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Am. J. Trop. Med. Hyg., 71(2), 2004, pp. 227-233
Copyright © 2004 by The American Society of Tropical Medicine and Hygiene

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GENETIC SUBSTRUCTURING WITHIN OESOPHAGOSTOMUM BIFURCUM (NEMATODA) FROM HUMAN AND NON-HUMAN PRIMATES FROM GHANA BASED ON RANDOM AMPLIFIED POLYMORPHIC DNA ANALYSIS

JOHANNA M. DE GRUIJTER, JUVENTUS ZIEM, JACO J. VERWEIJ, ANTON M. POLDERMAN, AND ROBIN B. GASSER
Department of Parasitology, Leiden University Medical Center, University of Leiden, Leiden, The Netherlands; University for Development Studies, Tamale, Ghana; Department of Veterinary Science, The University of Melbourne, Werribee, Victoria, Australia


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Random amplified polymorphic DNA (RAPD) was used to study genetic variation within Oesophagostomum bifurcum in Ghana. Four different decamer primers were used for the amplification of DNA from individual O. bifurcum adults (n = 41) from humans and non-human primates (including the Mona monkey, Patas monkey and Olive baboon) from different geographic regions. Analysis of the amplicons from all 41 nematodes by high resolution, denaturing polyacrylamide gel electrophoresis defined a total of 326 informative RAPD bands. Cluster analysis of the RAPD data (based on pairwise comparison of banding profiles) showed that O. bifurcum from humans was genetically distinct from O. bifurcum from the Mona and Patas monkeys, and from the Olive baboon. These findings clearly demonstrate the existence of population genetic substructuring within O. bifurcum from different primate hosts in Ghana, and raise interesting questions about host specificity, epidemiology (e.g., zoonotic transmission), and ecology of the different genotypes of O. bifurcum.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human infection with Oesophagostomum bifurcum (Nematoda: Strongylida) is recognized as a parasitic disease with major socioeconomic significance in northern Togo and Ghana.1,2 The infection causes pathologic effects that can result in two distinct clinical presentations.3,4 The uninodular disease, also referred to as the ‘Dapaong tumor’, presents as a painful, abdominal mass with a diameter of 2–11 cm, frequently adhering to the abdominal wall. The multinodular disease is associated with hundreds of peasized nodules in a thickened, edematous submucosa and subserosa of the large intestine. In spite of the serious health problems caused by O. bifurcum, there are major gaps in our knowledge of the epidemiology and transmission of human oesophagostomiasis.5 Although it has been proposed that some species of non-human primates can act as reservoir hosts for human oesophagostomiasis,6 there is a significant difference in the geographic distribution of the infection between human and non-human primates in Ghana (van Lieshout L and others, unpublished data). Also, there is some evidence based on morphologic studies that significant variation can occur in the length of adult parasites of O. bifurcum isolated from human and non-human primates.7 These observations have suggested population variation within O. bifurcum, and have stimulated investigations using molecular techniques into the genetic diversity within O. bifurcum from human and non-human primates.

We recently used a polymerase chain reaction (PCR)–based single-strand conformation polymorphism (SSCP) analysis to scan for nucleotide variability in the ribosomal second internal transcribed spacer (ITS-2) and part of the mitochondrial cytochrome c oxidase subunit 1 gene (pcox1) of O. bifurcum from human and Mona monkey hosts.8,9 While some nucleotide microheterogeneity (representing population variation) was detected in these studies, no genetic differentiation between O. bifurcum from humans and the Mona monkey was detectable. Nonetheless, it is possible that genetic variation does indeed exist within O. bifurcum from human and non-human primate hosts, but this is not adequately reflected in the ITS-2 and pcox1 regions because they represent only a minute part of the nuclear and mitochondrial genomes, respectively, and/or because they are not sufficiently variable in sequence to demonstrate any differentiation. It was thus concluded that another molecular approach, such as the random amplified polymorphic DNA (RAPD),10,11 could provide more variable genetic markers.

