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    Map of the Nile River and the Delta: (•) indicates sampling localities in which Biomphalaria snails were found. Note that because of their proximity, some of the collection localities fall under the same dots in the figure (see Table 2 for more details).

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    Different morphotypes of Biomphalaria and Helisoma duryi snails from Egypt. (A–D, F, G) Biomphalaria snails from El-Mattar village (Alexandria), El-Atwany (Aswan), Nahia (Giza), Kafr Hakeem (Giza), El-Sail Unit (Aswan), and Village of Abadii Valley (Aswan), respectively, (E) B. glabrata laboratory adapted strain from TBRI, (H and I) Helisoma duryi from Village of Abadii Valley and El-Sail Unit (Aswan).

  • View in gallery

    Representative results for species-specific PCR assays. Agarose gel electrophoresis of PCR products using species-specific primers to amplify B. alexandrina ITS1 (A), B. glabrata ITS2 (B), B. alexandrina ND1 (C), and B. glabrata ND1 (D). Lanes 1–10: Biomphalaria snails from 10 different field localities. Lane 11: B. alexandrina laboratory strain. Lane 12: B. glabrata laboratory strain. Lane M: 100-bp DNA ladder. The results shown were identical for all 248 individual snails assayed.

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A MOLECULAR SURVEY OF BIOMPHALARIA IN EGYPT: IS B. GLABRATA PRESENT?

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  • 1 Parasitology Department, Medical Research Institute, El-Hadara, Alexandria, Egypt; Department of Biology, University of New Mexico, Albuquerque, New Mexico; Department of Environmental Researches and Medical Malacology, Theodor Bilharz Research Institute (TBRI), Imbaba, Giza, Egypt

Two species of Biomphalaria are reported from Egypt, the indigenous Biomphalaria alexandrina and Biomphalaria glabrata, the latter believed to be introduced during the past few decades. Both are known to be excellent hosts of Schistosoma mansoni, the human-infecting blood fluke common in Egypt. Given the concerns regarding the spread of the exotic B. glabrata, this study was carried out to get a more current picture of the status of Biomphalaria in Egypt. Snail collections were undertaken during 2002–2003 from regions between Alexandria and Ismailia in the north of the Nile Delta, to as far south as Abu Simbel at Lake Nasser. Biomphalaria snails were found in 37 out of 76 sampled localities and were widely distributed in the Nile Delta and along the Nile as far south as Aswan. According to the results of species-specific polymerase chain reaction assays that sampled both nuclear and mitochondrial genomes, and according to DNA sequence data, all Biomphalaria collected during this survey were B. alexandrina. There was no evidence of the presence of B. glabrata or of hybridization of B. alexandrina with B. glabrata in the examined sites. The results were surprising given that some field-collected snails strongly resembled B. glabrata in both size and conchology and that previous survey work suggested B. glabrata had established in Egypt. Continued scrutiny to ascertain the possible presence of B. glabrata in Egypt is warranted. Also, the planorbid Helisoma duryi was detected in the Delta and as far south as Aswan, so it is important for Egyptian schistosomiasis workers to accurately distinguish this non-schistosome-transmitting snail from Biomphalaria.

INTRODUCTION

Schistosomiasis, the most important parasitic disease in Egypt, has plagued its people since ancient times.1,2 Control of schistosomiasis in Egypt was the goal of an ambitious 10-year program coordinated by the Egyptian Ministry of Health and Population and the U.S. Agency for International Development, which was concluded in 1998.3 Although significant progress was undeniably made in the control of schistosomiasis, particularly urinary schistosomiasis, one of the striking findings of this study was that in parts of Egypt, especially in the Nile Delta, the prevalence of the related schistosome, Schistosoma mansoni, remains quite high. Furthermore, S. mansoni is replacing Schistosoma haematobium in the Delta and has become well established in the past decade in Middle Egypt4 and has been reported in parts of Upper Egypt.56 Based on these considerations, it appears that S. mansoni is likely to remain of public health significance in Egypt for the foreseeable future.

