activecompounds vary widely as do the views of the community on the usefulness of the differentapproaches. The most common approach is the identification of pathways or essential func-tions that are unique to the parasite; these include trypanothione in T. brucei (Fairlamb andCerami, 1992; Krauth-Siegel and Coombs, 1999), the shikimate pathway in apicomplexanparasites (Roberts et al., 1999), mannitol metabolism in Eimeria spp. (Schmatz, 1989) andthe isoprenoid and protein biosynthetic pathways of the unique apicomplexan organelle, theapicoplast (Soldata, 1999; Coombs, 1999), to name but a few. The opportunities for usingthese molecules as unique targets have been reviewed recently (see references above) andso will not be dealt with in detail here. The development of transfection techniques thatallow genes to be knocked out in a number of the parasitic protozoa, provides a very usefultool for demonstrating that a target is essential and therefore a valid molecule for furtherresearch. One would anticipate that the availability of suitable targets is unlikely to be alimiting factor as further genome sequences are completed. The critical restraints are accessto libraries of compounds and the development of high throughput screening systems todetect active compounds. There are two approaches to circumvent these restraints, the firstis illustrated by the discovery of the shikimate pathway in apicomplexan parasites based onthe identification of EST and genomic sequence homologues of some of the enzymes. Asthis pathway is not present in mammals but was a target in plants for the development ofherbicides, a series of active compounds were already available and a considerable exper-tise in their chemistry. This allows a ‘hitch-hiking’ approach where new compounds can


be developed on the basis of the existence of a lead compound already in existence. Theapproach has a lot of advantages with the prospect of the identification of many other targetsfrom genome sequences. The second approach can be termed the ‘designer drug’ approach.It is based on identifying a target enzyme, solving the three-dimensional structure by crys-tallography and then using a modeling based approach to design specific inhibitors to theactive site. This is often aided by the availability of the structure of the host homologuesuch that specificity can also be addressed. To date, this approach has not yet borne fruit.It is worth considering, in principal, the approaches to drug development, if a library ofcompounds is available and there is access to a high throughput screening system. Usingcomparative genomics, unique parasite target genes can be identified but they need to beexpressed as active recombinant molecules and assay systems devised that can be used ina screening system. This approach could then identify active compounds (‘hits’), however,there is still the need to test whether the compound can enter the parasite and, in the case ofthe intracellular parasites the host cell as well aside from the issues of toxicity, residues andpharmacokinetics. As a result of these issues there is an argument for using whole organismscreening as the first line of approach as the hits will already have had to enter the cell tobe active and therefore avoided one of the restraints of the purely in vitro approach. Theseapproaches are costly in terms of time and resource and therefore lead to a reluctance toinvest when the return on the investment is likely to be low in the case of many parasiticdiseases because of the small market size in economic terms. This is particularly the casewith most tropical diseases where the cost of a new drug has to be low. In the near futurewe are likely to be faced with a whole array of promising targets delivered by the genomesequencing projects but may lack the means to fully exploit the opportunities.

7. Conclusions

Access to genome information on model helminths and protozoa opens up new frontiersfor advancing our knowledge of parasite biology and for improving diagnosis and control.New techniques, such as DNA microarrays and molecular beacons can be used to rapidlyexploit the new knowledge of parasite genomes. Veterinary parasitologists must embracemolecular knowledge and use the tools of molecular biology in our research, teaching andclinical work if the field is to advance, be respected, funded and be given an appropriateplace in the curriculum in our universities. It is our hope that in future meetings of WAAVPmore exposure will be given to dissecting advances and discussing future directions forresearch, separately in each of the many aspects of veterinary parasitology which are usingmolecular approaches.

References

Almeida, P.J.L.P., de Ndao, M., Meirvenne, N., van Geerts, S., 1997. Diagnostic evaluation of PCR in goatsexperimentally infacted with Trypanosoma vivax. Acta Tropica. 66 (1), 45–50.

Anon., 1998. Caenorhabditis elegans Sequencing Consortium. Genome sequence of the nematode C. elegans: aplatform for investigating biology. Science 282, 2012–2018.

Anon., 2000. http://genome.cvm.umn.edu/.


Arena, J.P., Liu, K.K., Paress, P.S., Schaeffer, J.M., Cully, D.F., 1992. Expression of a glutamate-activated chloridecurrent in Xenopus oocytes injected with Caenorhabditis elegans RNA: evidence for modulation by avermectin.Mol. Brain Res. 15, 339–348.

Awad-el-Kariem, F.M., Warhurst, D.C., McDonald, V., 1994. Detection and species identification ofCryptosporidium oocysts using a system based on PCR and endonuclease restriction. Parasitology 109, 19–22.

Barrett, M.P., Fairlamb, A.H., 1999. The biochemical basis of arsenical–diamidine cross resistance in Africantrypanosomes. Parasitol. Today 15, 136–140.

Bashiruddin, J.B., Camma, C., Rebêlo, E., 1999. Molecular detection of Babesia equi and Babesia caballi in horseblood by PCR amplification of part of the 16S rRNA gene. Vet. Parasitol. 84, 75–83.

Beech, R.N., Prichard, R.K., Scott, M.E., 1994. Genetic variability of the ∃-tubulin genes inbenzimidazole-susceptible and -resistant strains of Haemonchus contortus. Genetics 138, 103–110.