The method of RAPD is based on the screening of the entire genome without the need for any prior DNA sequence information. Although there has been some reservation about the reproducibility of results using this method,12–14 it has been applied effectively to investigate genetic variation within and among a range of species of parasitic nematodes.15–17 Importantly, RAPD results have proven to be reproducible when an increased stringency (i.e., an annealing temperature of >45°C) is used in the PCR and amplicons are subjected to denaturing polyacrylamide gel electrophoresis.18 In the present study, RAPD analysis was used for investigating genetic diversity among individual adults of O. bifurcum from humans, the Patas monkey (Erythrocebus patas), the Mona monkey (Cercopithecus mona) and the Olive baboon (Papio anubis) from different geographic regions in Ghana to establish whether population genetic substructuring exists within O. bifurcum.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Parasites and isolation of genomic DNA. Adult worms (n = 41) of O. bifurcum were obtained from human and non-human primate hosts from three different geographic regions in Ghana (Figure 1Go). The study was reviewed and approved by the Ministry of Health in Bolgatanga, Ghana and the Wildlife Division in Accra, Ghana. Informed consent for participation was obtained from all human adult participants and from parents of children of less than 15 years of age. Worms were obtained from the feces of infected patients after treatment with pyrantel pamoate, as described previously,1 whereas worms from non-human primate hosts were removed from the large intestine at necropsy. The worms were washed extensively in physiologic saline and then stored in 70% ethanol until used for isolation of DNA. Each specimen of O. bifurcum was identified morphologically using published keys and descriptions.7,19,20 Genomic DNA was isolated from in dividual worms by digestion with sodium dodecyl sulfate/ proteinase K,21 purification over spin columns (WizardTM DNA Clean-Up; Promega, Madison, WI), and eluting into 50 µL of water. Purification and isolation of DNA from the large intestinal content from non-infected hosts (i.e., control DNA samples) were carried out as described previously.22



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  		    FIGURE 1. Map of Ghana showing the geographic regions where Oesophagostomum bifurcum was collected from different primate hosts.

 
Enzymatic amplification and SSCP analysis. Since the sequence of the ITS-2 of ribosomal DNA (rDNA) allows the specific identification of O. bifurcum,23,24 all individual worms (n = 41) used in this study were subjected to mutation scanning analysis of this rDNA region8 to verify their identity prior to RAPD analysis. To do this, the ITS-2 was amplified by a PCR using primers NC1 (forward: 5'-ACGT CTGGTTCAGGGTTGTT-3') and NC2 (reverse: 5'-TTAG TTTCTTTTCCTCCGCT-3').21 Primers were end-labeled with {gamma}-33P-ATP (New England Nuclear Life Science Products, Boston, MA) using T4 polynucleotide kinase (Promega) prior to the PCR. The PCR amplification was performed in 50-µL volumes using 25 pmol of each primer, 250 µM of each dNTP, 3 mM MgCl2, and 2 units of Taq polymerase (Promega) under the following conditions: an initial denaturation at 94°C for five minutes; 30 cycles at 94°C for 30 seconds (denaturation), 55°C for 30 seconds (annealing), and 72°C for 30 seconds (extension); followed by a final extension at 72°C for five minutes in a thermocycler (Perkin Elmer Cetus, Nor-walk, CT). Samples without genomic DNA (no-DNA controls) were included in each amplification run. Also, control DNA samples derived from the contents of the large intestine from a non-infected human, a Mona monkey, a Patas monkey and an Olive baboon were subjected to the same amplification procedure as used for parasite DNA. In no case were products detectable in these control DNA samples (not shown).