One of the keys to understanding the present and future of S. mansoni in Egypt is to understand more about the freshwater snails that play an indispensable role in its transmission. Biomphalaria alexandrina has been historically implicated in transmission of S. mansoni in Egypt.7 All African Biomphalaria are relatively recent species, at most 5 million years old but probably much more recent.810 Biomphalaria alexandrina in particular is very closely related to several other African Biomphalaria species that compose a Nilotic species complex that is found in the Nile River basin.10 Biomphalaria alexandrina has historically been confined to the Nile Delta and separated from other Biomphalaria species by hundreds of kilometers, although there are reports of B. alexandrina from one location in Libya and in the Sudan between Khartoum and Kosti.11 There are some indications that a larger geographic range existed in wetter periods, as fossilized shells of B. alexandrina were detected in a Paleolithic site in the Egyptian Western Desert12 and in a Neolithic site in Sinai.13 However, at late Paleolithic sites in the Nile valley in Upper Egypt (Edfu and Esna), fossilized shells of other freshwater snails, including Bulinus truncatus, but not B. alexandrina, have been recovered,14 which may indicate that B. alexandrina was not present in Upper Egypt in that time. In very recent times, B. alexandrina appears to be expanding its range upstream in Egypt. In the late 1970s and 1980s, the snail was found at increasing distances upstream, as far as Aswan City and Abu Simbel at Lake Nasser, respectively.15,16 Changes in the hydrology of the Nile basin, controlled water flow, and new irrigation networks following construction of the Low and High Dams at Aswan, in 1902 and 1968, respectively, have been implicated in increasing appropriate habitats for the snail.15,17

The situation with respect to Biomphalaria in Egypt has become complicated in recent years by the introduction of B. glabrata.9,1822 This large snail is the most widespread and important intermediate host of S. mansoni in the Neotropics. In 1996, snails identified as Biomphalaria glabrata by conchological and morphologic criteria were found along many kilometers of irrigation canals and drains in Giza, in Qalyoubia Governorate in the south of the Nile Delta, and in Kafr El-Sheikh Governorate in the north-central Nile Delta.19 In 1999, snails considered to be hybrids between B. glabrata and the indigenous B. alexandrina were reported to be widespread throughout the Nile Delta.2022 Experimentally, both B. glabrata and hybrids were found to be susceptible to Egyptian strains of S. mansoni but showed lower susceptibilities than B. alexandrina. However, the duration of cercarial shedding was longer and the numbers of cercariae shed per snail were higher in B. glabrata and hybrid snails than in B. alexandrina.21 This may indicate that the introduced B. glabrata and the hybrid snails are more hazardous than B. alexandrina in the transmission of S. mansoni. In addition to complicating the epidemiology of schistosomiasis in Egypt, B. glabrata can also be viewed as an invasive exotic that threatens the integrity of the African aquatic biota.23

A series of detailed studies of the epidemiology of schistosomiasis in Egypt (see the review by El-Khoby and others24) give only minor consideration to the snail hosts.24 Surveys documenting the spread of B. alexandrina up the Nile were last done in the early 1990s.15 Given the changing ecological landscape of modern Egypt, the apparent invasion of another excellent schistosome snail host, and the limited survey information currently available, it is important to achieve a more current picture of the status of Biomphalaria in Egypt.

Most snail surveys studying Biomphalaria in Egypt have used morphologic methods for identification,16,19,20 and only one study used allozymes but did not consider B. glabrata.15 Using known nuclear and mitochondrial sequences for Biomphalaria spp. and newly developed polymerase chain reaction (PCR)-based assays,25 this study was carried out to identify the species of Biomphalaria present in Egypt, to assess the current distribution of B. alexandrina, B. glabrata, and their possible hybrids, and to examine further the nature and extent of hybridization if hybrids were found. Also, sequence data were used to assess the extent of genetic variation in B. alexandrina.

MATERIALS AND METHODS

Study area.

