Using Molecular Tools for Diagnosis in Veterinary Parasitology Am Comes, Jf Humbert, J Cabaret, L Élard

Using molecular tools for diagnosis in veterinary parasitology Am Comes, Jf Humbert, J Cabaret, L Élard

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Am Comes, Jf Humbert, J Cabaret, L Élard. Using molecular tools for diagnosis in veterinary parasitology. Veterinary Research, BioMed Central, 1996, 27 (4-5), pp.333-342. hal-00902426

HAL Id: hal-00902426 https://hal.archives-ouvertes.fr/hal-00902426 Submitted on 1 Jan 1996

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Using molecular tools for diagnosis in 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 ― The development of molecular biology has made available tools that identify parasites which are important in veterinary parasitology. PCR (polymerase chain reaction) is the most frequently used tool. Target sequences are ribosomal DNA and RNA as well as fragments derived from RAPD (random amplified polymorphic DNA). The most studied protozoan genera have been: Eimeria, Babesia, Theileria, Trypanosoma and Cryptosporidium. Trichinella species and ruminant Trichostrongylid parasites have been the most investigated helminths. The use of molecular tools for identification in veterinary parasitology, however, remains a research technique rather than one that is currently used in the field for diagnostic purposes. diagnosis / veterinary parasitology / molecular tool / polymerase chain reaction (PCR) I random amplified polymorphic DNA (RAPD)

Résumé ― 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 diagnostic des parasitoses animales. Parmi ces outils, la PCR (Polymerase chain reaction) est celui qui 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 travaux chez les protozoaires se rapporte aux genres suivants : Eimeria, Babesia, Theleria, Trypanosoma et 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, ces outils 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 Toxoplasma gondii (see reviews by Weiss, 1995, and Smits and Hartskeerl, The tools available in molecular biology 1995). The large volume of investigations on due to its as a have increased enormously over the last malaria, significance human has also resulted ten years and have improved the detec- major disease, tion and identification of microorganisms. in the production of PCR primers and In the field of human parasitology, for probes for the identification of various example, investigations of opportunistic Plasmodium species. In the field of veteri- diseases in AIDS patients have led to the nary parasitology, however, the results isolation of polymerase chain reaction are much less abundant and most have (PCR) primers that can be used for the been published only during the last two identification of Pneumocystis carinii and years. This is probably due to the exis- tence of classical techniques of identifica- TECHNICAL CHOICES AND SCREEN- tion (among the Trichostrongyle parasites ING OF TARGET SEQUENCES of ruminants for example) that have been considered to be in most satisfactory On rare occasions (9 references out of cases. 43), detections were based on the use of The aim of the present paper is to a hybridizing probe with highly repeated describe the array of available diagnostic sequences that did not require amplifica- tools for protozoa and helminths that are tion. These techniques of using hybridiza- of particular interest to veterinary para- tion without previous amplification are sitology, with attention being given to the less frequently employed since the devel- strategies that were used to construct opment of PCR. We will nevertheless them. We have also taken into account mention them in this paper. articles devoted to species and strain Actually, the vast majority of diagnostic characterization for three reasons. The procedures use PCR (37 articles out of first one is that a correct cannot diagnosis 43) as a first step in the detection of the be achieved without a species and, in very small amount of DNA present. This future, a strain identification. Thus, the amplification is performed either on ability to identify nematode strains resis- known sequences (PCR with a primer tant or susceptible to anthelmintics would size greater than or equal to 20 bases lead to a better diagnosis, and therefore that hybridize to known sequences) or better deworming strategies. The second randomly (random amplified polymorphic reason is that species characterization DNA (RAPD) with a primer size of 100 with molecular markers might be a first bases). The PCR products are directly step in the construction of diagnostic tools stained with ethidium bromide or cut by a (see our work on rabbit coccidiosis, Cere restriction enzyme and then stained with et al, in press). The last reason is that a ethidium bromide (PCR-Restriction frag- diagnosis could be achieved in a two-step ment length polymorphism [PCR-RFLP]) procedure. The isolation and numeration or transferred to a membrane for of parasites using ’classical’ techniques hybridization with a radioactive probe. would constitute the first The second step. Three types of sequences are used as would involve an exact iden- step species PCR targets: (i) ribosomal DNA or RNA tification in taxonomic groups using (13 references out of 43) that present molecular techniques where classical highly repeated sequences and thus per- techniques fail to achieve a sufficiently mit a sharp detection level. These accurate specific identification. For exam- sequences are generally highly pre- ple, the numeration of eggs or larvae of served, which may hinder any identifica- trichostrongyle nematodes of sheep is tion of phylogeneticalfy closely related easily achieved with methods based on species; (ii) sequences chosen after faecal examination, but the species identi- cloning, either for their specificity or for fication is very difficult with classical tech- the fact that they code for an antigen sur- niques (morphology and morphometry). face protein specific to the species candi- On the other hand, molecular techniques date for identification (7 references out of allow the species identification from eggs 43). Specificity is thus high in this case, or larvae but not the quantification. The whereas sensitivity is lower than might combination of the two approaches will normally be expected from the use of permit an efficient diagnosis: species repeated sequences; and (iii) sequences identification and intensity of infection. obtained after random primer amplification (15 references out of 43). The use of species directly from 1-5 g of faeces, RAPD furnishes numerous fragments that even in multispecific infections. Berriatua are potentially interesting for diagnosis. et al (1995) developed a similar procedure When these fragments incorporate highly using PCR-hybridization of repeated repeated sequences, a high level of sen- sequences on sheep coccidiae and sitivity can be achieved. As a stand-alone, achieved a sensitivity of less than ten puri- however, RAPD is a poor technique for fied oocysts. Another technique (Stucki et identification. al, 1993), based on the use of a 5S ribosomal RNA repeat unit, was also very efficient (up to two purified oocysts) for the IDENTIFICATION OF PROTOZOA detection of E tenella, a chicken parasite.

