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Trente ans de lutte contre l’onchocercose en Afrique de l’Ouest. Traitements larvicides et protection de l’environnement

 | 
Laurent Yaméogo
, 
Christian Levêque
, 
Jean-Marc Hougard

Références des articles présentés sur le cédérom / Papers references presented on CD-ROM

Cloning and characterization of an Onchocerca volvulus specific DNA sequence

Stefanie E.O. Meredith, Thomas R. Unnasch, Marc Karam, Willy F. Piessens et Dyann F. Wirth

Résumé

A cloned sequence, pOvs134, was isolated from a genomic library prepared from Onchocerca volvulus of savanna origin in the plasmid pUC9. pOvs134 hybridizes to all the geographic isolates of O. volvulus tested from both the New and the Old World, but not to the species Onchocerca gibsoni, Onchocerca gutturosa, Onchocerca ochengi, Onchocerca cervicalis, the filarial parasites Brugia malayi. or Dirofilaria immitis, nor to human or simuliid DNA. As little as 250 pg of DNA can be detected on a dot blot hybridization. suggesting that pOvs134 is sensitive enough to detect a single third stage larva. DNA sequence analysis of the inserted DNA of pOvs134 revealed that it consisted of twelve examples of a 149-bp repeat. The sequence of this repeat is strikingly similar to that of two O. volvulus genomic clones previously described, one of which has been reported to be specific for forest form O. volvulus. and one of which hybridizes to genomic DNA of several species of Onchocerca. These results suggest that the 149-bp repeat sequence is highly repeated in the genome of O. volvulus, and that variants of this repeat with different specificities exist.

Texte intégral

1(Received 30 December 1988; accepted 7 March 1989)

Introduction

2There are marked geographical variations in the spectrum of clinical manifestations associated with onchocerciasis. In West Africa the incidence of blindness due to sclerosing keratitis is much higher in savanna areas than in forest areas [1–3]. Although in other endemic areas of Africa and South and Central America the epidemiology and etiology of the disease may vary considerably, there are similarities in the ocular changes found in different areas. Studies on African onchocerciasis indicate that both the vector and parasite may consist of different forms, strains or species [4-9] and it has now been established that the main vectors of onchocerciasis in Africa are sibling species of the Simulium damnosum complex. Immunological and biochemical studies on Onchocerca volvulus have done little to clarify the possible existence of different strains, forms or subspecies, although allozyme studies have shown evidence of genetic variation in worms from different geographic areas [10,11].

3During the collection of baseline data, epidemiological studies or during the monitoring of control or intervention programs, it is important to be able to assess accurately the transmission of O. volvulus by vector species. Most onchocerciasis vectors are known to be zoophilie to some degree, and can thus be infected with animal onchocerca species which, as third stage larvae (L3), are morphologically indistinguishable from each other and from O. volvulus. At present, Onchocerca infections are detected by dissection of the vector, and the transmission of the disease is measured by the Annual Transmission Potential, but the presence of animal Onchocerca species can resuit in inaccurate transmission data. A reliable method for detecting infected vectors and distinguishing between the Onchocerca species as well as between the strains of O. volvulus is required, especially with the implementation of large scale Ivermectin trials in West Africa [1].

4Note: Nucleotide sequence data reported in this paper have been submitted to the Genbank Data Bank with the accession number J04659

5Abbreviations: NET, NaCl/EDTA/Tris buffer; SSC, NaCl/Nacitrate; TE. Tris/EDTA buffer; PBS, phosphate-buffered saline; RF, replicative form.

6Highly repeated DNA sequences which undergo rapid evolutionary change [13] have been successfully used to isolate species-specific probes for vectors and parasites [14-17]. Recently, Onchocerca-specific clones were described which hybridized strongly to O. volvulus, but also to some other Onchocerca species [18-19], and Erttmann et al. reported a DNA sequence specific for a forest form of O. volvulus [20]. We describe here a cloned DNA sequence which appears to be O. volvulus-specific, hybridizing with isolates of O. volvulus from forest and savanna areas of West Africa, Central Africa and Central America. Sequence analysis of this clone revealed that it consists of twelve examples of a 149bp repeated sequence. The sequence of this repeat is strikingly similar to those previously reported for both the O. volvulus forest form-specific clone reported by Erttmann et al. [20] and the Onchocerca-specific clone reported by Shah et al. [19]. These results suggest that this 149-bp sequence is highly repeated in the genome of O. volvulus, and that the repeats have evolved to include different members which demonstrate different levels of specificity.