Berasain, P., Carmona, C., Frangione, B., Dalton, J.P., Goni, F., 2000. Fasciola hepatica: parasite-secretedproteinases degrade all human IgG subclasses: determination of the specific cleavage sites and identificationof the immunoglobulin fragments produced. Exp. Parasitol. 94, 99–110.

Bishop, R., Allsopp, B., Spooner, P., Sohanpal, B., Morzaria, S., Gobright, E., 1995. Theileria: improved speciesdiscrimination using oligonucleotides derived from large-subunit ribosomal RNA sequences. Exp. Parasitol.80, 107–115.

Blackhall, W.J., 2000. Genetic variation and multiple mechanisms of anthelmintic resistance in Haemonchuscontortus. Ph.D. Thesis. McGill University, Montreal.

Blackhall, W., Liu, H.Y., Xu, M., Prichard, R.K., Beech, R.N., 1998a. Selection at a P-glycoprotein gene inivermectin- and moxidectin-selected strains of Haemonchus contortus. Mol. Biochem. Parasitol. 95, 193–201.

Blackhall, W.J., Pouliot, J.-F., Prichard, R.K., Beech, R.N., 1998b. Haemonchus contortus: selection at aglutamate-gated chloride channel gene in ivermectin- and moxidectin-selected strains. Exp. Parasitol. 90,42–48.

Blackhall, W.J., Prichard, R.K., Beech, R.N., 1998c. Allele frequency changes in a GABA receptor gene inivermectin- and moxidectin-selected strains of Haemonchus contortus. In: Proceedings of the 73rd AnnualMeeting of the American Society of Parasitologists, Kona, Hawaii, August 16–20 (Abstract 55).

Boulter, N.R., Hall, F.R., 2000. Immunity and vaccine development in bovine theilerioses. Adv. Parasitol. 44,41–97.

Boulter, N.R., Glass, E.J., Knight, P.A., Bell-Sakyi, L., Brown, C.G., Hall, R., 1995. Theileria annulata sporozoiteantigen fused to hepatitis B core antigen used in a vaccination trial. Vaccine 13, 1152–1160.

Boulter, N.R., Brown, C.G., Kirvar, E., Glass, E., Campbell, J., Morzaria, S., Nene, V., Musoke, A., d’Oliveira,C., Gubbels, M.J., Jongejan, F., Hall, F.R., 1998. Different vaccine strategies used to protect against Theileriaannulata. Ann. NY Acad. Sci. 849, 234–246.

Bronstein, I., Fortin, J., Stanley, P.E., Stewart, G.S.A.B., Kricka, L.J., 1994. Chemiluminescent and bioluminescentreporter gene assays. Anal. Biochem. 219, 169–181.

Brown, W.C., Palmer, G.H., 1999. Designing blood-stage vaccines against Babesia bovis and B. bigemina. Parasitol.Today 15, 275–281.

Brown, W.C., Logan, K.S., Wagner, G.G., Tetzlaff, C.L., 1991. Cell mediated immune responses to Babesia bovismerozoite antigens in cattle following infection with tick-derived or cultured parasites. Infect. Immun. 59,2418–2426.

Brown, W.C., McElwain, T.F., Ruef, B.J., Suarez, C.E., Shkap, V., Chitko-McKown, C.G., Tuo, W., Rice-Ficht,A.C., Palmer, G.H., 1996. Babesia bovis rhoptry-associated protein-1 is immunodominant for T-helper cellsof immune cattle and contains T-cell epitopes conserved among geographically distant B. bovis strains. Infect.Immun. 64, 3341–3350.

Brown, W.C., McElwain, T.F., Hotzel, I., Ruef, B.J., Rice-Ficht, A.C., Stich, R.W., Suarez, C.E., Estes, D.M.,Palmer, G.H., 1998. Immunodominant T-cell antigens and epitopes of Babesia bovis and Babesia bigemina.Ann. Trop. Med. Parasitol. 92, 473–482.

Burg, J.L., Grover, C.M., Pouletty, P., Boothroyd, J.C., 1989. Direct and sensitive detection of a pathogenicprotozoan Toxoplasma gondii, by polymerase chain reaction. J. Clin. Microbiol. 27, 1787–1792.

Buxton, D., Thomson, K., Maley, S., Wright, S., Bos, H.J., 1991. Vaccination of sheep with a live incompletestrain (S48) of Toxoplasma gondii and their immunity to challenge when pregnant. Vet. Record 129, 89–93.

Calder, J.A.M., Reddy, G.R., Chieves, L., Courtney, C.H., Littell, R., Livengood, J.R., Norval, R.A., Smith, C.,Dame, J.B., 1996. Monitoring Babesia bovis infections in cattle by using PCR-based tests. J. Clin. Microbiol.34, 2748–2755.


Callow, L.L., Dalgliesh, R.J., Vos, A.J.de, 1997. Development of effective living vaccines against bovine babesiosis– the longest field trial. Internet. J. Parasitol. 27, 747–767.

Carter, N.S., Fairlamb, A.H., 1993. Arsenical-resistant trypanosomes lack an unusual adenosine transporter. Nature361, 173–175.

Chapman, H.D., 1993. Resistance to anticoccidial drugs in fowl. Parasitol. Today 9, 159–162.Chung, E., Aldom, J.E., Carreno, R.A., Chagla, A.H., Kostrzynska, M., Lee, H., Palmateer, G., Trevors, J.T.,

Unger, S., Xu, R., De Grandis, S.A., 1999. PCR-based quantitation of Cryptosporidium parvum in municipalwater samples. J. Microbiol. Meth. 38, 119–130.

