J. Parasitol., 97(1), 2011, pp. 156–159 F American Society of Parasitologists 2011
Genotyping Natural Infections of Schistosoma mansoni in Biomphalaria alexandrina From Damietta, Egypt, with Comparisons to Natural Snail Infections From Kenya Wael M. Lotfy, Ben Hanelt*, Gerald M. MkojiÀ, and Eric S. Loker*, Parasitology Department, Medical Research Institute, Alexandria University, 165 EI-Horreya Avenue, Alexandria, Egypt, PO Box 21561; *Center for Evolutionary and Theoretical Immunology, Department of Biology, University of New Mexico, MSC03 2020, Albuquerque, New Mexico, 87131-0001; ÀCenter for Biotechnology Research and Development, Kenya Medical Research Institute, Mbagathi Road, P.O. Box 54840-00200, City Square, Nairobi, Kenya. e-mail:
[email protected] ABSTRACT: The distribution of Schistosoma genotypes among individuals in snail populations provides insights regarding the dynamics of transmission and compatibility between schistosome and snail hosts. A survey of Biomphalaria alexandrina from Damietta (Nile Delta, Egypt), an area subjected to persistent schistosomiasis control efforts, provided only 17 snails infected with Schistosoma mansoni (6.1% overall prevalence), each shown by microsatellite analysis to have a single genotype infection. By contrast, recent studies of uncontrolled S. mansoni transmission foci in Kenya revealed that 4.3% Biomphalaria pfeifferi and 20–25% Biomphalaria sudanica snails had multiple genotype infections. Compared with the 3 Kenyan populations, the Egyptian population of S. mansoni also showed a lesser degree of genetic variability and was genetically differentiated from them. We suggest that tracking of genotype diversity in infected snails could be further developed to serve as an additional and valuable independent indicator of efficacy of schistosomiasis control in Egypt and elsewhere.
Damietta, 1 of the few regions of the Nile Delta where we have been able to find S. mansoni infections in snails. We compare the results obtained with those from Biomphalaria pfeifferi and Biomphalaria sudanica snails infected with S. mansoni in Kenya, from locations where systematic control efforts have not been initiated. A comparison of the results of the 2 countries can provide a useful perspective on the impact of control programs and suggest other means for monitoring their progress. In 2006–2009, we carried out a malacological survey in the Nile Delta guided by the field teams of the Schistosomiasis Control Program of the Ministry of Health and Population (MoHP). During this survey, we discovered a focus of B. alexandrina infected with S. mansoni in El-Riyad Village, Kafr Saad, Damietta Governorate, in the northeastern part of the Nile Delta (31.402583uN, 31.7041uE). The focus was a slow-flowing, narrow canal used to drain wastewater from surrounding households. In August 2009, 277 B. alexandrina snails were collected from this canal. All snails were examined for infection by isolating them in 24-well plates placed under light for 48 hr. We found 17 snails infected with S. mansoni (6.1% prevalence). Infected snails that shed S. mansoni cercariae were fixed and stored in 95% ethanol. The S. mansoni prevalence among snails at this restricted focus was unusually high as compared with other reports from Egypt, regardless of the months of collection (Chu and Dawood, 1970; Allam et al., 1989; Ahmed et al., 2003). Shortly after we collected the Damietta snails, personnel of the Schistosomiasis Control Program sprayed niclosamide (molluscicide) in the water canal and dispensed praziquantel (PZQ) as a mass treatment to the surrounding human population. For comparison, we used data from previous studies in Kenya. Biomphalaria sudanica snails were collected from the shore of Lake Victoria at the Carwash (0.095867uS, 34.748594uE) or Asembo (0.188508uS, 34.387534uE) sites. In addition, B. pfeifferi snails were collected from