Bellec, Christian

Cuny, Gérard

Herder, Stéphane

Meredith, S.E.O.

Horizon / Pleins textes

Genomic fingerprinting of Onchocerca species 
using random amplified polymorphic DNA 
S. Herder1, C. BellecI, S. E. O. Meredith2, G. Cuny3 
Laboratoire d'Epidémiologie des Maladies à Vecteurs, Centre ORSTOM de Montpellier, Montpellier, France; 
Royal Tropical Institute (KIT), Amsterdam, The Netherlands; 
Laboratoire Rétrovirus-Parasites, Centre ORSTOM de Montpellier, Montpellier, France 
Abstract 
A method based on amplification of 
genomic DNA by decamer primers of random nucleo- 
tide sequence was used to obtain DNA fmgerprints 
from different species of the genus Onchocerca. Each of 
the 20 primers tested allowed a clear distinction be- 
tween the different species of the genus on an agarose 
gel. The technique offers the potential to construct spe- 
cies or strain specific probes and oligonucleotides PCR 
primers from the species specific fragments. A combi- 
nation of these primers or others could be useful as 
population markers. 
Introduction 
There are marked geographical variations 
in the spectrum of disease associated with onchocerciasis. 
Clinical manifestations ranging from dermal lesions to 
ocular manifestations including blindness can occur. In 
West Africa, the incidence of blindness is much higher in 
savannah areas than in forest areas (Anderson et al., 
1974; Prost et al., 1980). In forest areas, despite high 
prevalence rates and large numbers of microfilariae in the 
skin, the disease is generally less severe than in savanna 
areas. An exception is the pattern of disease in Sierra- 
Leone, where blinding onchocerciasis occurs in forest re- 
gions (McMahon et al., 1986). Most onchocerciasis vectors 
are known to be zoophilic, and can thus be infected with 
animal Onchocerca species whose third stage larvae (L3) 
are morphologically indistinguishable from each other 
and from O. volvulus. These non-volvulus Onchocerca 
larvae affect the accurate measurement of the transmis- 
sion of human onchocerciasis and thus, there is a real 
need for a method which can distinguish between the 
Onchocerca species as well as between strains of O. vol- 
vulus. This is especially important in areas where on- 
chocerciasis control programmes are developed and 
where mass distribution of ivermectin is planned. 
Accepted 18 April 1994 
Trop. Med. Parasitol.v45 (1r694) 199-202 
O Georg Thieme Verlag Stuttgart . New York 
q1mR 6.p-3?-2~4'2! h b 3 %  
Immunological and biochemical studies on 
O. volvulus have done little to clarify the possible exist- 
ence of distinct strains, although allozyme studies showed 
evidence of genetic variation (Cianchi et al., 1985; Flock- 
hart et al., 1986). However, recently, some DNA probes 
specific for O. volvulus have been described which belong 
to a tandemly repeated DNA sequence family (Meredith et 
al., 1989; Harnett et al., 1989). A polymerase chain reac- 
tion (PCR), based on the 150 bp repetitive DNA, has now 
been developed for the detection of all stages of O. vol- 
vulus (Meredith et al., 1991). The variability of the 150 bp 
family has been exploited to develop species and strain 
specific DNA probes and has shown that at least two 
genetic forms of O. volvulus do exist in West Africa (Ertt- 
mann et al., 1987; Erttmann et al., 1990). The high level 
of homology within this repeat family means that the 
species specificity of these probes is largely dependant on 
the hybridization and washing stringencies (Zimmermann 
et al.; 1992). 
A technique generating genomic fmger- 
prints by Random Amplified Polymorphic DNA (RAPD) 
based on the amplifcation by the Polymerase Chain Re- 