Conway, D.J., Cavanagh, D.R., Tanabe, K., Roper, C., Mikes, Z.S., Sakihama, N., Bojang, K.A., Oduola, A.M.,Kremsner, P.G., Arnot, D.E., Greenwood, B.M., McBride, J.S., 2000. A principal target of human immunity tomalaria identified by molecular population genetic and immunological analyses. Nat. Med. 6, 689–692.

Coombs, G.H., 1999. Biochemical peculiarities and drug targets in Cryptosporidium parvum: lessons from othercoccidian parasites. Parasitol. Today 15, 333–338.

Cully, D.F., Paress, P.S., Liu, K.K., Schaeffer, J.M., Arena, J.P., 1996. Identification of a Drosophila melano-gaster glutamate-gated chloride channel sensitive to the antiparasitic agent avermectin. J. Biol. Chem. 271,20187–20191.

Dadley-Moore, D.L., Lightowlers, M.W., Rothel, J.S., Jackson, D.C., 1999. Synthetic peptide antigens induceantibodies to Taenia ovis oncospheres. Vaccine 17, 1506–1515.

Dent, J.A., Smith, M.M., Vassilatis, D.K., Avery, L., 2000. The genetics of ivermectin resistance in Caenorhabditiselegans. In: Proceedings of the National Academy of Sciences of the United States of America, 1997,pp. 2674–2679.

Didier, A., Loor, F., 1996. The abamectin derivative ivermectin is a potent P-glycoprotein inhibitor. AnticancerDrugs 7, 745–751.

Dobson, R.J., LeJambre, L., Gill, J.H., 1996. Management of anthelmintic resistance: inheritance of resistanceand selection with persistent drugs. Int. J. Parasitol. 26, 993–1000.

d’Oliveira, C., van der Weide, M., Habela, M.A., Jacquiet, P., Jongejan, F., 1995. Detection of Theileria annulatain blood samples of carrier cattle by PCR. J. Clin. Microbiol. 33, 2665–2669.

d’Oliveira, C., Feenstra, A., Vos, H., Osterhaus, A.D., Shiels, B.R., Cornelissen, A.W., Jongejan, F., 1997. Inductionof protective immunity to Theileria annulata using two major merozoite surface antigens presented by differentdelivery systems. Vaccine 15, 1796–1804.

Dowling, S.C., Perryman, L.E., Jasmer, D.P., 1996. A Babesia bovis 225-kDa spherical body protein: localisationto the cytoplasmic face of infected erythrocytes after merozoite invasion. Infect. Immun. 64, 2618–2626.

Eisler, M.C., et al., 2000. Area-wide appraisal of drug resistance in trypanosomes infecting cattle in East andSouthern Africa. ICPTV Newsletter, Vol. 2, pp. 16–18.

Elard, L., Cabaret, J., Humbert, J.F., 1999. PCR diagnosis of benzimidazole-susceptibility or -resistance in naturalpopulations of the small ruminant parasite, Teladorsagia circumcincta. Vet. Parasitol. 80, 231–237.

Emery, D.L., 1981. Adoptive transfer of immunity to injection with Theileria parva between cattle twins. 1981Res. in Vet. Science. 30, 364–367.

Eysker, M., Ploeger, H.W., 2000. Value of present diagnostic methods for gastrointestinal nematode infections inruminants. Parasitology 120, S109–S119.

Fairlamb, A.H., Cerami, A., 1992. Metabolism and functions of trypanothione in the kinetoplastida. Annu. Rev.Microbiol. 46, 695–729.

Figueroa, J.V., Chieves, L.P., Johnson, G.S., Buening, G.M., 1993. Multiplex polymerase chain reaction-basedassay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood.Vet. Parasitol. 50, 69–81.

Fleming, J.T., Squire, M.D., Barnes, T.M., Tornoe, C., Matsuda, K., Ahnn, J., Fire, A., Sulston, J.E., Barnard,E.A., Sattelle, D.B., Lewis, J.A., 1997. Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, andunc-38 encode functional nicotinic acetylcholine receptor subunits. J. Neurosci. 17, 5843–5857.

Forrester, S.G., Hamdan, F.F., Prichard, R.K., Beech, R.N., 1999. Cloning, sequencing and developmentalexpression levels of a novel glutamate-gated chloride channel homologue in the parasitic nematode Haemonchuscontortus. Biochem. Biophys. Res. Commun. 254, 529–534.

Gasser, R.B., Monti, J.R., Zhu, X.Q., Chilton, N.B., Hung, G.C., Guldberg, P., 1997. Polymerase chainreaction-linked single-strand conformation polymorphism of ribosomal DNA to fingerprint parasites.Electrophoresis 18, 1564–1566.


Geary, T.G., Nulf, S.C., Favreau, M.A., Tang, L., Prichard, R.K., Hatzenbuhler, N.T., Shea, M.H., Alexander,S.J., Klein, R.D., 1992. Three beta-tubulin cDNAs from the parasitic nematode Haemonchus contortus. Mol.Biochem. Parasitol. 50, 295–306.

Geary, T.G., Sangster, N.C., Thompson, D.P., 1999a. Frontiers in anthelmintic pharmacology. Vet. Parasitol. 84,275–295.