Asao stream (0.33256uS, 34.999144uE) (Steinauer, Mwangi et al., 2008; Steinauer et al., 2009). Prevalence in the Egyptian focus in Damietta was also high when compared with the snail samples collected from the 3 Kenyan foci, i.e., 0.73% in Asao, 2.59% in Carwash, and 0.77% in Asembo (Steinauer, Mwangi et al., 2008), though each Kenyan habitat is much larger in size and harbors many more snails than the relatively restricted Damietta habitat. We used a different and direct approach for genotyping of S. mansoni infection in snails. The HotSHOT (Truett et al., 2000) method was used to prepare genomic DNA from whole individual B. alexandrina, including the intra-molluscan stages of S. mansoni. Molecular genotyping was done for all specimens using a total of 11 primer pairs for previously described microsatellite loci in the S. mansoni genome. Two multiplex PCR reactions were carried out (panels P17A and P22A). The P17A panel was a modification of the P17 panel described by Steinauer, Agola et al. (2008), where the number of loci was reduced from 7 to 6: C1 (AF325695), C5 (AF325698), D1 (AF202965), D2 (AF202966), D4 (AF202968), and D6 (L46951). The P22A panel is a modification of the P22 panel described by Steinauer, Agola et al. (2008), where the number of loci was reduced from 6 to 5: S1 (AI067617), S3 (AI395184), S6 (BF936409), D7 (M85305), and D8 (R95529). Primers were labeled with 6-FAM, HEX, and NED dyes (Applied Biosystems, Carlsbad, California). The reverse primer specific for each of the C1, C5, D1, D2, D4, and D6 loci was labeled with the HEX, 6FAM, NED, 6FAM, NED, and HEX dyes, respectively. On the other hand, the forward primer specific for each of S1, S3, S6, D7, and D8 was labeled with the HEX, HEX, 6FAM, 6FAM, and NED dyes, respectively. The QIAGEN Multiplex PCR Kit (Qiagen, Valencia, California) was used for PCR amplifications in 5.8-ml reactions according to the manufacturer’s directions. The thermal cycling profile included an initial denaturation
Molecular approaches, including the use of microsatellite markers, have become an increasingly important way to study the epidemiology of schistosomiasis, enabling more rigorous examination of schistosome population structure and genetic subdivision and response to control efforts. Among the applications of microsatellite and other molecular markers is the identification of Schistosoma genotypes among individual infected snails. The distribution of Schistosoma mansoni genotypes among snails can have significant consequences for the transmission dynamics of the parasite and on the distribution of genetic diversity of schistosomes among the definitive host population (Barral et al., 1996). Minchella et al. (1995) used a polymorphic repetitive mitochondrial DNA element (mtVNTR) to show that S. mansoni genetic diversity was overdispersed in naturally infected snail populations from Barreiro and Cabana, Brazil. In total, 57.2% of the Biomphalaria glabrata naturally infected with S. mansoni from 2 Brazilian foci harbored multiple infections, and some snails carried at least 9 parasite genotypes. Sire et al. (1999) used S. mansoni RAPDs markers in a study on 43 naturally infected B. glabrata from Grande Terre island of Guadeloupe and found that the large majority of snails (88.4%) harbored a single parasite genotype, and parasite intensity did not exceed 3 genotypes per snail. Eppert et al. (2002), in 2 malacological surveys, collected 84 B. glabrata snails naturally infected with S. mansoni from Dionisio, Brazil, a site known to have a very high percentage of infected snails. In that study, mtVNTR revealed the presence of multiple parasite genotypes in more than half of the infected B. glabrata snails, and the parasite intensity did not exceed 4 genotypes per snail. The distribution of parasite genotypes among snails is likely influenced by a number of factors, among them, chemotherapy-based