Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at the worldwide scale.

Population genetic studies are efficient for inferring the invasion history based on a comparison of native and invasive populations, especially when conducted at species scale. An expected outcome in invasive populations is variability loss, and this is especially true in self-fertilizing species. We here focus on the self-fertilizing Pseudosuccinea columella, an invasive hermaphroditic freshwater snail that has greatly expanded its geographic distribution and that acts as intermediate host of Fasciola hepatica, the causative agent of human and veterinary fasciolosis. We evaluated the distribution of genetic diversity at the largest geographic scale analysed to date in this species by surveying 80 populations collected during 16 years from 14 countries, using eight nuclear microsatellites and two mitochondrial genes. As expected, populations from North America, the putative origin area, were strongly structured by selfing and history and harboured much more genetic variability than invasive populations. We found high selfing rates (when it was possible to infer it), none-to-low genetic variability and strong population structure in most invasive populations. Strikingly, we found a unique genotype/haplotype in populations from eight invaded regions sampled all over the world. Moreover, snail populations resistant to infection by the parasite are genetically distinct from susceptible populations. Our results are compatible with repeated introductions in South America and flash worldwide invasion by this unique genotype/haplotype. Our study illustrates the population genetic consequences of biological invasion in a highly selfing species at very large geographic scale. We discuss how such a large-scale flash invasion may affect the spread of fasciolosis.

Sharing schistosomes: the elephant schistosome Bivitellobilharzia nairi also infects the greater one-horned rhinoceros (Rhinoceros unicornis) in Chitwan National Park, Nepal.

Because the digenetic trematode fauna of Nepal is poorly known, we began to search for schistosomes in and around Chitwan National Park (CNP) of southern Nepal. Both domestic and wild Indian elephants (Elephus maximus) are present, and we found one of two dung samples from wild elephants and 1 of 22 (4.5%) dung samples from domestic elephants to be positive for schistosome eggs. The morphology of the eggs and both cox1 and 28S sequences derived from the eggs/miracidia were consistent with Bivitellobilharzia nairi, reported here for the first time from Nepal. Also, 7 of 14 faecal samples from the Asian or greater one-horned rhinoceros (Rhinoceros unicornis) contained viable eggs indistinguishable from those of B. nairi. This identification was confirmed by comparison with both cox1 and 28S sequences from B. nairi eggs/miracidia derived from Nepalese and Sri Lankan elephants. This represents the first sequence-verified identification of a schistosome from any species of rhinoceros, and the first verified occurrence of a representative of Bivitellobilharzia (a genus of 'elephant schistosomes') in mammals other than elephants. Our work suggests that elephants and rhinos share B. nairi in CNP, even though these two members of the 'charismatic megafauna' belong to unrelated mammalian families. Their shared life style of extensive contact with freshwater habitats likely plays a role, although the snail intermediate host and mode of definitive host infection for B. nairi have yet to be documented. This report also supports Bivitellobilharzia as a monophyletic group and its status as a distinct genus within Schistosomatidae.

Molecular phylogenetics of the elephant schistosome Bivitellobilharzia loxodontae (Trematoda: Schistosomatidae) from the Central African Republic.

One of the most poorly known of all schistosomes infecting mammals is Bivitellobilharzia loxodontae. Nearly all of our available information about this species comes from the original description of worms that were obtained from an animal park-maintained elephant in Germany, probably a forest elephant Loxodonta cyclotis, originating from the present-day Democratic Republic of Congo. We obtained schistosome eggs from faecal samples from wild forest elephants from the Central African Republic. The eggs, which were similar in size and shape to those of described B. loxodontae, were sequenced for the 28S nuclear ribosomal gene and the mitochondrial cytochrome oxidase I (cox1) gene. In a phylogenetic analysis of 28S sequences, our specimens grouped closely with B. nairi, the schistosome from the Indian elephant Elephas maximus, to the exclusion of schistosomes from other genera. However, the eggs were genetically distinct (12% distance cox1) from those of B. nairi. We conclude the specimens we recovered were of B. loxodontae and confirm this is a distinct Bivitellobilharzia species. In addition to providing the first sequence data for B. loxodontae, this report also supports Bivitellobilharzia as a monophyletic group and gives the relative phylogenetic position of the genus within the Schistosomatidae. We also provide a review of the biology of this poorly known schistosome genus.