Materials and Methods

7Parasite material. The O. volvulus material was obtained either as nodules which had been cryopreserved in liquid nitrogen immediately after excision, or as worms which had been collagenase-digested from nodules freshly excised in the field and snap-frozen.

8The O. volvulus were from either a defined savanna area (Manambougou, Missira, Foura in Mali) or forest regions (Danane, Trompleu, Abraninouin, Ivory Coast; Bo, Sierra Leone) and from habitats of unknown definition in Zaire and Guatemala. O. gutturosa originating from England and O. cervicalis from South Carolina (U.S.A.) were received in propanol after excision from the hosts.

9Frozen nodules of O. gibsoni from Australia and O. ochengi from the Central African Republic were obtained from abattoirs.

10Brugia malayi microfilariae were obtained from jirds infected intraperitoneally [21], and Dirofilaria immitis microfilariae were provided by Dr. A. Scott (Johns Hopkins University, Baltimore).

11Isolation of DNA. The nodules which had been cryopreserved immediately after excision were allowed to thaw at 37°C. Nodules of human origin were thawed in a 0.1% SDS solution as a precaution against HIV infection. The parasites were then freed by collagenase digestion as described by Schulz-Key et al. [22] and carefully cleaned of any host tissue. The worms were then washed in PBS and NET buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl, pH 7.5) three times. Similarly, the cryopreserved or alcohol-preserved adults were carefully washed to remove host tissue.

12The parasites were freeze-thawed three times and homogenized with a mortar and pestle. The homogenate was then transferred to a 15 ml tube in NET buffer (1.8 ml NET per adult female worm). n-Lauryl sarcosine (Sigma, St. Louis, MO) and proteinase K (Boehringer Mannhein, Indianapolis, IA), were added to a final concentration of 1% (w/v) and 100 μg ml-1, respectively. The mixture was then incubated at 37°C for up to 6 h. Ribonuclease A (Sigma) was then added to a final concentration of 100 μg ml-1 and the solution was further incubated for 30 min at 37°C. The DNA was extracted with phenol/chloroform and dialyzed extensively against TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

13The yield of DNA varied from 3 μg from a small female to 13 |xg from a large female worm. The amount of host DNA contamination of the preparation was monitored as described previously [19] using an AluI like human sequence or calf thymus DNA (Sigma) to probe Southern blots.

14Blackfly material. DNA was extracted as described above from pooled Simulium sirbanum from Tienfala (Niger River, Mali) which had been either cryopreserved in liquid nitrogen or preserved in propanol.

15Construction of genomic libraries. Genomic libraries were constructed in the plasmid pUC 9; Sau 3A fragments of DNA pooled from O. volvulus from Mali were ligated into the BamHI site of the polylinker [19] for the O. volvulus ‘savanna’ library. Similarly, Sau3A restriction fragments of worms from Danane, Ivory Coast, were ligated into the BamHI site of pUC9 for the ‘forest’ library.

16The libraries were screened by differential hybridization of four replicate nitrocellulose filters of the ampicillin-resistant colonies from the library. The filters were air-dried and the DNA was denatured, fixed and neutralized according to Maniatis et al. [23]. The filters were probed with 32P-radiolabeled nick-translated genomic DNA from O. volvulus (savanna or forest), human DNA, O. gibsoni DNA and a cocktail of DNA from B. malayi, Brugia pahangi and D. immitis.

17Colonies were selected if they hybridized to O. volvulus DNA and not to DNA from humans, bovine Onchocerca species or the lymphatic filariae. A second screening of the selected O. volvulus colonies was carried out by probing replicate filters with nick-translated DNA from O. volvulus savanna and forest, and second species of animal Onchocerca. Colonies hybridizing strongly to one of the O. volvulus isolates and not to the other Onchocerca species were investigated further.

18Plasmids pOvs134, pOvs742 and pOvs745 from the savanna library, and pOvf4, pOvf7 and pOvf2 from the forest library, which appeared to hybridize specifically to O. volvulus genomic DNA, were colony-purified and the plasmid DNA containing the O. volvulus sequences was isolated [23].

19Southern and dot blot analysis. Genomic DNA was digested with a 5-fold excess of restriction endonuclease. The completeness of the digestion was monitored by including a lambda DNA standard in an aliquot of the digestion mixture.

