EXPERIMENTAL
PARASITOLOGY
73, 33.5-344
Onchocerca vo/vulus: to Identification
(191)
Application of the Polymerase Chain Reaction and Strain Differentiation of the Parasite
STEFANIE E.O. MEREDITH,* GABRIEL LANDO,~ AIAH A. GBAKIMA,~ PETER A. ZIMMERMAN,§"'AND THOMAS R. UNNASCH"7' “Division of Geographic Medicine, Department of Medicine, University of Alabama at Birmingham, University Station, Birmingham, Alabama 35294 U.S.A.; I’lJniversity Centre for Health Sciences, C.U.S.S., University of Yaounde, Yaounde, Cameroon: *Department of Medical Biology, University of Amsterdam, Meibergdreef 39, 1105 AZ, Amsterdam, The Netherlands; #Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106; and ,tNjala University, PMB Freetown, Sierra Leone MEREDITH,
S. E. O., LANDO,
G., GBAKIMA,
A. A., ZIMMERMAN,
P. A., AND UNNASCH,
T. R. 1991. Onchocerca volvulus: Application of the polymerase chain reaction to identification and strain differentiation of the parasite. Experimental Parasitology 73, 335-344. Previous studies have demonstrated that the genome of Onchocerca volvulus contains a variable tandemly repeated DNA sequence family with a unit length of 150 bp. The variability of the 150-bp family has been exploited to develop 0. volvulus strain and species specific DNA probes. Application of these DNA probes to the study of the epidemiologitally most significant life cycle stages of the parasite has been confounded by several obstacles. These include the relative insensitivity of some of the DNA probes and the difftculty in releasing genomic DNA from infective larvae and skin microfilariae in a form that may be directly detected by hybridization to the probes. DNA sequence comparison of 18 known examples of the 150-bp repeat has been used to develop two populations of degenerate ohgonucleotides. These oligonucleotides have been shown to support the amplification of the ISO-bp repeat family from Onchocerca DNA, using the polymerase chain reaction. Both strain and species specific members of the repeat family are faithfully amplified, allowing characterization of a parasite on the basis of hybridization of the PCR amplification products to the previously developed DNA probes. This method is shown to be applicable to all diagnostically important forms of the parasite, including adults, infective larvae, and skin microtilariae. In addition, the method is capable of detecting 0. volvulus infective larvae directly in extracts of blackfly vectors. Q IWI Academic press,rnc. INDEX DESCRIPTORS AND ABBREVIATIONS: Onchocerca volvulus; Nematode; DNA probes; Forest and Savannah strains; Polymerase chain reaction; Microtilariae; Infective larvae; Deoxyribonucleic acid (DNA); Base pairs (bp); Polymerase chain reaction (PCR); Dithiothreitol (DTT); Bovine serum albumin (BSA); Polyvinyl pyrrolidone (PVP); Sodium lauryl sulfate (SDS); Saline sodium citrate (SSC); Ethylene diaminetetraacetic acid (EDTA).
INTRODUCTION
Onchocerciasis, the second leading cause of infectious blindness, is endemic in subsaharan Africa and in Central and South America (Greene 1984). It is caused by the volvufus, filarial parasite Onchocerca which is transmitted to humans through the bite of Simuliid flies. In West Africa, the incidence of blindness due to infection with 0. volvulus is much higher in Savannah r To whom correspondence should be addressed.
than in forest regions (Remme et al. 1989; Dadzie et al. 1989). It is likely that this is partially due to differences in parasite populations in the two bioclimatic zones, since Savannah-derived parasites appear to be more pathogenic than forest-derived parasites in experimental models of 0. volvulusinduced ocular pathology (Duke and Anderson 1972). This fact, together with entomological (Duke et al. 1966) and biochemical (Flockhart et al. 1986) differences between parasites from different geographic regions, has led to the hypothesis 335 0014-4894/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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that different strains of the parasite exist in the forest and Savannah bioclimes of West Africa. The ability to distinguish 0. volvulus from other animal filariae and to further distinguish the forest and Savannah strain of the parasite has important implications for disease control. The Onchocerciasis Control Programme (OCP), a multiyear international program sponsored by the World Bank, World Health Organization, and the United Nations, has succeeded in markedly reducing transmission of 0. volvulus throughout large areas of West Africa (DeSole et al. 1990). Assessment of the effect of control programs relies upon measurement of the annual transmission potential, which in turn requires an accurate count of the number of vector blackflies carrying 0. volvulus infective larvae. Unfortunately, 0. volvulus infective larvae are morphologically indistinguishable from some species of animal filariae, which are also carried by the vector blackflies (Omar et al. 1979). This can lead to distortions in the measurement of the annual transmission potential. In addition, the OCP has concentrated the majority of its efforts in the Savannah regions of West Africa (Le Berre et al. 1990). In many of the areas in which the OCP has been operating, transmission of the parasite has virtually ceased (De Sole et al. 1990; Baker et al. 1990). In such regions, methods to detect new outbreaks of transmission, and to determine if the outbreaks are caused by the forest or Savannah strain of the parasite, are urgently needed. Recently, two DNA probes have been identified which are specific for 0. volvulus (Hamett et al. 1989; Meredith et al. 1989) as well as one for the forest (Erttmann et al. 1987) and one for the Savannah (Erttmann et al. 1990) strain of the parasite. Each of these DNA probes consists of specific members of a variable tandemly repeated DNA sequence family with a unit length of 150-bp. The 150-bp repeat is present in approximately 4000 copies in the haploid ge-
ET AL.