Schistosomes of small mammals from the Lake Victoria Basin, Kenya: new species, familiar species, and implications for schistosomiasis control.

Recent schistosomiasis control efforts in sub-Saharan Africa have focused nearly exclusively on treatment of humans with praziquantel. However, the extent to which wild mammals act as reservoirs for Schistosoma mansoni and therefore as sources of renewed transmission following control efforts is poorly understood. With the objective to study the role of small mammals as reservoir hosts, 480 animals belonging to 9 rodent and 1 insectivore species were examined for infection with schistosomes in Kisumu, in the Lake Victoria Basin, Kenya. Animals were collected from 2 sites: near the lakeshore and from Nyabera Marsh draining into the lake. A total of 6.0% of the animals captured, including 5 murid rodent species and 1 species of shrew (Crocidura olivieri) were infected with schistosomes. Four schistosome species were recovered and identified using cox1 DNA barcoding: S. mansoni, S. bovis, S. rodhaini and S. kisumuensis, the latter of which was recently described from Nyabera Marsh. Schistosoma mansoni and S. rodhaini were found infecting the same host individual (Lophuromys flavopunctatus), suggesting that this host species could be responsible for the production of hybrid schistosomes found in the area. Although the prevalence of S. mansoni infection in these reservoir populations was low (1.5%), given their potentially vast population size, their impact on transmission needs further study. Reservoir hosts could perpetuate snail infections and favour renewed transmission to humans once control programmes have ceased.

A new approach to characterize populations of Schistosoma mansoni from humans: development and assessment of microsatellite analysis of pooled miracidia.

OBJECTIVES: To develop and assess a microsatellite technique to characterize populations of Schistosoma mansoni from humans. METHODS: For each of five patients, we calculated the allele count and frequency at 11 loci for several pools of miracidia (50 and 100), and compared these to population values, determined by amplifying microsatellites from 186 to 200 individual miracidia per patient. RESULTS: We were able to detect up to 94.5% of alleles in pools. Allele count and frequency strongly and significantly correlated between singles and pools; marginally significant differences (P < 0.05) were detected for one patient (pools of 50) for allele frequencies and for two patients (pools of 100) for allele counts. Kato-Katz egg counts and number of alleles per pool did not co-vary, indicating that further direct comparisons of the results from these two techniques are needed. CONCLUSIONS: Allele counts and frequency profiles from pooling provide important information about infection intensity and complexity, beyond that obtained from traditional methods. Although we are not advocating use of pooling to replace individual genotyping studies, it can potentially be useful in certain applications as a rapid and cost effective screening method for studies of S. mansoni population genetics, or as a more informative way to quantify and characterize human worm populations.

Schistosoma kisumuensis n. sp. (Digenea: Schistosomatidae) from murid rodents in the Lake Victoria Basin, Kenya and its phylogenetic position within the S. haematobium species group.

Schistosoma kisumuensis n. sp. is described based on 6 adult males and 2 adult females collected from the circulatory system of 3 murid rodent species, Pelomys isseli, Mastomys natalensis, and Dasymys incomtus. Specimens were collected from a single location, Nyabera Swamp, in Kisumu, Kenya in the Lake Victoria Basin. This new species is morphologically similar to members of the S. haematobium group, currently represented by 8 species parasitizing artiodactyls and primates, including humans. Schistosoma kisumuensis differs from these species by producing relatively small Schistosoma intercalatum-like eggs (135.2 x 52.9 microm) with a relatively small length to width ratio (2.55). Comparison of approximately 3000-base-pair sequences of nuclear rDNA (partial 28S) and mtDNA (partial cox1, nad6, 12S) strongly supports the status of S. kisumuensis as a new species and as a sister species of S. intercalatum. The cox1 genetic distance between these two species (6.3%) is comparable to other pairwise comparisons within the S. haematobium group. Separation of the Congo River and Lake Victoria drainage basins is discussed as a possible factor favoring the origin of this species.

Schistosomes in the southwest United States and their potential for causing cercarial dermatitis or 'swimmer's itch'.

