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Using molecular tools for diagnosis in veterinaryparasitology

Am Comes, Jf Humbert, J Cabaret, L Élard

To cite this version:Am Comes, Jf Humbert, J Cabaret, L Élard. Using molecular tools for diagnosis in veterinary para-sitology. Veterinary Research, BioMed Central, 1996, 27 (4-5), pp.333-342. <hal-00902426>

Review article

Using molecular tools for diagnosisin veterinary parasitology

AM Comes, JF Humbert J Cabaret L Élard

Station de pathologie aviaire et de parasitologie, Centre de recherches de Tours, Inra,37380 Nouzilly, France

(Received 20 November 1995; accepted 5 March 1996)

Summary &horbar; The development of molecular biology has made available tools that identify parasiteswhich are important in veterinary parasitology. PCR (polymerase chain reaction) is the most fre-

quently used tool. Target sequences are ribosomal DNA and RNA as well as fragments derivedfrom RAPD (random amplified polymorphic DNA). The most studied protozoan genera have been:Eimeria, Babesia, Theileria, Trypanosoma and Cryptosporidium. Trichinella species and ruminantTrichostrongylid parasites have been the most investigated helminths. The use of molecular toolsfor identification in veterinary parasitology, however, remains a research technique rather than onethat is currently used in the field for diagnostic purposes.

diagnosis / veterinary parasitology / molecular tool / polymerase chain reaction (PCR) I ran-dom amplified polymorphic DNA (RAPD)

Résumé &horbar; Les outils moléculaires pour le diagnostic en parasitologie vétérinaire. Le déve-loppement de la biologie moléculaire a permis de mettre au point de nouveaux outils pour le dia-gnostic des parasitoses animales. Parmi ces outils, la PCR (Polymerase chain reaction) est celuiqui est le plus utilisé. Les séquences cibles les plus fréquentes sont les ADN et les ARN riboso-miques ainsi que des fragments obtenus par amorçage aléatoire (RAPD). L’essentiel des travauxchez les protozoaires se rapporte aux genres suivants : Eimeria, Babesia, Theleria, Trypanosomaet Cryptosporidium. Chez les helminthes, les trichines ainsi que les trichostrongles gastro-intesti-naux ont fait l’objet de nombreuses publications. De l’ensemble de ces travaux, il ressort que l’ap-plication de ces outils pour le diagnostic de routine n’est pas encore évidente. En revanche, cesoutils de diagnostic seront certainement très utiles à des fins de recherche.

diagnostic / parasitologie vétérinaire / outil moléculaire / polymerase chain reaction (PCR) /random amplified polymorphic DNA (RAPD)

*

Correspondence and reprints

INTRODUCTION

The tools available in molecular biologyhave increased enormously over the lastten years and have improved the detec-tion and identification of microorganisms.In the field of human parasitology, forexample, investigations of opportunisticdiseases in AIDS patients have led to theisolation of polymerase chain reaction(PCR) primers that can be used for theidentification of Pneumocystis carinii and

Toxoplasma gondii (see reviews byWeiss, 1995, and Smits and Hartskeerl,1995). The large volume of investigationson malaria, due to its significance as amajor human disease, has also resultedin the production of PCR primers andprobes for the identification of variousPlasmodium species. In the field of veteri-

nary parasitology, however, the resultsare much less abundant and most have

been published only during the last twoyears. This is probably due to the exis-

tence of classical techniques of identifica-tion (among the Trichostrongyle parasitesof ruminants for example) that have beenconsidered to be satisfactory in most

cases.

The aim of the present paper is to

describe the array of available diagnostictools for protozoa and helminths that areof particular interest to veterinary para-sitology, with attention being given to thestrategies that were used to constructthem. We have also taken into account

articles devoted to species and straincharacterization for three reasons. Thefirst one is that a correct diagnosis cannotbe achieved without a species and, in

future, a strain identification. Thus, theability to identify nematode strains resis-tant or susceptible to anthelmintics wouldlead to a better diagnosis, and thereforebetter deworming strategies. The secondreason is that species characterizationwith molecular markers might be a firststep in the construction of diagnostic tools(see our work on rabbit coccidiosis, Cereet al, in press). The last reason is that a

diagnosis could be achieved in a two-stepprocedure. The isolation and numerationof parasites using ’classical’ techniqueswould constitute the first step. The secondstep would involve an exact species iden-tification in taxonomic groups usingmolecular techniques where classicaltechniques fail to achieve a sufficientlyaccurate specific identification. For exam-ple, the numeration of eggs or larvae oftrichostrongyle nematodes of sheep is

easily achieved with methods based onfaecal examination, but the species identi-fication is very difficult with classical tech-

niques (morphology and morphometry).On the other hand, molecular techniquesallow the species identification from eggsor larvae but not the quantification. Thecombination of the two approaches willpermit an efficient diagnosis: speciesidentification and intensity of infection.