control operations that have the effect of reducing parasite egg input into the environment. Egypt has sustained a large national control program for a prolonged period and succeeded in reducing morbidity to very low levels. The overall prevalence of S. mansoni in the Nile Delta has declined from 38.6% in 1983 to 1.5% in 2006 (WHO, 2001, 2007). In addition to undertaking 1–2 rounds of mass chemotherapy per year in ‘‘hot spot’’ areas, the program also implements snail control using chemical molluscicides in active transmission sites, improves village environmental conditions, provides safe water to endemic villages, and enforces health education and behavioral changes (WHO, 2001, 2005, 2007). The aim of the present work is to document the genetic structure and number of S. mansoni genotypes per infected snail in Biomphalaria alexandrina from DOI: 10.1645/GE-2537.1 156
RESEARCH NOTES
TABLE I. Number of S. mansoni per infected snail in the different studied populations. Number of genotypes/snail Populations (N) Damietta (17) Asao (46) Carwash (28) Asembo (20)
One (%)
Two (%)
Three (%)
Four (%)
17 44 21 16
0 1 (2.2) 4 (14.3) 4 (20)
0 1 (2.2) 2 (7.1) 0
0 0 1 (3.6) 0
(100) (95.7) (75) (80)
step at 94 C for 5 min, followed by 35 cycles of 45 sec at 94 C, annealing temperature specific for each multiplex panel (52 C for P17, 48 C for P22), 30 sec at 72 C, and a final step of 7 min at 72 C using an Eppendorf Mastercycler Systems thermocycler (EppendorfH, Hamburg, Germany). Multiplexed PCR products were diluted in N,N9-dimethyl formamide with GenescanH–500 (ROX 500) as an internal size standard and genotyped using an ABI3100 automated sequencer (Applied Biosystems) and scored with GeneMapperH v. 4.0 (Applied Biosystems) software. All genotype calls were verified manually. As a check on the sensitivity of our direct extractions from infected snails, we made artificial mixtures of genomic DNA from 2 infected snails, in the volumetric proportions of 95:5, 75:25, and 50:50, and also subjected them to microsatellite analysis. All mixed preparations recovered the expected peaks known to be present in the individual samples used to make the mixtures. The relative magnitude of each peak was proportional to the amount of the DNA added (data not shown). This indicates the technique is reliable for detection of microsatellites contributed by at least 2 S. mansoni genotypes against a background of B. alexandrina DNA. This approach may be of value for field surveys because it is simple, fast, and relatively inexpensive as compared to techniques requiring passage of
157
cercariae into mice, which may also induce bias due to the host selective pressure (Loverde et al., 1985), or result in death of the mouse. The approach we used may encounter difficulties in locations where multiple genotypes per snail are present because it will not enable individual genotypes to be sorted out. Also, it may underestimate the number of genotypes present in complex infections, though this was not a factor in the present study. Our results suggest that 1 approach to conserve resources for future efforts is to preserve both the shedding snail and, separately, cercariae derived from it. One could first genotype the parasites from the snail and, if found to be complex in composition, then individual cercariae could be genotyped. All 17 infected B. alexandrina snails collected from Damietta showed single genotype infections with S. mansoni (Table I). In previous studies of infected snails from Kenya (Steinauer, Agola et al., 2008; Steinauer et al., 2009), 46 naturally infected B. pfeifferi were recovered from 1 stream (Asao), only 2 of which had multiple genotype infections, 1 with 2 and 1 with 3 S. mansoni genotypes. From the Carwash site in Kisumu, on the Lake Victoria shoreline, 28 infected B. sudanica snails were found, 7 with multiple genotype infections (25%), i.e., 4 with 2, 2 with 