Five microliters of each PCR product were mixed with 3 µL of loading buffer (1 mM EDTA, 0.25% bromophenol blue, 0.25% xylene cyanole, 30% glycerol) and subjected to electrophoresis on ethidium bromide–stained 2% agarose gels using TBE buffer (65 mM Tris-HCl, 27 mM boric acid, 1 mM EDTA, pH 9.0; Bio-Rad, Hercules, CA) and {phi}X-174 DNA digested with Hae III (Promega) as a size marker. The SSCP analysis was then carried out as described previously.25 Ten microliters of each PCR product were mixed with an equal volume of loading dye (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanole). After denaturation at 95°C for five minutes and snap-cooling on a freeze block (–20°C), individual samples (2 µL) were loaded into the wells of a 0.4 mm–thick, non-denaturing gel (0.5 x mutation detection enhancement [MDE]; FMC BioProducts, Rockland, ME), and subjected to electrophoresis in a conventional sequencing apparatus (BaseRunner; IBI, New Haven, CT). The conditions for electrophoresis (35 watts for five hours at 18°C) were standardized for optimum resolution of bands, and the gel concentration was as recommended by the manufacturer. After electrophoresis, gels were dried on to blotting paper and subjected to autoradiography using RP1 film (Agfa, Mortsel, Belgium).

Amplicons representing variable SSCP profiles were purified over spin columns (WizardTM PCR-Prep; Promega) and sequenced in both orientations by automated sequencing (Big Dye Chemistry; Applied Biosystems, Foster City, CA) using the same primers as for the primary PCR. Sequences were aligned manually and compared with the ITS-2 sequence of O. bifurcum from Togo and Ghana recorded previously.24,26

Random amplified polymorphic DNA analysis. The RAPD analysis was performed in 25 µL reaction volumes containing 125 pmol of {gamma}33P-end-labeled primer (Operon Technologies, Alameda, CA), 250 µM of each dNTP, 3 mM MgCl2, 2 units of Taq polymerase (Promega), and ~1 ng of genomic DNA. The cycling profile consisted of 35 cycles at 94°C for two minutes (denaturation), 48°C for two minutes (annealing), and 72°C for two minutes (extension) in a thermocycler (Perkin Elmer Cetus). Control samples without DNA were included in each PCR run, and DNA isolated from the large intestinal content from non-infected hosts (i.e., human, Mona monkey, Patas monkey, and Olive baboon) was subjected to the same amplification procedure as used for parasite DNA. For agarose gel electrophoresis, individual RAPD products (10 µL) were loaded on 2% agarose gels and subjected to electrophoresis at 80 volts for two hours using TBE buffer, stained with ethidium bromide, and then detected using an Imago Compact Imaging System (Isogen Life Science, Maarssen, The Netherlands). A 100-base pair ladder (Promega) was included on all gels. For denaturing gel electrophoresis (which achieved a substantially higher resolution than agarose gel electrophoresis), RAPD amplicons (25 µL) were mixed with 15 µL of loading dye, denatured at 94°C for 15 minutes, and snap-cooled on ice. Subsequently, samples (1.3 µL) were loaded on to a 5% denaturing polyacrylamide gel and subjected to electrophoresis at 55 watts for two hours and 15 minutes at 55°C using TBE buffer. Gels were dried on to blotting paper and exposed to RP1 film. The pGEM marker (Promega) was used as a size standard on every gel.

Statistical and cluster analyses were carried out using the software program Free-Tree (available at www.natur.cuni.cz/flegr/programs).27 Similarity coefficients were calculated according to the method of Nei and Li,28 and an unrooted den-drogram was constructed using the unweighted pair group method using arithmetic averages. Statistical support for the dendrogram was obtained by bootstrapping using 200 resamplings, and bootstrap values >80% were considered significant.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Verification of the identity of individual O. bifurcum by SSCP analysis. Agarose gel electrophoresis showed that there was no detectable size difference in the ITS-2 amplicons within or between O. bifurcum from human and non-human primates. Autoradiographic exposure of the agarose gels indicated the specificity of the PCR products and conditions in that each product appeared as a single band, and no non-specific background bands were detectable. The SSCP analysis of the 41 O. bifurcum samples revealed 14 different profiles, each consisting of 2–4 strong bands and 1–4 additional weak bands. Subsequent sequencing of amplicons representing each of the 14 profiles revealed five polymorphic nucleotide positions (99, 105, 112, 117, and 162) that were consistent with some recorded previously for the ITS-2 of O. bifurcum from humans from Togo (positions recorded in relation to the sequence with accession number Y11733).24 Thus, there was no unequivocal sequence difference in the ITS-2 between any of the 41 O. bifurcum individuals from Ghana and those from previous studies in Togo and Ghana,24,26 providing genetic evidence that all specimens represented O. bifurcum.

