Transcriptional profiling of Bulinus globosus provides insights into immune gene families in snails supporting the transmission of urogenital schistosomiasis.

Schistosomiasis, urogenital and intestinal, afflicts 251 million people worldwide with approximately two-thirds of the patients suffering from the urogenital form of the disease. Freshwater snails of the genus Bulinus (Gastropoda: Planorbidae) serve as obligate intermediate hosts for Schistosoma haematobium, the etiologic agent of human urogenital schistosomiasis. These snails also act as vectors for the transmission of schistosomiasis in livestock and wildlife. Despite their crucial role in human and veterinary medicine, our basic understanding at the molecular level of the entire Bulinus genus, which comprises 37 recognized species, is very limited. In this study, we employed Illumina-based RNA sequencing (RNAseq) to profile the genome-wide transcriptome of Bulinus globosus, one of the most important intermediate hosts for S. haematobium in Africa. A total of 179,221 transcripts (N50 = 1,235) were assembled and the benchmarking universal single-copy orthologs (BUSCO) was estimated to be 97.7%. The analysis revealed a substantial number of transcripts encoding evolutionarily conserved immune-related proteins, particularly C-type lectin (CLECT) domain-containing proteins (n = 316), Toll/Interleukin 1-receptor (TIR)-containing proteins (n = 75), and fibrinogen related domain-containing molecules (FReD) (n = 165). Notably, none of the FReDs are fibrinogen-related proteins (FREPs) (immunoglobulin superfamily (IgSF) + fibrinogen (FBG)). This RNAseq-based transcriptional profile provides new insights into immune capabilities of Bulinus snails, helps provide a framework to explain the complex patterns of compatibility between snails and schistosomes, and improves our overall understanding of comparative immunology.

Bulinus snails in the Lake Victoria Basin in Kenya: Systematics and their role as hosts for schistosomes.

The planorbid gastropod genus Bulinus consists of 38 species that vary in their ability to vector Schistosoma haematobium (the causative agent of human urogenital schistosomiasis), other Schistosoma species, and non-schistosome trematodes. Relying on sequence-based identifications of bulinids (partial cox1 and 16S) and Schistosoma (cox1 and ITS), we examined Bulinus species in the Lake Victoria Basin in Kenya for naturally acquired infections with Schistosoma species. We collected 6,133 bulinids from 11 sites between 2014-2021, 226 (3.7%) of which harbored Schistosoma infections. We found 4 Bulinus taxa from Lake Victoria (B. truncatus, B. tropicus, B. ugandae, and B. cf. transversalis), and an additional 4 from other habitats (B. globosus, B. productus, B. forskalii, and B. scalaris). S. haematobium infections were found in B. globosus and B. productus (with infections in the former predominating) whereas S. bovis infections were identified in B. globosus, B. productus, B. forskalii, and B. ugandae. No nuclear/mitochondrial discordance potentially indicative of S. haematobium/S. bovis hybridization was detected. We highlight the presence of Bulinus ugandae as a distinct lake-dwelling taxon closely related to B. globosus yet, unlike all other members of the B. africanus species group, is likely not a vector for S. haematobium, though it does exhibit susceptibility to S. bovis. Other lake-dwelling bulinids also lacked S. haematobium infections, supporting the possibility that they all lack compatibility with local S. haematobium, thereby preventing widespread transmission of urogenital schistosomiasis in the lake's waters. We support B. productus as a distinct species from B. nasutus, B. scalaris as distinct from B. forskalii, and add further evidence for a B. globosus species complex with three lineages represented in Kenya alone. This study serves as an essential prelude for investigating why these patterns in compatibility exist and whether the underlying biological mechanisms may be exploited for the purpose of limiting schistosome transmission.

Host preference of field-derived Schistosoma mansoni is influenced by snail host compatibility and infection status.