Geary, T.G., Thompson, D.P., Klein, R.D., 1999b. Mechanism-based screening: discovery of the next generationof anthelmintics depends upon more basic research. Int. J. Parasitol. 29, 105–112.

Geerts, S., Holmes, P.H., Diall, O., Eisler, M.C., 2001. African bovine trypanosomosis: the problem of drugresistance. Trends in Parasitol. 17, 25–29.

Gill, J.H., Lacey, E., 1998. Avermectin resistance in trichostrongylid nematodes. Int. J. Parasitol. 28, 863–877.Gill, J.H., Kerr, C.A., Shoop, W.L., Lacey, E., 1998. Evidence of multiple mechanisms of avermectin resistance

in Haemonchus contortus — comparison of selection protocols. Int. J. Parasitol. 28, 783–789.Gubbels, J.M., de Vos, A.P., van der Weide, M., Viseras, J., Schouls, L.M., de Vries, E., Jongejan, F., 1999.

Simultaneous detection of bovine Theileria and Babesia species by reverse line blot hybridisation. J. Clin.Microbiol. 37, 1782–1789.

Harper, G.S., Hibbs, A.R., East, I.J., Waltisbuhl, D.J., Jorgensen, W.K., Riddles, P.W., 1996. Babesia bovis:biosynthesis and localisation of 12D3 antigen in bovine erythrocytes. Int. J. Parasitol. 26, 1255–1262.

Harrison, G.B., Heath, D.D., Dempster, R.P., Gauci, C., Newton, S.E., Cameron, W.G., Robinson, C.M., Lawrence,S.B., Lightowlers, M.W., Rickard, M.D., 1996. Identification and cDNA cloning of two novel low molecularweight host-protective antigens from Taenia ovis oncospheres. Int. J. Parasitol. 26, 195–204.

Harrison, G.B., Shakes, T.R., Robinson, C.M., Lawrence, S.B., Heath, D.D., Dempster, R.P., Lightowlers, M.W.,Rickard, M.D., 1999. Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccineformulated with saponin or selected adjuvants. Vet. Immunol. Immunopathol. 70, 161–172.

Hejmadi, M.V., Jagannathan, S., Delany, N.S., Coles, G.C., Wolstenholme, A.J., 2000. l-glutamate binding sitesof parasitic nematodes: an association with ivermectin resistance. Parasitology 120, 535–545.

Hines, S.A., Palmer, G.H., Jasmer, D.P., McGuire, T.C., McElwain, T.F., 1992. Neutralisation sensitive merozoitesurface antigens of Babesia bovis encode members of a polymorphic gene family. Mol. Biochem. Parasitol.55, 85–94.

Jacobs, D.E., Zhu, X.Q., Gasser, R.B., Chilton, N.B., 1997. PCR-based methods for identification of potentiallyzoonotic ascaridoid parasites of the dog, fox and cat. Acta Trop. 68, 191–200.

Jagannathan, S., Laughton, D.L., Critten, C.L., Skinner, T.M., Horoszok, L., Wolstenholme, A.J., 1999.Ligand-gated chloride channel subunits encoded by the Haemonchus contortus and Ascaris suum orthologuesof the Caenorhabditis elegans gbr-2 (avr-14) gene. Mol. Biochem. Parasitol. 103, 129–140.

Jasmer, D.P., Reduker, D.W., Hines, S.A., Perryman, L.E., McGuire, T.C., 1992. Surface epitope localisation andgene structure of a Babesia bovis 44 kDa variable merozoite surface protein. Mol. Biochem. Parasitol. 55,75–84.

Joachim, A., Daugschies, A., Christensen, C.M., Bjorn, H., Nansen, P., 1997. Use of random amplified polymorphicDNA-polymerase chain reaction for the definition of genetic markers for species and strains of porcineOesophagostomum. Parasitol. Res. 83, 646–654.

Johnson, K.S., Harrison, G.B., Lightowlers, M.W., O’Hoy, K.L., Cougle, W.G., Dempster, R.P., Lawrence, S.B.,Vinton, J.G., Heath, D.D., Rickard, M.D., 1989. Vaccination against ovine cysticercosis using a definedrecombinant antigen. Nature 338, 585–587.

Johnson, J.D., Butcher, P.D., Savva, D., Holliman, R.E., 1993. Application of the polymerase chain reaction tothe diagnosis of human toxoplasmosis. J. Infect. 26, 147–158.

Kamerbeek, J., Schouls, L., Kolk, A., van Agterveld, M., van Soolingen, D., Kuijper, S., Bunschoten, A.,Molhuizen, H., Shaw, R., Goyal, M., van Embden, J., 1997. Simultaneous detection and strain differentiationof mycobacterium tuberculosis for diagnosis and epidemiology. J. Clin. Microbiol. 35, 907–914.

Katakura, K., Lubinga, C., Chitambo, H., Tada, Y., 1997. Decettion of Trypanosoma congolense and T. bruceisubspecies in cattle in Zambia by polymerase chain reaction from blood collected on a filter paper. Parasitol.Res. 83, 241–245.

Klei, T.R., 1997. Immunological control of gastrointestinal nematode infections. Vet. Parasitol. 72, 507–516.Krauth-Siegel, R.L., Coombs, G.H., 1999. Enzymes of parasite thiol metabolism as drug targets. Parasitol. Today

15, 404–408.