3, and 1 with 4 S. mansoni genotypes. Additionally, of 20 naturally infected B. sudanica snails from Asembo Bay, Lake Victoria, 4 were infected with multiple genotype infections (20%), each with 2 S. mansoni genotypes. The distribution of schistosome infections within a snail population is influenced by genetic, behavioral, and ecological processes (Anderson and Gordon, 1982; Eppert et al., 2002) and stochastic events. The overall prevalence of infection at the Damietta site was relatively high, and no multiple genotype infections were recovered, suggesting that susceptibility in the natural population is relatively high, i.e., multiple genotype infections might be expected if only a small proportion of the snail population was susceptible to infection (Eppert et al., 2002). Further study is needed to determine if the lack of multiple genotype infections is indicative of active responses occurring within the snail, mediated by host responses to super-infection, or by established parasites in response to competitors. Certainly, a number of studies suggest multiple schistosome
TABLE II. Distribution of the different alleles observed in the 11 studied loci of the S. mansoni populations from Egypt (EG) and Kenya (KE). Loci S6
D7
S3
S1
D8
D2
C5
C1
D6
D4
D1
3 3 4 5
7 9 8 7
Number of observed alleles Damietta (EG) Asao (KE) Carwash (KE) Asembo (KE)
5 10 8 8
10 13 16 12
5 5 6 4
3 9 10 7
7 16 16 13
1 3 3 2
10 28 28 17
8 11 13 9
4 17 18 16
HO Damietta (EG) Asao (KE) Carwash (KE) Asembo (KE)
0.65 0.84 0.87 0.92
0.65 0.82 0.79 0.88
0.47 0.59 0.56 0.42
0.65 0.55 0.49 0.54
0.88 0.86 0.85 0.79
0.00 0.08 0.05 0.08
0.88 0.80 0.74 0.71
0.88 0.82 0.92 0.67
0.47 0.94 0.95 0.88
0.18 0.47 0.56 0.75
0.59 0.84 0.72 0.83
HE Damietta (EG) Asao (KE) Carwash (KE) Asembo (KE)
0.63 0.78 0.81 0.83
0.78 0.86 0.88 0.86
0.44 0.60 0.65 0.44
0.52 0.64 0.59 0.57
0.70 0.88 0.85 0.85
0.00 0.08 0.05 0.08
0.86 0.81 0.85 0.80
0.82 0.87 0.87 0.86
0.56 0.92 0.92 0.92
0.16 0.53 0.59 0.66
0.74 0.79 0.69 0.74
Unbiased HE Damietta (EG) Asao (KE) Carwash (KE) Asembo (KE)
0.65 0.79 0.82 0.85
0.81 0.87 0.89 0.87
0.45 0.61 0.66 0.45
0.53 0.65 0.60 0.59
0.72 0.88 0.86 0.87
0.00 0.08 0.05 0.08
0.89 0.82 0.86 0.81
0.84 0.88 0.88 0.88
0.58 0.93 0.93 0.94
0.17 0.53 0.60 0.67
0.76 0.80 0.70 0.76
0.02 0.00 0.27 0.83
0.55 0.88 0.58 0.30
0.58 0.10 0.00 0.00
0.00 0.72 0.10 0.14
– 0.99 1.00 0.83
0.01 0.02 0.48 0.92
0.80 0.01 0.83 0.20
0.93 0.05 0.93 0.52
0.98 0.71 0.82 0.86
0.82 0.40 0.87 0.78
Chi-square tests for HWE (P) Damietta (EG) Asao (KE) Carwash (KE) Asembo (KE)
0.57 0.00 0.40 0.17
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THE JOURNAL OF PARASITOLOGY, VOL. 97, NO. 1, FEBRUARY 2011
TABLE III. Genetic variability of the Egyptian (EG) and Kenyan (KE) S. mansoni populations. Schistosoma mansoni populations Damietta (EG) (n 5 17)
Asao (KE) (n 5 49)
Carwash (KE) (n 5 39)
Asembo (KE) (n 5 24)
11 90.91 5.73 ± 0.89 0.57 ± 0.09 0.56 ± 0.08 0.58 ± 0.09
11 100 11.27 ± 2.19 0.69 ± 0.08 0.71 ± 0.07 0.71 ± 0.07
11 100 11.82 ± 2.22 0.68 ± 0.08 0.70 ± 0.07 0.71 ± 0.08
11 100 9.09 ± 1.47 0.68 ± 0.07 0.69 ± 0.07 0.71 ± 0.08
Loci Polymorphic loci (%) MNA ± SD Mean HO ± SD Mean HE ± SD Mean unbiased HE ± SD
TABLE IV. Pairwise population differentiation statistics for different S. mansoni populations from Egypt (EG) and Kenya (KE). FST estimates Schistosoma mansoni populations Damietta (EG) Damietta (EG) Damietta (EG) Asao (KE) Asao (KE) Carwash (KE)
.Asao (KE) .Carwash (KE) .Asembo (KE) .Carwash (KE) .Asembo (KE) .Asembo (KE)
RST estimates
FST
Lin FST
P*
RST
Lin RST
P*
0.169 0.170 0.194 0.005 0.003 0.000 (20.002){
0.204 0.206 0.240 0.005 0.003 0.000
0.010 0.010 0.010 0.030 0.200 0.430
0.372 0.279 0.370 0.000 (20.008){ 0.000 (20.013){ 0.000 (20.019){
0.592 0.387 0.586 0.000 0.000 0.000
0.010 0.010 0.010 0.300 0.320 0.390
* Probability values based on 99 permutations. { Negative FST and RST converted to zero.