Schistosome parasites cause a chronic inflammatory disease in humans, and recent studies have emphasized the importance of control programs for understanding the aquatic phases of schistosomiasis transmission. The host-seeking behavior of larval schistosomes (miracidia) for their snail intermediate hosts plays a critical role in parasite transmission. Using field-derived strains of Kenyan snails and parasites, we tested two main hypotheses: (1) Parasites prefer the most compatible host, and (2) parasites avoid hosts that are already infected. We tested preference to three Biomphalaria host snail taxa (B. pfeifferi, B. sudanica, and B. choanomphala), using allopatric and sympatric Schistosoma mansoni isolates and two different nonhost snail species that co-occur with Biomphalaria, Bulinus globosus, and Physa acuta. We also tested whether schistosomes avoid snail hosts that are already infected by another trematode species and whether competitive dominance played a role in their behavior. Preference was assessed using two-way choice chambers and by visually counting parasites that moved toward competing stimuli. In pairwise comparisons, we found that S. mansoni did not always prefer the more compatible snail taxon, but never favored an incompatible host over a compatible host. While parasites preferred B. pfeifferi to the nonhost species B. globosus, they did not significantly prefer B. pfeifferi versus P. acuta, an introduced species in Kenya. Finally, we demonstrated that parasites avoid infected snails if the resident parasite was competitively dominant (Patagifer sp.), and preferred snails infected with subordinates (xiphidiocercariae) to uninfected snails. These results provide evidence of "fine tuning" in the ability of schistosome miracidia to detect hosts; however, they did not always select hosts that would maximize fitness. Appreciating such discriminatory abilities could lead to a better understanding of how ecosystem host and parasite diversity influences disease transmission and could provide novel control mechanisms to improve human health.

Comparative Vectorial Competence of Biomphalaria sudanica and Biomphalaria choanomphala, Snail Hosts of Schistosoma mansoni, From Transmission Hotspots In Lake Victoria, Western Kenya.

Schistosoma mansoni, which causes human intestinal schistosomiasis, continues to be a major public health concern in the Lake Victoria basin in western Kenya, with Biomphalaria sudanica (a shoreline inhabiting snail) and Biomphalaria choanomphala (a deep-water snail) playing roles in transmission. A recent study showed that B. sudanica was abundantly present near all study villages on the lakeshore, but B. choanomphala was significantly more abundant near villages known to be persistent transmission hotspots. The present study investigated the relative compatibility of B. sudanica and B. choanomphala with S. mansoni. A reciprocal cross-infection experiment used young adult F1 generation B. sudanica and B. choanomphala that were exposed to either 1, 5, or 10 sympatric or allopatric human-derived S. mansoni miracidia. Three weeks post-exposure (PE) and weekly thereafter, the snails were counted and screened for schistosome cercariae, and at 7 wk PE, total cercariae shed during a 2 hr period by each infected snail was determined. Pre-patent periods for S. mansoni in both B. sudanica and B. choanomphala were similar, and most snails in all exposure combinations started shedding cercariae 5 wk PE. Prevalences were significantly higher in B. choanomphala (12.2-80.9%) than in B. sudanica (5.2-18.6%) at each dose, regardless of whether miracidia were of an allopatric or a sympatric source (P < 0.0001). Overall, the odds of a snail becoming infected with 5 or 10 miracidia were significantly higher than the odds of being infected with 1 miracidium, (P < 0.0001), and fewer cercariae were produced by snails exposed to single as compared to 5 or 10 miracidia. On average, B. choanomphala produced more cercariae ( = 458, SD = 414) than B. sudanica ( = 238, SD = 208) (P < 0.0001). These results suggest that B. choanomphala is more compatible with S. mansoni than B. sudanica. Though B. choanomphala can be found in shallow shoreline waters, it is, for the most part, a deeper-water taxon. Because dredging is a relatively inefficient means of sampling, B. choanomphala is likely underestimated with respect to its population size, the number of S. mansoni-positive snails, and its role in maintaining transmission.

Transmission sites for Schistosoma haematobium and Schistosoma bovis identified in localities within the Athi River basin of Kenya using a PCR-RFLP assay.