Cercarial dermatitis or swimmer's itch results when cercariae of schistosomes penetrate human skin and initiate inflammatory responses. The parasites typically die in the skin but in some cases may persist and infect other organs. Cercarial dermatitis is caused by a complex and poorly known assemblage of schistosome species, and can occur in any location where people come in contact with water bodies harbouring schistosome-infected snails. In North America, most cases are reported from the upper Midwest. In south-western USA, this phenomenon has not been well studied, and it is not known which schistosome species are present, or if cercarial dermatitis occurs with any regularity. As part of our ongoing studies of schistosome diversity, using morphological traits and sequence data to differentiate species, we have thus far identified eight schistosome genetic lineages from snails from New Mexico and Colorado. We have investigated two cercarial dermatitis outbreaks, one occurring in Stubblefield Lake in northern New Mexico, and one in Prospect Lake in the heart of Colorado Springs, Colorado. The New Mexico outbreak involved either one or two different avian schistosome species, both transmitted by physid snails. The Colorado outbreak was due to Trichobilharzia brantae, a species transmitted by geese and the snail Gyraulus parvus. These outbreaks are in contrast to those in northern states where schistosomes infecting snails of the family Lymnaeidae are more often responsible for outbreaks. Our survey suggests that dermatitis-causing schistosomes are not rare in the southwest, and that there are plenty of opportunities for dermatitis outbreaks to occur in this region.

Microsatellite typing reveals strong genetic structure of Schistosoma mansoni from localities in Kenya.

Genetic diversity and population structure of seven populations of Schistosoma mansoni sampled in Kenya were assessed using five microsatellite markers. The mean number of alleles per locus, expected heterozygosity in Hardy-Weinberg equilibrium and pairwise F(ST) values ranged from 5.2 to 10.7, 0.5-0.8 and 3.6-27.3%, respectively. These data reveal that S. mansoni populations in Kenyan have relatively high levels of genetic diversity and is significantly differentiated. Our data combined with information on biogeography support the hypothesis that the strong genetic structure in Kenyan schistosomes is as a result of limited gene flow and large population sizes. Resistance to anthelminthics has not been reported among the Kenyan schistosomes, we hypothesize that this is probably due to the very little gene flow among populations, thereby limiting opportunities for the spread of rare alleles that might confer resistance to the drugs.

A neotropical snail host of Schistosoma mansoni introduced into Africa and consequences for the schistosomiasis transmission Biomphalaria tenagophila in Kinshasa (Democratic Republic of Congo).

Malacological surveys carried out in the early 1970s in water bodies of the Kinshasa area, Lower Zaire (Democratic Republic of Congo), showed the appearance of a Biomphalaria species which was identified as Biomphalaria camerunensis. In 1976, other surveys confirmed the presence of the species in several sites and showed numerous infected snails with Schistosoma mansoni, demonstrating for the first time an active transmission of the parasite responsible of the intestinal schistosomiasis in this area. The most recent malacological sampling was carried out by one of us in 1994 in Mangungu River and revealed the presence of apparently the same snail species. However, conchological, anatomical and molecular studies showed that this snail may be considered as an introduced neotropical species, B. tenagophila. To our knowledge, this is the second example of the introduction of a neotropical snail host of schistosomes into Africa.

Digenean-gastropod host associations inform on aspects of specific immunity in snails.

Gastropod immunology is informed importantly by the study of the frequent encounters snails endure with digeneans (digenetic trematodes). One of the hallmarks of gastropod-digenean associations is their specificity: any particular digenean parasite species is transmitted by a limited subset of snail taxa. We discuss the nature of this specificity, including its immunological basis. We then review studies of the model gastropod Biomphalaria glabrata indicating that the baseline responses of snails to digeneans can be elevated in a specific manner. Studies incorporating molecular and functional approaches are then highlighted, and are further suggestive of the capacity for specific gastropod immune responses. These studies have led to the compatibility polymorphism hypothesis: the interactions between diversified fibrinogen-related proteins (FREPs) and diverse carbohydrate-decorated polymorphic parasite antigens determine recognition and trigger specific immunity. Complex glycan structures are also likely to play a role in the host specificity typifying snail-digenean interactions. We conclude by noting the dynamic and consequential interactions between snails and digeneans can be considered as drivers of diversification of digenean parasites and in the development and maintenance of specific immunity in gastropods.

Structure of two FREP genes that combine IgSF and fibrinogen domains, with comments on diversity of the FREP gene family in the snail Biomphalaria glabrata.

