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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

Design of Onchocerca DNA probes based upon analysis of a repeated sequence family

Peter A. Zimmerman, Laurent Toe et Thomas R. Unnasch

Résumé

Repeated DNA sequences have been instrumental in the development of DNA probes for many different parasites. Isolation of such DNA probes has generally been accomplished by differential screening of genomic libraries with total genomic DNA preparations. In the current work, a rational design strategy is presented for the development of oligonucleotide probes based upon repeated sequence families. A repeated sequence family present in the genome of Onchocerca parasites, designated O-150, has been amplified from various samples of genomic DNA using PCR. DNA sequence analysis of the resulting PCR products demonstrated that the sequences may be arranged into clusters within which the individual sequences are identical or nearly identical. Differences among the cluster consensus sequences have been exploited to explain the specificities of previously isolated O-150 based probes and to develop two new oligonucleotide probes. One of these probes hybridizes specifically to Onchocerca volvulus O-150 PCR products, while the second hybridizes specifically to O-150 PCR products from the closely related bovine parasite O. ochengi. These oligonucleotide probes have been used to characterize Onchocerca infective larvae isolated from wild caught infected flies in West Africa. Because repeated sequence families are a common feature of most genomes, including those of parasites, this method should be applicable to the rational design of oligonucleotide probes for other parasitic infections.

Note de l’éditeur

Correspondence address: Thomas Unnasch, Division of Geographie Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Tel.: (205) 934-1630; Fax: (205) 933-5671.

Texte intégral

1Abbreviations: SSC, saline sodium citrate; SDS, sodium lauryl sulfate; PCR, polymerase chain reaction; ATP, annual transmission potential; TE, Tris/EDTA buffer; OVS2 Onchocerca volvulus specific oligonucleotide probe; OCH Onchocerca ochengi specific oligonucleotide probe.

2Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank™ data base with the accession numbers L04875 through L04898.

3(Received 6 July 1992; accepted 3 December 1992)

Introduction

4Onchocerca volvulus, the filarial parasite which is the causative agent of river blindness, is transmitted by blackflies of the Simulium damnosum species complex [1]. Although O. volvulus is an obligate human parasite, most of the other members of the genus Onchocerca are parasites of ungulates [2]. In West Africa, several species of Onchocerca exist which are parasites of the endemic ungulate species. One of these animal parasites, O. ochengi, has particular relevance to ongoing efforts to control onchocerciasis. The Onchocerciasis Control Programme (OCP), an international effort to eliminate onchocerciasis as a public health problem, monitors the effectiveness of its efforts by measurement of the annual transmission potential (ATP). Calculation of the ATP involves estimation of the number of parous flies found to be carrying O. volvulus infective larvae in a given area. However, O. ochengi is transmitted by the same members of the S. damnosum complex that transmit O. volvulus [3], and the infective stages of the two species are difficult or impossible to distinguish morphologically [3,4]. The presence of O. ochengi can thus adversely affect calculation of the ATP in areas where both O. volvulus and O. ochengi are endemic. The ability to characterize individual infective larvae using DNA probes that hybridize specifically to O. volvulus has the potential of overcoming this problem. However, the practical use of O. volvulus specific probes would be enhanced by the development of a DNA probe specific for O. ochengi, as it would allow unambiguous positive identification of infective larvae of both species.

5In the past several years, DNA probes have been isolated which demonstrate varying degrees of specificity for Onchocerca parasites [5-9]. Some of these probes are specific for the genus Onchocerca, while others are specific for O. volvulus, or for distinct strains of O. volvulus. All of these probes contain specific members of a tandemly repeated DNA sequence family with a unit length of 150 bp found in the Onchocerca genome. This family has been designated O-150. DNA sequence analysis of the O-150 family demonstrated that individual examples of O-150 tended to group into distinct clusters. Within each of these clusters, the individual repeats were found to be identical, or nearly identical (ref. 10 and P. Zimmerman, Ph.D thesis). This finding is consistent with the hypothesis that such repeated sequences are subject to mechanisms of concerted evolution [11].

