IN VIVO AND IN VITRO ENCYSTMENT OF ECHINOCHASMUS LILIPUTANUS CERCARIAE AND BIOLOGICAL ACTIVITY OF THE METACERCARIAE

Aim To explore if Echinochasmus liliputanus cercariae can develop into metacercariae both invivo and in vitro and biological activity of the metacercariae,then to determine the effects of silver ni-trate on eereariae encystation in vitro.Methods Cereariae of Echinochasmus liliputanus from Bellamyaaeruginosa snails treated with or without silver nitrate were used to infected goldfish,the second inter-mediate host,or to incubated in many different solutions for 24 h to record the encystation rates.Themetacercariae formed both in vivo and in vitro were then used to infected New Zealand rabbits to testtheir infectivity to its definitive hosts or to exeyst in 0.1% sodium deoxycholate excystation medium at37℃ for 1 h.Results In vivo encystment of cercariae occurred in the gills of goldfish.However,thecercariae were also able to encyst in vitro in Locke’s solution,NaCl solution,artificial gastric juice orhuman gastric juice with eneystation rates of 74.28 %,44.94 %,8.37 % and 10.79 %.0.7×-1.2×Locke’s or 0.7%-1.2% NaCl solution was shown to be appropriate for in vitro encystment to occurwithin 24 hour,however,full- strength Locke’s solution was shown to be optimal.The one- day- oldencysted metacereariae formed in vivo showed 88.53 % exeystation when treated in 0.1% sodium de-oxycholate exeystation medium at 37℃ for 1h.The metacercariae formed in vitro,however,showed88.60% and 84.95% excystation for normal and abnormal ones respectively.While abnormal cysts atroom temperature usually die within 10 days,about 70% normal cyst,both in vivo and in vitro,canstill excyst after stored in 0.5×Locke’s at 4℃ for 3 mouths.Cysts formed in vivo and in vitro were e-qually infective to rabbits.1 uM silver nitrate had a dramatic effect on the cercariae encysting in vitro.When treated with silver nitrate,the cereariae encystation rates decreased to 16.25% in Locke’s solu-tion and 6.69% in NaCl solution,however,the encystment was largely restored wben the cercariaewere washed to remove Ag+.Conclusion The finding of E.liliputanus cercariae encysting in vitro,especially in human gastric juice,might be helpful in elucidating mechanisms of the definitive hosts in-fected by the cercariae directly.The encystment of the cercariae in vitro could be inhibited when thecercariae were treated with 1 uM silver nitrate.As silver nitrate binds to the papillae,especially to theciliated papillae,on the cercarial surface,it is suggested that papillar chemoreceptors may be involved inencystment of the cercariae.

AUTORADIOGRAPHIC STUDY OF THE ATTRITION OF MIGRATING SCHISTOSOMULA IN THE SKIN OF MICE II.MICE CHALLENGED WITH NORMAL S.MANSONI CERCARIAE AFTER IMMUNIZATION WITH HIGHLY IRRADIATED CERCARIAE

The migratory pattern and attrition of75Se-labelled S.mansoni challenge cercariae in CF1mice immunized 0,1,3, or 5 times with 12kR-or 48kR-irradiated cercariae were investigatedby compressed organ autoradiography.Mice were percutaneously inoculated with 500-1000 cer-cariae for each immunization and with a known count of approximately 90 cercariae at adifferent site for challenge.The skin,lungs,liver,other organs,and catrcass were processedfor autoradiography.Hepatic and mesenteric perfusions for worm collection were alsosampled from day 19 through 34.In naive tnice,25% of challenge cercariae died in theskin.Skin attrition of challenge cercariae in mice immunized with 12kR-irradiated cercariaewas 43% with 1 immunization,54% with 3,and 58% with 5,whereas in mice immunized with48kR-irradiated cercariae,skin,attrition was48% with 1,63% with 3,and 76% with 5 immunni-zations.In all immunized mice,a high percentage of migrating sehistosomula died in the skinwithin 24 hours of a challenge infection.Utilizing autoradiography,it was found that thesilver foci created by single labelled worms were difficult to differentiate from those of pairedworms.This study documents the induction of acquired resistance against a challenge infectionin the skin by vaccination with highly irradiated cercariae,with the degree of attemtation ofimmunizing cercariae and the number of immunizations being closely related to the attritiotn.rate in the skin.Finally,we discuss the contradictory views concerning the site of attritiotn ofchallenge cercariae.