Evaluation of RAPD primers and analysis on agarose gels. Originally, 40 individual decamer primers (OPA1–OPA20 and OPB1–OPB20) were tested for their ability to amplify PCR products from genomic DNA (~1 ng) from individual adults of O. bifurcum. Different annealing temperatures (45°C, 48°C, 50°C, 52°C, and 55°C) were tested, and 48°C achieved the highest amplification efficiency. Increasing the annealing temperature beyond 50°C resulted in a substantially decreased efficiency. Four primers (OPA-10, OPB-1, OPB-6, and OPB-8) that gave reproducible and discrete banding patterns on agarose gels (using a PCR annealing temperature of 48°C) were selected for further RAPD analysis of O. bifurcum individuals. Amplicons for each of these four primers consisted of 2–7 strong bands ranging from 0.2 to 1.2 kb in size. Profiles for each primer (using 10 selected DNA samples) were shown to be reproducible on different days and in different laboratories (i.e., in the Department of Parasitology, Leiden University Medical Center, The Netherlands, and the Department of Veterinary Science, The University of Melbourne, Australia).

Analysis by RAPD on high-resolution, denaturing poly-acrylamide gels. The RAPD analysis was performed on all 41 individual adults of O. bifurcum (20 from humans, 9 from the Mona monkey, 8 from the Patas monkey, and 4 from the Olive baboon) from different geographic locations in Ghana (Figure 1Go). Analysis (using primers OPA-10, OPB-1, OPB-6, and OPB-8) on denaturing polyacrylamide gels gave reproducible results (i.e., banding profiles) for all individuals on consecutive days, using the same amplicons and different amplicons produced on different days. An example of the reproducibility of RAPD banding profiles for primer OPB-1 is shown in Figure 2Go. No products were amplified from the no DNA samples. Some amplicons were produced from samples containing DNA isolated from large intestinal contents from non-infected hosts (i.e., human, Mona monkey, Patas monkey, or Olive baboon), but none of them were the same in their position on gels as those produced from any of the O. bifurcum individuals examined. Using primers OPA-10, OPB-1, OPB-6, and OPB-8, we detected 320 polymorphic bands and 6 monomorphic bands (i.e., bands present in all 41 O. bifurcum samples analyzed) ranging in size from 50 to 700 base pairs. Amplification with primer OPA-10 produced the most bands (n = 109) compared with primers OPB-1 (n = 79), OPB-6 (n = 74), and OPB-8 (n = 64). Using primer OPB-1, we detected a polymorphic band of ~300 base pairs that was common to all 20 O. bifurcum individuals from humans, but was absent from all 21 O. bifurcum specimens from non-human primates examined. Subsequently, for each of the 41 O. bifurcum samples subjected to analysis, the presence or absence of each of the 326 polymorphic bands was recorded, and a binary data matrix of the data was constructed. Based on these data, similarity coefficients were calculated and a dendrogram was constructed (Figure 3Go). Bootstrap values (greater than 80%) supported three main clusters. Cluster I comprised all 20 O. bifurcum individuals isolated from humans, cluster II included all 9 O. bifurcum isolated from the Mona monkey as well as all 8 isolated from the Patas monkey, and cluster III comprised all 4 O. bifurcum individuals isolated from the Olive baboon. Similarity coefficients among individual O. bifurcum within clusters I, II, and III ranged from 0.33 to 0.63, 0.39 to 0.73, and 0.39 to 0.51, respectively. With the exception of the grouping of samples M8 and M9 (representing O. bifurcum isolated from the Mona monkey) and that of PMD1 and PMD7 (representing O. bifurcum isolated from the Patas monkey) within cluster II, there was no significant bootstrap support for subclustering within either cluster I, II, or III. Also, there was no evidence for clustering according to geographic origin of each host species.