We searched for Biomphalaria snails during 2002–2003 in the area between Alexandria and Ismailia in the north of Egypt and along the course of the Nile as far south as Abu Simbel at Lake Nasser. The specific goals of this study were to assess the possible presence of B. glabrata in Egypt, to begin to determine how widely this exotic snail might be distributed, and to learn if B. glabrata was possibly hybridizing with the indigenous species, B. alexandrina. Our goals were best served by obtaining samples of Biomphalaria from as many different localities around Egypt as possible, with an emphasis on areas where B. glabrata had previously been reported, such as in Giza, Qalyoubia, and Kahr El-Sheikh Governorates. Our goals were not to assess levels of infection with S. mansoni, to provide a comprehensive survey of where Biomphalaria snails can or cannot be found throughout Egypt, to obtain extensive samples from each positive locality, or to assess the relative or absolute abundance of Biomphalaria spp. at the various collection localities we sampled. Because Biomphalaria populations are often difficult to locate, we enlisted the assistance of local experts in the Snail Control Section, Ministry of Health and Population, who knew where to find snails of this genus. We were also assisted in location of Biomphalaria populations by Dr. Fouad Yousif of the Theodor Bilharz Research Institute (TBRI). In addition, to increase the breadth of our sampling, we also searched for snails in water bodies we encountered along the roads we traveled in the Delta and along the Nile. Our standard approach for searching for Biomphalaria in a particular habitat was to walk along the shoreline and directly inspect vegetation and other objects in the water for snails. We also used a long-handled wire-meshed net to scoop snails from the water or vegetation. Each habitat was searched for a minimum of 1 hour by at least two experienced snail collectors. A total of 76 sites were searched, most of which were irrigation and drainage systems in the Nile Delta and Valley. A Garmin e-Trex Summit GPS (global positioning system) was used to mark the geographic coordinates of each field snail population.

Specimen collection.

At least 20 Biomphalaria were collected from each location, but in some cases Biomphalaria snails were rare and we were unable to collect this number. Collected samples were preserved in 70% ethanol, which was replaced at least two times. Snails were stored in 70% ethanol at 4°C. As indicated below, assuming they were available, at least six snails per population were singled out for molecular study, and the remaining snails were retained in our collections should they be needed for future study. In addition to the field samples, B. alexandrina and B. glabrata laboratory reference stocks from TBRI were included in this study. The nuclear first internal transcribed spacer (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (ND1) sequences for these two laboratory stocks (GenBank AY622607–AY622610) were reported by Lotfy and others.25 It is important to note that the laboratory strain of B. glabrata was originally collected from the field from Qalyoubia Governorate, Egypt, in 1999 by Dr. Yousif and maintained in his laboratory.

Conchological measurements.

Shell height and diameter were measured, and number of whorls was counted for each studied snail. If possible, snails chosen for molecular analysis were among the largest snails of each population, as B. glabrata and putative hybrids are larger than B. alexandrina.19

DNA extraction.

Snail DNA was extracted from the muscular head-foot region by the alkalinelysis (HotSHOT) method described by Truett and others, in a final volume of 400 μL of storage buffer.26

Species-specific PCR.

For 33 of the 37 localities positive for Biomphalaria, at least six snails were examined with species-specific PCR. The number of snails sampled was increased in localities suspected to harbor B. glabrata or hybrids based on the size or appearance of the shell. For the remaining four localities, the total number of snails we were able to find was less than six, so we sampled all the available specimens. The species-specific PCR technique for B. alexandrina and B. glabrata was developed, tested, and reported by Lotfy and others.25 The purpose of the technique is to amplify in a sensitive and specific way either ITS or ND1 of either B. alexandrina or B. glabrata. The primers designed to amplify B. alexandrina ITS1 or ND1 did not amplify B. glabrata ITS or ND1 under any conditions tested, nor did ITS2 or ND1 primers developed for B. glabrata amplify from any B. alexandrina template tested.25 The primers succeeded in detection of appropriate target DNA in amounts as low as 0.226 ng, even when mixed with nontarget DNA in 1,000-fold excess.25 Individual snails that are one of these two species and not hybrids can be easily identified in this way. If an individual was a hybrid of or a descendent of a hybrid, then PCR products would be expected using primer pairs (particularly ITS primers) devised for both species.