20The digest products were then size-fractionated by electrophoresis on an agarose gel, and the gel was used to prepare a Southern blot, as previously described [24], Nick-translated DNA probes (2-3 x 106 cpm (100 ng DNA)-1) were hybridized to the filters. Hybridization was performed at 42°C in a solution containing 50% formamide, 5 x Denhart’s solution, 2.5 x SSC, 0.1% SDS and 200μg/ml-1 herring sperm DNA, and three washes of 30 min each in 0.1 x SSC (150 mM NaCl, 15 mM sodium citrate), 0.1% SDS at 50°C.

21Dot blot hybridizations were carried out on nitrocellulose filters by spotting dilutions of genomic DNA onto the filter, air-drying it and then denaturing the DNA for 1 min in 0.5 M NaOH and neutralizing twice for 5 min in 1 M Tris, 1.5 M NaCl, pH 8. Before hybridization, the filters were baked at 70°C for 2 h.

22Dot blots on the nylon membrane Gene Screen Plus (Du Pont Company, Boston, MA) were performed according to the manufacturers directions using a manifold vacuum filtration apparatus (Biorad, Richmond, CA) and the filter hybridized in a solution containing 10% dextran sulphate at 65°C.

23The sensitivity of the selected plasmids was assessed by spotting genomic DNA from savanna O. volvulus, and forest O. volvulus in a doubling dilution series from 500 ng to 0.244 ng onto Gene Screen Plus membrane and probing the filter with the purified EcoRI-HindIII fragment of the insert of plasmid pOvs134.

24DNA sequence analysis. The inserted DNA of pOvs134 was subcloned into the bacteriophage vectors M13Mp18 and M13Mp19, and doublestranded replicative form (RF) DNA was prepared. The RF DNA was then used to prepare a set of clones containing nested deletions of the pOvs134 insert using the exonuclease BAL 31, as previously described [25]. The DNA sequence of the nested deletions was then determined using the dideoxynucleotide termination method [26]. The entire sequence of the insert, in both orientations, was obtained from the overlapping deletions.

Results

25DNA isolation. The isolation of high molecular weight DNA from Onchocerca parasites proved to be quite difficult. In order to avoid the problem of host contamination of the parasite DNA reported by Perler et al. [18], it was necessary to digest the nodular material with collagenases. However, the thawing and the subsequent long incubation of the material in collagenase resulted in degradation of the parasite DNA. If the incubation time was minimized by frequent changes (2-3-hourly) of collagenase and constant agitation. DNA of reasonable quality could be obtained. The best quality DNA was obtained from the worms digested in the field and snap-frozen. High-molecular-weight DNA was also obtained from the worms preserved in propanol, although the quality of DNA did deteriorate with increasing time of conservation.

26Identification of Onchocerca-specific sequences from the genomic libraries. 800 clones of the savanna library, and 600 clones from the forest O. volvulus library were screened with nick-translated genomic DNA from O. volvulus, humans, B. malayi, B. pahangi, D. immitis and various bovine or equine Onchocerca species. Clones which hybridized strongly to the O. volvulus DNA, but not to the other DNAs were selected for further investigation. The plasmids pOvs134, pOvs742 and pOvs745 were isolated from the savanna library, and pOvf2, pOvf4 and pOvf7 were isolated from the forest library.

27Specificity and sensitivity. To further examine the specificity of the six putatively O. volvulus-specific clones, the inserted DNA of each clone was used to probe Southern blots of genomic DNA of various filarial species. Using this stringent test, only one of the six clones, pOvs134, proved to be specific for O. volvulus. As is shown in Fig. 1, pOvs134 hybridizes strongly to O. volvulus DNA from both savanna and forest isolates, as well as to O. volvulus from Guatemala. The probe did not hybridize to genomic DNA from Onchocerca gibsoni, Onchocerca ochengi, Onchocerca gutturosa, O. cervicalis, or human or vector blackfly DNA.

Fig. 1. Specificity. Approximately 200 ng and 400 ng of genomic DNA from O. volvulus isolates (O.v.). and from other Onchocerca species was spotted onto a nitrocellulose filter which was hybridized to the purified insert of pOvs134.

28Southern blot analysis was then used to confirm the specificity of pOvs134 for O. volvulus DNA. As is shown in Fig. 2, pOvs134 hybridized strongly to genomic DNA from two isolates of O. volvulus, but did not hybridize to DNA from O. gutturosa, O. ochengi, O. cervicalis, O. gibsoni, B. malayi or D. immitis.

TABLE I. Specificity of the three related probes at different stringencies

TABLE I. Specificity of the three related probes at different stringencies

Each of the three related probes was hybridized to Southern blots containing the DNA samples shown, and the blots were washed at the temperatures indicated in the table.