BACKGROUND: The epidemiology of human urinary schistosomiasis caused by Schistosoma haematobium can be complicated by the presence of ruminant schistosomiasis caused, primarily by S. bovis. The two schistosome species may be transmitted by the same Bulinus species, they may occur sympatrically in the same habitat, and their cercariae are very similar in morphology and therefore, difficult to tell them apart. Screening of snails collected from freshwater habitats for schistosome infections is often used to identify transmission sites or to evaluate success or failure of interventions. However, pin-pointing sites involved in S. haematobium transmission can be complicated by the presence of other mammalian schistosomes such as the bovine schistosome, which is a fairly common parasite. A PCR-RFLP method targeting a unique segment of the second internal transcribed spacer (ITS2) region of the ribosomal DNA (rDNA) in the schistosomes was used to identify mammalian schistosome cercariae shed by bulinid snails collected from endemic freshwater habitats located within Machakos county in south-eastern Kenya, with the aim to identify the transmission sites and assess the distribution each of the parasite species in the study area. RESULTS: A total of 5,034 bulinid snails were collected from 41 different sites and screened for schistosome infections, and out of these, 43 (<1%) were found to be shedding mammalian schistosome cercariae. On analysis using the Polymerase chain reaction- Restriction Fragment Length Polymorphisms (PCR-RFLP) assay, cercariae from 32 snails were identified as S. haematobium while cercariae from 11 snails turned out to be S. bovis. Only two sites out of 40 namely Kisukioni and Katiwa, were active transmission sites. Both sites were active transmission sites for both S. haematobium and S. bovis. The assay reliably identified and distinguished between S. haematobium and S. bovis cercariae, even when only a few cercariae (5-10) were present in the sample, or when the parasite DNA concentrations were as low as five pico grammes (5pg). The FTA® paper offered a more reliable way of collecting, transporting and storing DNA material, and the samples. CONCLUSION: The PCR-based assay can potentially be used to support schistosomiasis control efforts, in epidemiological studies of urinary schistosomiasis, or in transmission ecology studies of S. haematobium and S. bovis.

Antagonism between parasites within snail hosts impacts the transmission of human schistosomiasis.

Human disease agents exist within complex environments that have underappreciated effects on transmission, especially for parasites with multi-host life cycles. We examined the impact of multiple host and parasite species on transmission of the human parasite Schistosoma mansoni in Kenya. We show S. mansoni is impacted by cattle and wild vertebrates because of their role in supporting trematode parasites, the larvae of which have antagonistic interactions with S. mansoni in their shared Biomphalaria vector snails. We discovered the abundant cattle trematode, Calicophoron sukari, fails to develop in Biomphalaria pfeifferi unless S. mansoni larvae are present in the same snail. Further development of S. mansoni is subsequently prevented by C. sukari's presence. Modeling indicated that removal of C. sukari would increase S. mansoni-infected snails by two-fold. Predictable exploitation of aquatic habitats by humans and their cattle enable C. sukari to exploit S. mansoni, thereby limiting transmission of this human pathogen.

A Search for Snail-Related Answers to Explain Differences in Response of Schistosoma mansoni to Praziquantel Treatment among Responding and Persistent Hotspot Villages along the Kenyan Shore of Lake Victoria.

Following a 4-year annual praziquantel (PZQ) treatment campaign, the resulting prevalence of Schistosoma mansoni was seen to differ among individual villages along the Kenyan shore of Lake Victoria. We have investigated possible inherent differences in snail-related aspects of transmission among such 10 villages, including six persistent hotspot (PHS) villages (≤ 30% reduction in prevalence following repeated treatments) located along the west-facing shore of the lake and four PZQ-responding (RESP) villages (> 30% prevalence reduction following repeated treatment) along the Winam Gulf. When taking into account all sampling sites, times, and water hyacinth presence/absence, shoreline-associated Biomphalaria sudanica from PHS and RESP villages did not differ in relative abundance or prevalence of S. mansoni infection. Water hyacinth intrusions were associated with increased B. sudanica abundance. The deeper water snail Biomphalaria choanomphala was significantly more abundant in the PHS villages, and prevalence of S. mansoni among villages both before and after control was positively correlated with B. choanomphala abundance. Worm recoveries from sentinel mice did not differ between PHS and RESP villages, and abundance of non-schistosome trematode species was not associated with S. mansoni abundance. Biomphalaria choanomphala provides an alternative, deepwater mode of transmission that may favor greater persistence of S. mansoni in PHS villages. As we found evidence for ongoing S. mansoni transmission in all 10 villages, we conclude that conditions conducive for transmission and reinfection occur ubiquitously. This argues for an integrated, basin-wide plan for schistosomiasis control to counteract rapid reinfections facilitated by large snail populations and movements of infected people around the lake.

The diverse echinostomes from East Africa: With a focus on species that use Biomphalaria and Bulinus as intermediate hosts.