The details of this technique are as follows: one primer pair (AITS1F1 and AITS1R1) was designed to amplify a 316-bp fragment of B. alexandrina ITS1, while a second primer pair (GITS2F1 and GITS2R1) was designed to amplify an approximately 361-bp fragment of B. glabrata ITS2. Primer pairs were also designed to amplify a 265-bp fragment of ND1 for each species: AND1F2 and AND1R2 for B. alexandrina, and GND1F2 and GND1R2 for B. glabrata (Table 1). The volume of each amplification reaction was 20 μL with 4 μL (approximately 200 ng) of DNA, 0.8 mM dNTPs, 2 mM Mg Cl2, 0.5 μM of each primer, 0.5 unit Taq DNA Polymerase (Promega, Madison, WI), and buffer. For B. alexandrina ITS1 and B. glabrata ITS2 primers (AITS1F1, AITS1R1, GITS2F1, and GITS2R1), the thermocycler (Whitman Biometry T Gradient, Labrepco, Horsham, PA) was programmed as follows, with a 1°C per second rate of change: 1 cycle of 95°C for 1 minute, 62°C for 2 minutes, and 74°C for 1 minute 30 seconds, followed by 30 cycles of 95°C for 30 seconds, 62°C for 30 seconds, 74°C for 1 minute 30 seconds, plus a final extension step for 7 minutes. For ND1, the conditions were identical, except the annealing temperature was 49°C for the B. alexandrina AND1F2 and AND1R2 primers and 60°C for the B. glabrata GND1F2 and GND1R2 primers. Amplified products were analyzed by electrophoresis in 1.5% agarose gels and detected by staining with ethidium bromide. All PCR reactions and other molecular analyses, including DNA sequencing (see below), were undertaken the Biology Department, University of New Mexico.

DNA sequencing.

DNA sequencing was carried out to confirm the species-specific PCR diagnosis and to study any possible intraspecific variations among the different populations. At least one snail from each population was studied by DNA sequencing. The number was increased from localities suspected to harbor B. glabrata or hybrids. Two DNA regions were amplified and sequenced: nuclear rDNA internal transcribed spacer 1 (ITS1, 550–552 bp; GenBank AY628810-AY628859) and partial mitochondrial NADH dehydrogenase subunit 1 (ND1, 446 bp; GenBank AY628767-AY628809). Primers used to amplify ITS1 were ITS1.2F and ITS1.2R, and mitochondrial DNA ND1 was amplified by using SND1F2 and SND1R4 primers.27 PCR products were purified using PCR Microcon columns (Millipore, Billerica, MA) and sequenced on both strands at least once using an Applied Bio-systems 3100 automated sequencer (BigDye terminator cycle sequencing kit, ABI, Foster City, CA).

During the survey work, snails readily identifiable as Helisoma duryi based on conchological criteria11 were found at some localities. To provide an alternative confirmation that these snails were of the genus Helisoma and not unusual Biomphalaria specimens, we amplified and sequenced the ITS1 region (using ITS1.2F and ITS1.2R primers) of two such individuals, one from the Nile Delta and one from Upper Egypt (Mutubas in Kafr El-Sheikh and El-Sail Unit in Aswan City, respectively). These sequences were deposited in Gen-Bank (AY628860–AY628861).

RESULTS

Biomphalaria snails were found in 37 of 76 sites searched and were found as far south as Aswan City (Figure 1). Although our primary purpose was not to delineate where Biomphalaria snails were not found, it is nonetheless noteworthy that we did not find Biomphalaria in two important potential snail habitats, around the High Dam (Western Port area) or in Lake Nasser at Abu Simbel (Old Port area and Manshiat El-Noba Pocket).

Biomphalaria from Egypt exhibit strikingly different morphotypes (Figure 2), with some snails, such as those from Kafr Hakeem (Figure 2D) having a diameter and height comparable to laboratory B. glabrata (20.75 ± 1.26 mm, 5.85 ± 0.17 mm versus 18.00 ± 1.67 mm, 5.33 ± 0.54 mm, respectively). Other snails, such as those from El-Sail Unit (Figure 2F) are small in diameter (10.50 ± 0.55 mm) but thick (4.55 ± 0.23 mm) and may be confused with small H. duryi (Figures 2H and 2I), as the diameter and height of H. duryi in the same location were 17.83 ± 3.54 mm and 7.68 ± 1.71 mm, respectively.

A total of 248 snails from 37 sites were studied by using species-specific PCR. Representative results of the species-specific PCR assays are shown in Figure 3. According to the results of these assays, all the Biomphalaria snails collected from the field were B. alexandrina, and there was no evidence of the presence of B. glabrata or of hybrids between B. alexandrina and B. glabrata (Table 2). Positive and negative controls (using laboratory strains of each species) gave the expected results in all assays.