Fig. 2. Southern blot analysis of 1.5 μg of genomic DNA from Onchocerca and filarial species digested with RsaI and probed with the purified insert of pOvs134. MW. molecular weight markers (λ HindIII, Ox HaeIII); O.v.F, O. volvulus forest (Ivory Coast); O.v.S, O. volvulus savanna (Mali); O.gu. O. gutturosa: O.o. O. ochengi; O.c. O. cervicalis; O.g. O. gibsoni; B.m. B. malayi; D.i. D. immitis.

29In order to test if the DNA sequences recognized by pOvs134 were restricted to a sub-population of O. volvulus, a Southern blot containing O. volvulus genomic DNA samples from several geographically distinct isolates was probed with pOvs134. As shown in Fig. 3, pOvs134 hybridized to O. volvulus genomic DNA from parasites isolated from the Ivory Coast, Sierra Leone, and Mali. These results, together with those shown in Fig. 1, suggest that the sequence recognized by pOvs134 is present in O. volvulus from both the savanna and forest regions of West Africa, as well as in isolates from the New World.

30The sensitivity of clone pOvs134 was assessed by dot-blotting decreasing amounts of O. volvulus genomic DNA from the savanna and forest regions of West Africa. As shown in Fig. 4, pOvs134 was able to detect as little as 250 pg of O. volvulus genomic DNA.

31Sequence organization. Hybridization of pOvs134 to complete RsaI digests of O. volvulus DNA revealed two bands approximately 320 and 170 bp in size (Fig. 5). Partial digestion of the O. volvulus DNA gave a ladder pattern, suggesting that pOvs134 contains a sequence that is tandemly repeated in the genome (Fig. 5).

32DNA sequence analysis of pOvs134. The DNA sequence of the inserted DNA of pOvs134 was determined as described in Materials and Methods. The insert of pOvs134 was found to be 1779 bp in length. It consisted entirely of 12 repeats of a 149-bp sequence which were joined head to tail. Examination of the DNA sequence of pOvs134 for similarities to other known sequences showed that the repeat was similar to both the Onchocerca specific clone pUOv3 described by Shah et al. [19], as well as the O. volvulus forest formspecific clone pFS-1 described by Erttmann et al. [20]. The sequence of the twelve repeats found in pOvs134 is given in Fig. 6, as well as the sequences of pUOv3 and pFS-1. A consensus sequence derived from the fifteen known examples of the repeat is also shown. Residues in individual repeats which differ from the consensus are outlined.

Fig. 3. Southern blot analysis of different isolates of O. volvulus genomic DNA digested with Rsal and probed with purified insert of pOvs134. Lanes 2, 4.6 and 8 had 1.5 μg DNA and lanes 3 and 5 had less parasite DNA due to slight contamination with host DNA. MW, molecular weight markers; O.v.F10 and O.v.F11, O. volvulus forest isolates from Ivory Coast; O.v. Sierra Leone, O. volvulus forest isolate from Sierra Leone; O.V.S3, 12 and 4, O. volvulus savanna isolates from Mali.

Fig. 4. Sensitivity. Genomic DNA from O. volvulus forest (O.v. Forest), O. volvulus savanna (O.v. Savanna), and O. ochengi were spotted in doubling dilutions from 500 ng to 0.244 ng onto Gene Screen Plus membrane and probed with nicktranslated pOvs134. Filter exposure of 90 min.

33Copy number of the 149-bp repeat in the genome. An estimation of the copy number of pOvs134 in the genome was made by dot-blotting genomic DNA, pOvs134 and pUC9 onto Gene Screen Plus filters in dilutions from 100 to 0.1 ng, and hybridizing the filter to the nick-translated insert of pOvs134. As is shown in Fig. 7, approximately 1% of the O. volvulus genomic DNA appears to be related to the repeat found in pOvs134. Assuming a haploid genome size of 8 x107 bp [27], this resuit suggests that the repeat is present in around 4500 copies per haploid genome.

34Specificity of the related DNA probes at different stringencies. The discovery that the DNA probes pOvs134, pFS-1 and puOv3 are variations of a 149-bp sequence which is highly repeated in the genome of O. volvulus suggested that the specificity of these probes might be affected by the stringency of hybridization. To test if this was the case, Southern blots containing O. volvulus forest and savannah form DNA, as well as O. gibsoni were probed with the three probes. The blots were then washed at 50°C and 55°C. pOvs134 retained its specificity and sensitivity at both temperatures. In contrast, pFS-1 lost its specificity at 50°C, and pUOV3 appeared to lose all sensitivity at 55°C (Table I).