action (PCR) of random DNA fragments has recently been 
developed. In RAPD mapping, decamer oligonucleotide 
primers of arbitrary sequence but with a GC content of 50 % 
or higher are used to amplie fragments of genomic DNA 
(Williams et al., 1990; Welsh and McClelland, 1990). 
RAPD has been successfully applied to the analysis of 
genomic DNA variation of several organisms (Hadrys et 
al., 1992; Klein-Lankhorst et al., 1991; Crowhurst et al., 
1991; Mazurier et al., 1992). Species and strains can be 
distinguished by comparing polymorphisms in genomic 
fingerprints (Welsh et al., 1991). The particular advan- 
tages of RAPD technology is that small amounts (nano- 
grams) of genomic DNA are needed and, unlike PCR, no 
prior knowledge of the genome' subjected to analysis is 
required. 
We describe here the adaptation of the 
RAPD assay to the genus Onchocerca, using a set of 20 
oligonucleotide decamer primers. By comparing the h- 
gerprints generated by the RAPD method, O. volvulus, O. 
gutturosa, O. lienalis, O. gibsonì and O. ocheizgì can be 
identified and distinguished from each other by the 
specific polymorphic patterns. 
4 
O. K.S.T.O.U. Fonds Documentaire 
f l " !  4A52-8 
... , .  
200 Trop. Med. Parasitol. 45 (1 994) S. Herder, C. Bellec, S. E. O. Meredith, G. Cuny - 
M l 2 3 4 5 6 7 8 9  
Fig. 1 Single primer PCR on genomic DNA from different species. 
Primerr OPB-4 was used to amplify genomic DNA from human (lane 2), 
Dirofi/aria immitis (lane 3), O. gutturosa (lane 4), O. lienalis (lane 5), 
O. gibsoni (lane 6), O. ochengi (lane 7) and1 two different O. volvulus 
isolates from Cameroon (lane 8) and from Sierra-Leone (lane 9). 
M: Molecular Weight Marker, 0X-174 Hae 111 digest. Lane 1: negative 
control. 
Fig. 2 Single primer PCR on O. volvulus genomic DNA from differ- 
ent origins. Genomic DNA from O. volvulus (two different worms 
from the same nodule) coming from Cameroon (4 foci: Touboro, 
Poli, Bafia and Sa'a) and from Sierra-Leone (1 focus) were amplified 
with primer OPB-4. 
M: Molecular Weight Marker, 0X-174 Hae 111 digest. 
Lane 1: negative control 
Lane 2: worm 1 from Touboro 
Lane 3: worm 2 from Touboro 
Lane 4: worm 1 from Poli 
Lane 5: worm 2 from Poli 
Lane 6: worm 1 from Bafia 
Lane 7: worm 2 from Bafia 
Lane 8: worm 1 from Sa'a 
Lane 9: worm 2 from Sa'a 
Lane IO:  worm 1 from Sierra Leone 
Lane 11: worm 2 from Sierra Leone 
Materials and methods 
Nodules containing 0. voEvulzis were excised 
and either conserved directly in absolute alcohol or collagenase- 
digested (Schultz-Key et al., 1977) and the worms preserved in 
absolute alcohol. The nodules were from savannah areas 
(Touboro and Poli, Cameroon), degraded forest areas (ßafia and 
Sa'a, Cameroon) and the isolated worms from forest regions of 
West Africa (Lunsar and Njala, Sierra-Leone). Dirofilaria immitis, 
Table 1 Nucleotide sequence of primers OPB-O1 to OPB-20 
(Kit 8, Operon technologies Inc.). 
Primer Nucleotide sequence 
OPB-O1 
OPB-O3 
OPB-04 
OPB-O5 
OPB-06 
OPB-07 
OPB-O8 
OPB-O9 
OPB-10 
OPB-I 1 
OPB-12 
OPB-13 
OPB-I4 
OPB-15 
OPB-I 6 
OPB-17 
OPB-18 
OPB-19 
OPB-20 
5' GTllCGCTCC 3' 
5' CATCCCCCTG 3' 
5' GGACTGGAGT 3' 
5' TGCGCCClTC 3' 
5' TGCTCTGCCC 3' 
5' GGTGACGCAG 3' 
5' GTCCACACGG 3' 
5' TGGGGGACTC 3' 
5' CTGCTGGGAC 3' 
5' GTAGACCCGT 3' 
5' CCTTGACGCA 3' 
5' TTCCCCCGCT 3' 
5' TCCGCTCTGG 3' 
5' GGAGGGTGTT3' 
5' TllGCCCGGA 3' 
5' AGGGAACGAG 3' 
5' CCACAGCAGT 3' 
5' ACCCCCGAAG 3' 
5' GGACCCTTAC 3' 
O. gutturosa, O. lienalis. O. gibsoni, and O. ochengi DNA were 
obtained from pooled worms as described previously (Meredith 
et al., 1989). The O. volvulus DNA was extracted from pooled 
worms as described by Meredith et al. (1989). PCR reactions 