Echinostomes are a diverse group of digenetic trematodes that are globally distributed. The diversity of echinostomes in Africa remains largely unknown, particularly in analyses using molecular markers. Therefore, we were interested in the composition and host usage patterns of African echinostomes, especially those that also use schistosome transmitting snails as intermediate hosts. We collected adults and larval stages of echinostomes from 19 different localities in East Africa (1 locality in Uganda and 18 in Kenya). In this study we provide locality information, host use, museum vouchers, and genetic data for two loci (28S and nad1) from 98 samples of echinostomes from East Africa. Combining morphological features, host use information, and phylogenetic analyses we found 17 clades of echinostomes in East Africa. Four clades were found to use more than one genus of freshwater snails as their first intermediate hosts. We also determined at least partial life cycles (2 of the 3) of four clades using molecular markers. Of the 17 clades, 13 use Biomphalaria or Bulinus as a first intermediate host. The overlap in host usage creates opportunities for competition, including against human schistosomes. Thus, our study can be used as a foundation for future studies to ascertain the interactions between schistosomes and echinostomes in their respective intermediate hosts.

Complete mitochondrial and rDNA complex sequences of important vector species of Biomphalaria, obligatory hosts of the human-infecting blood fluke, Schistosoma mansoni.

Using high throughput Illumina sequencing technology, we determined complete sequences for the mitochondrial genome (mitogenome) and nuclear ribosomal DNA (rDNA) complex for three African freshwater snail taxa within the genus Biomphalaria, B. pfeifferi, B. sudanica and B. choanomphala, and for two laboratory strains of B. glabrata originating from the Neotropics. Biomphalaria snails are obligate vectors of the blood fluke Schistosoma mansoni, a major etiologic agent of human intestinal schistosomiasis. Our data show that mitogenomes from African and Neotropical Biomphalaria are highly conserved. With respect to rDNA, the two internal transcribed spacers (ITS1 and 2) were found to be highly variable whereas the three ribosomal RNA genes (28S, 5.8S and 18S rRNA) exhibited no or very limited variation. Our analyses reveal that the two taxa inhabiting Lake Victoria, B. sudanica and B. choanomphala, are very similar to one another relative to the similarity either shows to B. pfeifferi or B. glabrata. This new sequence information may prove useful for developing new markers for snail identification, environmental detection/monitoring purposes or for tracking epidemiology and snail dependencies of S. mansoni in endemic areas. It also provides new information pertinent to still unresolved questions in Biomphalaria systematics and nomenclature.

A Comparison of Kenyan Biomphalaria pfeifferi and B. Sudanica as Vectors for Schistosoma mansoni, Including a Discussion of the Need to Better Understand the Effects of Snail Breeding Systems on Transmission.

In Kenya, schistosomes infect an estimated 6 million people with >30 million people at risk of infection. We compared compatibility with, and ability to support and perpetuate, Schistosoma mansoni of Biomphalaria pfeifferi and Biomphalaria sudanica, 2 prominent freshwater snail species involved in schistosomiasis transmission in Kenya. Field-derived B. pfeifferi (from a stream in Mwea, central Kenya) and B. sudanica (from Nawa, Lake Victoria, in western Kenya) were exposed to S. mansoni miracidia isolated from fecal samples of naturally infected humans from Mwea or Nawa. Juvenile (<6 mm shell diameter), young adult (6-9 mm), and adult snails (>9 mm) were each exposed to a single miracidium. Schistosoma mansoni developed faster and consistently had higher infection rates (39.6-80.7%) in B. pfeifferi than in B. sudanica (2.4-21.5%), regardless of the source of S. mansoni or the size of the snails used. Schistosoma mansoni from Nawa produced higher infection rates in both B. pfeifferi and B. sudanica than did S. mansoni from Mwea. Mean daily cercariae production was greater for B. pfeifferi exposed to sympatric than allopatric S. mansoni (583-1,686 vs. 392-1,232), and mean daily cercariae production among B. sudanica were consistently low (50-590) with no significant differences between sympatric or allopatric combinations. Both non-miracidia-exposed and miracidia-exposed B. pfeifferi had higher mortality rates than for B. sudanica, but mean survival time of shedding snails (9.3-13.7 wk) did not differ significantly between the 2 species. A small proportion (1.5%) of the cercariae shedding B. pfeifferi survived up to 40 wk post-exposure. Biomphalaria pfeifferi was more likely to become infected and to shed more cercariae than B. sudanica, suggesting that the risk per individual snail of perpetuating transmission in Kenyan streams or lacustrine habitats may differ considerably. High infection rates exhibited by the preferential self-fertilizing B. pfeifferi relative to the out-crossing B. sudanica point to the need to investigate further the role of host breeding systems in influencing transmission of schistosomiasis by snail hosts.