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  		    FIGURE 2. Reproducibility of random amplified polymorphic DNA profiles for three adult specimens (a–c) of Oesophagostomum bifurcum. Primer OPB-1 was used. Denaturing polyacrylamide gel electrophoresis of amplicons produced on different days (1 or 2) was carried out on different days.

 


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  		    FIGURE 3. Dendrogram based on cluster analysis of random amplified polymorphic DNA data for 41 individual Oesophagostomum bifurcum adults from humans (cluster I), the Mona or Patas monkeys (cluster II), and the Olive baboon (cluster III) from Ghana. Similarity coefficients were calculated according to the method of Nei and Li.28 The branch lengths represent the genetic distances between the individuals, and the numbers on branches are bootstrap values (using 200 resamplings).

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Random amplified polymorphic DNA has been widely used as a genetic screening method29–32 because it is rapid, relatively simple to perform, and requires only a small amount (1–20 ng) of genomic DNA. In addition, it requires no genome sequence information prior to analysis, and can be applied to complex DNA of any origin.10,11 The RAPD banding patterns can be affected by a number of factors, for example, the quality and quantity of template DNA, concentration of reagents, use of different thermocyclers, and/or co-migration of non-homologous fragments.12–14,33–35 However, the effect of these factors on the resultant banding patterns is largely due to the low annealing temperatures (25–35°C) used in the PCR. Thus, the use of increased annealing temperatures (45–55°C), as is the case in the present study, increases the stringency of the PCR and ensures the reproducibility of RAPD results.18,36 Also, the analysis of single-stranded RAPD products on denaturing polyacrylamide gels achieves a much better resolution of DNA fragments compared with analysis of double-stranded RAPD products on agarose gels, and improves reproducibility of banding patterns.18,37,38 These findings are supported by the results of the present study.

The main objective of this study was to investigate the genetic composition of O. bifurcum isolated from human and different species of non-human primates from Ghana by RAPD analysis. Together with morphologic study, SSCP-based analysis of the ITS-2 region demonstrated that all individuals included in this investigation represented O. bifurcum. Subsequent RAPD analysis, using primers OPA-10, OPB-1, OPB-6, and OPB-8, showed a relatively high degree of polymorphism among all individuals of O. bifurcum (n = 41) examined (associated with a total of 320 polymorphic bands). Cluster analysis of the RAPD profile data (considering all polymorphic bands) showed that O. bifurcum represented three distinct groups, namely those from humans, those from the Mona or the Patas monkey, and those from the Olive baboon. This result demonstrates clearly the existence of population genetic substructuring within the species O. bifurcum according to host species, and that O. bifurcum from human and non-human primates comprises genetically distinct groups. There was evidence that O. bifurcum from the Mona monkey was genetically distinct from O. bifurcum from the Patas monkey, although the boostrap support was not strong (which may relate to the limited sample sizes). The fact that O. bifurcum from humans (e.g., samples H30-H43) and from the Patas monkey (e.g., samples PMN2, PMN6, and PMN8) from geographic region 1 grouped into different clusters (i.e., clusters I and II, respectively) (Figures 1Go and 3Go) showed that there was no association between O. bifurcum genotype and the geographic origin of the host species based on the RAPD data. This was also indicated for O. bifurcum from the Patas monkey (e.g., samples PMD1-PMD3) and from the Olive baboon (samples B2-B5) from region 2, which related to clusters II and III, respectively (Figures 1Go and 3Go).