Upon exposure to infection with digenetic trematodes such as Echinostoma paraensei, the freshwater snail Biomphalaria glabrata produces increased quantities of hemolymph lectins, some of which are unique polypeptides containing both immunoglobulin superfamily (IgSF) and fibrinogen domains. These unusual lectins have been termed fibrinogen-related proteins (FREPs), and recognize and precipitate digenean antigens. We here report 11 distinct FREP-encoding sequences from B. glabrata, and provide the complete genomic sequence for two of the most frequently recovered FREPs. The unique juxtaposition of IgSF and fibrinogen domains, previously known only from incomplete cDNAs, is confirmed. Sequences corresponding to known peptides derived from FREPs from hemolymph were found in one of these genes. Both genes contain four exons, the first encodes a putative signal peptide, the second and third a portion of an IgSF-type loop, and the fourth a fibrinogen domain. Cysteines, postulated to form an intrachain loop, are present in the IgSF domain and are separated from one another by 78 or 79 residues. The IgSF sequences most closely resemble V (variable)-type Ig domains, based on canonical and hydrophobic residues and predicted secondary structure. Some minor differences in genomic fragments isolated for each of the two sequences were noted and may represent allelic variants. The results may be of relevance in understanding the role of B. glabrata in transmission of Schistosoma mansoni, a digenean parasite that infects nearly 100 million people in the tropics.

Carbohydrate-binding plasma proteins from the gastropod Biomphalaria glabrata: strain specificity and the effects of trematode infection.

As lectins are believed to mediate non-self-recognition in molluscs, carbohydrate-binding proteins (CBP) from the circulating plasma of the gastropod Biomphalaria glabrata were harvested by affinity chromatography using six different monosaccharides as ligands. Pools of plasma were derived from B. glabrata of either the M line strain, which is susceptible to infection with the PR1 strain of the digenetic trematode Schistosoma mansoni, or from the 13-16-R1 strain, which is resistant to infection. For each strain, plasma was obtained from control snails and from snails exposed to infection 1 or 8 days previously with S. mansoni or the related digenean, Echinostoma paraensei, which is able to develop in either host strain. For control snails, only minor interstrain differences were noted. In M line snails exposed 8 days previously to either parasite, marked changes in CBP populations were observed. Only E. paraensei infections produced comparable alterations in 13-16-R1 snails. The most conspicuous changes noted were the increased production of 80-120 kDa CBP in both strains, 150-210 kDa in 13-16-R1 snails, and 190-210 kDa in M line snails. The results demonstrate 1) interstrain differences in CBP, particularly following exposure to trematodes; 2) that infection provokes increased production and diversity of CBP that bind with greater affinity to the columns; and 3) that snails of the same strain respond differently to the two parasites used.

Plasma components which mediate cellular defences in the gastropod mollusc Biomphalaria glabrata.

Blood plasmas from certain strains of Biomphalaria glabrata are known to have components which facilitate a hemocyte-effected cytotoxic response against encapsulated Schistosoma mansoni sporocysts. The possible identity of the factor(s) has been investigated. Sporocysts placed in snail plasma rapidly acquire a wide variety of host plasma antigens, at least some of which are displayed on the parasite surface. Plasmas from strains of snail resistant to the parasite agglutinate fixed sporocysts, while plasmas from susceptible strains fail to do so. Fixed sporocysts incubated in plasma bind selectively a subpopulation of plasma antigens; some are bound uniquely in resistant plasma. Another resembles a hemagglutinin from snail plasma. These and other recently acquired data are discussed in light of increasing evidence for defensive roles of multivalent lectins.

In vitro encounters between Schistosoma mansoni primary sporocysts and hemolymph components of susceptible and resistant strains of Biomphalaria glabrata.

Encounters between axenically transformed sporocysts of the Puerto Rican 1 strain of Schistosoma mansoni and hemocytes and plasmaaa components of susceptible "M line" and resistant 10-R2 and 13-16-1 strains of Biomphalaria glabrata were studied in vitro. After 24 hours of incubation in susceptible hemolymph components, 94.4% of 36 observed sporocysts retained normal structure, whereas 9l.6% of 48 sporocysts were destroyed after 24 hours of incubation in resistant hemolymph components. Sporocysts preincubated for 6 hours in susceptible plasm were destroyed when subsequently incubated with resistant hemocytes in susceptible plasma. Sporocysts preincubated in resistant plasma were not destroyed when subsequently incubated with susceptible hemocytes and plasma. Susceptible hemocytes preincubated in resistant plasma, and then incubated for 24 hours with sporocysts in the presence of susceptible plasma were incapable of inflicting damage. The simultaneous incubation of sporocysts with susceptible hemocytes and resistant plasma produced variable results. The predominant trend was sporocyst destruction in five replicates and lack of substantial damage in six replicates. Results of these and previous experiments imply that differences between susceptible and resistant strains of snails in their ability to destroy sporocysts in vitro can be attributed primarily to differences in their hemocytes, although plasma factors may alter these responses in some cases. The results also suggest that if sporocysts do acquire a coat of host plasma components, this strategy is by itself insufficient to account for the persistence of sporocysts in susceptible snails.