were performed in 25 pl of 10 mM Tris-HC1 (pH 9,0), 50 mM KCl, 
0.1 % Triton X-100, 1,5 mM MgC12, 0,2 mM of each dNTP, 20 
picomoles of decamer primer(s) as given in Table 1 (Kit ß, 
W D T M  primer kits, Operon Technologies Inc.), 25 ng template 
DNA and 1 unit Taq polymerase (Promega). Amplification was 
carried out in a thermocycler (Techne PHC-3) programmed for 45 
cycles of 1 minute at  92"C, 1 minute at 36°C and 2 minutes at 
72 "C. Amplification products were resolved electrophoretically 
on 2 %  agarose gel, revealed by ethidium bromide and photo- 
graphed under U. V. light (Sambrook et al., 1989). 
Abbreviations 
Deoxyribonucleic acid (DNA); random amplified 
polymorphic DNA (RAPD); base pairs (bp): polymerase chain 
reaction (PRC); sodium lauryl sulfate (SDS); operon primer kit B 
(OPB). 
Results 
First, we investigated the potential of 
RAF'D in differentiating at the species level. Genomic DNA 
from different fdarial species was amplified by use of a 
single decamer primer; 20 primers were tested ín these 
experiments and in each case, at least one specific frag- 
ment was obtained for each Onchocerca DNA used. Fig. 1 
shows the amplifications with primer OPB-4 on DNA from 
Human, D. immitis, O. gutturosa, O. lienalis, O. gìbsoni, 
O. ochengi and two of O. volvulus isolates from Cameroon 
and Sierra-Leone. Depending on the primer used, several 
bands can be seen, ranging from 1.5 Kb to 150 bp. Some 
of the bands are common between the dif€erent species 
and some are unique to one species. Preliminary inves- 
tigations were also carried out to determine whether the 
RAPD patterns generated by these primers (OPB-1 to OPB- 
20) could be useful in strain differentiation within O. vol- 
vulus. 
Dop. Med. Parasitol. 45 (1994) 201 - Genomic fingerprinting of Onchocerca species 
DNA from different O. volvulus worms (4 
isolates from Cameroon and 1 from Sierra-Leone) were 
amplified with different primers: Fig. 2 shows these am- 
plifications with primer OPB-4; this figure is representa- 
tive of all the amplification profiles obtained with the 19 
other primers. The patterns observed for the different 
worms are very similar with some differences represented 
only by low intensity bands. To increase the chance of 
detecting intraspecific variation, pairs of oligonucleotides 
were tested. As an example, amplifications of O. volvulus 
genomic DNA with two primers (OPB-7 and 8) are pres- 
ented in Fig. 3: the combination of two primers (Lane 2) 
result in the appearance of new amplified DNA fragments 
compared to ampliíìcation with single primer (Lane 1 and 3). 
Discussion 
RAPD is useful in differentiating at genus, 
species, and intraspecific level (Hadrys et al., 1992). 
Specific RAPD markers have been identified at genus level 
and species level in irisis (Arnold et al., 1991) and tomato 
(Klein-Lankhorst et al., 1991), strain level in mouse 
(Welsh et al., 19911, "cultivar" level in broccoli and cauli- 
flower (Hu and Quiros, 1991) and clone level in fungi 
(Smith et al., 1992). Divergence of even a fraction of one 
percent between two genomes often results in a different 
fmgerprinting and thus allow strains of almost any or- 
ganism to be distinguished (Welsh et al., 1991). 
In this study, fragments of genomic DNA 
from various species of the genus Onchocerca were ob- 
tained by amplification with short primers of arbitrary 
nucleotide sequence. The polymorphic patterns allowed a 
clear distinction between the different species by RAPD 
with numerous different primers. When standardized re- 
action conditions were used, these results were highly 
reproducible. Clear differences on a simple agarose gel 
can be observed, unlike the conventional PCR assay de- 