Also, among all 37 sites, all sequence data obtained were consistent with those of B. alexandrina. Each ITS1 and ND1 sequence was identical or nearly identical to the laboratory reference stock of B. alexandrina, and they were easily distinguished from B. glabrata sequences. The sequence chromatograms were closely scrutinized for evidence of both species’ DNA, as might be expected in hybrids, but no mixed peaks were found, even for the many nucleotide sites (18 substitutions and 14 indels in ITS1; 70 substitutions in ND1) at which B. alexandrina and B. glabrata showed differences.

Remarkably, there were few intraspecific differences among the B. alexandrina sequences obtained from the 37 field sites. For both ITS1 and ND1, sequences from 35 of the 37 sites were identical to sequences for the laboratory B. alexandrina. For ITS1, the sequences obtained from Abu Homos (Behira) and El-Mahallah El-Kubra (Gharbia) differed by one indel (2 bp) and one transversional substitution. For ND1, the sequence from Rosetta (Behira) differed by two transitions and one transversion, and the sequence from El-Atwany (Aswan) differed by two transitions. Thus, very little phylogenetic signal was present in the data.

Snails conchologically identified as H. duryi were found at Kafr El-Sheikh (Northwestern Delta), Ismailia (Eastern Delta), Qalyoubia (Southern Delta), and Aswan (Upper Egypt). ITS1 sequences of these snails from Kafr El-Sheikh and Aswan confirmed their identity as Helisoma. These sequences were much more similar to the sequences of H. trivolvis (GenBank AY030403), the only Helisoma ITS1 sequence available, than to sequences of any Biomphalaria species.

DISCUSSION

According to the current results, Biomphalaria snails remain common in Egypt, being collected across the width and length of the Delta, and southwards along the Nile River as far as Aswan City. In contrast to the study of Sattman and Kinzelbach,16 we did not find Biomphalaria in Lake Nasser, but we are quick to note that we were able to search the shoreline of the lake for snails only at Abu Simbel. A complete lack of Biomphalaria from the vast reaches of Lake Nasser is hard to imagine, particularly given that Biomphalaria pfeifferi, Biomphalaria sudanica, and even Biomphalaria alexandrina have been reported from northern Sudan.28 The lack of Biomphalaria at Abu Simbel could also be due to the effective control measures and the continuous monitoring carried out by the snail control field workers of the Egyptian Ministry of Health and Population in these locations.

The different morphotypes exhibited by B. alexandrina in Egypt may be a consequence of the conditions prevailing in the different environments they occupy (Figure 2).29 These morphologic variations pose difficulties in species identification.29 The species-specific PCR assays developed by Lotfy and others proved useful for differentiation between B. alexandrina and B. glabrata.25 The results of these species-specific PCR assays and of ITS1 and ND1 sequencing data show that the Biomphalaria snails collected from the field were B. alexandrina. We found no evidence for the presence of B. glabrata or of hybrids of B. alexandrina with B. glabrata, even though 19 of our samples (including 8 samples from Giza, Qalyoubia, and Kafr El-Sheikh Governorates) came from the Nile Delta or nearby, where both B. glabrata and hybrids had been previously reported.19,20

Although B. glabrata was not detected among our collections, the past introduction of this species was confirmed by a previous molecular study.9 Also, the snails collected by Dr. Yousif (TBRI) from Qalyoubia in 1999 and maintained in his laboratory were confirmed to be B. glabrata by species-specific PCR (Figure 3) and DNA sequencing (AY622608, AY622610). Our results do not exclude the current presence of B. glabrata in Egypt, but they do suggest that B. glabrata is not common. Also, after the presence of B. glabrata in the Nile Delta was reported,1822 the Snail Control Section in the Egyptian Ministry of Health and population was alerted and reacted strongly by applying molluscicides in putative B. glabrata habitats. In the current study, most of the localities previously reported to harbor B. glabrata in Egypt were searched and found negative. The ND1 sequence of the Egyptian laboratory strain of B. glabrata was also compared with the many neotropical B. glabrata ND1 sequences reported by DeJong and others,27 who found these sequences to be highly variable. Interestingly, the Egyptian laboratory strain of B. glabrata has an ND1 sequence that is identical to that of three common laboratory strains of Brazilian heritage (10-R2, 13-16-R1, and M-line; GenBank AY198032, AY198034, AY198035). This finding supports the suggestion that the introduction of B. glabrata into Egypt was the result of the accidental release of laboratory snails.18