TECHNICAL CHOICES AND SCREEN-ING OF TARGET SEQUENCES

On rare occasions (9 references out of43), detections were based on the use ofa hybridizing probe with highly repeatedsequences that did not require amplifica-tion. These techniques of using hybridiza-tion without previous amplification areless frequently employed since the devel-opment of PCR. We will neverthelessmention them in this paper.

Actually, the vast majority of diagnosticprocedures use PCR (37 articles out of43) as a first step in the detection of the

very small amount of DNA present. Thisamplification is performed either onknown sequences (PCR with a primersize greater than or equal to 20 basesthat hybridize to known sequences) orrandomly (random amplified polymorphicDNA (RAPD) with a primer size of 10 0bases). The PCR products are directlystained with ethidium bromide or cut by arestriction enzyme and then stained withethidium bromide (PCR-Restriction frag-ment length polymorphism [PCR-RFLP])or transferred to a membrane for

hybridization with a radioactive probe.Three types of sequences are used as

PCR targets: (i) ribosomal DNA or RNA(13 references out of 43) that presenthighly repeated sequences and thus per-mit a sharp detection level. These

sequences are generally highly pre-served, which may hinder any identifica-tion of phylogeneticalfy closely relatedspecies; (ii) sequences chosen aftercloning, either for their specificity or forthe fact that they code for an antigen sur-face protein specific to the species candi-date for identification (7 references out of43). Specificity is thus high in this case,whereas sensitivity is lower than mightnormally be expected from the use ofrepeated sequences; and (iii) sequencesobtained after random primer amplifica-

tion (15 references out of 43). The use ofRAPD furnishes numerous fragments thatare potentially interesting for diagnosis.When these fragments incorporate highlyrepeated sequences, a high level of sen-sitivity can be achieved. As a stand-alone,however, RAPD is a poor technique foridentification.

IDENTIFICATION OF PROTOZOA

Eimeria spp

This genus has undergone a great deal ofinvestigation as its morphological charac-terization has often been misleading and areal need exists for the reliable identifica-

tion of the particular species harboured bychicks, rabbits, sheep, goats and cattle.Three papers (Cere et al, 1995;MacPherson and Gajadhar, 1993;Procunier and Barta, 1993) describe theuse of RAPD which provide evidence ofcharacteristic fragments of species oreven isolates. These fragments are notreally helpful for diagnosis because, whenseveral species are found within a studiedsample, the fragments obtained afteramplification are different from thoseobserved in cases of single-species infec-tion. Single-species fragments might be ofdiagnostic interest under particular condi-tions (Cere et al, in press) as we havedemonstrated for the Eimeria species ofrabbits. This new technique is primarilybased on RAPD (fig 1) to identify a frag-ment of potential use for diagnosis. Thefragment obtained is cloned, then labelledwith digoxigenin (Boehringer), and its

extremities are sequenced. Finally, fromthe obtained sequences, two primers (20bases) are chosen in order to amplify thisfragment by PCR. We can detect, usingPCR-hybridization with the labelled probe,less than ten oocysts of a particular

species directly from 1-5 g of faeces,even in multispecific infections. Berriatuaet al (1995) developed a similar procedureusing PCR-hybridization of repeatedsequences on sheep coccidiae andachieved a sensitivity of less than ten puri-fied oocysts. Another technique (Stucki etal, 1993), based on the use of a 5S riboso-mal RNA repeat unit, was also very effi-cient (up to two purified oocysts) for thedetection of E tenella, a chicken parasite.

Trypanosoma spp

In two studies (Dirie et al, 1993; Waitumbiand Murphy, 1993), fragments character-izing species or isolates of Trypanosoma,a parasite of ruminants and pigs, werefound using RAPD. It was, however, nec-

essary to purify the parasites from thehost blood before amplification and themethod needs further refinements before

being used as a diagnostic tool.