6The fact that variation within such repeated sequence families is constrained suggests the possibility of a rational approach to the design of additional DNA probes with defined specificities. To accomplish this, the repeat population in question could be amplified using the polymerase chain reaction (PCR) and the composition of the amplified population analyzed by DNA sequence analysis. Comparison of DNA sequence data from different parasite populations might then be used to design oligonucleotides that were specific to a given parasite population. As a test of this method, sequences of the O-150 family from the forest and savanna strains of O. volvulus and from O. ochengi have been compared. The sequence analysis provides an explanation of the observed specificities of previously identified O. volvulus species and strain specific DNA probes. The sequence data have been utilized to design an oligonucleotide probe which hybridizes specifically to O. ochengi PCR products, as well as an oligonucleotide that recognizes PCR products from both forest and savanna strains of O. volvulus, but not from O. ochengi. Since repeated noncoding DNA sequences appear to be a common feature of eucaryotic genomes, this method should be applicable to the rational design of DNA probes for the diagnosis of other parasitic infections.

Materials and Methods

7Parasite materials. Onchocercomata were surgically removed from infected humans (O. volvulus) and cattle (O. ochengi) and preserved in isopropanol. Adult parasites were surgically removed from the surrounding host tissue and DNA purified as previously described [12]. Individual infective larvae were isolated from infected Simulium damnosum s.l. and treated to release their DNA as previously described [13]. Herring sperm DNA (1 µg) was added to these preparations as a carrier and the parasite DNA further purified by adsorption to a glass slurry, following the manufacturer's protocol (Bio 101, La Jolla, CA). The DNA was eluted from the glass slurry into 100 µl of TE.

8Polymerase chain reaction, cloning of PCR amplification products and DNA sequence analysis. The repeated sequence family O-150 was amplified from 10 ng samples of purified adult stage genomic DNA, or from 25 µl of the purified larval DNA preparations, as previously described [13]. The primers utilized in the PCR were derived from the most conserved region of the O-150 repeat, as judged by a comparison of the sequence of the 17 known examples of the repeat determined prior to the experiments described in this manuscript [13]. The primers were synthesized with sufficient degeneracy so that all known examples of the O-150 repeat were represented in the primer population. Furthermore, the primers were synthesized to contain synthetic HindIII sites, to facilitate subsequent sequence analysis. Analysis of the previously characterized members of the O-150 family [5-9] demonstrated that no known member contained a HindIII site, nor were there any sequences found within the known examples of the O-150 family which could be easily mutated to form a HindIII site. Finally, genomic Southern blot analysis suggested that HindIII sites were rare or absent in the O-150 array [6]. The sequence of the primers used in the PCR were 5ʹ CCCAAGCTTGATTYTTCCGRCGAAXARCGC 3ʹ and 5ʹ CCCAAGCTTGCXRTRTAAATXTGXAAATTC 3ʹ, where R = A or G, Y = C or T and X = A,G,C or T. PCR amplification of the O-150 repeat family was carried out as previously described [13]. The amplification products were treated with HindIII, and multimers of the 150-bp monomeric unit were separated by electrophoresis on a 10% polyacrylamide gel. Monomers from each sample were electroeluted from polyacrylamide gel slices using the Elutrap System (Schleicher and Schuell, Keene, NH). The purified monomers were cloned into the single stranded bacteriophage vector M13mp19 and the DNA sequence of individual monomers determined using standard procedures [14]. Clustering of sequences was done visually following transformation of the sequence data into geometric symbols, as previously described [10] and by maximum parsimony methods [15]. Both methods produced essentially identical clustering.

9Oligonucleotide hybridization. PCR products, prepared as described above, were separated on a 2% agarose gel and transferred to a nylon membrane (Hybond N, Amersham, Arlington Heights, IL). The transferred DNA was immobilized by UV crosslinking (Stratalinker, Stratagene, La Jolla Ca.) and the blot prehybridized in a solution containing 5 x SSC/ 20 mM sodium phosphate, pH 7.0/ 10 x Denhardt’s (0.2% Ficoll 400/ 0.2% polyvinylpyrrolidone/ 0,2% bovine serum albumin)/ 7% SDS/ 100 µg ml-1 herring sperm DNA.