nome of 0. volvulus (Meredith et al. 1989). However, application of these DNA probes to the epidemiologically most significant life cycle stages has proven difficult for two reasons. First, some of the DNA probes are not sensitive enough to detect the genomic DNA present in a single infective larvae (Erttmann et al. 1990). Also, both infective larvae and skin microfilariae have proven to be resistant to simple treatments to free their genomic DNA in a form which may be directly detected by the DNA probes (McReynolds et al. 1991). In this report, a method is described by which strain and species specific DNA sequences may be amplified from 0. volvulus genomic DNA using the polymerase chain reaction (PCR) (Saiki et al. 1988). The amplified sequences may be characterized on the basis of hybridization to the strain and species specific DNA probes. This method may be used to characterize all diagnostically important forms of the parasite, including infective larvae, skin microfilariae, and adults. In addition, the method is capable of detecting 0. volvulus infective larvae directly in vector blackflies carrying the parasite. MATERIALS
AND METHODS
Parasite material. Adult 0. volvulus, obtained by nodulectomy from infected individuals residing throughout West Africa, were freed from surrounding host tissue by collagenase digestion as previously described (Engelbrecht and Schulz-Key 1984). Infective larvae were prepared by experimental infection of laboratory bred flies, as previously described (Ham et al. 1979). Microtilariae were isolated from skin snips, using standard procedures (Kirkwood et a/. 1983). Adult 0. ochengi, 0. dukei, and 0. gibsoni were freed from nodular tissue by collagenase digestion, and 0. gutterosa and 0. armillata adults were removed from host tissue by dissection. Parasites and infected flies were preserved by storage in either liquid nitrogen or propanol. Preparation of extracts for PCR. Genomic DNA was prepared from adult parasites isolated from individual nodules as previously described (Meredith et al. 1989). Isolated skin microtilaria or infective larvae were transferred by a micropipet into a solution of 10 nu%4Tris-HCI (pH 8.0), 1 mM EDTA, 100 &ml pro-
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teinase K, and placed at 56°C for 1 hr. Dithiothreitol GATGACCTATGACATATAA-3’. Membranes (DTT) was added to a final concentration of 20 mM, probed with the oligonucleotide were hybridized at and the samples were heated to 100°Cfor 30 min. The 55°C in a solution containing 5X SSC, 20 mM Na,PG, samples were then subjected to three cycles of freez- (pH 7.0), 0.2% Ficoll 500, 0.2% BSA, 0.2% PVP, 7% ing and thawing, and the parasites collected by cen- SDS, 100 p&ml salmon sperm DNA, and the oligonutrifugation at 14,OOOgfor 5 min. cleotide, labeled as previously described (Sambrook et Whole tiles were homogenized in a solution contain- al. 1989).The blots were washed at 55°C in 3X SSC, 10 ing 10 mM Tris-HCI (pH 7.5), 5 mM MgC12, 0.32 M mM Na,PO, (pH 7.0), 0.2% Ficoll 500, 0.2% BSA, sucrose, and 1% Triton X100. The homogenate was 0.2% PVP, 5% SDS for 1 hr, followed by I hr at 62°C subjected to three cycles of freezing and thawing, and in a solution containing 1X SSC, 1% SDS. DTT was added to a tinal concentration of 10 mM. The homogenate was incubated at 50°C for 1 hr, and proRESULTS teinase K added to a final concentration of 100 t&ml. The sample was then incubated at 56°C for a minimum of 30 min, and up to 3 hr, and was subjected to cenAs a first step in developing a method trifugation at 12,OOOgfor 5 min. The supematant was incorporating PCR in a protocol to identify removed to a new tube, diluted IO-fold, and heated to and characterize 0. volvulus, it was neces100°C for 10 min. sary to develop conditions under which it PCR amplification. PCR amplifications were carried out in a solution containing 10 mM Tris-HC1 (pH 8.3), was possible to reliably amplify the 150-bp 50 mM KCI, 6 mM MgCI,, 0.01% gelatin, 200 pLM sequence family from all isolates of the pardATP, dGTP, dCTP, and TTP, 200 urn of each of the asite. In order to accomplish this, the DNA two amplification primers, 2.5 units of Taq DNA polysequences of the 18 known examples of the merase (U.S. Biochemical, Cleveland, OH U.S.A.), 150-bp repeat family were compared (Harand either 10 ng of purified genomic DNA or the parasite material prepared as described above. The nett et al. 1989; Meredith et al. 1989; Erttmann et al. 1987,199O;Shah et al. 1987). As primers used in the amplification were 5’GATTYTTCCGRCGAAXARCGC-3’ and 5’shown in Fig. 1, it was evident that the 150GCXRTRTAAATXTGXAAATTC-3’ where R = A or bp sequence family consists of relatively G, Y = C or T, and X = A, G, C, or T. Unless otherwise noted, the reactions were subjected to 20 more conserved and less conserved recycles of amplification, with each cycle consisting of 2 gions. The most conserved region was used to generate two degenerate populations of min at 94”C, 2 min at 37°C and 30 set at 72°C. Hybridizafion and blotting. Amplification reactions oligonucleotides, as indicated in Fig. 1A. were separated by agarose gel electrophoresis and Since the 150-bp repeat family is organized transferred to a nylon support membrane (Hybond, in tandem arrays in the genome of 0. volAmersham) as previously described (Erttmann et al. vulus (Meredith et al. 1989), it was ex1990). Membranes probed with the forest specific pected that use of these oligonucleotides as probe pFS-1, or the Savannah specific probe pSSl-BT were hybridized at 42°C in a solution containing 50% primers in a PCR would result in the ampliformamide, 5X SSC, 0.5% SDS, 0.1% Ficoll500,0.1% fication of a ladder of monomers and mulBSA, 0.1% PVP, 0.5% nonfat dry milk, and the approtimers of the 150-bp repeat (Fig. IB). As priate DNA probe, labeled as previously described (Erttmann et al. 1990). Filters probed with pFS-1 were shown in Fig. 2A, monomers and multimers washed in 0.05X SSC and 0.5% SDS at 56°C and those of the 150-bp repeat were amplified from 0. probed with pSS-1BT were washed at 52°C in 0.05X volvulus from both forest and Savannah reSCC and 0.5% SDS. These conditions have previously gions of West Africa. In similar experibeen shown to maximize the sensitivity and specificity ments, the 150-bp repeat family has been of pFS-1 and pSS-IBT, respectively (Erttmann er al. successfully amplified from every one of 1987; Erttmann et al. 1990). Because the 0. volvulus specific probe pOVS134 more than 160 0. volvulus isolates tested contained sequences homologous to the oligonuclefrom both West Africa and South and Cenotides used to prime the PCR (Meredith et al. 1989),an tral America (data not shown). The primers oligonucleotide consisting of sequences located bealso supported the amplification of 150-bp tween the primer sites of pOVS134 was prepared and used as a species specific probe. The sequence of this repeat related sequences from 0. ochengi, 0. gibsoni, and 0. gutterosa (Fig. 2A). oligonucleotide was 5’-AAATTGATTATTAACA-
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ET AL.
FIG. 1. Development of oligonucleotide primers for PCR amplification of the Onchocerca l50-bp repeat family. The DNA sequence of the 18 characterized members of the 150-bp repeat family are aligned in A. The sequences and location of the primers used in the amplification reaction (labeled PCR) are shown above the aligned repeats. The sequence and location of the species specific oligonucleotide described in the text (labeled OVS) is shown below the aligned repeats. Arrows indicate 5’-3’ polarity of the oligonucleotides in relation to the repeat sequences. In A “-” indicates identity to the sequence shown in the first repeat, and “0” indicates a single base pair deletion. B illustrates the monomeric and multimeric products predicted to arise from amplification of the 150-bp tandem repeat family using the primers depicted in A.