Kwa, M.S.G., Kooyman, F.N., Boersema, J.H., Roos, M.H., 1993. Effect of selection for benzimidazole resistancein Haemonchus contortus on ∃-tubulin isotype 1 and isotype 2 genes. Biochem. Biophys. Res. Commun. 191,413–419.

Kwa, M.S.G., Veenstra, J.G., Roos, M.H., 1994. Benzimidazole resistance in Haemonchus contortus is correlatedwith a conserved mutation at amino acid 200 in ∃-tubulin isotype I. Mol. Biochem. Parasitol. 63, 299–303.

Kwa, M.S.G., Veenstra, J.G., Van Dijk, M., Roos, M.H., 1995. Beta-tubulin genes from the parasitic nematodeHaemonchus contortus modulate drug resistance in Caenorhabditis elegans. J. Mol. Biol. 246, 500–510.

Kwa, M.S.G., Okoli, M.N., Schulz-Key, H., Okongkwo, P.O., Roos, M.H., 1998. Use of P-glycoprotein gene probesto investigate anthelmintic resistance in Haemonchus contortus and comparison with Onchocerca volvulus. Int.J. Parasitol. 28, 1235–1240.

Kyes, S., Craig, A.G., Marsh, K., Newbold, C.I., 1993. Plasmodium falciparum: a method for the amplificationof S antigens and its application to laboratory and field samples.. Exptl. Parasitol. 77, 473–483.

Lally, N.C., Jenkins, M.C., Dubey, J.P., 1996. Development of a polymerase chain reaction assay for the diagnosisof neosporosis using the Neospora caninum 14-3-3 gene. Mol. Biochem. Parasitol. 75, 169–178.

Laughton, D.L., Amar, M., Thomas, P., Towner, P., Harris, P., Lunt, G.G., Wolstenholme, A.J., 1994. Cloning of aputative inhibitory amino acid receptor subunit from the parasitic nematode Haemonchus contortus., Receptors& Channels. 2, 155–163.

Le Jambre, L.F., Lenane, I.J., Wardrop, A.J., 1999. A hybridisation technique to identify anthelmintic resistancegenes in Haemonchus. Int. J. Parasitol. 29, 1979–1985.

Lewis, J.A., Wu, C.H., Levine, J.H., Berg, H., 1980. Levamisole-resistant mutants of the nematode Caenorhabditiselegans appear to lack pharmacological acetylcholine receptors. Neuroscience 5, 967–989.

Liddell, S., Knox, D.P., 1998. Extracellular and cytoplasmic Cu/Zn superoxide dismutases from Haemonchuscontortus. Parasitology 116, 383–394.

Lightowlers, M.W., Lawrence, S.B., Gauci, C.G., Young, J., Ralston, M.J., Maas, D., Health, D.D., 1996a.Vaccination against hydatidosis using a defined recombinant antigen. Parasite Immunol. 18, 457–462.

Lightowlers, M.W., Rolfe, R., Gauci, C.G., 1996b. Taenia saginata: vaccination against cysticercosis in cattle withrecombinant oncosphere antigens. Exp. Parasitol. 84, 330–338.

Lightowlers, M.W., Flisser, A., Gauci, C.G., Heath, D.D., Jensen, O., Rolfe, R., 2000. Vaccination againstcysticercosis and hydatid disease. Parasitol. Today 16, 191–196.

Lin, M.-H., Chen, T.C., Kuo, T.T., Tseng, C.C., Tseng, C.P., 2000. Real-time PCR for quantitative detection ofToxoplasma gondii. J. Clin. Microbiol. 38, 4121–4125.

Lincke, C.R., The, I., van Groenigen, M., Borst, P., 1992. The P-glycoprotein gene family of Caenorhabditiselegans. Cloning and characterization of genomic and complementary DNA sequences. J. Mol. Biol. 228,701–711.

Lubega, G.W., Klein, R.D., Geary, T.G., Prichard, R.K., 1994. Haemonchus contortus: the role of two beta-tubulingene subfamilies in the resistance to benzimidazole anthelmintics. Biochem. Pharmacol. 47, 1705–1715.

Machado, R.Z., McElwain, T.F., Pancracio, H.P., Freschi, C.R., Palmer, G.H., 1999. Babesia bigemina:immunisation with purified rhoptries induces protection against acute parasitaemia. Exp. Parasitol. 93, 105–108.

Mahan, S.M., Allsop, B., Kocan, K.M., Palmer, G.H., Jongejan, F., 1999. Vaccine strategies for Cowdria ruminatuminfections and their applications to other Ehrlichial infections. Parasitol. Today 15, 290–294.

Mahoney, D.F., 1986. In: Morrison, W.I. (Ed.), The Ruminant Immune System in Health and Disease. CambridgeUniversity Press, Cambridge, pp. 539–545.

Majiwa, P.A., Maina, M., Waitumbi, J.N., Mihok, S., Zweygarth E., 1993. Trypanosoma (Nannomonas)congolense: molecular characterization of a new genotype from Tsavo, Kenya. Parasitol. 106, 151–162.

Martin, R.J., Robertson, A.P., Bjorn, H., Sangster, N.C., 1997. Heterogeneous levamisole receptors: asingle-channel study of nicotinic acetylcholine receptors from Oesophagostomum dentatum. Eur. J. Pharmacol.322, 249–257.