Interestingly, the infection of humans with O. bifurcum appears to be restricted to the extreme northern regions of Togo and Ghana, where at least 250,000 people are infected.1,4,39 The non-human primates in this geographic area are also infected but have significantly decreased in numbers over the last decades. In other locations further south in Ghana (e.g., Mole National Park and Baobeng-Fiema; Figure 1Go), non-human primates remain numerous and in close contact with human settlements. There, the prevalence of infection in the non-human primates is high but humans have been found not to be infected.22 To date, there is no explanation for this fascinating observation. The results of the present study (i.e., the existence of genetically distinct groups of O. bifurcum in a number of different host species) suggest that the parasite of non-human primate hosts in Togo and Ghana may be unable to infect or be inefficient in infecting the human host. Although the latter proposal is supported (to some extent) by an experimental study,40 showing that non-human primates appear to be poorly susceptible to infection with O. bifurcum from humans, it cannot yet be ruled out that transmission of the parasite does not occur between humans and non-human primates in Togo and Ghana, and/or that at least some non-human primates may represent a natural reservoir for human infection in these countries. These proposals still require testing. Also, it remains unclear why O. bifurcum from humans is restricted to the far northern regions of Togo and Ghana.

The definition and characterization of genetic markers for the differentiation of human O. bifurcum from non-human primate O. bifurcum is of significance for addressing epidemiologic and ecologic questions. In this study, RAPD analysis using primer OPB-1 showed one polymorphic band that was specific to O. bifurcum from humans. In future work, DNA of this band should be cloned and sequenced. Primers designed specifically to this band sequence will be evaluated in the PCR for the specific identification of O. bifurcum from humans. Such a specific PCR assay could be used to assess (by amplifying O. bifurcum egg DNA from the feces from the host) whether non-human primates from Ghana can harbor the "human genotype" of O. bifurcum and/or to undertake ecologic studies of this genotype. Clearly, a better understanding of the transmission patterns of O. bifurcum could assist in the effective control of this important parasite. In addition, amplified fragment length polymorphism analysis41 will be conducted to further examine genetic substructuring within O. bifurcum, to define additional genetic markers specific for O. bifurcum from different species of non-human primate hosts and from humans.

Received December 10, 2003. Accepted for publication February 25, 2004.

Acknowledgments: We thank Y.G. Abs EL-Osta and A.J. Nisbet (Department of Veterinary Science, The University of Melbourne), H. Roberts (Department of Statistical Science, University College, London, United Kingdom), and L. Dijkshoorn (Department of Infectious Diseases, Leiden University Medical Center) for assistance and discussions. We are grateful to Neil Chilton for comments on the manuscript. We also thank the following individuals who provided, or assisted in providing, some of the samples used in this study: M. Adu-Nsiah (Wildlife Division, Accra, Ghana), V. Asigri (Parasitic Diseases Research Laboratory, Tamale, Ghana), D. Laar and S. Amponsah (Ghana), and J. Blotkamp (The Netherlands).

Financial support: This study was supported by the Dutch Foundation for the Advancement of Tropical Research (WOTRO-NWO), the Australian Research Council, and the Collaborative Research Program of The University of Melbourne.

Authors’ addresses: Johanna M. de Gruijter, Jaco J. Verweij and Anton M. Polderman, Department of Parasitology, Leiden University Medical Center, University of Leiden, PO Box 9600, 2300 RC Leiden, The Netherlands, Telephone: 31-71-526-5081, Fax: 31-71-526-6907, E-mails: j.m.de_gruijter{at}lumc.nl, j.j.verweij{at}lumc.nl, and a.m.polderman{at}lumc.nl. Juventus Ziem, University for Development Studies, PO Box 967, Tamale, Ghana, Fax: 00-233-7122777. Robin B. Gasser, Department of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia, Telephone: 61-3-9731-8283, Fax: 61-3-9731-2366, E-mail: robinbg{at}unimelb.edu.au.

Reprint requests: Johanna M. de Gruijter, Department of Parasitology, Leiden University Medical Center, University of Leiden, PO Box 9600, 2300 RC Leiden, The Netherlands.


REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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