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IDENTIFY
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FIG. 2. Hybridization specificity of the strain and species specific DNA probes when tested on PCR amplification products. PCR amplification reactions were prepared as described under Materials and Methods. The products were used to prepare three identical Southern blots. A, Ethidium bromide staining of an agarose gel used to prepare the blots. B, Blot of PCR products hybridized with the species specific oligonucleotide. C, Blot of PCR products hybridized with pFS-1. D, Blot of PCR products hybridized with pSS-IBT. In each panel, Lane 1 is the products of Onchocerca vo/vulus from Liberia; Lane 2, products of 0. volvu/us from Sierra Leone, Lane 3, products of 0. volvulus from Mali; Lane 4, products of 0. vo/vulus from Bas-Zaire; Lane 5, products from 0. gibsoni; Lane 6, products from 0. ochengi; Lane 7, products from 0. gutterosa. The PCR reaction in the lane labeled “H” contained 100 ng human DNA as well as negative PCR.
Because the 150-bp repeat family was amplified from both forest and Savannah 0. volvulus, as well as from other species of Onchocerca, it was necessary to further characterize the amplification products by hybridization to the strain and species specific DNA probes. It was possible that biases and errors arising during the amplitication process could result in a population of amplified repeat sequences that differed qualitatively and quantitatively from the repeat population found in the genomic DNA. This could result in a loss in specificity or sensitivity when the PCR products were hybridized to the strain and species specific DNA probes. To see if this was the case, Southern blots of PCR amplification products from several different strains and species of Onchocerca were hybridized with a species specific oligonucleotide derived from the sequence of pOVS134 (illustrated in Fig. l), as well as with the strain specific probes pFS-1 and pSS- IBT. The species specific oligonucleotide hybridized to PCR products from four isolates of 0. volvulus,
but not to PCR products of 0. gibsoni, 0. and 0. gufterosa (Fig. 2B). The forest specific probe pFS-1 hybridized to isolates from forest regions in Liberia and Sierra Leone, but not to an isolate from the Savannah region of Mali (Fig. 2C). In contrast, the Savannah specific probe pSS-1BT hybridized to the Mali Savannah PCR products, but not to PCR products from the forest isolates (Fig. 2D). Interestingly, PCR products from an isolate from Bas-Zaire, a forested region in which a high level of blinding onchocerciasis exists (WHO Expert Committee 1987) were also recognized specifically by pSS-1BT (Fig. 2). This suggests that the parasite population found in this exceptional region may be more closely related to the Savannah strain than the forest strain. The oligonucleotides shown in Fig. 1 were derived primarily from 0. volvulus specific members of the 150-bp repeat family. Therefore, by increasing the stringency of the amplification reaction, it seemed that it might be possible to limit amplification of
ochengi,
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ET AL.
the repeat family to 0. volvufus. To test this hypothesis, reactions were performed at increasing annealing temperatures from 42 to 65°C. As shown in Table I, the oligonucleotides ceased to prime the amplification of the 0. gutterosa repeat family at an annealing temperature of 50°C. However, the oligonucleotides continued to prime the amplification of the 150-bp repeat families of 0. volvulus, 0. gibsoni, 0. dukei, and 0. ochengi up to an annealing temperature of 63°C. At 65°C the primers no longer supported the amplification of the 150-bp repeat family from any of the DNA samples. The ultimate use of the strain and species specific DNA probes will be to characterize skin microfilariae and infective larvae. It was therefore necessary to devise a simple method which would make the genomic DNA present in these stages available to serve as a suitable template for PCR. Proteinase K treatment of infective larvae or microfilariae under reducing conditions, followed by a heat treatment, was found to be successful in accomplishing this goal. As shown in Fig. 3, treatment of isolated skin microfilariae and infective larvae under these conditions, followed by PCR, resulted in the amplitication of monomers and dimers of the 150-bp repeat. Experiments
bQ 1857s 1060929’
FIG. 3. Amplification of the 150-bp family from 0. vo/vu/us infective larvae and skin microfilariae. PCR reactions were carried out on approximately five skin microfilariae and five infective larvae, as described under Materials and Methods. The products were separated on a 2% agarose gel. A Southern blot prepared from the gel was then hybridized with the 0. volvu/us specific probe. Panel L3, PCR of infective larvae; Panel mf, PCR of skin microtilaria. In each panel, the reactions labeled “ + ” contained parasite material, while the lane labeled “ - ” did not.
were then undertaken to examine if this technique could be adapted to detect infective larvae directly within infected vector blackflies. Homogenization of 0. volvulus experimentally infected flies, followed by treatment of the extracts as described under Materials and Methods, resulted in the amplification of monomers of the 150-bp repeat (Fig. 4). The amplification products
TABLE I Effect of Annealing Temperature on PCR Amplification of the 150-bp Repeat Family Annealing temperature of PCR Species 0. volvulus 0. ochengi 0. gibsoni 0. dukei 0. guttorosa 0. armillata Setaria species B. malayi Simulium damnosum sl
42°C
50°C
55°C
63°C
65°C
+ + + +
+ + + +
+ + + +
+ + + +
-
-
-
-
-
-
Human Note. PCR reactions were performed as described under Materials and Methods with the exception that the annealing temperature was varied as indicated in the table. A “ + ” indicates that amplification of the 150-bp repeat family was obtained from a given genomic DNA sample at a given annealing temperature.
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tion of PCR and currently available DNA probes. This assay may be used to characterize 0. volvulus adults, microtilariae, and infective larvae, as well as S. dumnosum carrying infective larvae. The assay should therefore provide the ability to monitor the effects of control measures targeted at all 0. volvulus life cycle stages throughout west Africa. Incorporation of PCR into a protocol employing the strain and species specific DNA probes of 0. volvulus confers several advantages. First, as shown in Fig. 2, PCR FIG. 4. Amplification of the 1%bp repeat family leads to amplification of both strain and from extracts of Simulium damnosum. Extracts prespecies members of the 1%bp repeat fampared from individual infected and uninfected blackily. Comparison of the relative signals genflies were subjected to PCR as described under Materials and Methods, except that the number of cycles erated by the form specific DNA probes to was increased to 25. The products were analyzed by 10 ng of genomic DNA, and to the PCR agarose gel electrophoresis. Lanes labeled “ - ” con- products generated from an equivalent tained products from extracts of uninfected flies, and amount of genomic DNA, suggests that the lanes labeled ” + ” contained products from extracts of infected flies. The lane labeled “0” contained PCR form specific DNA sequences are amplified at least loo-fold by the procedure described products from a reaction containing no extract. above (data not shown). Although some of produced from the experimentally infected the DNA probes are theoretically sensitive flies hybridized to both the Savannah spe- enough to detect single microtilariae or incific and species specific probes (data not fective larvae (e.g., Harnett et al. 1989; shown). In contrast, no amplification was Meredith et al. 1989) others are not (Erttseen in extracts prepared in the same man- mann et al. 1990). Inclusion of an amplification step in the protocol surmounts this ner from uninfected flies (Fig. 4). Initial experiments using this technique difficulty. Second, reliable methods to diproduced variable yields of the amplifica- rectly identify and characterize skin microtion products from infected flies. In some filariae or infective larvae have not been cases, the amplification products were not reported. In contrast, as shown in Fig. 3, visible in the ethidium bromide stained gel, inclusion of a PCR amplification step rebut were only detectable following hybrid- sults in easily detectable signals from either ization to the species specific probe. Fur- microfilaria or infective larvae. A third advantage of using PCR as part of a method to ther investigation revealed that unidentified substances present in the blackfly inhibited characterize 0. volvulus infective larvae or PCR amplification. Dilution of the homoge- microfilaria results from the fact that prodnates at least lo-fold prior to PCR ampliti- ucts of a single PCR are easily subdivided. cation was found to overcome this inhibi- Therefore simultaneous analysis of a single tion, resulting in consistent amplification of sample can be performed with a number of different probes. For example, it will be the 150-bp repeat from infected blackflies. possible to determine if an individual blackDISCUSSION fly is infected with 0. volvulus, and to deThe results presented above describe a termine the strain of the parasite, while practical assay to both identify and charac- avoiding the difficulties inherent in sequenterize 0. volvulus, based upon a combina- tially probing a single filter.