Fig. 5. Southern blot analysis of 1.5 μg of genomic DNA digested with RsaI to completion, (lane 1 and 2) or partially digested (lane 3) and probed with purified insert of pOvs134. MWM, molecular weight markers; O.c, O. cervicalis; O.v.S, O. volvulus savanna; O.v.S p, O. volvulus savanna partial digest.

Discussion

35There is a real need for a method of distinguishing between the savanna and forest forms of O. volvulus, and also for differentiating O. volvulus infective larvae from other Onchocerca species. The Onchocerciasis Control Programme (OCP) in West Africa is committed to reducing the level of onchocerciasis in savanna regions to a level where it is no longer a public health problem [28]. Until very recently this has been primarily a vector control program and, despite considerable success, problems remain with reinvading infective Aies and occasional new outbreaks of infection [29]. Thus it is important to be able to identify the origin or form of the parasite in order to assess the risks and plan appropriate control strategies. Erttmann et al. reported identifying a DNA sequence, pFS-1, which appears to be specific for the forest form of O. volvulus [20]. An O. volvulus savanna-specific sequence has not yet been isolated.

Fig. 6. DNA sequence of the inserted DNA of pOvs134. The DNA sequence of the inserted DNA was determined as described in Materials and Methods. The sequence of each of the twelve repeats of pOvs134 described in the text are labeled Ovsl-12. The sequence of the forest form specific clone pFS-1 is labeled pFS-1 [20], and the two repeat units found in the Onchocerca specifie clone pUOV3 [19] are labeled Ov3A and Ov3B. A consensus sequence derived from the fifteen examples of the repeat is labeled CONS. Residues within individual repeats which differ from the consensus are outlined.

36The necessity to distinguish between L3 of O. volvulus and other Onchocerca species arises because domestic animais are commonly infected with Onchocerca species. In Africa, for example, cattle are known to carry several different Onchocerca species [30]. Although the vectors are not known for all species, it has been demonstrated that O. ochengi and O. gutturosa develop to infective L3 indistinguishable from O. volvulus in S. damnosum s.l. and S. vorax [31,32] while Simulium and Culicoides species are the incriminated vectors for the other Onchocerca species. In the OCP the transmission is estimated by measuring the Annual Transmission Potential [13]. The presence of infective larvae of animal origin can affect the accuracy of the transmission assessments. This is not only of importance in areas where control measures are in operation, but also in areas where trials of Ivermectin are being undertaken and an accurate assessment of the effect of the drug on transmission is crucial. Although pOvs134 still needs to be tested for cross-hybridization to the closely related species Onchocerca dukei, Onchocerca hamoni, and Onchocerca schulzkeyi, it could be valuable as a means of distinguishing O. volvulus infective larvae from other morphologically similar species. Used in combination with a clone such as the forest-specific O. volvulus sequence described by Erttmann et al. [20], pOvs134 could also be useful in distinguishing between savanna and forest O. volvulus strains in vectors, or in human hosts.

Fig. 7. Copy number. Genomic DNA from O. volvulus savanna and forest. plasmid pOvs134 and pUC9 were spotted onto Gene Screen Plus membrane and probed with the EcoRI- HindIII purified insert of pOvs134.1-100 ng. 2-50 ng. 3-10 ng. 4-5 ng. 6-0.5 ng. 7-0.1 ng.

37Evolution within the genus Onchocerca is thought to be relatively recent, specially in the line of Onchocerca of African Bovidae to which O. volvulus belongs [33]. The relatedness of Onchocerca species is confirmed by rRNA sequence data [34] and is evident from the difficulties encountered in isolating species-specific repetitive DNA sequences. In this study, out of hundreds of clones only one was found to be specific for O. volvulus, while Erttmann et al. screened over 20000 clones in order to identify one clone that appeared to be specific for the forest form of O. volvulus [20]. Surprisingly, DNA sequence analysis of pOvs134 demonstrated that it was very similar to both the Onchocerca-specific clone pUOV3 isolated by Shah et al. [19], as well as the O. volvulus forest form-specific clone isolated by Erttmann et al. [20]. Comparison of the DNA sequences of these three clones may allow oligonucleotide sequences to be identified whose specificités as probes will be less dependent upon the conditions of hybridization.