Theileria spp

Three papers discuss the use of molecu-

lar tools for this parasite which affectsruminants, mostly bovines. Tanaka et al(1993) utilized primers and a probederived from a gene encoding a 32-kDaintraerythrocytic piroplasm surface proteinof T sergenti. Their method was sufficient-ly sensitive to detect four parasites permicrolitre of blood with a 10-pL sample.D’Oliveira et al (1995) reported the detec-tion, using PCR, of Theileria annulata in

blood samples of carrier cattle. The assayemployed primers specific for the geneencoding the 30-kDa major merozoite sur-face antigen of this species. The specifici-ty and sensitivity of this PCR assay arevery good (three parasites per microlitreof blood). Allsopp et al (1993) developedanother technique based on PCR using

oligonucleotide probes which detectedsmall subunit ribosomal RNA sequences(srRNA). These probes were efficient atdiscriminating between the six different

species harboured by cattle. One investi-gation has increased the accuracy of theidentification of isolates or strains usingRAPD (Bishop et al, 1993).

Babesia spp

Figueroa et al (1994) set up a PCR-baseddiagnostic assay to detect B bovis inchronically-infected cattle. The targetsequence was a gene encoding a 60-kDamerozoite surface protein. The level ofsensitivity was high as the PCR productwas detected in blood samples containingapproximately three B bovis-infected ery-throcytes (20 pL of packed cells with aparasitemia of 0.000 001 %). Carson et al(1994) used RAPD to reveal markers ofspecies and isolates for B bovis and Bbigemina.

Cryptosporidium spp

Awad-El-Kariem et al (1994) used amplifi-cation by PCR of an 18 srRNA fragment,followed by digestion with restrictionenzymes, to identify three species ofCryptosporidia: C parvum, C muris and Cbaileyi. Another study (Johnson et al,1993) compared the efficacy of twomolecular techniques (srDNA and

hybridization of repetitive DNA probes)with a classical technique (immunofluo-rescence assay, IFA) to detect the pres-ence of Cryptosporidia. The best compro-mise between sensitivity and rapidity ofresponse was found when PCR was

used. Webster et al (1993), using primersadapted to C parvum, obtained good sen-sitivity, as samples of only 20 purifiedoocysts were able to be detected. Whenthis PCR is coupled with an immunomag-netic particle separation of faecal sam-ples, 80-90 oocysts per gram of bovinefaeces can be detected. This representsan increase in sensitivity over convention-al diagnostic methods based on faecalexamination (Webster et al, 1996).

Other protozoa

A specific DNA probe and PCR amplifica-tion were proposed by Ho et al (1994) todetect Trichomonas infection in cattle.

Wheeler et al (1990) demonstrated that aPCR test developed for the detection ofToxoplasma gondii in human and murinetissues can be used for the diagnosis ofovine toxoplasmosis. Good results wereobtained when detecting Perkinsus mari-nus in the oyster Crassostrea virginica byMarsh et al (1995); the parasites wereidentified and the level of infection was

semi-quantified for the first time.

IDENTIFICATION OF HELMINTHS

Cestodes

Echinococcus multilocularis is the onlyspecies to be studied thus far due to itszoonotic significance. Gootstein andMowatt (1991) characterized this speciesusing PCR-hybridization. Bretagne et al(1993b) established efficient diagnostictools using another target sequence (U1snRNA gene), and could thus detect aslittle as one egg in 4 g of fox faeces.

Trematodes

The detection of five larval stages ofFasciola hepatica within intermediarysnail hosts was possible with a probederived from an rRNA sequences

(Shubkin et al, 1992). Utilization of thesecond internal transcribed spacer (ribo-somal RNA) of adult flukes (Adlard et al,1993) permitted the identification of threespecies of Fasciolidae: F hepatica, Fgigantica and F magna.

Nematodes

The majority of the investigations concerntrichostrongyles or Trichinella spp. In

order to characterize the Trichinellae

species, two strategies were followed.The first one was based on the utilizationof repeated sequences. Dupouy-Carnet etal (1991) using PCR, identified T spiralisin mouse. Dick et al (1992) using otherprimers could identify pig isolates with agood level of sensitivity. It was possible toidentify an isolate from a single larva, fol-lowing digestion or in situ in the muscletissue. Recently, several references in theliterature have demonstrated the utility ofRAPD in identifying Trichinella species(Bandi et al, 1993a, b, 1995; Dupouy-Camet et al, 1994; Tighe et al, 1994).These results represent only preliminarysteps in the construction of a reliable