The extremely low amount of variation among the B. alexandrina sequences suggests that this species may have had a restricted geographic range in the past and may now be expanding its range. However, because the sequence variability is so low, there is very little phylogeographic signal to indicate the direction of B. alexandrina expansion. If B. alexandrina has expanded southwards from the Delta, as has been supported by allozyme data,15 then the most diversity would be expected in the Delta, with the least amount of diversity in Upper Egypt. In our study, three of the four variant sequences were from the Delta, one was from Upper Egypt, and no variant sequences were found in Middle Egypt. The fact that 18 of our 37 sampled populations were from Middle and Upper Egypt and only one variant sequence was detected provides at least weak support for the southward expansion hypothesis.

Helisoma duryi is also an introduced snail in Egypt, in this case from North America. It has been studied in field trials as a competitor of intermediate hosts of schistosomes in Egypt.30 It was first found in Egypt in 1980 and 1981 a few kilometers north of Cairo.31,32 A 1993 study described this snail’s range extension further north into the Nile Delta,33 but more recent descriptions of the distribution are not available in the literature. Our results indicate the distribution of H. duryi has further extended as it was found in Kafr El-Sheikh (north-central Delta), in Ismailia (eastern Delta), and more surprisingly it was found in Aswan City. A negative correlation between H. duryi and schistosomiasis snail hosts, Biomphalaria and Bulinus, has previously been noted in Egypt,33 so H. duryi may negatively affect the distribution of Biomphalaria snails in the future. During the field activities of this study, it was noticed that some Egyptian field workers misidentified H. duryi as B. glabrata. Similar reports of misidentification of H. duryi as Biomphalaria in other parts of the world have been documented.34 Helisoma duryi is distinguishable from Biomphalaria by having a higher shell, more angular whorls, a flat surface within the umbilicus, and a deeply concave upper side.35,36 Previous published reports of B. glabrata from Egypt do not represent misidentified H. duryi because the two species are conchologically distinct and readily told apart by experienced workers. Furthermore, previous reports of Egyptian B. glabrata refer to anatomically distinct features such as a renal ridge possessed by B. glabrata but not by H. duryi.19 Further studies are needed to evaluate the distribution of H. duryi in Egypt and its impact on the aquatic biota.

In conclusion, this survey found no evidence for B. glabrata in Egypt, so if it is present, it is uncommon. Biomphalaria alexandrina does remain common, and no evidence for hybridization with B. glabrata was found. Further monitoring of this situation is warranted.

Table 1

Species-specific primers used in the current study

Amplified regionForward primerReverse primer
B. alexandrinaAITS1F1AITS1R1
    ITS1 species-specific primers5′-TTG CTA TCG ACG ATA ACA GCA C-3′5′-AGG GGC ATA GGT ACC CTG GAA C-3′
B. glabrataGITS2F1GITS2R1
    ITS2 species-specific primers5′-CTG CTG GTG TTA TGG GTT TCC C-3′5′-CCG ATC TGA GGT CGG AGA TTA A-3′
B. alexandrinaAND1F2AND1R2
    ND1 species-specific primers5′-TTG AAT AAT TTA TCC TAG TAT TTA TAG TG-3′5′-GAA ATA TAA TTG AAT AAC TTG TTA A-3′
B. glabrataGND1F2GND1R2
    ND1 species-specific primers5′-CTG AGT AAT CTA CCC TAG TGT TTA TTG CA-3′5′-AA AAT ATG ACG GAA TAC CCT TTA AG-3′
Table 2