Loads of trematodes: discovering hidden diversity of paramphistomoids in Kenyan ruminants.

Paramphistomoids are ubiquitous and widespread digeneans that infect a diverse range of definitive hosts, being particularly speciose in ruminants. We collected adult worms from cattle, goats and sheep from slaughterhouses, and cercariae from freshwater snails from ten localities in Central and West Kenya. We sequenced cox1 (690 bp) and internal transcribed region 2 (ITS2) (385 bp) genes from a small piece of 79 different adult worms and stained and mounted the remaining worm bodies for comparisons with available descriptions. We also sequenced cox1 and ITS2 from 41 cercariae/rediae samples collected from four different genera of planorbid snails. Combining morphological observations, host use information, genetic distance values and phylogenetic methods, we delineated 16 distinct clades of paramphistomoids. For four of the 16 clades, sequences from adult worms and cercariae/rediae matched, providing an independent assessment for their life cycles. Much work is yet to be done to resolve fully the relationships among paramphistomoids, but some correspondence between sequence- and anatomically based classifications were noted. Paramphistomoids of domestic ruminants provide one of the most abundant sources of parasitic flatworm biomass, and because of the predilection of several species use Bulinus and Biomphalaria snail hosts, have interesting linkages with the biology of animal and human schistosomes to in Africa.

Development of a Loop Mediated Isothermal Amplification for Diagnosis of Ascaris lumbricoides in Fecal Samples.

Ascaris lumbricoides is a nematode parasite that causes the common tropical infection ascariasis in humans. It is also considered among the neglected tropical diseases. Diagnosis relies mainly on microscopy-based methods which are laborious, are limited by low sensitivity, and require high expertise. We have developed a loop mediated isothermal amplification (LAMP) for diagnosis of ascariasis in fecal samples, based on the first internal transcribed (ITS-1) spacer region of the ribosomal DNA. We used Primer Explorer V4 software to design primers. Ascaris adult and ova were obtained from naturally infected school children, whose parents/guardians gave consent for their participation in the study. Genomic DNA was extracted using alkaline lysis method and amplified by LAMP at 63°C for 45 minutes. LAMP products were visualized by naked eyes after adding SYBR Green dye and also on agarose gel. LAMP successfully and reliably detected Ascaris DNA from a single egg and in fecal samples. The assay specifically detected Ascaris DNA without amplifying DNA from ova of other parasites which commonly coexist with A. lumbricoides in feces. The developed LAMP assay has great potential for use in ascariasis diagnosis at the point of care and in low infection intensity situation that characterize control and elimination campaigns.

Relative compatibility of Schistosoma mansoni with Biomphalaria sudanica and B. pfeifferi from Kenya as assessed by PCR amplification of the S. mansoni ND5 gene in conjunction with traditional methods.

BACKGROUND: Schistosoma mansoni is hosted by several species of Biomphalaria spp. snails in Africa. We were interested in determining if there were differences in compatibility of S. mansoni with Biomphalaria sudanica from Lake Victoria, or with B. pfeifferi from streams and smaller water bodies in Kenya. Does this parasite develop with equal efficiency in both snail species, and does this have implications for transmission in different habitat types? METHODS: Primers for PCR amplification of the S. mansoni ND5 gene were designed and tested for sensitivity and specificity. We exposed laboratory-reared B. sudanica and field-derived B. pfeifferi to single miracidium infections and at 1, 2, 4, 8, 16 and 24 days post-exposure (dpe), snails were extracted for the PCR assay. Snails were also shed for cercariae and/or dissected prior to extraction. Additionally, B. sudanica and B. pfeifferi were collected from field locations and tested with the PCR assay. RESULTS: The ND5 PCR assay was sensitive (>0.1 fg S. mansoni genomic DNA) and allowed S. mansoni to be differentiated from other relevant schistosome species or snails. The number of PCR positive snails at 1-4 dpe was higher for B. pfeifferi than for B. sudanica, but not significantly so (P = 0.052). From 8-24 dpe, more B. pfeifferi harbored successfully developing parasites (positive by both dissection and PCR) than did B. sudanica (P = 0.008). At 40 dpe, more B. pfeifferi than B. sudanica shed cercariae or harbored dissection positive/PCR positive infections (P < 0.001). Both immature and failed (dissection negative but PCR positive) S. mansoni infections could also be detected in naturally infected snails of both species. CONCLUSIONS: The PCR assay detected S. mansoni infections in snails exposed to one miracidium for one day. Both B. sudanica and B. pfeifferi supported full development of S. mansoni, but B. pfeifferi was more compatible, with significantly more dissection positive/PCR positive or shedding infections, and significantly fewer failed infections (dissection negative/PCR positive). This highlights the relatively lower compatibility of B. sudanica with S. mansoni, and suggests the factors responsible for incompatibility and how they might affect transmission of S. mansoni in habitats like Lake Victoria deserve additional study.