10Oligonucleotides were labeled using either [32P-γ]ATP and polynucleotide kinase, following standard protocols [16], or with digoxigenin using the Genius 3 labeling kit (Boehringer Mannheim, Indianapolis, IN), following the manufacturer’s protocol. Control experiments indicated that the two labeling methods were equivalent in specificity and sensitivity. Labeled oligonucleotides were added to the blot in the prehybridization buffer described above supplemented with dextran sulfate to a final concentration of 10% and hybridization allowed to continue overnight. Blots were washed once for 30 min in a solution containing 3 x SSC, 10 mM sodium phosphate (pH 7.0), 10 x Denhardt’s solution and 5% SDS, followed by a second 30 minute wash in 1 x SSC and 1%SDS. For blots probed with OVS2, hybridization was carried out at 42°C and washing was carried out at 50°C. For blots probed with OCH hybridization and washing were carried out at 53°C. Signals on blots hybridized with radioactively labeled probes were visualized by autoradiography. Positive signals on blots hybridized with the digoxigenin labeled probes were developed according to the manufacturer’s instructions.

Results

11As a first step in the rational development of new oligonucleotide probes, it was necessary to determine the DNA sequence of a large number of PCR amplification products derived from the O-150 family. To obtain a PCR product population that was as representative of the O-150 family as possible, the primer populations utilized in the PCR were degenerate and the annealing temperature used was low enough to allow extension from imperfectly matched primers. For this reason, sequence data collected from the regions from which the primers were derived could not be considered reliable. Thus, analysis of the sequence data were restricted to the 106 nucleotides of the O-150 sequence located between the primer sites.

12One potential difficulty in this approach was the possibility that PCR induced mutations in the product population might complicate the interpretation of the data. To assess the frequency that PCR induced errors occurred in this System, PCR products derived from the plasmid pOVS134 were analyzed. pOVS134 is a clone derived from O. volvulus genomic DNA that contains 12 tandemly linked monomers of the O-150 family [8]. PCR amplifications were carried out with pOVS134 as an experimental template, using conditions identical to those utilized to amplify the O-150 family directly from O. volvulus genomic DNA. The amplification products produced from pOVS134 were then cloned into M13mp19, as described in Materials and Methods. Twenty clones were selected at random and their DNA sequence determined. When the sequence of these clones were compared to that of pOVS134, only 1 transition mutation was found to have occurred. Thus, the PCR induced mutation rate per site in this System could be estimated at approximately 4 x 10-4. This is approximately two orders of magnitude less than variation seen between individual repeat units (see Fig. 1). It was therefore concluded that PCR generated artifacts would not significantly affect the process of DNA sequence analysis for the purpose of oligonucleotide probe development.

Fig. 1. Consensus sequences for the O-150 clusters present in O. volvulus and O. ochengi. The sequence data shown represent the 106 nucleotides of the O-150 family located between the PCR primer sites. Clustering of individual O-150 monomers was accomplished as described in Materials and Methods. Consensus sequences were determined by choosing the modal nucleotide at each position. L1-L6, cluster consensus sequences for the clusters found in O. volvulus from Liberia (rainforest strain), M1-M3, consensus sequences for the clusters identified in Mali O. volvulus (savanna strain), Z1-Z4, cluster consensus sequences identified in O. volvulus from Zaire (savanna strain), G1-G3, cluster consensus sequences from Guatemalan O. volvulus, B1-B5, cluster consensus sequences from Brazilian O. volvulus and O1-O3, cluster consensus sequences found in O. ochengi. Freq., the relative proportion that individual sequences belonging to a given cluster are represented in the O-150 population of a given isolate as a whole. OVS2, the sequence of the O. volvulus specific oligonucleotide OVS2 and OCH, the sequence of the O. ochengi specific oligonucleotide OCH. Cluster consensus sequences identical to OVS2 are indicated by shading; the cluster consensus sequence identical to OCH is indicated by open symbols. Rectangles;, A, ■, G, ●, C and ♦, T.