Summary of molecular results obtained for Biomphalaria snails from Egypt

Species-specific PCR (ITS1 and ND1)
for B. alexandrinafor B. glabrataDNA sequences
Location of snail populationNPositiveNegativePositiveNegativeITS1 (N)*ND1 (N)†
* GenBank Accession Numbers: AY628810–AY628859.
† GenBank Accession Numbers: AY628767–AY628809.
Alexandria
    El-Mattar village (31°11.469′N, 29°57.413′E)66006B. alexandrina (1)B. alexandrina (1)
Behira
    Rosetta (31°24.797′N, 30°25.624′E)44004B. alexandrina (1)B. alexandrina (1)
    Kafr El-Dawwar (31°11.821′N, 30°4.109′E)66006B. alexandrina (1)B. alexandrina (1)
    Kafr El-Dawwar (31°11.752′N, 30°4.272′E)11110011B. alexandrina (5)B. alexandrina (1)
    Damanhur (31°08.649′N, 30°22.004′E)66006B. alexandrina (1)B. alexandrina (1)
    Abu Homos (31°06.458′N, 30°17.687′E)66006B. alexandrina (1)B. alexandrina (1)
Gharbia
    El-Mahallah El-Kubra (31°05.840′N, 31°08.943′E)66006B. alexandrina (1)B. alexandrina (1)
    Tanta (30°47.867′N, 30°57.266′E)66006B. alexandrina (1)B. alexandrina (1)
    El-Santa (30°44.997′N, 31°07.447′E)66006B. alexandrina (1)B. alexandrina (1)
Kafr El-Sheikh
    Sidi Salem (31°16.588′N, 30°47.881′E)11110011B. alexandrina (1)B. alexandrina (1)
Dakahlia
    Gamasa (31°25.691′N, 31°33.111′E)66006B. alexandrina (1)B. alexandrina (1)
Ismailia
    El-Manayef (30°32.735′N, 32°12.610′E)66006B. alexandrina (1)B. alexandrina (1)
Qalyoubia
    Kafr Shokr (30°33.614′N, 31°15.422′E)66006B. alexandrina (1)B. alexandrina (1)
    Shubra Shehab (30°16.697′N, 31°6.938′E)66006B. alexandrina (1)B. alexandrina (1)
    El-Barada (30°13.900′N, 31°9.409′E)66006B. alexandrina (1)B. alexandrina (1)
    Kalyoub (30°13.896′N, 31°9.469′E)66006B. alexandrina (1)B. alexandrina (1)
Giza
    Kafr Hakeem (30°4.850′N, 31°6.983′E)55005B. alexandrina (1)B. alexandrina (1)
    Nahia (30°3.773′N, 31°8.554′E)17170017B. alexandrina (6)B. alexandrina (6)
    El-Saff (29°45.447′N, 31°18.041′E)66006B. alexandrina (4)B. alexandrina (1)
Fayoum
    Etsa (29°14.156′N, 30°48.971′E)66006B. alexandrina (1)B. alexandrina (1)
Bani Sweif
    Nasser (29°10.747′N, 31°9.134′E)11110011B. alexandrina (1)B. alexandrina (1)
Menia
    Abeuha (27°57.450′N, 30°49.804′E)66006B. alexandrina (1)B. alexandrina (1)
    Abu-Korkas El-Balad (27°55.904′N, 30°49.981′E)11110011B. alexandrina (1)B. alexandrina (1)
Sohag
    El-Kother (26°34.991′N, 31°46.200′E)66006B. alexandrina (1)B. alexandrina (1)
    Needa (26°36.524′N, 31°43.568′E)44004B. alexandrina (1)B. alexandrina (1)
Qena
    Dandara (26°9.989′N, 32°39.078′E)66006B. alexandrina (1)B. alexandrina (1)
    Dandara (26°9.265′N, 32°39.792′E)66006B. alexandrina (1)B. alexandrina (1)
Luxor
    Naga El-Wehda, Nile River (25°37.567′N, 32°35.237′E)66006B. alexandrina (1)B. alexandrina (1)
    Naga El-Wehda (25°36.325′N, 32°33.961′E)11110011B. alexandrina (1)B. alexandrina (1)
Aswan
    El-Atwany, Edfu (25°00.949′N, 32°53.055′E)66006B. alexandrina (1)B. alexandrina (1)
    Village of Abadii Valley, Edfu (25°00.344′N, 32°55.778′E)66006B. alexandrina (1)B. alexandrina (1)
    Village of Abadii Valley, Edfu (24°59.911′N, 32°55.875′E)66006B. alexandrina (1)B. alexandrina (1)
    El-Nozel village, Edfu (24°59.210′N, 32°54.036′E)66006B. alexandrina (1)B. alexandrina (1)
    Azbet El-Basaaly, Edfu (24°56.966′N, 32°55.088′E)66006B. alexandrina (1)B. alexandrina (1)
    El-Sabeel, Kom Ombo (24°28.821′N, 32°55.383′E)66006B. alexandrina (1)B. alexandrina (1)
    Kassel, Kom Ombo (24°28.796′N, 32°55.690′E)11001B. alexandrina (1)B. alexandrina (1)
    El-Sail Unit, Aswan City, Nile River (24°06.743′N, 32°53.903′E)66006B. alexandrina (2)B. alexandrina (2)
Figure 1.
Figure 1.