Field-derived Schistosoma mansoni and Biomphalaria pfeifferi in Kenya: a compatible association characterized by lack of strong local adaptation, and presence of some snails able to persistently produce cercariae for over a year.

BACKGROUND: Schistosoma mansoni is widely distributed in sub-Saharan Africa with Biomphalaria pfeifferi being its most widespread and important snail intermediate host. Few studies have examined the compatibility of field-derived B. pfeifferi snails with S. mansoni miracidia derived from human hosts. We investigated compatibility (as defined by shedding of cercariae following exposure to miracidia) of two isolates of S. mansoni from school children from Asao (western Kenya) and Mwea (central Kenya) with B. pfeifferi collected directly from Asao stream or the Mwea rice fields. METHODS: We exposed snails from both regions to four different doses of miracidia (1, 5, 10 and 25) from sympatric or allopatric S. mansoni, and maintained them in a shaded, screened out-of-doors rearing facility in Kisian, in western Kenya. Both snail survival and the number of snails that became infected were monitored weekly. This was done for 25 weeks post-exposure (PE). Those infected snails which survived beyond this period were monitored until they all died. RESULTS: Although overall survival of Mwea snails maintained in western Kenya was generally low, both sympatric and allopatric combinations of parasites and snails exhibited high compatibility (approximately 50% at a dose of one miracidium per snail), with an increase in infection rates as the miracidial dose was increased (P < 0.002). Schistosomes were no more compatible with sympatric than allopatric snails, nor were snails less compatible with sympatric than allopatric schistosomes. Snail mortality increased significantly with dose of miracidia (P < 0.05). Approximately 3% of Asao snails exposed to a low dose of sympatric miracidia (1 or 5) continued to shed cercariae for as long as 58 weeks post exposure. CONCLUSIONS: There were no significant local adaptation effects for either schistosomes or snails. Also, the existence of "super-survivor" snails is noteworthy for its implications for current control initiatives that mostly rely on mass drug administration (MDA). Long-term shedders could provide an ongoing source of cercariae to initiate human infections for many months, suggesting care is required in considering how human MDA treatments are timed. Future control programs should incorporate means to eliminate infected snails to complement chemotherapy interventions in controlling schistosomiasis.

No apparent reduction in schistosome burden or genetic diversity following four years of school-based mass drug administration in mwea, central kenya, a heavy transmission area.

BACKGROUND: Schistosomiasis is a debilitating neglected tropical disease that infects over 200 million people worldwide. To combat this disease, in 2012, the World Health Organization announced a goal of reducing and eliminating transmission of schistosomes. Current control focuses primarily on mass drug administration (MDA). Therefore, we monitored transmission of Schistosoma mansoni via fecal egg counts and genetic markers in a typical school based MDA setting to ascertain the actual impacts of MDA on the targeted schistosome population. METHODS: For 4 years, we followed 67 children enrolled in a MDA program in Kenya. Infection status and egg counts were measured each year prior to treatment. For 15 of these children, for which there was no evidence of acquired resistance, meaning they became re-infected following each treatment, we collected microsatellite genotype data from schistosomes passed in fecal samples as a representation of the force of transmission between drug treatments. We genotyped a total of 4938 parasites from these children, with an average of 329.2 parasites per child for the entire study, and an average of 82.3 parasites per child per annual examination. We compared prevalence, egg counts, and genetic measures including allelic richness, gene diversity (expected heterozygosity), adult worm burdens and effective number of breeders among time points to search for evidence for a change in transmission or schistosome populations during the MDA program. FINDINGS: We found no evidence of reduced transmission or schistosome population decline over the course of the program. Although prevalence declined in the 67 children as it did in the overall program, reinfection rates were high, and for the 15 children studied in detail, schistosome egg counts and estimated adult worm burdens did not decline between years 1 and 4, and genetic diversity increased over the course of drug treatment. INTERPRETATION: School based control programs undoubtedly improve the health of individuals; however, our data show that in an endemic area, such a program has had no obvious effect on reducing transmission or of significantly impacting the schistosome population as sampled by the children we studied in depth. Results like these, in combination with other sources of information, suggest more integrated approaches for interrupting transmission and significantly diminishing schistosome populations will be required to achieve sustainable control.