38The titration experiment described above suggests that the 149-bp repeat unit is present in approximately 4500 copies per haploid genome. This fact, when considered in light of the discovery that three independently isolated clones with differing specificites for Onchocerca DNA appear to contain variations of this 149-bp repeat, suggests that members of the 149-bp repeat family have diverged during the course of the evolution of the genus Onchocerca. This process has resulted in the production of different versions of the repeat unit. Some of the repeats, such as those found in pOvs134, have diverged to the point that they are now specific for O. volvulus, and at least one version appears to be found only in the forest form of O. volvulus. In contrast, some of the repeats, such as those found in pUOV3, are still able to recognize repeats in different species of Onchocerca. This situation is similar to that found with minisatellite sequences in the human genome, where closely related minisatellite DNA probes recognize different repeat sequence families within the genome [35,36]. By analogy to other highly repeated DNA sequences found in other species, it is unlikely that evolution of the 149-bp repeat family will be hindered by functional constraints. It may therefore be possible to use the sequence of examples of the 149-bp repeat to define the evolutionary relationship between closely related species of Onchocerca, as well as the different forms of O. volvulus, that would be difficult to elucidate using more slowly evolving DNA sequences.

Acknowledgements

39We are indebted to the following people for providing material: Dr. R. Baker and Mr. D. Boakye of the OCP, Dr. J. Comley, Dr. M.C. Henry, Dr. E. James, Dr. J. McMahon, Dr. A. Scott, Dr. S. Townson; and to Dr. S. Aldritt, Dr. A. Comeau, Dr. R. Patel, and Dr. J. Shah for invaluable advice and technical assistance. Mr. G. Schoone printed the figures, Dr. G. van Eys, Dr. P. Wright, and Dr. P. Cutting gave helpful advice on the manuscript and Mr. V. van Eysinga edited the document.

40This work was supported by the UNDP/WORLD BANK/WHO Special Programme for Research and Training in Tropical Diseases. D.F.W. is a Burroughs-Wellcome Scholar.

Bibliographie

References

1 World Health Organization (1987) WHO Expert Committee on Onchocerciasis. Technical Report Series No. 752. WHO, Geneva.

2 Anderson, J., Fuglsang, H., Hamilton, P.J.S. and Marshall. T.F. de C. (1974) Studies on onchocerciasis in the United Cameroon Republic. II. Comparison of onchocerciasis in rain forest and Sudan savanna. Trans. R. Soc. Trop. Med. Hyg. 68, 209-222.

3 Prost. A., Rougement, A. and Omar, M.S. (1980) Caractères épidémiologiques, cliniques et biologiques des onchocercoses de savane et de forêt en Afrique Occidentale. Ann. Parasitol. 55, 347-355.

4 McCrae, A.W.R. (1969) Ecology and speciation in African blackflies. Biol. J. Linn. Soc. 1, 43-39.

5 Lewis, D.J. and Duke, B.O.L. (1966) Onchocerca-Simulium complexes, II. Variation in West African female Simulium damnosum. Ann. Trop. Med. Parasitol. 60, 337-346.

6 Crosskey, R.W. (1960) A taxonomic study of the larvae of West African Simuliidae (Diptera:Nematocerca) with comments on the morphology of the larval blackfly head. Bull. Br. Mus. Nat. His. (Ent.) 10, 1-74.

7 Duke, B.O.L., Lewis, D.J. and Moore, P.J. (1966) Onchocerca-Simulium complexes, 1. Transmission of forest and Sudan savanna strains of Onchocerca volvulus from Cameroon by Simulium damnosum of various West African bioclimatic zones. Ann. Trop. Med. Parasitol. 60, 318-335.

8 Duke, B.O.L. (1967) Onchocerca-Simulium complexes, IV. Transmission of a variant of a forest strain of Onchocerca volvulus. Ann. Trop. Med. Parasitol. 61, 326-331.

9 De Leon, J.R. and Duke, B.O.L. (1966) Experimental studies on the transmission of Guatemalan and West African strains of O. volvulus by Simulium ochraceum, S. metallicum and S. callidum. Trans. R. Soc. Trop. Med. Hyg. 60, 735-752.

10 Ciacanchi, R., Karam, M., Henry, M.C., Villani, F., Kumlien, S. and Bullini. L. (1985) Preliminary data on the genetic differentiation of Onchocerca volvulus in Africa (Nematoda: Filarioda). Acta Trop. 42, 341-351.