Eimeria spp Trypanosoma spp

This genus has undergone a great deal of In two studies (Dirie et al, 1993; Waitumbi investigation as its morphological charac- and Murphy, 1993), fragments character- terization has often been misleading and a izing species or isolates of Trypanosoma, real need exists for the reliable identifica- a parasite of ruminants and pigs, were tion of the particular species harboured by found using RAPD. It was, however, nec- chicks, rabbits, sheep, goats and cattle. essary to purify the parasites from the Three papers (Cere et al, 1995; host blood before amplification and the MacPherson and Gajadhar, 1993; method needs further refinements before Procunier and Barta, 1993) describe the being used as a diagnostic tool. use of RAPD which provide evidence of characteristic fragments of species or even isolates. These fragments are not Theileria spp really helpful for diagnosis because, when several are within a studied species found Three papers discuss the use of molecu- sample, the fragments obtained after lar tools for this parasite which affects amplification are different from those ruminants, mostly bovines. Tanaka et al observed in cases of single-species infec- (1993) utilized primers and a probe tion. Single-species fragments might be of derived from a gene encoding a 32-kDa diagnostic interest under particular condi- intraerythrocytic piroplasm surface protein tions (Cere et al, in press) as we have of T sergenti. Their method was sufficient- demonstrated for the Eimeria species of ly sensitive to detect four parasites per rabbits. This new technique is primarily microlitre of blood with a 10-pL sample. based on RAPD (fig 1) to identify a frag- D’Oliveira et al (1995) reported the detec- ment of potential use for diagnosis. The tion, using PCR, of Theileria annulata in fragment obtained is cloned, then labelled blood samples of carrier cattle. The assay with digoxigenin (Boehringer), and its employed primers specific for the gene extremities are sequenced. Finally, from encoding the 30-kDa major merozoite sur- the obtained sequences, two primers (20 face antigen of this species. The specifici- bases) are chosen in order to amplify this ty and sensitivity of this PCR assay are fragment by PCR. We can detect, using very good (three parasites per microlitre PCR-hybridization with the labelled probe, of blood). Allsopp et al (1993) developed less than ten oocysts of a particular another technique based on PCR using oligonucleotide probes which detected species harboured by cattle. One investi- small subunit ribosomal RNA sequences gation has increased the accuracy of the (srRNA). These probes were efficient at identification of isolates or strains using discriminating between the six different RAPD (Bishop et al, 1993). Babesia spp Other protozoa

Figueroa et al (1994) set up a PCR-based A specific DNA probe and PCR amplifica- diagnostic assay to detect B bovis in tion were proposed by Ho et al (1994) to chronically-infected cattle. The target detect Trichomonas infection in cattle. sequence was a gene encoding a 60-kDa Wheeler et al (1990) demonstrated that a merozoite surface protein. The level of PCR test developed for the detection of sensitivity was high as the PCR product Toxoplasma gondii in human and murine can be for the of was detected in blood samples containing tissues used diagnosis ovine Good results were approximately three B bovis-infected ery- toxoplasmosis. obtained when Perkinsus mari- throcytes (20 pL of packed cells with a detecting nus in the Crassostrea parasitemia of 0.000 001 %). Carson et al oyster virginica by Marsh et al the were (1994) used RAPD to reveal markers of (1995); parasites identified and the level of infection was species and isolates for B bovis and B for the first time. bigemina. semi-quantified