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In the experiments described above, the amplification of the 150-bp repeat was primed using oligonucleotides derived from the most conserved region of the repeat sequence. This region was chosen to ensure that the 150-bp repeat family was amplified from all isolates of 0. volvulus. However, these primers are also capable of directing the amplification of the 150-bp repeat sequence from other species of Onchocerca. Since it was not possible to confine the amplification reaction to 0. voZvulus by increasing the stringency of the reaction, it was necessary to further characterize the PCR products on the basis of their ability to hybridize to the species specific DNA probe. It may be possible to devise oligonucleotides which will limit the amplification of the 150-bp repeat only to 0. volvulus. Design of such oligonucleotides will require a greater understanding of the sequence diversity of 150-bp repeat in animal Onchocerca, particularly 0. gibsoni, 0. ochengi, and 0. dukei. The data presented above demonstrate that it is possible to obtain amplification of the 150-bp repeat family from isolated skin microfilariae, isolated infective larvae, or 0. volvulus infected blackflies. Use of isolated parasites is compatible with the monitoring protocols currently being used by the Onchocerciasis Control Programme. These involve either dissection of wild caught blackflies or incubation of skin snips from putatively infected individuals, followed by examination of the culture media for liberated microtilariae. These protocols have the advantage that the parasite number can be quantitated in infected blackflies or humans. However, in regions with control programs in place, the percentage of blackflies infected with the parasite is likely to be low. This is especially true in regions where ivermectin is used as a control measure (Taylor et al. 1990), and in regions where active vector control has been terminated (Le Berre et al. 1990). In this case, it may be more practical to rely upon mass
ET AL.
screening programs, rather than on the dissection of individual flies. The results presented above demonstrate that dissection is not necessary in order to obtain amplification of the 150-bp repeat family from 0. volvulus infected flies. It may therefore be possible to modify the assay to amplify the 150-bp repeat family from homogenates containing both infected and uninfected flies. The amplification products could then be further characterized on the basis of their ability to hybridize to the strain and species specific probes. Such an assay will enable large numbers of flies to be rapidly examined in areas where the percentage of infected flies is low. One difficulty that may arise in adapting this technique to a mass screening protocol is the presence of substances inhibitory to the PCR reactions in blackfly homogenates. In the experiments shown in Fig. 4, extracts were prepared from individual flies, and the extracts were diluted significantly prior to PCR amplitication. Although this method results in amplification of the 150-bp repeat, it may not be practical when extracts prepared from large numbers of flies are to be assayed. Recently, a simple method has been described to isolate genomic parasite DNA from extracts of Wuchereria bancrofti infected mosquitoes, followed by PCR amplification (Dissanayake et al. 1991). This purification step eliminates the inhibitors of PCR found in mosquito extracts. It may be possible to adapt this method to detection of 0. volvulus infective larvae in extracts prepared from large numbers of flies. As mentioned in the introduction, the forest and Savannah strains of the parasite may be distinguished on the basis of several different criteria. However, the division of forest and Savannah strains is not absolute. For example, high prevalences of blinding onchocerciasis exist in heavily forested regions in Zaire and Sierra Leone (WHO Expert Committee, 1987). The existence of such exceptional isolates has lead to the suggestion that 0. volvulus might be more
PCR TO IDENTIFY
properly classified into blinding and nonblinding forms (Brown et al. 1990). In light of this controversy, it is interesting to note that the DNA probe results described above suggest that the exceptional isolate from Bas-Zaire is most like parasites found in Savannah regions, where the prevalence of blindness is also high. Use of the strain specific probes to characterize other such exceptional isolates, together with an examination of parasites from individuals with clinically well-characterized forms of the disease, may be useful in furthering our understanding of the differences between the strains of 0. volvulus. ACKNOWLEDGMENTS We thank Drs. G. Wahl, 0. Bain, M. C. Henry, and B. M. Greene for providing material used in this study; Drs. N. Lang-Unnasch and D. 0. Freedman for critical reading of the manuscript; and Sherry Wozniak for help in the preparation of the manuscript. This investigation received support from the UNDPWORLD Bank/WHO Special Programme for research and training in Tropical Diseases Project I.D. 890307 and the Science and Technology Program of the Commission of the European Communities Grant Number ISSN208.
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L., POOLE, C. B., AND WILLIAMS, S. A. 1991. Filarial DNA Probes in Jakarta. Parasitology Today, in press. MEREDITH, S. E. O., UNNASCH, T. R., KARAM, M., PIESSENS, W. F., AND WIRTH, D. F. 1989. Cloning and characterization of an Onchocerca volvulus specific DNA sequence. Molecular and Biochemical Parasitology 36, I-10. OMAR, M. S., DENKE, A. M., AND RAYBOULD, J. N. 1979. The development of Onchocerca ochengi (Nematoda: Filarioidea) to the infective stage in Simulium damnosum s.1. with a note on the histochemical staining of the parasite. Tropenmedizin Und Parasitologie 30, 157-162. MCREYNOLDS,
REMME, J., DADZIE, K. Y., ROLLAND, A., AND THYLEFORS, B. 1989. Onchocerciasis and intensity
of infection in the community. I. West African savanna. Tropical Medicine and Parasitology 40, 340347. SAIKI,
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S. J., HIGUCHI, R. G., HORN, G. T., K. B., AND ERLICH, H. A. 1988. Primer-
directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487491. SAMBROOK,
J.,
FRITSCH,
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MANIATIS,
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1989. Molecular Cloning: A Laboratory Manual. Second ed., pp. 11.31-11.33. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. SHAH,
J. S.,
KARAM,
M.,
PIESSENS,
WIRTH, D. F. 1987. Characterization
W.