11 Flockart, H.A., Cibulskis, R.E., Karam. M. and Albiez, E.J. (1986) Onchocerca volvulus: enzyme polymorphism in relation to the differentiation of forest and savannah strains of this parasite. Trans. R. Soc. Trop. Med. Hyg. 80, 285-292.

12 Flavell. R. (1982) Sequence amplification, deletion and rearrangement: major sources of variation during species divergence. In: Genome Evolution (Dover, G.A. and Flavell, R.B., eds.), pp. 301-323. Academic Press, New York.

13 Walsh, J.F., Davies, J.B., Le Berre, R. and Garms, R. (1978) Standardization of criteria for assessing the effect of Simulium control in onchocerciasis control programmes. Trans. R. Soc. Med. Hyg. 72, 675-676.

14 Gale, K.R. and Crampton, J.M. (1987) DNA probes for species identification of mosquitoes in the Anopheles gambiae complex. Med. Vet. Entomol. 1, 127-136.

15 Collins, F.H., Hendes, M.A., Rasmussen, M.O., Mehaffey, P.C., Besansky, N.J. and Finnerty, V. (1987) A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex. Am. J. Trop. Med. Hyg. 37, 37–41.

16 Barker, Jr. R.H., Suebsaeng, H., Rooney, W., Alecrim, G., Dourado, H.V. and Wirth, D.F. (1986) Specific DNA probe for the diagnosis of Plasmodium falciparum malaria. Science 231, 434-436.

17 Sim, B.K.L., Piessens, W.F. and Wirth, D.F. (1986) A DNA probe cloned in E. coli for the identification of Brugia malayi. Mol. Biochem. Parasitol. 19, 117-123.

18 Perler, F.B. and Karam, M. (1986) Cloning of two Onchocerca volvulus repeated DNA sequences. Mol. Biochem. Parasitol. 21, 171-178.

19 Shah, J., Karam, M., Piessens, W.F. and Wirth, D.F. (1987) Characterization of an Onchocerca specific clone from Onchocerca volvulus. Am. J. Trop. Med. Hyg. 37, 376-384.

20 Erttmann, K.D., Unnasch, T.R., Greene, B.M., Albiez, E.J., Boateng, J., Denke, A.M., Ferraroni, J.J., Karam, M., Shulz-Key, H. and Williams, P.N. (1987) A DNA sequence specific for forest form Onchocerca volvulus. Nature 327, 415-417.

21 Shah, J.S., Lamontagne. L., Unnasch. T.R., Wirth, D.F. and Piessens. W.F. (1986) Characterization of a ribosomal DNA clone of B. malayi. Mol. Biochem. Parasitol. 19, 67–75.

22 Schulz-Key, H., Albiez. E.J. and Buttner. D.W. (1977) Isolation of living adult Onchocerca volvulus front nodules. Tropenmed. Parasitol. 28, 428-430.

23 Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory. Cold Spring Harbor, New York.

24 Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517.

25 Unnasch, T.R., Gallin. M.Y., Soboslay, P.T., Erttmann. K.D. and Greene. B.M. (1988) Isolation and characterization of expression cDNA clones encoding antigens of Onchocerca volvulus infective larvae. J. Clin. Invest. 82, 262-269.

26 Sanger. F.; Nicklen. S. and Coulson. A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467.

27 McReynolds. L.A., De Simone. S.M. and Williams, S.A. (1986) Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi. Proc. Natl. Acad. Sci. USA 83, 797-801.

28 Walsh. J. (1986) River blindness: a gamble pays off. Science 231, 922-925.

29 Walsh. J.F., Davies. J.B. and Garms. R. (1981) Further studies on the reinvasion of the onchocerciasis control program by Simulium damnosum s.l.: the effects of an extension of control activities into Southern Ivory Coast during 1979. Tropenmed. Parasitol. 32, 269–273.

30 Ogunrinade, A.F. (1980) Bovine onchocerciasis in Nigeria. Ann. Trop. Med. Parasitol. 74, 367-368.

31 Omar, M.S., Denke, A.M. and Reybould, J.N. (1979) The development of Onchocerca ochengi (Nematoda: Filarioidea) to the infective stage in Simulium damnosum s.l. with a note on the histochemical staining of the parasite. Tropenmed. Parasitol. 30, 157-162.

32 Mwaiko, G.L. (1981) The development of Onchocerca gutturosa Neuman to infective stage in Simulium vorax Pomeroy. Tropenmed. Parasitol. 32, 276-277.

33 Bain, O. (1981) Le Genre Onchocerca; hypothèses sur son évolution et clé dichotomique des éspèces. Ann. Parasitol. 56, 503-526.