IDENTIFICATION OF HELMINTHS Cryptosporidium spp

Awad-El-Kariem et al (1994) used amplifi- Cestodes cation by PCR of an 18 srRNA fragment, followed with restriction by digestion Echinococcus multilocularis is the only to three of enzymes, identify species species to be studied thus far due to its Cryptosporidia: C parvum, C muris and C zoonotic significance. Gootstein and baileyi. Another study (Johnson et al, Mowatt (1991) characterized this species 1993) compared the efficacy of two using PCR-hybridization. Bretagne et al molecular techniques (srDNA and (1993b) established efficient diagnostic hybridization of repetitive DNA probes) tools using another target sequence (U1 with a classical technique (immunofluo- snRNA gene), and could thus detect as rescence assay, IFA) to detect the pres- little as one egg in 4 g of fox faeces. ence of Cryptosporidia. The best compro- mise between sensitivity and rapidity of response was found when PCR was Trematodes used. Webster et al (1993), using primers to obtained sen- adapted C parvum, good The detection of five larval stages of sitivity, as samples of only 20 purified Fasciola hepatica within intermediary were able to be detected. When oocysts snail hosts was possible with a probe this PCR is with an coupled immunomag- derived from an rRNA sequences netic particle separation of faecal sam- (Shubkin et al, 1992). Utilization of the ples, 80-90 oocysts per gram of bovine second internal transcribed spacer (ribo- faeces can be detected. This represents somal RNA) of adult flukes (Adlard et al, an increase in sensitivity over convention- 1993) permitted the identification of three al diagnostic methods based on faecal species of Fasciolidae: F hepatica, F examination (Webster et al, 1996). gigantica and F magna. Nematodes either as adult or infective larvae. Similar work was done by Chandrasekharan et al (1994), who described the utilization of a The majority of the investigations concern repeated sequence to identify Dirofilaria or Trichinella spp. In trichostrongyles A filaria in infected order to characterize the Trichinellae repens. single dog blood and a third two were followed. samples single stage species, strategies larvae in have been detected. The first one was based on the utilization mosquitoes of repeated sequences. Dupouy-Carnet et al PCR, identified (1991) using T spiralis CONCLUSION in mouse. Dick et al (1992) using other primers could identify pig isolates with a good level of sensitivity. It was possible to PCR has obviously been the major molec- identify an isolate from a single larva, fol- ular biological technique for identification lowing digestion or in situ in the muscle in the last few years. The ability to detect tissue. Recently, several references in the very small quantities of target material literature have demonstrated the utility of and the absence of the need to use RAPD in identifying Trichinella species radioactive elements are two of the (Bandi et al, 1993a, b, 1995; Dupouy- advantages of PCR compared with Camet et al, 1994; Tighe et al, 1994). hybridization techniques. It is not, howev- These results represent only preliminary er, evident that is use will spread to rou- steps in the construction of a reliable tine diagnosis in veterinary laboratories. diagnostic tool. Among the There are several drawbacks to the rou- Trichostrongyles, Beh et al (1989) demon- tine use of such a technique. First of all, strated that is was possible to utilize contamination of the laboratory environ- repeated sequences in order to identify ment has to be rigorously controlled and the different species without using PCR. this implies numerous controls for quality Christensen et al (1994a) isolated other diagnoses. The enormous amplification repeated sequences that were able to dis- achieved by PCR implies that a very small criminate between four genera and were contamination may appear significant. capable of detecting as few as 25 eggs. A Means of avoiding contamination have specific Haemonchus placei probe has been presented by Altwegg (1995) and been isolated (Christensen et al, 1994b). Carrino and Lee (1995). The first sugges- Other papers refer to the diagnosis of tion relates to the organization of the labo- these species based on ribosomal DNA or ratory space for the different stages of RNA, or RAPD fragments. Zarlenga et al diagnosis. DNA and RNA extraction, and (1994) as well as other studies from the analyses of PCR products (by elec- Australia (Campbell et al, 1995; Chilton et trophoresis for example), should be pro- al, 1995; Gasser et al, 1994; Hoste et al, cessed in different parts of the laboratory, 1995; Stevenson et al, 1995) have shown with specific materials restricted to each the utility of ribosomal DNA or RNA for process. The second suggestion relates strongyle identification. Identification is to the use of Uracil DNA Glycosylase possible on a single egg. We have found (UDG) for the preamplification sterilization other specific markers using RAPD in our of the PCR products (Longo et al, 1990). laboratory (Humbert and Cabaret, 1994; During PCR, dUTP are incorporated in Jacquiet et al, 1995). From these specific the synthetized fragments. Before the fol- markers we isolated probes in order to lowing PCR, the amplification mixture identify three species of Haemonchus should be systematically treated with the enzyme (UDG) that cuts the ADN frag- taxonomy to ecology or epidemiology) ments in which dUTP are incorporated. rather than for day-to-day diagnosis in Any contamination resulting from a previ- veterinary laboratories. These tools will ous amplification may then be avoided. probably be of interest primarily in the For the detection of contaminants, nega- diagnosis of zoonotic diseases such as tive controls must be realized at each step Trichinellosis, Echinococosis and of the PCR preparation (eg, isolation of Cysticercosis. the sample, extraction of the DNA). PCR can fail due to the inhibition of specific amplification (false negative). Thus, the ACKNOWLEDGMENTS use of positive controls is necessary to increase confidence in PCR negative A Comes was funded on an INRA postdoctor- results. 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