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chocerca-specific DNA clone from Onchocerca vu/us. American Journal of Tropical Medicine Hygiene 31, 376384.
voland
TAYLOR, H. R., PACQU~, M., MUNOZ, B., AND GREENE, B. M. 1990. Impact of mass treatment of
onchocerciasis with ivermectin on the transmission of infection. Science 250, 116-l 18. WHO EXPERT COMMITTEE ON ONCHOCERCIASIS. Third Report 1987. WHO Technical Report Series 752, pp. U-16. World Health Organization, Geneva. Received 25 February 1991; accepted with revision 10 June 1991
PARASITOLOGY
73, 33.5-344
Onchocerca vo/vulus: to Identification
(191)
Application of the Polymerase Chain Reaction and Strain Differentiation of the Parasite
STEFANIE E.O. MEREDITH,* GABRIEL LANDO,~ AIAH A. GBAKIMA,~ PETER A. ZIMMERMAN,§"'AND THOMAS R. UNNASCH"7' “Division of Geographic Medicine, Department of Medicine, University of Alabama at Birmingham, University Station, Birmingham, Alabama 35294 U.S.A.; I’lJniversity Centre for Health Sciences, C.U.S.S., University of Yaounde, Yaounde, Cameroon: *Department of Medical Biology, University of Amsterdam, Meibergdreef 39, 1105 AZ, Amsterdam, The Netherlands; #Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106; and ,tNjala University, PMB Freetown, Sierra Leone MEREDITH,
S. E. O., LANDO,
G., GBAKIMA,
A. A., ZIMMERMAN,
P. A., AND UNNASCH,
T. R. 1991. Onchocerca volvulus: Application of the polymerase chain reaction to identification and strain differentiation of the parasite. Experimental Parasitology 73, 335-344. Previous studies have demonstrated that the genome of Onchocerca volvulus contains a variable tandemly repeated DNA sequence family with a unit length of 150 bp. The variability of the 150-bp family has been exploited to develop 0. volvulus strain and species specific DNA probes. Application of these DNA probes to the study of the epidemiologitally most significant life cycle stages of the parasite has been confounded by several obstacles. These include the relative insensitivity of some of the DNA probes and the difftculty in releasing genomic DNA from infective larvae and skin microfilariae in a form that may be directly detected by hybridization to the probes. DNA sequence comparison of 18 known examples of the 150-bp repeat has been used to develop two populations of degenerate ohgonucleotides. These oligonucleotides have been shown to support the amplification of the ISO-bp repeat family from Onchocerca DNA, using the polymerase chain reaction. Both strain and species specific members of the repeat family are faithfully amplified, allowing characterization of a parasite on the basis of hybridization of the PCR amplification products to the previously developed DNA probes. This method is shown to be applicable to all diagnostically important forms of the parasite, including adults, infective larvae, and skin microtilariae. In addition, the method is capable of detecting 0. volvulus infective larvae directly in extracts of blackfly vectors. Q IWI Academic press,rnc. INDEX DESCRIPTORS AND ABBREVIATIONS: Onchocerca volvulus; Nematode; DNA probes; Forest and Savannah strains; Polymerase chain reaction; Microtilariae; Infective larvae; Deoxyribonucleic acid (DNA); Base pairs (bp); Polymerase chain reaction (PCR); Dithiothreitol (DTT); Bovine serum albumin (BSA); Polyvinyl pyrrolidone (PVP); Sodium lauryl sulfate (SDS); Saline sodium citrate (SSC); Ethylene diaminetetraacetic acid (EDTA).
INTRODUCTION
Onchocerciasis, the second leading cause of infectious blindness, is endemic in subsaharan Africa and in Central and South America (Greene 1984). It is caused by the volvufus, filarial parasite Onchocerca which is transmitted to humans through the bite of Simuliid flies. In West Africa, the incidence of blindness due to infection with 0. volvulus is much higher in Savannah r To whom correspondence should be addressed.
than in forest regions (Remme et al. 1989; Dadzie et al. 1989). It is likely that this is partially due to differences in parasite populations in the two bioclimatic zones, since Savannah-derived parasites appear to be more pathogenic than forest-derived parasites in experimental models of 0. volvulusinduced ocular pathology (Duke and Anderson 1972). This fact, together with entomological (Duke et al. 1966) and biochemical (Flockhart et al. 1986) differences between parasites from different geographic regions, has led to the hypothesis 335 0014-4894/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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that different strains of the parasite exist in the forest and Savannah bioclimes of West Africa. The ability to distinguish 0. volvulus from other animal filariae and to further distinguish the forest and Savannah strain of the parasite has important implications for disease control. The Onchocerciasis Control Programme (OCP), a multiyear international program sponsored by the World Bank, World Health Organization, and the United Nations, has succeeded in markedly reducing transmission of 0. volvulus throughout large areas of West Africa (DeSole et al. 1990). Assessment of the effect of control programs relies upon measurement of the annual transmission potential, which in turn requires an accurate count of the number of vector blackflies carrying 0. volvulus infective larvae. Unfortunately, 0. volvulus infective larvae are morphologically indistinguishable from some species of animal filariae, which are also carried by the vector blackflies (Omar et al. 1979). This can lead to distortions in the measurement of the annual transmission potential. In addition, the OCP has concentrated the majority of its efforts in the Savannah regions of West Africa (Le Berre et al. 1990). In many of the areas in which the OCP has been operating, transmission of the parasite has virtually ceased (De Sole et al. 1990; Baker et al. 1990). In such regions, methods to detect new outbreaks of transmission, and to determine if the outbreaks are caused by the forest or Savannah strain of the parasite, are urgently needed. Recently, two DNA probes have been identified which are specific for 0. volvulus (Hamett et al. 1989; Meredith et al. 1989) as well as one for the forest (Erttmann et al. 1987) and one for the Savannah (Erttmann et al. 1990) strain of the parasite. Each of these DNA probes consists of specific members of a variable tandemly repeated DNA sequence family with a unit length of 150-bp. The 150-bp repeat is present in approximately 4000 copies in the haploid ge-
ET AL.
nome of 0. volvulus (Meredith et al. 1989). However, application of these DNA probes to the epidemiologically most significant life cycle stages has proven difficult for two reasons. First, some of the DNA probes are not sensitive enough to detect the genomic DNA present in a single infective larvae (Erttmann et al. 1990). Also, both infective larvae and skin microfilariae have proven to be resistant to simple treatments to free their genomic DNA in a form which may be directly detected by the DNA probes (McReynolds et al. 1991). In this report, a method is described by which strain and species specific DNA sequences may be amplified from 0. volvulus genomic DNA using the polymerase chain reaction (PCR) (Saiki et al. 1988). The amplified sequences may be characterized on the basis of hybridization to the strain and species specific DNA probes. This method may be used to characterize all diagnostically important forms of the parasite, including infective larvae, skin microfilariae, and adults. In addition, the method is capable of detecting 0. volvulus infective larvae directly in vector blackflies carrying the parasite. MATERIALS
AND METHODS
Parasite material. Adult 0. volvulus, obtained by nodulectomy from infected individuals residing throughout West Africa, were freed from surrounding host tissue by collagenase digestion as previously described (Engelbrecht and Schulz-Key 1984). Infective larvae were prepared by experimental infection of laboratory bred flies, as previously described (Ham et al. 1979). Microtilariae were isolated from skin snips, using standard procedures (Kirkwood et a/. 1983). Adult 0. ochengi, 0. dukei, and 0. gibsoni were freed from nodular tissue by collagenase digestion, and 0. gutterosa and 0. armillata adults were removed from host tissue by dissection. Parasites and infected flies were preserved by storage in either liquid nitrogen or propanol. Preparation of extracts for PCR. Genomic DNA was prepared from adult parasites isolated from individual nodules as previously described (Meredith et al. 1989). Isolated skin microtilaria or infective larvae were transferred by a micropipet into a solution of 10 nu%4Tris-HCI (pH 8.0), 1 mM EDTA, 100 &ml pro-
PCR TO IDENTIFY
0.