genotypes can develop in individual snails of other Biomphalaria species (Eppert et al., 2002), so this aspect of the biology of S. mansoni–B. alexandrina associations deserves further study. Population genetic analyses were done by using GenAlEx v. 6.3 (http:// www.anu.edu.au/BoZo/GenAlEx; Peakall and Smouse, 2006). For the panel of 11 microsatellite loci, we included 17 S. mansoni genotypes from Damietta, 49 from Asao, 39 from Carwash, and 24 from Asembo. Except for locus D2 from Damietta, all loci were polymorphic, and the number of alleles per locus varied from 1 to 28. Private alleles (alleles unique to 1 population) were present in all populations. The Pearson’s chi-square test showed that some of the loci in the 4 different populations deviated from Hardy-Weinberg equilibrium (HWE) (Table II). These deviations are likely caused by small effective population size or may indicate that some of the parasites initiating the different infections were related, which may not be surprising if relatively few definitive hosts were contributing eggs to infect snails. The S. mansoni population from Damietta showed the lowest percentage of polymorphic loci, mean number of alleles/locus (MNA), mean observed heterozygosity (HO), mean expected heterozygosity (HE), and mean unbiased HE when compared with the 3 Kenyan populations (Table III). This suggests that the genetic variability in the studied Egyptian population of S. mansoni is smaller than that of the Kenyan populations. The genetic differentiation for the 4 populations was assessed by FST, linearized FST (Lin FST), RST, and Lin RST, and analyzed by AMOVA. All these indicators suggest the presence of genetic differentiation between the Egyptian population and each one of the Kenyan populations, and the P-values of AMOVA were significant (0.01 for each, based on 99 permutations). Relatively little microsatellite differentiation was found among the 3 Kenyan populations (Table IV). In all our pairwise population comparisons, the RST values were higher than the FST values, indicating a contribution of stepwise mutations to genetic differentiation. One possible explanation for both the single genotype infections and smaller degree of genetic variability noted in the Egyptian samples is the ongoing national schistosomiasis control program, which has conceivably achieved significant reductions in the genetic variability of S. mansoni through its persistent lowering of prevalence and intensity in humans and snails. The difficulty we experienced in finding infected snails in Egypt further supports this supposition. In contrast, in Kenya, estimates indicate that more than 10% of the human population is infected with schistosomiasis (Toomey, 1986; Brooker et al., 2009), with transmission occurring unimpeded across broad geographic areas. The present results
argue for more studies to further explore the possibility that schistosome genotypic diversity, including within individual infected snails, can serve as a valuable indicator of the progress of Schistosoma control programs, to go along with more traditional measures of prevalence in either human or snail populations. We would like to thank the field teams of the Schistosomiasis Control Program of the Ministry of Health and Population in the Damietta Governorate for their help in the malacological survey, and George Rosenberg from the Molecular Biology Facility at the University of New Mexico, supported by National Institutes of Health (NIH) grant 1P20RR18754 from the IDeA program of the National Center for Research Resources. This work was supported by funds provided by the US-Egypt Joint Science and Technology Fund, grant BIO9-005-002 (W.M.L. and E.S.L.). This research was supported by the Kenya Medical Research Institute (KEMRI) and is published with the approval of the Director, KEMRI.