Masake, R.A., Majiwa, P.A.O., Moloo, S.K., Makau, J.M., Njuguna, J.T., Maina, M., Kabata, J., ole-MoiYoi,O.K., Nantulya, V.M., 1997. Sensitive and specific detection of Trypanosoma vivax using the polymerase chainreaction.. Exp. Parasitol. 85 (2), 193–205.

Mäser, P., Sutterlin, C., Kralli, A., Kaminsky, R., 1999. A nucleoside transporter from Trypanosoma brucei involvedin drug resistance. Science 285, 242–244.

Masiga, D.K., Smyth, A.J., Hayes, P., Bromidge, T.J., Gibson, W.C., 1992. Sensitive detection of trypanosomesin tsetse files by DNA amplification.. Internet. J. Parasitol. 22 (7), 909–918.


McElwain, T.F., Perryman, L.E., Musoke, A.J., McGuire, T.C., 1991. Molecular characterization andimmunogenicity of neutralization-sensitive Babesia bigemina merozoite surface proteins.. Mol. Biochem.Parasitol. 47, 213–222.

McKeand, J.B., 1998. Molecular diagnosis of parasitic nematodes. Parasitology 117, S87–S96.McKeever, D.J., Taracha, E.L., Innes, E.L., MacHugh, N.D., Awino, E., Goddeeris, B.M., Morrison, W.I., 1994.

Adoptive transfer of immunity to Theileria parva in the CD8+ fraction of responding efferesnt lymph.. Proc.Nat. Acad. Sci. U.S.A. 91, 1959–1963.

McKeever, D.J., Taracha, E.L., Morrison, W.I., Musoke, A.J., Morzaria, S.P., 1999. Protective immune mechanismsagainst Theileria parva: evolution of vaccine development strategies. Parasitol. Today 15, 263–267.

McLauchlin, J., Pedraza-Diaz, S., Amar-Hoetzeneder, C., Nichols, G.L., 1999. Genetic characterisation ofCryptosporidium strains from 218 patients with diarrhea diagnosed as having sporadic Cryptosporidiosis.J. Clin. Microbiol. 37, 3153–3158.

McNamara, J.J., Mohammed, G., Gibson, W.C., 1994. Trypanosoma (Nannomonas) godfreyi sp. nov. from tsetseflies in The Gambia: biological and biochemical Characterization. Parasitology 109, 497–509.

Molento, M.B., Prichard, R.K., 1999. Effects of the multidrug-resistance–reversing agents verapamil and CL 344,099 on the efficacy of ivermectin or moxidectin against unselected and drug-selected strains of Haemonchuscontortus in jirds (Meriones unguiculatus). Parasitol. Res. 85, 1007–1011.

Morgan, U.M., Thompson, R.C., 1998. Molecular detection of parasitic protozoa. Parasitology. 117 (Suppl.),S73–85.

Morrison, W.I., Taracha, E.L., McKeever, D.J., 1995. Theileriosis: progress towards vaccine development throughunderstanding immune responses to the parasite. Vet. Parasitol. 57, 177–187.

Morrison, W.I., McKeever, D.J., 1998. Immunology of infections with Theileria parva in cattle. Chem. Immunol.70, 163–185.

Muller, N., Zimmermann, V., Hentrich, B., Gottstein, B., 1996. Diagnosis of Neospora caninum and Toxoplasmagondii infection by PCR and DNA hybridisation immunoassay. J. Clin. Microbiol. 34, 2850–2852.

Munn, E.A., 1997. Rational design of nematode vaccines: hidden antigens. Int. J. Parasitol. 27, 359–366.Musoke, A., Morzaria, S., Nkonge, C., Jones, E., Nene, V., 1992. A recombinant sporozoites surface antigen of

Theileria parva induces protection in cattle. Proc. Natl. Acad. Sci. USA 89, 514–518.Nene, V., Musoke, A., Gobright, E., Morzaria, S., 1996. Conservation of the sporozoites p67 vaccine antigen in

cattle-derived Theileria parva stocks with different cross-immunity profiles. Infect. Immun. 64, 2056–2061.Newton, S.E., Munn, E.A., 1999. The development of vaccines against gastrointestinal nematode parasites,

particularly Haemonchus contortus. Parasitol. Today 15, 116–122.Nutman, T.B., Zimmerman, P.A., Kubofcik, J., Kostyu, D.D., 1994. A universally applicable diagnostic assay

approach to filarial and other infections. Parasitol. Today 10, 239–243.Paiement, J.P., Leger, C., Ribeiro, P., Prichard, R.K., 1999a. Haemonchus contortus: effects of glutamate,

ivermectin and moxidectin on pharyngeal pump activity in unselected and ivermectin-selected adults.Exp. Parasitol. 92, 193–198.

Paiement, J.P., Prichard, R.K., Ribeiro, P., 1999b. Haemonchus contortus: characterization of a glutamate bindingsite in unselected and ivermectin-selected larvae and adults. Exp. Parasitol. 92, 32–39.

Palmer, G.H., Rurangirwa, K.M., Kocan, K.M., Brown, W.C., 1999. Molecular basis for vaccine developmentagainst the Ehrlichial pathogen Anaplasma marginale. Parasitol. Today 15, 281–286.