Non-invasive sampling of schistosomes from humans requires correcting for family structure.

For ethical and logistical reasons, population-genetic studies of parasites often rely on the non-invasive sampling of offspring shed from their definitive hosts. However, if the sampled offspring are naturally derived from a small number of parents, then the strong family structure can result in biased population-level estimates of genetic parameters, particularly if reproductive output is skewed. Here, we document and correct for the strong family structure present within schistosome offspring (miracidia) that were collected non-invasively from humans in western Kenya. By genotyping 2,424 miracidia from 12 patients at 12 microsatellite loci and using a sibship clustering program, we found that the samples contained large numbers of siblings. Furthermore, reproductive success of the breeding schistosomes was skewed, creating differential representation of each family in the offspring pool. After removing the family structure with an iterative jacknifing procedure, we demonstrated that the presence of relatives led to inflated estimates of genetic differentiation and linkage disequilibrium, and downwardly-biased estimates of inbreeding coefficients (FIS). For example, correcting for family structure yielded estimates of FST among patients that were 27 times lower than estimates from the uncorrected samples. These biased estimates would cause one to draw false conclusions regarding these parameters in the adult population. We also found from our analyses that estimates of the number of full sibling families and other genetic parameters of samples of miracidia were highly intercorrelated but are not correlated with estimates of worm burden obtained via egg counting (Kato-Katz). Whether genetic methods or the traditional Kato-Katz estimator provide a better estimate of actual number of adult worms remains to be seen. This study illustrates that family structure must be explicitly accounted for when using offspring samples to estimate the genetic parameters of adult parasite populations.

Polymorphism associated with the Schistosoma mansoni tetraspanin-2 gene.

A vaccine against schistosomiasis would contribute significantly to reducing the 3-70 million disability-adjusted life years lost annually to the disease. Towards this end, inoculation with the large extracellular loop (EC-2) of Schistosoma mansoni tetraspanin-2 protein (Sm-TSP-2) has proved effective in reducing worm and egg burdens in S. mansoni-infected mice. The EC-2 loop of Schistosoma japonicum TSP-2, however, has been found to be highly polymorphic, perhaps diminishing the likelihood that this antigen can be used for vaccination against this species. Here, we examine polymorphism of the EC-2 of Sm-TSP-2 in genetically unique worms derived from six individuals from Kisumu, Kenya.

Reduced susceptibility to praziquantel among naturally occurring Kenyan isolates of Schistosoma mansoni.

BACKGROUND: The near exclusive use of praziquantel (PZQ) for treatment of human schistosomiasis has raised concerns about the possible emergence of drug-resistant schistosomes. METHODOLOGY/PRINCIPAL FINDINGS: We measured susceptibility to PZQ of isolates of Schistosoma mansoni obtained from patients from Kisumu, Kenya continuously exposed to infection as a consequence of their occupations as car washers or sand harvesters. We used a) an in vitro assay with miracidia, b) an in vivo assay targeting adult worms in mice and c) an in vitro assay targeting adult schistosomes perfused from mice. In the miracidia assay, in which miracidia from human patients were exposed to PZQ in vitro, reduced susceptibility was associated with previous treatment of the patient with PZQ. One isolate ("KCW") that was less susceptible to PZQ and had been derived from a patient who had never fully cured despite multiple treatments was studied further. In an in vivo assay of adult worms, the KCW isolate was significantly less susceptible to PZQ than two other isolates from natural infections in Kenya and two lab-reared strains of S. mansoni. The in vitro adult assay, based on measuring length changes of adults following exposure to and recovery from PZQ, confirmed that the KCW isolate was less susceptible to PZQ than the other isolates tested. A sub-isolate of KCW maintained separately and tested after three years was susceptible to PZQ, indicative that the trait of reduced sensitivity could be lost if selection was not maintained. CONCLUSIONS/SIGNIFICANCE: Isolates of S. mansoni from some patients in Kisumu have lower susceptibility to PZQ, including one from a patient who was never fully cured after repeated rounds of treatment administered over several years. As use of PZQ continues, continued selection for worms with diminished susceptibility is possible, and the probability of emergence of resistance will increase as large reservoirs of untreated worms diminish. The potential for rapid emergence of resistance should be an important consideration of treatment programs.