34 Gill, L., Hardman, N., Chappell, L., Hu Qu, L., Nicoloso, M. and Bachellerie, J.P. (1988) Phylogeny of Onchocerca volvulus and related species deduced from rRNA sequence comparisons. Mol. Biochem. Parasitol. 28, 9-76.

35 Jeffreys. A.J., Wilson, V. and Thein, S.L. (1985) Hypervariable ‘minisatellite’ regions in human DNA. Nature 314, 67-73.

36 Jeffreys, A.J., Wilson, V., Kelly, R., Taylor, B.A. and Bulfield, G. (1987) Mouse DNA ‘fingerprints’: analysis of chromosome localization and germ-line stability of hypervariable loci in recombinant inbred strains. Nucleic Acids Res. 15, 2823-2836.

Table des illustrations

Légende Fig. 1. Specificity. Approximately 200 ng and 400 ng of genomic DNA from O. volvulus isolates (O.v.). and from other Onchocerca species was spotted onto a nitrocellulose filter which was hybridized to the purified insert of pOvs134.
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Titre TABLE I. Specificity of the three related probes at different stringencies
Légende Each of the three related probes was hybridized to Southern blots containing the DNA samples shown, and the blots were washed at the temperatures indicated in the table.
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Légende Fig. 2. Southern blot analysis of 1.5 μg of genomic DNA from Onchocerca and filarial species digested with RsaI and probed with the purified insert of pOvs134. MW. molecular weight markers (λ HindIII, Ox HaeIII); O.v.F, O. volvulus forest (Ivory Coast); O.v.S, O. volvulus savanna (Mali); O.gu. O. gutturosa: O.o. O. ochengi; O.c. O. cervicalis; O.g. O. gibsoni; B.m. B. malayi; D.i. D. immitis.
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Légende Fig. 3. Southern blot analysis of different isolates of O. volvulus genomic DNA digested with Rsal and probed with purified insert of pOvs134. Lanes 2, 4.6 and 8 had 1.5 μg DNA and lanes 3 and 5 had less parasite DNA due to slight contamination with host DNA. MW, molecular weight markers; O.v.F10 and O.v.F11, O. volvulus forest isolates from Ivory Coast; O.v. Sierra Leone, O. volvulus forest isolate from Sierra Leone; O.V.S3, 12 and 4, O. volvulus savanna isolates from Mali.
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Légende Fig. 4. Sensitivity. Genomic DNA from O. volvulus forest (O.v. Forest), O. volvulus savanna (O.v. Savanna), and O. ochengi were spotted in doubling dilutions from 500 ng to 0.244 ng onto Gene Screen Plus membrane and probed with nicktranslated pOvs134. Filter exposure of 90 min.
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Légende Fig. 5. Southern blot analysis of 1.5 μg of genomic DNA digested with RsaI to completion, (lane 1 and 2) or partially digested (lane 3) and probed with purified insert of pOvs134. MWM, molecular weight markers; O.c, O. cervicalis; O.v.S, O. volvulus savanna; O.v.S p, O. volvulus savanna partial digest.
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Légende Fig. 6. DNA sequence of the inserted DNA of pOvs134. The DNA sequence of the inserted DNA was determined as described in Materials and Methods. The sequence of each of the twelve repeats of pOvs134 described in the text are labeled Ovsl-12. The sequence of the forest form specific clone pFS-1 is labeled pFS-1 [20], and the two repeat units found in the Onchocerca specifie clone pUOV3 [19] are labeled Ov3A and Ov3B. A consensus sequence derived from the fifteen examples of the repeat is labeled CONS. Residues within individual repeats which differ from the consensus are outlined.
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Légende Fig. 7. Copy number. Genomic DNA from O. volvulus savanna and forest. plasmid pOvs134 and pUC9 were spotted onto Gene Screen Plus membrane and probed with the EcoRI- HindIII purified insert of pOvs134.1-100 ng. 2-50 ng. 3-10 ng. 4-5 ng. 6-0.5 ng. 7-0.1 ng.
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Auteurs

Department of Tropical Public Health. 665 Huntington Avenue, Boston MA 02115. U.S.A.

Onchocerciasis Control Programme, Ouagadougou, Burkina Faso

Department of Tropical Public Health, Boston, MA, U.S.A.

Department of Tropical Public Health, Boston, MA, U.S.A.

© IRD Éditions, 2003

Conditions d’utilisation : http://www.openedition.org/6540