Map of the Nile River and the Delta: (•) indicates sampling localities in which Biomphalaria snails were found. Note that because of their proximity, some of the collection localities fall under the same dots in the figure (see Table 2 for more details).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 1; 10.4269/ajtmh.2005.73.131

Figure 2.
Figure 2.

Different morphotypes of Biomphalaria and Helisoma duryi snails from Egypt. (A–D, F, G) Biomphalaria snails from El-Mattar village (Alexandria), El-Atwany (Aswan), Nahia (Giza), Kafr Hakeem (Giza), El-Sail Unit (Aswan), and Village of Abadii Valley (Aswan), respectively, (E) B. glabrata laboratory adapted strain from TBRI, (H and I) Helisoma duryi from Village of Abadii Valley and El-Sail Unit (Aswan).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 1; 10.4269/ajtmh.2005.73.131

Figure 3.
Figure 3.

Representative results for species-specific PCR assays. Agarose gel electrophoresis of PCR products using species-specific primers to amplify B. alexandrina ITS1 (A), B. glabrata ITS2 (B), B. alexandrina ND1 (C), and B. glabrata ND1 (D). Lanes 1–10: Biomphalaria snails from 10 different field localities. Lane 11: B. alexandrina laboratory strain. Lane 12: B. glabrata laboratory strain. Lane M: 100-bp DNA ladder. The results shown were identical for all 248 individual snails assayed.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 1; 10.4269/ajtmh.2005.73.131

*

Address correspondence to Eric S. Loker, Department of Biology, MSC03 2020, University of New Mexico, Albuquerque, New Mexico 87131-0001. E-mail: esloker@unm.edu

Authors’ addresses: Wael M. Lotfy, Parasitology Department, Medical Research Institute, 165 El-Horreya Avenue, El-Hadara, Alexandria, Egypt, E-mail: waelotfy@hotmail.com. Randall J. DeJong, Department of Biology, MSC03 2020, University of New Mexico, Albuquerque, New Mexico, 87131-0001, E-mail: rjdejong@unm.edu. Dr. Ahmed Abdel-Kader, Department of Environmental Researches and Medical Malacology, Theodor Bilharz Research Institute (TBRI), P.O. Box 30, Imbaba, Giza, Egypt, E-mail: abdelkaderahawary@yahoo.com. Eric S. Loker, Department of Biology, MSC03 2020, University of New Mexico, Albuquerque, New Mexico, 87131-0001, E-mail: esloker@unm.edu.

Acknowledgments: Sincere thanks are given to Prof. Dr. Fouad Yousif (Department of Environmental Researches and Medical Malacology, Theodor Bilharz Research Institute) for his kind assistance during the fieldwork and for supplying us with the laboratory reference stocks of B. alexandrina and B. glabrata. Special thanks are given to Ms. Brandee S. Black (Department of Biology, University of New Mexico) for her contribution to the laboratory activities of this work and to Mr. Mike T. Friggens (Department of Biology, University of New Mexico) for creating the map of Egypt. The authors thank also the staff of the Snail Control Section, Ministry of Health and Population, Egypt, for their help in the field and the Molecular Biology Facility at the University of New Mexico for their valuable assistance during the laboratory studies.

Financial support: This work was supported by the U.S-Egypt Joint Science and Technology Board, grant no. BIO6-002-017.

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Author Notes

Reprint requests: Eric S. Loker, Department of Biology, MSC03 2020, University of New Mexico, Albuquerque, New Mexico, 87131-0001, Telephone: 505-277-2496 or 9740, Fax: 505-277-0304, E-mail: esloker@unm.edu.
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