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teinase K, and placed at 56°C for 1 hr. Dithiothreitol GATGACCTATGACATATAA-3’. Membranes (DTT) was added to a final concentration of 20 mM, probed with the oligonucleotide were hybridized at and the samples were heated to 100°Cfor 30 min. The 55°C in a solution containing 5X SSC, 20 mM Na,PG, samples were then subjected to three cycles of freez- (pH 7.0), 0.2% Ficoll 500, 0.2% BSA, 0.2% PVP, 7% ing and thawing, and the parasites collected by cen- SDS, 100 p&ml salmon sperm DNA, and the oligonutrifugation at 14,OOOgfor 5 min. cleotide, labeled as previously described (Sambrook et Whole tiles were homogenized in a solution contain- al. 1989).The blots were washed at 55°C in 3X SSC, 10 ing 10 mM Tris-HCI (pH 7.5), 5 mM MgC12, 0.32 M mM Na,PO, (pH 7.0), 0.2% Ficoll 500, 0.2% BSA, sucrose, and 1% Triton X100. The homogenate was 0.2% PVP, 5% SDS for 1 hr, followed by I hr at 62°C subjected to three cycles of freezing and thawing, and in a solution containing 1X SSC, 1% SDS. DTT was added to a tinal concentration of 10 mM. The homogenate was incubated at 50°C for 1 hr, and proRESULTS teinase K added to a final concentration of 100 t&ml. The sample was then incubated at 56°C for a minimum of 30 min, and up to 3 hr, and was subjected to cenAs a first step in developing a method trifugation at 12,OOOgfor 5 min. The supematant was incorporating PCR in a protocol to identify removed to a new tube, diluted IO-fold, and heated to and characterize 0. volvulus, it was neces100°C for 10 min. sary to develop conditions under which it PCR amplification. PCR amplifications were carried out in a solution containing 10 mM Tris-HC1 (pH 8.3), was possible to reliably amplify the 150-bp 50 mM KCI, 6 mM MgCI,, 0.01% gelatin, 200 pLM sequence family from all isolates of the pardATP, dGTP, dCTP, and TTP, 200 urn of each of the asite. In order to accomplish this, the DNA two amplification primers, 2.5 units of Taq DNA polysequences of the 18 known examples of the merase (U.S. Biochemical, Cleveland, OH U.S.A.), 150-bp repeat family were compared (Harand either 10 ng of purified genomic DNA or the parasite material prepared as described above. The nett et al. 1989; Meredith et al. 1989; Erttmann et al. 1987,199O;Shah et al. 1987). As primers used in the amplification were 5’GATTYTTCCGRCGAAXARCGC-3’ and 5’shown in Fig. 1, it was evident that the 150GCXRTRTAAATXTGXAAATTC-3’ where R = A or bp sequence family consists of relatively G, Y = C or T, and X = A, G, C, or T. Unless otherwise noted, the reactions were subjected to 20 more conserved and less conserved recycles of amplification, with each cycle consisting of 2 gions. The most conserved region was used to generate two degenerate populations of min at 94”C, 2 min at 37°C and 30 set at 72°C. Hybridizafion and blotting. Amplification reactions oligonucleotides, as indicated in Fig. 1A. were separated by agarose gel electrophoresis and Since the 150-bp repeat family is organized transferred to a nylon support membrane (Hybond, in tandem arrays in the genome of 0. volAmersham) as previously described (Erttmann et al. vulus (Meredith et al. 1989), it was ex1990). Membranes probed with the forest specific pected that use of these oligonucleotides as probe pFS-1, or the Savannah specific probe pSSl-BT were hybridized at 42°C in a solution containing 50% primers in a PCR would result in the ampliformamide, 5X SSC, 0.5% SDS, 0.1% Ficoll500,0.1% fication of a ladder of monomers and mulBSA, 0.1% PVP, 0.5% nonfat dry milk, and the approtimers of the 150-bp repeat (Fig. IB). As priate DNA probe, labeled as previously described (Erttmann et al. 1990). Filters probed with pFS-1 were shown in Fig. 2A, monomers and multimers washed in 0.05X SSC and 0.5% SDS at 56°C and those of the 150-bp repeat were amplified from 0. probed with pSS-1BT were washed at 52°C in 0.05X volvulus from both forest and Savannah reSCC and 0.5% SDS. These conditions have previously gions of West Africa. In similar experibeen shown to maximize the sensitivity and specificity ments, the 150-bp repeat family has been of pFS-1 and pSS-IBT, respectively (Erttmann er al. successfully amplified from every one of 1987; Erttmann et al. 1990). Because the 0. volvulus specific probe pOVS134 more than 160 0. volvulus isolates tested contained sequences homologous to the oligonuclefrom both West Africa and South and Cenotides used to prime the PCR (Meredith et al. 1989),an tral America (data not shown). The primers oligonucleotide consisting of sequences located bealso supported the amplification of 150-bp tween the primer sites of pOVS134 was prepared and used as a species specific probe. The sequence of this repeat related sequences from 0. ochengi, 0. gibsoni, and 0. gutterosa (Fig. 2A). oligonucleotide was 5’-AAATTGATTATTAACA-
338
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ET AL.
FIG. 1. Development of oligonucleotide primers for PCR amplification of the Onchocerca l50-bp repeat family. The DNA sequence of the 18 characterized members of the 150-bp repeat family are aligned in A. The sequences and location of the primers used in the amplification reaction (labeled PCR) are shown above the aligned repeats. The sequence and location of the species specific oligonucleotide described in the text (labeled OVS) is shown below the aligned repeats. Arrows indicate 5’-3’ polarity of the oligonucleotides in relation to the repeat sequences. In A “-” indicates identity to the sequence shown in the first repeat, and “0” indicates a single base pair deletion. B illustrates the monomeric and multimeric products predicted to arise from amplification of the 150-bp tandem repeat family using the primers depicted in A.
PCR TO
IDENTIFY
0.