LITERATURE CITED AHMED, A. H., A. RUPPEL, AND R. M. RAMZY. 2003. A longitudinal study of schistosome intermediate host snail populations and their trematode infection in certain areas of Egypt. Journal of the Egyptian Society of Parasitology 33: 201–217. ALLAM, A. F., L. M. ABOU-BASHA, A. SALEM, N. F. LOUTFY, AND H. K. BASSIOUNY. 1989. Prevalence of the larval trematodes in Biomphalaria alexandrina in the vicinity of Alexandria. Journal of the Medical Research Institute 10: 51–62. ANDERSON, R. M., AND D. M. GORDON. 1982. Processes influencing the distribution of parasite numbers within host populations with special emphasis on parasite-induced host mortalities. Parasitology 85: 373–398. BARRAL, V., S. MORAND, J. P. POINTIER, AND A. THE´RON. 1996. Distribution of schistosome genetic diversity within naturally infected Rattus rattus detected by RAPD markers. Parasitology 113: 511–517. BROOKER, S., N. B. KABATEREINE, J. L. SMITH, D. MUPFASONI, M. T. MWANJE, O. NDAYISHIMIYE, N. J. LWAMBO, D. MBOTHA, P. KARANJA, C. MWANDAWIRO ET AL. 2009. An updated atlas of human helminth infections: The example of East Africa. International Journal of Health Geographics 8: 42. CHU, K. Y., AND I. K. DAWOOD. 1970. Cercarial transmission seasons of Schistosoma mansoni in the Nile Delta area. Bulletin of the World Health Organization 42: 575–580.
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EPPERT, A., F. A. LEWIS, C. GRZYWACZ, P. COURA-FILHO, I. CALDAS, AND D. J. MINCHELLA. 2002. Distribution of schistosome infections in molluscan hosts at different levels of parasite prevalence. Journal of Parasitology 88: 232–236. LOVERDE, P. T., J. DE WALD, D. J. MINCHELLA, S. C. BOSSHARDT, AND R. T. DAMIAN. 1985. Evidence for host-induced selection in Schistosoma mansoni. Journal of Parasitology 71: 297–301. MINCHELLA, D. J., K. M. SOLLENBERGER, AND C. PEREIRA DE SOUZA. 1995. Distribution of schistosome genetic diversity within molluscan intermediate hosts. Parasitology 111: 217–220. SIRE, C., P. DURAND, J. POINTER, AND A. THERON. 1999. Genetic diversity and recruitment pattern of Schistosoma mansoni in a Biomphalaria glabrata snail population: A field study using random amplified polymorphic DNA markers. Journal of Parasitology 85: 436–441. STEINAUER, M. L., L. E. AGOLA, I. N. MWANGI, G. M. MKOJI, AND E. S. LOKER. 2008. Molecular epidemiology of Schistosoma mansoni: A robust, high-throughput method to assess multiple microsatellite markers from individual miracidia. Infection, Genetics and Evolution 8: 68–73. ———, B. HANELT, L. E. AGOLA, G. M. MKOJI, AND E. S. LOKER. 2009. Genetic structure of Schistosoma mansoni in western Kenya: The effects of geography and host sharing. International Journal for Parasitology 39: 1353–1362.
159
———, I. N. MWANGI, G. M. MAINA, J. M. KINUTHIA, M. W. MUTUKU, E. L. AGOLA, B. MUNGAI, G. M. MKOJI, AND E. S. LOKER. 2008. Interactions between natural populations of human and rodent schistosomes in the Lake Victoria region of Kenya: A molecular epidemiological approach. PLoS Neglected Tropical Diseases 2: e222. TOOMEY, G. 1986. Kenyan community fights schistosomiasis. IDRC Reports 3: 8. TRUETT, G. E., P. HEEGER, R. L. MYNATT, A. A. TRUETT, J. A. WALKER, AND M. L. WARMAN. 2000. Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). BioTechniques 29: 52–53. WORLD HEALTH ORGANIZATION (WHO). 2001. Report of the informal consultation on schistosomiasis in low transmission areas: Control strategies and criteria for elimination, London, 10–13 April 2000. Document WHO/CDS/CPE/SIP/2001.1. World Health Organization, Geneva, Switzerland. WHO. 2005. Report of the scientific working group meeting on schistosomiasis meeting report. 14–16 November 2005. World Health Organization, Geneva, Switzerland. ———. 2007. Report of an inter-country meeting on strategies to eliminate schistosomiasis from the Eastern Mediterranean region. 6–8 November 2007, Muscat, Oman. World Health Organization, Geneva, Switzerland.