scribed for O. volvulus (Meredith et al., 1991) that neces- 
sitate Southern blot analysis and hybridization. One im- 
portant problem in Onchocerciasis control strategies and 
surveillance is the identification of Onchocerca species 
infecting the Simulium vector. The RAPD technique is not 
suitable for identifkation of the parasites in the fly since 
polymorphic pattern of the vector DNA will be superposed 
with those of the parasite, making interpretation of results 
difficult. However it shows great potential for the genera- 
tion of species specific probes from the fragments 
generated, and species specific PCRs could be developed 
from the unique fragments for direct identification of On- 
chocerca species in vectors. 
The primers used in this study do not show 
clear distinctions between the different isolates of O. vol- 
vulus tested. However, considering the number of RAPD 
primers available it is more than likely that clear differen- 
tiation between isolates can be obtained with other 
primers. Thus, a combination of these or other random 
primers could prove to be useful population markers. 
Further investigation of large number of isolates of O. 
volvulus is needed to evaluate the potential of RAPD in 
studies on population genetics. This technique may allow 
the selection of species or strain specific sequences as 
potential probes more easily than the classical approach. 
M 1 2 3 '  
1353 
1078 
872 
603 
31 O 
281 
Fig. 3 Comparison of patterns 
generated with single and mixed 
primers (O. volvu-lus from Sierra- 
Leone). 
M: Molecular Weight Marker, 
OX-174 Hae 111 digest. 
Lane 1 : genomic DNA amplified 
with primer OPE-7. 
Lane 2: genomic DNA amplified 
with primer OPB-7 and OPE-8. 
Lane 3: genomic DNA amplified 
with primer OPB-8. 
Such probes could improve the direct identification of 
Onchocerca infected vector (by hybridization or PCR) and 
the specificity would not be dependant on the stringency 
conditions. 
It is important to be able to identify the 
origin or form of the parasite (L3 of different species of 
the genus Onchocerca or different strains of O. voZuulus) 
in order to plan appropriate onchocerciasis control strate- 
gies. The results obtained on the correlation between 
classification of O. uoluulus by DNA probes and epidemio- 
logic patterns of onchocercal blindness (Zimmerman et 
al., 19921, support the hypothesis of strain association 
with pathogenicity. 
RAPD analysis is interesting because very 
small amounts of genomic DNA are subjected to PCR and 
the amplification products are resolved on agarose gel. 
Consequently, for field application, there is no need for 
utilisation of radioactivity. Polymorphisms generated by 
this assay are useful as genetic markers when strictly 
standardized reaction conditions are used (Hadrys et al., 
19921, which guarantee reproducible amplification pro- 
ducts. 
Acknowledgements . 
We would like to thank Drs. M. Boussinesq and 
J. P. Chippaux for providing material and epidemiological data 
and Dr. J. Prod'hon for support of this work. We are also grateful 
to Dr. J. P. Aussel for excellent technical assistance, Pr. Dorchies 
and Dr. C. Bevilaqua (Ecole Vétérinaire de Toulouse) for the 
O. lienalis control sample. We also thank Dr. R. Hartskeerl and 
G. van der Vliet for critical reading of the manuscript. 
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6531-6535 
Dr. S. Herder 
Laboratoire d'Epidémiologie des Maladies à Vecteurs 
Centre ORSTOM de Montpellier, 911 
Av. Agropolis, BP 5045 
F-34032 Montpellier 
France 


Genomic fingerprinting of Onchocerca species using random amplified polymorphic DNA

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