PCR-RFLP analysis of the ITS2 region to identify Schistosoma haematobium and S. bovis from Kenya.

Schistosoma haematobium, primarily a human parasite, and the closely related Schistosoma bovis from ruminants, are sympatric in many African countries such as Kenya. Because these two species 1) can inhabit the same Bulinus snails, 2) may be found in the same freshwater habitat, and 3) have morphologically similar cercariae, better means are needed to tell them apart. The second internal transcribed spacer (ITS2) region of the ribosomal gene complex (rDNA) of recent Kenyan isolates of both species was sequenced and found to be a 98% match. The S. bovis sequences were nearly identical (99%) to conspecific sequences from Niger; the S. haematobium sequences were nearly identical (99%) to conspecific sequences from Egypt, Mali, and Niger. Restriction fragment length polymorphism (RFLP) analysis of a 480 base pair (bp) PCR product containing the ITS2 region using two restriction enzymes, Taq1 and Sau3A1, yielded species-specific fragment patterns that allowed successful identification of a single S. haematobium cercaria. The protocol outlined here is useful in providing a rapid, one-day identification of S. haematobium (and likely S. bovis) cercariae (the infective larval stage) and/or other life cycle stages in a basic molecular biology laboratory. By helping to determine whether schistosome-infected bulinid snails in a particular body of water are transmitting a human or an animal schistosome, or both, this analysis will aid in disease control and in ongoing epidemiological studies.

Control of schistosome-transmitting snails in Kenya by the North American crayfish Procambarus clarkii.

Snail-transmitted trematode parasites such as schistosomes and liver flukes assume considerable medical and veterinary significance in tropical Africa. We have observed a strong negative association between the presence of medically important pulmonate snails and the crayfish Procambarus clarkii in freshwater habitats in Kenya. This crayfish, introduced into Kenya around 1970, readily consumes these snails in the laboratory. Field enclosure experiments indicate that crayfish exert a significant negative impact on the abundance of Biomphalaria pfeifferi, the intermediate host of the human blood fluke Schistosoma mansoni. It is likely that P. clarkii will continue to spread naturally in Kenya and that schistosome-transmitting snails will be excluded or reduced in numbers where crayfish are present. Procambarus clarkii may represent an alternative, biological means of snail control in East Africa.

Impact of the crayfish Procambarus clarkii on Schistosoma haematobium transmission in Kenya.

The Louisiana red swamp crayfish, Procambarus clarkii, which was introduced into east Africa in the 1950s or 1960s, has since widely dispersed. Previous work by our group has shown that P. clarkii can reduce populations of the molluscan intermediate hosts of human schistosomes through predatory and competitive interactions. Here, we investigate whether crayfish can reduce populations of Bulinus africanus and consequently, Schistosoma haematobium prevalence in school children. Children from 6 primary schools in the Machakos and Kitui Districts of Kenya were selected for study. Schools were divided into 3 experimental-control pairs. At experimental schools, crayfish were introduced into nearby aquatic habitats harboring Bulinus africanus snails and serving as S. haematobium transmission sites. Snail habitats near control schools did not receive crayfish. Six months after crayfish introduction, all infected children were treated with praziquantel. Children were then monitored quarterly for 2 years, at which time infection and reinfection rates were compared statistically between the paired schools. In one such pair, crayfish failed to establish, resulting in neither snail control nor a reduction in transmission. At the second pair of schools, the numbers of snails were decreased by the presence of crayfish, but a clear difference in infection rates in children could not be detected, primarily because drought conditions kept overall transmission rates low. At the third school pair, crayfish established well in experimental habitats, snail numbers decreased precipitously, and children at the experimental school were significantly less likely to acquire S. haematobium infections than children at the control school. Our results indicate that under certain environmental circumstances, P. clarkii exerts a significant impact on the transmission of human schistosomiasis in Kenya. Important questions remain regarding the impact of P. clarkii on Kenyan freshwater ecosystems, not the least of which is its potential to significantly influence the epidemiology of schistosomiasis in east Africa.