Introgressive hybridization of human and rodent schistosome parasites in western Kenya.

Hybridization and introgression can have important consequences for the evolution, ecology and epidemiology of pathogenic organisms. We examined the dynamics of hybridization between a trematode parasite of humans, Schistosoma mansoni, and its sister species, S. rodhaini, a rodent parasite, in a natural hybrid zone in western Kenya. Using microsatellite markers, rDNA and mtDNA, we showed that hybrids between the two species occur in nature, are fertile and produce viable offspring through backcrosses with S. mansoni. Averaged across collection sites, individuals of hybrid ancestry comprised 7.2% of all schistosomes collected, which is a large proportion given that one of the parental species, S. rodhaini, comprised only 9.1% of the specimens. No F1 individuals were collected and all hybrids represented backcrosses with S. mansoni that were of the first or successive generations. The direction of introgression appears highly asymmetric, causing unidirectional gene flow from the rodent parasite, S. rodhaini, to the human parasite, S. mansoni. Hybrid occurrence was seasonal and most hybrids were collected during the month of September over a 2-year period, a time when S. rodhaini was also abundant. We also examined the sex ratios and phenotypic differences between the hybrids and parental species, including the number of infective stages produced in the snail host and the time of day the infective stages emerge. No statistical differences were found in any of these characteristics, and most of the hybrids showed an emergence pattern similar to that of S. mansoni. One individual, however, showed a bimodal emergence pattern that was characteristic of both parental species. In conclusion, these species maintain their identity despite hybridization, although introgression may cause important alterations of the biology and epidemiology of schistosomiasis in this region.

Interactions between natural populations of human and rodent schistosomes in the Lake Victoria region of Kenya: a molecular epidemiological approach.

BACKGROUND: Schistosoma mansoni exists in a complex environmental milieu that may select for significant evolutionary changes in this species. In Kenya, the sympatric distribution of S. mansoni with S. rodhaini potentially influences the epidemiology, ecology, and evolutionary biology of both species, because they infect the same species of snail and mammalian hosts and are capable of hybridization. METHODOLOGY/PRINCIPAL FINDINGS: Over a 2-year period, using a molecular epidemiological approach, we examined spatial and temporal distributions, and the overlap of these schistosomes within snails, in natural settings in Kenya. Both species had spatially and temporally patchy distributions, although S. mansoni was eight times more common than S. rodhaini. Both species were overdispersed within snails, and most snails (85.2% for S. mansoni and 91.7% for S. rodhaini) only harbored one schistosome genotype. Over time, half of snails infected with multiple genotypes showed a replacement pattern in which an initially dominant genotype was less represented in later replicates. The other half showed a consistent pattern over time; however, the ratio of each genotype was skewed. Profiles of circadian emergence of cercariae revealed that S. rodhaini emerges throughout the 24-hour cycle, with peak emergence before sunrise and sometimes immediately after sunset, which differs from previous reports of a single nocturnal peak immediately after sunset. Peak emergence for S. mansoni cercariae occurred as light became most intense and overlapped temporally with S. rodhaini. Comparison of schistosome communities within snails against a null model indicated that the community was structured and that coinfections were more common than expected by chance. In mixed infections, cercarial emergence over 24 hours remained similar to single species infections, again with S. rodhaini and S. mansoni cercarial emergence profiles overlapping substantially. CONCLUSIONS/SIGNIFICANCE: The data from this study indicate a lack of obvious spatial or temporal isolating mechanisms to prevent hybridization, raising the intriguing question of how the two species retain their separate identities.