VOhUhS
339
FIG. 2. Hybridization specificity of the strain and species specific DNA probes when tested on PCR amplification products. PCR amplification reactions were prepared as described under Materials and Methods. The products were used to prepare three identical Southern blots. A, Ethidium bromide staining of an agarose gel used to prepare the blots. B, Blot of PCR products hybridized with the species specific oligonucleotide. C, Blot of PCR products hybridized with pFS-1. D, Blot of PCR products hybridized with pSS-IBT. In each panel, Lane 1 is the products of Onchocerca vo/vulus from Liberia; Lane 2, products of 0. volvu/us from Sierra Leone, Lane 3, products of 0. volvulus from Mali; Lane 4, products of 0. vo/vulus from Bas-Zaire; Lane 5, products from 0. gibsoni; Lane 6, products from 0. ochengi; Lane 7, products from 0. gutterosa. The PCR reaction in the lane labeled “H” contained 100 ng human DNA as well as negative PCR.
Because the 150-bp repeat family was amplified from both forest and Savannah 0. volvulus, as well as from other species of Onchocerca, it was necessary to further characterize the amplification products by hybridization to the strain and species specific DNA probes. It was possible that biases and errors arising during the amplitication process could result in a population of amplified repeat sequences that differed qualitatively and quantitatively from the repeat population found in the genomic DNA. This could result in a loss in specificity or sensitivity when the PCR products were hybridized to the strain and species specific DNA probes. To see if this was the case, Southern blots of PCR amplification products from several different strains and species of Onchocerca were hybridized with a species specific oligonucleotide derived from the sequence of pOVS134 (illustrated in Fig. l), as well as with the strain specific probes pFS-1 and pSS- IBT. The species specific oligonucleotide hybridized to PCR products from four isolates of 0. volvulus,
but not to PCR products of 0. gibsoni, 0. and 0. gufterosa (Fig. 2B). The forest specific probe pFS-1 hybridized to isolates from forest regions in Liberia and Sierra Leone, but not to an isolate from the Savannah region of Mali (Fig. 2C). In contrast, the Savannah specific probe pSS-1BT hybridized to the Mali Savannah PCR products, but not to PCR products from the forest isolates (Fig. 2D). Interestingly, PCR products from an isolate from Bas-Zaire, a forested region in which a high level of blinding onchocerciasis exists (WHO Expert Committee 1987) were also recognized specifically by pSS-1BT (Fig. 2). This suggests that the parasite population found in this exceptional region may be more closely related to the Savannah strain than the forest strain. The oligonucleotides shown in Fig. 1 were derived primarily from 0. volvulus specific members of the 150-bp repeat family. Therefore, by increasing the stringency of the amplification reaction, it seemed that it might be possible to limit amplification of
ochengi,
340
MEREDITH
ET AL.
the repeat family to 0. volvufus. To test this hypothesis, reactions were performed at increasing annealing temperatures from 42 to 65°C. As shown in Table I, the oligonucleotides ceased to prime the amplification of the 0. gutterosa repeat family at an annealing temperature of 50°C. However, the oligonucleotides continued to prime the amplification of the 150-bp repeat families of 0. volvulus, 0. gibsoni, 0. dukei, and 0. ochengi up to an annealing temperature of 63°C. At 65°C the primers no longer supported the amplification of the 150-bp repeat family from any of the DNA samples. The ultimate use of the strain and species specific DNA probes will be to characterize skin microfilariae and infective larvae. It was therefore necessary to devise a simple method which would make the genomic DNA present in these stages available to serve as a suitable template for PCR. Proteinase K treatment of infective larvae or microfilariae under reducing conditions, followed by a heat treatment, was found to be successful in accomplishing this goal. As shown in Fig. 3, treatment of isolated skin microfilariae and infective larvae under these conditions, followed by PCR, resulted in the amplitication of monomers and dimers of the 150-bp repeat. Experiments
bQ 1857s 1060929’
FIG. 3. Amplification of the 150-bp family from 0. vo/vu/us infective larvae and skin microfilariae. PCR reactions were carried out on approximately five skin microfilariae and five infective larvae, as described under Materials and Methods. The products were separated on a 2% agarose gel. A Southern blot prepared from the gel was then hybridized with the 0. volvu/us specific probe. Panel L3, PCR of infective larvae; Panel mf, PCR of skin microtilaria. In each panel, the reactions labeled “ + ” contained parasite material, while the lane labeled “ - ” did not.
were then undertaken to examine if this technique could be adapted to detect infective larvae directly within infected vector blackflies. Homogenization of 0. volvulus experimentally infected flies, followed by treatment of the extracts as described under Materials and Methods, resulted in the amplification of monomers of the 150-bp repeat (Fig. 4). The amplification products
TABLE I Effect of Annealing Temperature on PCR Amplification of the 150-bp Repeat Family Annealing temperature of PCR Species 0. volvulus 0. ochengi 0. gibsoni 0. dukei 0. guttorosa 0. armillata Setaria species B. malayi Simulium damnosum sl
42°C
50°C
55°C
63°C
65°C
+ + + +
+ + + +
+ + + +
+ + + +
-
-
-
-
-
-
Human Note. PCR reactions were performed as described under Materials and Methods with the exception that the annealing temperature was varied as indicated in the table. A “ + ” indicates that amplification of the 150-bp repeat family was obtained from a given genomic DNA sample at a given annealing temperature.
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0.
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tion of PCR and currently available DNA probes. This assay may be used to characterize 0. volvulus adults, microtilariae, and infective larvae, as well as S. dumnosum carrying infective larvae. The assay should therefore provide the ability to monitor the effects of control measures targeted at all 0. volvulus life cycle stages throughout west Africa. Incorporation of PCR into a protocol employing the strain and species specific DNA probes of 0. volvulus confers several advantages. First, as shown in Fig. 2, PCR FIG. 4. Amplification of the 1%bp repeat family leads to amplification of both strain and from extracts of Simulium damnosum. Extracts prespecies members of the 1%bp repeat fampared from individual infected and uninfected blackily. Comparison of the relative signals genflies were subjected to PCR as described under Materials and Methods, except that the number of cycles erated by the form specific DNA probes to was increased to 25. The products were analyzed by 10 ng of genomic DNA, and to the PCR agarose gel electrophoresis. Lanes labeled “ - ” con- products generated from an equivalent tained products from extracts of uninfected flies, and amount of genomic DNA, suggests that the lanes labeled ” + ” contained products from extracts of infected flies. The lane labeled “0” contained PCR form specific DNA sequences are amplified at least loo-fold by the procedure described products from a reaction containing no extract. above (data not shown). Although some of produced from the experimentally infected the DNA probes are theoretically sensitive flies hybridized to both the Savannah spe- enough to detect single microtilariae or incific and species specific probes (data not fective larvae (e.g., Harnett et al. 1989; shown). In contrast, no amplification was Meredith et al. 1989) others are not (Erttseen in extracts prepared in the same man- mann et al. 1990). Inclusion of an amplification step in the protocol surmounts this ner from uninfected flies (Fig. 4). Initial experiments using this technique difficulty. Second, reliable methods to diproduced variable yields of the amplifica- rectly identify and characterize skin microtion products from infected flies. In some filariae or infective larvae have not been cases, the amplification products were not reported. In contrast, as shown in Fig. 3, visible in the ethidium bromide stained gel, inclusion of a PCR amplification step rebut were only detectable following hybrid- sults in easily detectable signals from either ization to the species specific probe. Fur- microfilaria or infective larvae. A third advantage of using PCR as part of a method to ther investigation revealed that unidentified substances present in the blackfly inhibited characterize 0. volvulus infective larvae or PCR amplification. Dilution of the homoge- microfilaria results from the fact that prodnates at least lo-fold prior to PCR ampliti- ucts of a single PCR are easily subdivided. cation was found to overcome this inhibi- Therefore simultaneous analysis of a single tion, resulting in consistent amplification of sample can be performed with a number of different probes. For example, it will be the 150-bp repeat from infected blackflies. possible to determine if an individual blackDISCUSSION fly is infected with 0. volvulus, and to deThe results presented above describe a termine the strain of the parasite, while practical assay to both identify and charac- avoiding the difficulties inherent in sequenterize 0. volvulus, based upon a combina- tially probing a single filter.