Growth of Biomphalaria glabrata populations in the presence of the ampullariid snails Pila ovata, Lanistes carinatus and Marisa cornuarietis.

Adults of three ampullariid species were examined for their ability to affect the population growth of Biomphalaria glabrata (M line strain) under laboratory conditions in which food (lettuce) was supplied ad libitum over an eleven week interval. Lanistes carinatus and Pila ovata were obtained from Kenya, and Marisa cornuarietis originated from the Dominican Republic. The presence of either 3 or 9 L. carinatus or 3 M. cornuarietis per 40 l aquarium did not reduce the population size of B. glabrata below levels attained in control aquaria lacking ampullariids. The presence of 9 M. cornuarietis adults significantly reduced B. glabrata populations by week 10 of the experiment. Presence of 3 or 9 P. ovata per aquarium significantly reduced B. glabrata numbers below control levels starting at week four of the experiment. Aquaria with 9 P. ovata were observed to have fewer B. glabrata egg masses than control aquaria, but the number of egg masses observed was not significantly reduced in aquaria with 3 P. ovata or with 3 or 9 L. carinatus or M. cornuarietis. However, the presence of 3 or 9 adults of each ampullariid species resulted in a significant increase in the percentage of such egg masses that disappeared prior to expected hatching date and that were presumed to have been consumed. The results of his laboratory study suggest that further investigation of the role of ampullariids as biological control agents for pulmonate snails in sub-Saharan Africa should focus on P. ovata.

Consumption of Biomphalaria glabrata egg masses and juveniles by the ampullariid snails Pila ovata, Lanistes carinatus and Marisa cornuarietis.

Lanistes carinatus and Pila ovata from Kenya and Marisa cornuarietis from the Dominican Republic were examined for their ability to consume egg masses and juveniles of Biomphalaria glabrata (M line strain) under laboratory conditions. Adults of all three ampullariid species consumed all egg masses presented to them over a five day period of observation. Juvenile P. ovata consumed significantly more egg masses than juveniles of M. cornuarietis or L. carinatus. Both adult and juvenile ampullariids generally ate more egg masses when maintained under a temperature regime (25-32 degrees C) approximating conditions in coastal Kenya than at 13-25 degrees C which approximates the temperature regime near Nairobi, Kenya. Egg masses attached to floating 'refugia' were not attacked, apparently reflecting the difficulty experienced by ampullariids in reaching free-floating objects. Adults of each ampullariid species ate approximately 25% of all lariids in reaching free-floating objects. Adults of each ampullariid species ate approximately 25% of all 1.5 +/- 0.5 mm B. glabrata juvenile snails presented to them. P. ovata adults consumed significantly more 3.0 +/- 0.5 mm juveniles than adults of the other two species. B. glabrata egg masses and juveniles were consumed even though lettuce was continually present in experimental aquaria. Implications of these results for biological control studies in East Africa are discussed.

A simple method of DNA extraction for Eimeria species.

A new, simple method is described for extracting DNA from coccidia (Eimeriidae) oocysts. In our hands this method works well for all Eimeria oocysts and, presumably, will work equally well for oocysts of other coccidia genera. This method combines the two steps of breaking oocyst and sporocyst walls, and dissolving the sporozoite membrane in one step. This greatly simplifies the currently used DNA extraction procedures for Eimeria species and overcomes the disadvantages of existing DNA extraction methods based on glass-bead grinding and sporozoite excystation procedures. Because all the procedures are done in a 1.5-ml microfuge tube, which minimizes the loss of DNA in the extraction procedures, this method is especially suitable for samples with small number of oocysts. In addition, this method directly lyses the oocyst and sporocyst walls as well as the sporozoite membrane in a continuous incubation; therefore, it does not require the sporozoites to be alive. The results of PCR experiments indicate that this method generates better quality of DNA than what the existing glass-bead grinding method does for molecular analysis, and is suitable for both large or small number (<10(2) oocysts) of living or dead oocyst samples.