13Examination of the raw DNA sequence data derived from the O-150 family suggested that variability within the family was constrained (ref. 10, and P. Zimmerman, Ph.D thesis). Thus, the individual monomers present in each isolate could be arranged into 3-6 clusters. Within these clusters, the sequence of individual monomers were identical or nearly identical. Consensus sequences for the clusters found in 5 different geographic isolates of O. volvulus and O. ochengi are shown in Fig. 1. The consensus sequences were derived from a total of 145 individual examples of O-150. Examination of this data revealed that differences exist between O. volvulus and O. ochengi and between the forest and savanna strains of O. volvulus. For example, the L1 through L3 clusters are found in the Liberian rainforest isolate of O. volvulus, but not in any other O. volvulus isolate, or in O. ochengi. Comparison of this sequence to the previously identified DNA probes demonstrated that the O. volvulus forest strain specific DNA probe pFS-1 [6] contained a repeat which was most homologous to the L3 cluster and was closely related to the L1 and L2 clusters. The specificity of pFS-1 is thus likely to be due to its homology with repeats that are members of these forest specific clusters, which together comprise 66% of the O-150 family present in the Liberian rainforest isolate. In a similar fashion, the O. volvulus specific probe pOVS134 [8] contained sequences similar to the L6, M2, M3, Z4, G3 and B3-5 clusters, but did not contain sequences similar to the clusters found in O. ochengi.

14The sequence data presented in Fig. 1 thus provided an explanation for the specificities of pFS-1 and pOVS134, 2 DNA probes isolated by conventional screening methods. Unfortunately, it is not possible to utilize pOVS134 to characterize PCR products of O-150, since it contains sequences homologous to the primers used in the PCR. However, the data presented above provides information necessary to develop new oligonucleotide probes that could be used to classify O-150 derived PCR products. For example, the data presented in Fig. 1 identified a sequence family common to all 6 isolates of O. volvulus. This is represented by the L4, Ml, Z1, G1 and B1 clusters shown in Fig. 1. The consensus sequences of these clusters were identical, with the exception of the B1 cluster consensus sequence, which differed from the others by a single G to A transition at position 2. Furthermore, this cluster was not found in either O. ochengi (Fig. 1) or Onchocerca gibsoni (data not shown). In a similar manner, the O1 family of O. ochengi contained an insertion of 11 bp located between bases 53 and 54 (Fig. 1). This insertion was not found in any of the O. volvulus sequences. Based upon this analysis, 2 different oligonucleotides were constructed, as indicated in Fig. 1. The oligonucleotide OVS2, consisting of nucleotides spanning positions 42-64 of the O. volvulus conserved cluster, was predicted to hybridize to all isolates of O. volvulus, but not to O. ochengi. Similarly, the oligonucleotide designated OCH, which was derived from the sequence of nucleotides 47-64 in the 01 cluster and which contained the 11bp insertion specific to this cluster, was predicted to be specific for O. ochengi.

15To test the specificity of these probes, the oligonucleotides were labeled as described in Materials and Methods and hybridized to PCR products from 3 standard isolates of the forest strain of O. volvulus, 3 standard isolates of the savanna strain of O. volvulus and 4 O. ochengi isolates. The oligonucleotide OVS2 was found to hybridize specifically to all of the standard O. volvulus isolates, but to none of the O. ochengi isolates (Fig. 2B, lanes labeled C, VF and VS). In contrast, the oligonucleotide OCH hybridized to the O. ochengi PCR products, but not to the PCR products derived from the O. volvulus standard isolates (Fig. 2C, lanes labeled C, VF and VS).

16In a recent study testing the ability of O. volvulus strain specific DNA probes to predict the pathogenic potential of different parasite populations, it was found that a small proportion of the samples tested hybridized to neither the forest strain specific probe pFS-1, nor to the savanna strain specific probe pSS-1BT [12]. This suggested that these isolates contained a repeat population distinct from that found in the parasites which were used to develop the previously identified probes. To test the ability of the rationally designed oligonucleotide OVS2 to detect these samples, PCR products from 6 of the double negative isolates were hybridized with OVS2. PCR products from all of these isolates hybridized to OVS2 (Fig. 2B, lanes labeled VU). In contrast, none of the isolates were detected by the O. ochengi oligonucleotide OCH (Fig. 2C, lanes labeled VU). Similar experiments have demonstrated that all of the O. volvulus isolates previously found to be negative with both strain specific probes were recognized by OVS2 (data not shown).