diagnostic tool. Among the

Trichostrongyles, Beh et al (1989) demon-strated that is was possible to utilizerepeated sequences in order to identifythe different species without using PCR.Christensen et al (1994a) isolated otherrepeated sequences that were able to dis-criminate between four genera and were

capable of detecting as few as 25 eggs. Aspecific Haemonchus placei probe hasbeen isolated (Christensen et al, 1994b).Other papers refer to the diagnosis ofthese species based on ribosomal DNA orRNA, or RAPD fragments. Zarlenga et al(1994) as well as other studies fromAustralia (Campbell et al, 1995; Chilton etal, 1995; Gasser et al, 1994; Hoste et al,1995; Stevenson et al, 1995) have shownthe utility of ribosomal DNA or RNA forstrongyle identification. Identification is

possible on a single egg. We have foundother specific markers using RAPD in ourlaboratory (Humbert and Cabaret, 1994;Jacquiet et al, 1995). From these specificmarkers we isolated probes in order toidentify three species of Haemonchus

either as adult or infective larvae. Similar

work was done by Chandrasekharan et al(1994), who described the utilization of arepeated sequence to identify Dirofilariarepens. A single filaria in infected dogblood samples and a single third stagelarvae in mosquitoes have been detected.

CONCLUSION

PCR has obviously been the major molec-ular biological technique for identificationin the last few years. The ability to detectvery small quantities of target materialand the absence of the need to use

radioactive elements are two of the

advantages of PCR compared withhybridization techniques. It is not, howev-

er, evident that is use will spread to rou-tine diagnosis in veterinary laboratories.There are several drawbacks to the rou-

tine use of such a technique. First of all,contamination of the laboratory environ-ment has to be rigorously controlled andthis implies numerous controls for qualitydiagnoses. The enormous amplificationachieved by PCR implies that a very smallcontamination may appear significant.Means of avoiding contamination havebeen presented by Altwegg (1995) andCarrino and Lee (1995). The first sugges-tion relates to the organization of the labo-ratory space for the different stages ofdiagnosis. DNA and RNA extraction, andthe analyses of PCR products (by elec-trophoresis for example), should be pro-cessed in different parts of the laboratory,with specific materials restricted to eachprocess. The second suggestion relatesto the use of Uracil DNA Glycosylase(UDG) for the preamplification sterilizationof the PCR products (Longo et al, 1990).During PCR, dUTP are incorporated inthe synthetized fragments. Before the fol-lowing PCR, the amplification mixtureshould be systematically treated with the

enzyme (UDG) that cuts the ADN frag-ments in which dUTP are incorporated.Any contamination resulting from a previ-ous amplification may then be avoided.For the detection of contaminants, nega-tive controls must be realized at each stepof the PCR preparation (eg, isolation ofthe sample, extraction of the DNA). PCRcan fail due to the inhibition of specificamplification (false negative). Thus, theuse of positive controls is necessary toincrease confidence in negative PCRresults. These controls are realized with

one fragment which is coamplified withthe fragment of interest (Cone et al,1992). Bretagne et al (1993a) demonstrat-ed in their diagnosis of Toxoplasmagondii, the interest of having such aninternal control incorporated in the PCRmixture.

The cost of diagnosis is the seconddrawback of PCR. Although PCR identifi-cation costs are being progressivelyreduced, they remain higher than those ofimmunological reactions (immunofluores-cence, etc) and histological procedures.

A final drawback is the absence of

quantification. One unique work (Marsh etal, 1995) did attempt to estimate the inten-sity of parasitation. In the case of strongyleinfections, intensity of parasitation is esti-

mated by classical techniques (faecal eggcounts for example) and PCR is only ofparticular use for the identification of thestrongyle species either from the eggs orlarvae. The identification of blood parasito-sis is much more complex and requires thequantification of parasite DNA before it can

constitute a real alternative to classical

diagnosis. Competition between an addedcontrol nucleic acid (internal control) andthe native nucleic acid of interest could be

exploited in order to quantify the PCR sig-nal (Gilliland et al, 1990).

The use of molecular biology tools forparasite identification will therefore contin-ue to be used in research activities (from

taxonomy to ecology or epidemiology)rather than for day-to-day diagnosis in

veterinary laboratories. These tools willprobably be of interest primarily in the

diagnosis of zoonotic diseases such as

Trichinellosis, Echinococosis and

Cysticercosis.

ACKNOWLEDGMENTS

A Comes was funded on an INRA postdoctor-ate grant and L Elard on a PhD grant from theRegion Centre.

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