Pepin, J., Milord, F., 1994. The treatment of human African trypanosomiasis. Adv. Parasitol. 33, 1–47.Pipano, E., 1995. Live vaccines against hemoparasitic diseases in livestock. Vet. Parasitol. 57, 213–231.Plancarte, A., Flisser, A., Gauci, C.G., Lightowlers, M.W., 1999. Vaccination against Taenia solium cysticercosis

in pigs using native and recombinant oncosphere antigens. Int. J. Parasitol. 29, 643–647.Pouliot, J.F., L’heureux, F., Liu, Z., Prichard, R.K., Georges, E., 1997. Ivermectin: reversal of

P-glycoprotein-associated multidrug resistance by ivermectin. Biochem. Pharmacol. 53, 17–25.Preston, P.M., Hall, F.R., Glass, E.J., Campbell, J.D., Darghouth, M.A., Ahmed, J.S., Shiels, B.R., Spooner, R.L.,

Jongejan, F., Brown, C.G., 1999. Innate and adaptive immune responses co-operate to protect cattle againstTheileria annulata. Parasitol. Today 15, 268–274.

Prichard, R., 1997. Application of molecular biology in veterinary parasitology. Vet. Parasitol. 71, 155–175.Ramaswamy, K., 1999. Progress towards development of naked DNA vaccines against parasitic infections. J. Vet.

Parasitol. 13, 1–12.


Redmond, D.L., Knox, D.P., Newlands, G., Smith, W.D., 1997. Molecular cloning and characterisation of adevelopmentally regulated putative metallopeptidase present in a host protective extract of Haemonchuscontortus. Mol. Biochem. Parasitol. 85, 77–87.

Rehman, A., Jasmer, D.P., 1999. Defined characteristics of cathepsin B-like proteins from nematodes: inferredfunctional diversity and phylogenetic relationships. Mol. Biochem. Parasitol. 102, 297–331.

Reifenberg, J.M., Solano, P., Duvallet, G., Guisance, D., Simpore, J., Cuny, G., 1997. Molecular characterizationof trypanosome isolates from naturally infected domestic animals in Burkina, Faso. Vet. Parasitol. 71, 251–262.

Roberts, C.W., Finnerty, J., Johnson, J.J., Roberts, F., Kyle, D.E., Krell, T., Coggins, J.R., Coombs, G.H., Milhous,W.J., Tzipori, S., Ferguson, D.J.P., Chakrabarti, D., McLeod, R., 1999. Shikimate pathway in apicomplexanparasites. Nature 397, 219–220.

Rohrer, S.P., Birzin, E.T., Eary, C.H., Schaeffer, J.M., Shoop, W.L., 1994. Ivermectin binding sites in sensitiveand resistant Haemonchus contortus. J. Parasitol. 80, 493–497.

Rothel, J.S., Waterkeyn, J.G., Strugnell, R.A., Wood, P.R., Seow, H.F., Vadolas, J., Lightowlers, M.W., 1997.Nucleic acid vaccination of sheep: use in combination with a conventional adjuvanted vaccine against Taeniaovis. Immunol. Cell Biol. 75, 41–46.

Rutti, B., Lienhard, R., Brossard, M., 1991. In: Dusbabek, F., Bukava, V., Modern Acarology, Vol. 1. Proceedingsof the VIII International Congress on Acarology. SPB Academic Publishing, pp. 95–102.

Sangster, N.C., Redwin, J.M., Bjorn, H., 1998a. Inheritance of levamisole and benzimidazole resistance in anisolate of Haemonchus contortus. Int. J. Parasitol. 28, 503–510.

Sangster, N.C., Riley, F.L., Wiley, L.J., 1998b. Binding of [3H]m-aminolevamisole to receptors inlevamisole-susceptible and -resistant Haemonchus contortus. Int. J. Parasitol. 28, 707–717.

Sangster, N.C., Bannan, S.C., Weiss, A.S., Nulf, S.C., Klein, R.D., Geary, T.G., 1999. Haemonchus contortus:sequence heterogeneity of internucleotide binding domains from P-glycoproteins. Exp. Parasitol. 91, 250–257.

Schmatz, D.M., 1989. The mannitol cycle — a new metabolic pathway in the coccidian. Parasitol. Today 5,205–210.

Schnieder, T., Heise, M., Epe, C., 1999. Genus-specific PCR for the differentiation of eggs or larvae fromgastrointestinal nematodes of ruminants. Parasitol. Res. 85, 895–898.

Shiels, B.R., D’Oliveira, C., McKellar, S., Ben-Miled, L., Kawazu, S., Hide, G., 1995. Selection of diversity atputative glycosylation sites in the immunodominant merozoite/piroplasm surface antigen of Theileria parasites.Mol. Biochem. Parasitol. 72, 149–162.

Shirley, M.W., Bedrnik, P., 1997. Live attenuated vaccines against Avian coccidiosis: success with precocious andegg-adapted lines of Eimeria. Parasitol. Today 13, 481–484.

Shoop, W.L., 1993. Ivermectin resistance. Parasitol. Today 9, 154–159.Skuce, P.J., Redmond, D.L., Liddell, S., Stewart, E.M., Newlands, G.F., Smith, W.D., Knox, D.P., 1999. Molecular

cloning and characterization of gut-derived cysteine proteinases associated with a host protective extract fromHaemonchus contortus. Parasitology 119, 405–412.

Skuce, P.J., Stewart, E.M., Smith, W.D., Knox, D.P., 1999. Cloning and characterization of glutamatedehydrogenase (GDH) from the gut of Haemonchus contortus. Parasitology 118, 297–304.