Fig. 2. Test of the specificity of the rationally designed oligonucleotides OVS2 and OCH. PCR products, prepared as described in Materials and Methods, were separated on a 2% agarose gel and used to prepare a Southern blot. (A) Ethidium bromide staining pattern of the gel; (B) blot hybridized with OVS2 and (C) blot hybridized with OCH. In each panel, lanes labeled C, PCR products from O. ochengi, VF, PCR products from O. volvulus rainforest strain isolates, VS, O. volvulus PCR products from savanna strain isolates and VU, O. volvulus PCR products from isolates that did not hybridize to either strain specific DNA probe. Isolates were classified based on the 2 step classification procedure previously described [12]. Control reactions without added parasite DNA resulted in no detectable product, either by ethidium bromide staining, or by hybridization to either probe (data not shown).

17As an initial test of the practical utility of these newly designed oligonucleotide probes, the OVS2 and OCH oligonucleotides were used to characterize infective larvae dissected from Simulium damnosum s.l. captured in the Banafing IV River basin in Mali and in the Bandama Blanc basin in northern Côte d’Ivoire. These waterways had been under intensive vector control for over a decade, resulting in the interruption of O. volvulus transmission. In 1990, vector control was terminated in this region of the control area and, as expected, S. damnosum reappeared at breeding sites (1992 Report of the Joint Program Committee of the Onchocerciasis Control Programme in West Africa, Document JPC13.2, World Health Organization). Subsequent surveillance of fly populations along the Banifing IV and Bandama Blanc identified several parous flies carrying Onchocerca infective larvae, resulting in a calculated annual transmission potential far in excess of what was predicted by the current prevalence of O. volvulus infection in these areas. Because of this fact, larvaciding was resumed at these foci (1992 Report of the Joint Program Committee of the Onchocerciasis Control Programme in West Africa, Document JPC13.2, World Health Organization). However, the disparity in the epidemiological assessment and the calculated ATP presented the possibility that the infected flies were in fact carrying larvae of O. ochengi. To test this hypothesis, infective larvae isolated from wild caught flies collected from the Banifing IV and Bandama Blanc basins were subjected to analysis using the DNA probes. To obtain larvae for this study, a total of 500 flies were dissected. Of these 500 flies, 350 were found to be parous. In the parous group, 13 infected flies were found, which together contained a total of 23 larvae. From the 13 infected flies collected, amplification of the O-150 repeat family was successfully obtained from 10 samples. The 10 PCR positive samples were then hybridized with the OVS2 and OCH oligonucleotides. None of the 10 samples tested hybridized to the O. volvulus specific oligonucleotide probe OVS2 (Fig. 3A). In contrast, 7 of these samples hybridized to OCH, (Fig. 3B, lanes 2,3,5,7,8,10 and 11) while 3 samples hybridized weakly or not at all to the O. ochengi specific oligonucleotide (Fig. 3B, lanes 4,6 and 9). The results of this study thus suggested that none of the larvae collected from this focus were O. volvulus and that the majority were O. ochengi. As a resuit of these findings, larviciding has been suspended in these waterways (1992 Report of the Joint Program Committee of the Onchocerciasis Control Programme in West Africa, Document JPC13.2, World Health Organization).

Fig. 3. Hybridization of Infective Larvae from individual wild caught Simulium damnosum s.l. from the Banifing IV and Bandama Blanc river basins. Preparation of larvae, amplification by the PCR and hybridization to the species specific oligonucleotides were as described in Materials and Methods. (A) Blot hybridized with OVS2 and (B) Blot hybridized with OCH. In each panel, lane 1, PCR positive control (10 ng pOVSl34 as template DNA), lanes 2-11, PCR products from infective larvae dissected from individual infected flies from the Banifing IV and Bandama Blanc river basins, lane 12, O. ochengi standard isolate, lane 13, O. volvulus rainforest strain standard isolate and lane 14, O. volvulus savanna strain standard isolate. Control reactions without added parasite DNA gave no detectable product (data not shown).