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In the experiments described above, the amplification of the 150-bp repeat was primed using oligonucleotides derived from the most conserved region of the repeat sequence. This region was chosen to ensure that the 150-bp repeat family was amplified from all isolates of 0. volvulus. However, these primers are also capable of directing the amplification of the 150-bp repeat sequence from other species of Onchocerca. Since it was not possible to confine the amplification reaction to 0. voZvulus by increasing the stringency of the reaction, it was necessary to further characterize the PCR products on the basis of their ability to hybridize to the species specific DNA probe. It may be possible to devise oligonucleotides which will limit the amplification of the 150-bp repeat only to 0. volvulus. Design of such oligonucleotides will require a greater understanding of the sequence diversity of 150-bp repeat in animal Onchocerca, particularly 0. gibsoni, 0. ochengi, and 0. dukei. The data presented above demonstrate that it is possible to obtain amplification of the 150-bp repeat family from isolated skin microfilariae, isolated infective larvae, or 0. volvulus infected blackflies. Use of isolated parasites is compatible with the monitoring protocols currently being used by the Onchocerciasis Control Programme. These involve either dissection of wild caught blackflies or incubation of skin snips from putatively infected individuals, followed by examination of the culture media for liberated microtilariae. These protocols have the advantage that the parasite number can be quantitated in infected blackflies or humans. However, in regions with control programs in place, the percentage of blackflies infected with the parasite is likely to be low. This is especially true in regions where ivermectin is used as a control measure (Taylor et al. 1990), and in regions where active vector control has been terminated (Le Berre et al. 1990). In this case, it may be more practical to rely upon mass
ET AL.
screening programs, rather than on the dissection of individual flies. The results presented above demonstrate that dissection is not necessary in order to obtain amplification of the 150-bp repeat family from 0. volvulus infected flies. It may therefore be possible to modify the assay to amplify the 150-bp repeat family from homogenates containing both infected and uninfected flies. The amplification products could then be further characterized on the basis of their ability to hybridize to the strain and species specific probes. Such an assay will enable large numbers of flies to be rapidly examined in areas where the percentage of infected flies is low. One difficulty that may arise in adapting this technique to a mass screening protocol is the presence of substances inhibitory to the PCR reactions in blackfly homogenates. In the experiments shown in Fig. 4, extracts were prepared from individual flies, and the extracts were diluted significantly prior to PCR amplitication. Although this method results in amplification of the 150-bp repeat, it may not be practical when extracts prepared from large numbers of flies are to be assayed. Recently, a simple method has been described to isolate genomic parasite DNA from extracts of Wuchereria bancrofti infected mosquitoes, followed by PCR amplification (Dissanayake et al. 1991). This purification step eliminates the inhibitors of PCR found in mosquito extracts. It may be possible to adapt this method to detection of 0. volvulus infective larvae in extracts prepared from large numbers of flies. As mentioned in the introduction, the forest and Savannah strains of the parasite may be distinguished on the basis of several different criteria. However, the division of forest and Savannah strains is not absolute. For example, high prevalences of blinding onchocerciasis exist in heavily forested regions in Zaire and Sierra Leone (WHO Expert Committee, 1987). The existence of such exceptional isolates has lead to the suggestion that 0. volvulus might be more
PCR TO IDENTIFY
properly classified into blinding and nonblinding forms (Brown et al. 1990). In light of this controversy, it is interesting to note that the DNA probe results described above suggest that the exceptional isolate from Bas-Zaire is most like parasites found in Savannah regions, where the prevalence of blindness is also high. Use of the strain specific probes to characterize other such exceptional isolates, together with an examination of parasites from individuals with clinically well-characterized forms of the disease, may be useful in furthering our understanding of the differences between the strains of 0. volvulus. ACKNOWLEDGMENTS We thank Drs. G. Wahl, 0. Bain, M. C. Henry, and B. M. Greene for providing material used in this study; Drs. N. Lang-Unnasch and D. 0. Freedman for critical reading of the manuscript; and Sherry Wozniak for help in the preparation of the manuscript. This investigation received support from the UNDPWORLD Bank/WHO Special Programme for research and training in Tropical Diseases Project I.D. 890307 and the Science and Technology Program of the Commission of the European Communities Grant Number ISSN208.
REFERENCES BAKER, R. H. A., GUILLET, P., S~KI?TI?LI, A., POUDIOUGO, P., BOAKYE, D., WILSON, M. D., AND BISSAN, Y. 1990. Progress in controlling the reinvasion of windborne vectors into the western area of the Onchocerciasis control Programme in West Africa. Philosophical Transactions of the Royal Society of London B328, 73 l-750. BROWN, K. R., DUKE, B. 0. L., GINGER, C. D., Gutteridge, W. E., LE BERRE, R., OVERBOSCH, D., REMME, J., SAMBA, E. M., VAN DER KAAY, H. J., AND WALSH, J. F. 1990. Conclusions and recommendations of the symposium on Onchocerciasis (O-Now!). Acta Leidensia 59, 443456. DADZIE, K. Y., REMME, J., ROLLAND, A., AND THYLEFORS, B. 1989. Ocular onchocerciasis and intensity of infection in the community. II. West African rainforest foci of the vector Simulium yahense. Tropical Medicine and Parasitology 40, 348-354. DESOLE, G., REMME, J., AND DADZIE, K. Y. 1990. Epidemiological impact of vector control. I. Incidence and changes in prevalence and intensity of Onchocerca volvulus infection. Acta Leidensia 59, 119-126.
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