Smith, H.V., 1998. Detection of parasites in the environment. Parasitology 117, S113–S141.Smith, W.D., 1999. Prospects for vaccines of helminth parasites of grazing ruminants. Int. J. Parasitol. 29, 17–24.Smith, T.S., Graham, M., Munn, E.A., Newton, S.E., Knox, D.P., Coadwell, W.J., McMichael-Phillips, D., Smith,

H., Smith, W.D., Oliver, J.J., 1997. Cloning and characterization of a microsomal aminopeptidase from theintestine of the nematode Haemonchus contortus. Biochim. Biophys. Acta 1338, 295–306.

Smooker, P.M., Steeper, K.R., Drew, D.R., Strugnell, R.A., Spithill, T.W., 1999. Humoral responses in micefollowing vaccination with DNA encoding glutathione S-transferase of Fasciola hepatica: effects of mode ofvaccination and the cellular compartment of antigen expression. Parasite Immunol. 21, 264–357.

Soldata, D., 1999. The apicoplast as a potential therapeutic target in Toxoplasma and other apicomplexan parasites.Parasitol. Today 15, 5–7.

Spano, F., Putignani, L., McLauchlin, J., Casemore, D.P., Crisanti, A., 1997. PCR–RFLP analysis of theCryptosporidium oocysts wall protein (COWP) gene discriminates between C. wrairi and C. parvum isolatesof human and animal origin. FEMS Microbiol. Lett. 150, 209–217.

Talvik, H., Christensen, C.M., Joachim, A., Roepstorff, A., Bjorn, H., Nansen, P., 1997. Prepatent periods ofdifferent Oesophagostomum spp. isolates in experimentally infected pigs. Parasitol. Res. 83, 563–568.


Vet, J.A., Majithia, A.R., Marras, S.A., Tyagi, S., Dube, S., Poiesz, B.J., Kramer, F.R., 1999. Multiplex detectionof four pathogenic retroviruses using molecular beacons. In: Proceedings of the National Academy of Science,Vol. 96, USA, pp. 6394–6399.

Wang, G.T., Ranjan, S., Hirschlein, C., Simkins, K., 1999. Experimental inducement of H. contortus resistanceto macrocyclic lactones. In: Proceedings of the 17th International Conference on World Association for theAdvancement of Veterinary Parasitology, Copenhagen, August 15–19 (Abstract b.1.04).

Willadsen, P., 1997. Novel vaccines for ectoparasites. Vet. Parasitol. 71, 209–222.Willadsen, P., Riding, G.A., McKenna, R.V., Kemp, D.H., Tellam, R.L., Nielsen, J.N., Lahnstein, J., Cobon, G.S.,

Gough, J.M., 1989. Immunologic control of a parasitic arthropod. Identification of a protective antigen fromBoophilus microplus. J. Immunol. 143, 1346–1351.

Willadsen, P., Bird, P., Cobon, G.S., Hungerford, J., 1995. Commercialisation of a recombinant vaccine againstBoophilus microplus. Parasitology 110, 43–50.

Willadsen, P., Jongejan, F., 1999. Immunology of the tick-host interaction and the control of ticks and tick-bornediseases. Parasitol. Today 15, 258–262.

Williams, S.A., Johnston, D.A., 1999. Helminth genome analysis: the current status of the filarial and Schistosomegenome projects. Filarial Genome Project, Schistosome Genome Project. Parasitology 118 (Suppl.), S19–S38.

Williams, S.A., Lizotte-Waniewski, M.R., Foster, J., Guiliano, D., Daub, J., Scott, A.L., Slatko, B., Blaxter, M.L.,2000. The Filarial Genome Project: analysis of the nuclear mitochondrial and endosymbiont genomes of Brugiamalayi. Int. J. Parasitol. 30, 411–419.

Wright, I.G., Casu, R., Commins, M.A., Dalrymple, B.P., Gale, K.R., Goodger, B.V., Riddles, P.W., Waltisbuhl,D.J., Abetz, I., Berrie, D.A., et al., 1992. The development of a recombinant Babesia vaccine. Vet. Parasitol.44, 3–13.

Xu, M., Molento, M., Blackhall, W., Ribeiro, P., Beech, R., Prichard, R., 1998. Ivermectin resistance in nematodesmay be caused by alteration of P-glycoprotein homologue. Mol. Biochem. Parasitol. 91, 327–335.

Yamage, M., Flechtner, O., Gottstein, B., 1996. Neospora caninum: specific oligonucleotide primers for thedetection of brain cyst DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). J.Parasitol. 82, 272–279.

Zajac, A.M., Sangster, N.C., Geary, T.G., 2000. Why veterinarians should care more about parasitology. Parasitol.Today 16, 504–506.

Zarlenga, D.S., Gasbarre, L.C., Boyd, P., Leighton, E., Lichtenfels, J.R., 1998. Identification and semi-quantitationof Ostertagia ostertagi eggs by enzymatic amplification of ITS-1 sequences. Vet. Parasitol. 77, 245–257.

Zarlenga, D.S., Chute, M.B., Martin, A., Kapel, C.M.O., 1999. A multiplex PCR for unequivocal differentiationof all encapsulated and non-encapsulated genotypes of Trichinella. Int. J. Parasitol. 29, 1859–1867.

Documents

The role of molecular biology in veterinary parasitology