Discussion

18Highly repeated DNA sequence families are a common feature of eucaryotic genomes and such sequence families appear to evolve at a more rapid rate than do single copy, noncoding sequences [17]. Such sequences have been utilized to develop DNA probes specific for a variety of parasitic organisms [18-22]. In general, these probes have been identified using conventional techniques, such as differential screening of libraries with labeled total genomic DNA. The results presented above demonstrate that it is possible to rationally design oligonucleotide probes with a desired specificity, based upon such repeated sequence families. This process involves amplification of a given repeated sequence family using PCR, followed by examination of the composition of the PCR amplified product population by DNA sequence analysis. Differences found between the PCR product populations arising from different parasite strains or species may then be exploited in the design of oligonucleotides with defined specificities.

19For this method of probe design to be effective, the PCR products sampled by DNA sequencing must yield an accurate picture of the composition of the PCR product population as a whole. If variation within a repeat population was unconstrained, the population of amplified products would have a very high complexity. If this were the case, it would be necessary to determine the DNA sequence of a very large number of PCR products in order to obtain an accurate picture of the population of PCR amplified products. Fortunately, it is likely that the variation within such sequences is in fact under some constraint. For example, in the case of the O-150 family of Onchocerca, defined clusters of sequences are found to exist. Between 3 and 6 such clusters exist in each isolate of Onchocerca examined to date. Within these clusters, the individual sequences are identical, or nearly identical. Such clustering appears to be a common feature of repeated sequence families, having been noted in the Alu and minisatellite repeat families of humans [23] as well as in the C repeats of rabbits [24] and in the CR1 repeated sequence families in birds [25]. It is thought that this clustering arises through the action of mechanisms of concerted evolution, which act to homogenize the individual members of the sequence family within a given genome [11]. Because of this constraint, it is possible to gain an accurate picture of the composition of a given repeat population by analyzing a relatively limited number of repeat units. This information can then be used to design oligonucleotides with the required specificities.

20It should be noted that the DNA sequence data used in the rational design process will usually be based upon a limited number of parasite isolates. This is due to the fact that performing such a detailed analysis on a large number of parasite isolates would be technically prohibitive. In this sense, a rational design strategy is similar to the approach of differentially screening genomic libraries with labeled total genomic DNA. In the latter case, the initial analysis is limited by the number of parasite isolates used to prepare the genomic DNA used to construct and screen the libraries. In both cases, it is therefore necessary to test additional parasite isolates not used in the initial development of the probes, in order to obtain an accurate estimate of their sensitivity and specificity.

21The O. volvulus specific oligonucleotide developed in the present study provides a useful addition to the previously identified species specific probes [7,8]. For example, the plasmid probe pOVS134 contains 12 complete examples of the O-150 repeat. Therefore it contains sequences homologous to those used to prime the PCR amplification of the O-150 family. Thus, pOVS134 cannot be used to classify parasite isolates following PCR amplification of the O-150 family, a procedure that is necessary in our hands in order to reliably classify individual L3 by DNA probe analysis [13]. However, probably the more important of the oligonucleotides developed in the current study is OCH, which appears to be specific for O. ochengi. Since O. volvulus and O. ochengi are co-endemic in the control area of the OCP, are transmitted by the same species of S. damnosum s.l. and are often morphologically indistinguishable, O. ochengi presents a challenge to the accurate estimation of the ATP for O. volvulus. This problem is likely to become more acute as active vector control is phased out within the OCP control area. In such areas, it is expected that the blackfly population will rapidly increase and the presence of O. ochengi in cattle found in these areas will lead to a concomitant increase in the transmission of O. ochengi. The results presented above demonstrate that the species speeific oligonucleotides, when used in conjunction with a general scheme to amplify the O-150 family by PCR, are capable of classifying individual infective larvae dissected from S. damnosum s.l.. The ability to determine if a given focus of infected flies is carrying O. volvulus or O. ochengi will thus have value in efficiently allocating scarce resources during the final years of the OCP’s operation, as well as in the years following the end of large scale vector control efforts in the OCP area.

Acknowledgements

22The authors would like to thank Dr. Stefanie Meredith and the members of the OCP Vector Control and Epidemiology units for providing parasite material and Dr. E.M. Samba, director of the OCP for supporting this research. We also thank the UAB oligonucleotide synthesis core facility for synthesizing the oligonucleotides used in this study. This work was supported by the Special Programme for Research and Training in Tropical Diseases of the World Health Organization, project number 890307.

Bibliographie

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25 Chen, Z.Q., Ritzel, R.G., Lin, C.C. and Hodgetts, R.B. (1991) Sequence conservation in avian CR 1: an interspersed repetitive DNA family evolving under functional constraints. Proc. Natl. Acad. Sci. USA 88, 5814-5818.

Table des illustrations

Légende Fig. 1. Consensus sequences for the O-150 clusters present in O. volvulus and O. ochengi. The sequence data shown represent the 106 nucleotides of the O-150 family located between the PCR primer sites. Clustering of individual O-150 monomers was accomplished as described in Materials and Methods. Consensus sequences were determined by choosing the modal nucleotide at each position. L1-L6, cluster consensus sequences for the clusters found in O. volvulus from Liberia (rainforest strain), M1-M3, consensus sequences for the clusters identified in Mali O. volvulus (savanna strain), Z1-Z4, cluster consensus sequences identified in O. volvulus from Zaire (savanna strain), G1-G3, cluster consensus sequences from Guatemalan O. volvulus, B1-B5, cluster consensus sequences from Brazilian O. volvulus and O1-O3, cluster consensus sequences found in O. ochengi. Freq., the relative proportion that individual sequences belonging to a given cluster are represented in the O-150 population of a given isolate as a whole. OVS2, the sequence of the O. volvulus specific oligonucleotide OVS2 and OCH, the sequence of the O. ochengi specific oligonucleotide OCH. Cluster consensus sequences identical to OVS2 are indicated by shading; the cluster consensus sequence identical to OCH is indicated by open symbols. Rectangles;, A, ■, G, ●, C and ♦, T.
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Légende Fig. 2. Test of the specificity of the rationally designed oligonucleotides OVS2 and OCH. PCR products, prepared as described in Materials and Methods, were separated on a 2% agarose gel and used to prepare a Southern blot. (A) Ethidium bromide staining pattern of the gel; (B) blot hybridized with OVS2 and (C) blot hybridized with OCH. In each panel, lanes labeled C, PCR products from O. ochengi, VF, PCR products from O. volvulus rainforest strain isolates, VS, O. volvulus PCR products from savanna strain isolates and VU, O. volvulus PCR products from isolates that did not hybridize to either strain specific DNA probe. Isolates were classified based on the 2 step classification procedure previously described [12]. Control reactions without added parasite DNA resulted in no detectable product, either by ethidium bromide staining, or by hybridization to either probe (data not shown).
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Légende Fig. 3. Hybridization of Infective Larvae from individual wild caught Simulium damnosum s.l. from the Banifing IV and Bandama Blanc river basins. Preparation of larvae, amplification by the PCR and hybridization to the species specific oligonucleotides were as described in Materials and Methods. (A) Blot hybridized with OVS2 and (B) Blot hybridized with OCH. In each panel, lane 1, PCR positive control (10 ng pOVSl34 as template DNA), lanes 2-11, PCR products from infective larvae dissected from individual infected flies from the Banifing IV and Bandama Blanc river basins, lane 12, O. ochengi standard isolate, lane 13, O. volvulus rainforest strain standard isolate and lane 14, O. volvulus savanna strain standard isolate. Control reactions without added parasite DNA gave no detectable product (data not shown).
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Auteurs

Division of Geographic Medicine, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
Department of Biology, Case Western Reserve University, Cleveland, OH, USA
Laboratory of Parasitic Diseases, National Institutes of Health, Bethesda, MD, USA.

Onchocerciasis Control Programme in West Africa, Bouake, Ivory Coast

© IRD Éditions, 2003

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