Effects of albendazole,artesunate,praziquantel and miltefosine,on Opisthorchis viverrini cercariae and mature metacercariae

Objective:To explore larvicidal effects of anthelmintic drugs on Opisthorchis viverrini(O.viverrini) for alternative approach to interrupting its cycle for developing a field-based control program.Methods:The larvicidal activities of albendazole(A1),artesunate(Ar),praziquantel(Pzq) and miltefosine(Mf) on O.viverrini cercariae and mature metacercariae were investigated.Lethal concentrations(LC_(50) and LC_(95)) of these drugs were determined.Mature metacercariae previously exposed to various concentrations of the drugs were administered to hamsters.Worms were harvested 30 d post infection and worm recovery rates calculated.Al,Ar,Pzq and Mf produced morphological degeneration and induced shedding tails of cercariae after 24 h exposure.Results:The LC_(50) and LC_(95) of Al,Ar,Pzq and Mf on cercariae were 0.720 and 1.139,0.350 and 0.861,0.017 and 0.693,and 0.530 and 1.134 ppm,respectively.LC_(50) and LC_(95) of Ar on mature metacercariae were 303.643 and 446.237 ppm and of Mf were 289.711 and 631.781 ppm,respectively but no lethal effect in Pzq-and Al-treated groups(up to 1 ppt).No worms were found in hamsters administered Pzq-treated metacercariae.The adult worms from Al-treated metacercariae were significantly bigger in size compared to the control group(P<0.05).Fecundity and body width were greater in adults from Mf-treated metacercariae compared to the control group(P<0.05).Conclusions:The larvicidal effects of these drugs were high efficacy to O.viverrini cercariae but lesser efficacy to metacercariae.It should be further studied with the eventual aim of developing a field-based control program.

Ultraviolet-attenuated cercariae ofSchistosoma japonicum fail to effectively induce a Th1 response in spite of up-regulating expression of cytotoxicity-related genes in C57BL/6 mice

Objective： To better understand the reason that Schistosoma japonicurn （S. japonicum） ultraviolet （UV）- radiated cercariae could not induce high level of protection in C57BL/6 mice. Methods： Microarray technology was performed to investigate the gene transcription profile in skin draining lymph nodes （sdLNs） at 1 w after exposure to attenuated cercariae （AC） or normal cercariae （NC） of S. japonicum in C57BL/6 mice. The expressions of some representative genes were further confirmed by real-time PCR. Subsequently, the expressions of Th1/Th2 cytokine genes, cytotoxicity-related genes, as well as co-stimulator genes in spleens from AC-vaccinated and NC- infected mice were analyzed by real-time PCR at w 3 and 6 post-exposure. Results： The gene expressions of Th1 cytokines, including interferon-y （IFN-γ）, interleukin （IL）-12 and tumor necrosis factor-α （TNF-α） in the sdLNs were significantly lower in AC-vaccinated mice than in NC-infected mice. Furthermore, the gene expressions of Th1- and Th2- cytokines, including IFN-γ, IL-12, TNF-α, IL-4 and IL-10, in the spleens from AC-vaccinated mice showed little changes at w 3 and 6 post-vaccination. In addition, cytotoxicity-related molecules including granzyme A, granzyme B, granzyme K, perforin 1 and Fas L were up-regulated from the early stage of vaccination, and peaked at the 3rd w after vaccination with UV-AC. Conclusion： UV-AC of S. japonicum could not ef- fectively induce a Thl response in C57BL/6 mice, which may be an explanation for the low protection against parasite challenge, and the role played by up-regulated expression of cytotoxicity-related genes in mice needs to be further investigated.

Characterization of randomly amplified polymorphic DNA (RAPD) fragments revealing clonal variability in cercariae of avian schistosome <i>Trichobilharzia szidati</i>(Trematoda: Schistosomatidae)

Recently we applied randomly amplified polymorphic DNA (RAPD) fingerprinting to detect clonal variability among individual cercariae within daughter sporocysts and rediae of 10 digenean trematodes (Platyhelminthes: Trematoda). The most variable RAPD patterns were obtained for Schistosomatidae representative-avian schistosome Trichobilharzia szidati. In this work, 50 polymorphic DNA fragments of approximately 300-1500 bp from RAPD patterns of individual T. szidati cercariae were cloned and sequenced. As a result genomic DNA sequences (total length of approximately 41,000 bp) revealing clonal variability in T. szidati cercariae were obtained and analyzed. The analysis indicated that these sequences contained tandem, inverted and dispersed repeats as well as regions homological to retroelements of two human parasites, Schistosoma mansoni and S. japonicum. Tandem and inverted repeats constituted 8.9% and 22.1% respectively, while the percentage of dispersed repeats was 21.0%. The average content of these components was 41.7% with the average AT content being 59.0%. About 40% of sequences included regions ranging in length from 96 to 1005 bp which displayed amino acid homology with open reading frame pol products of S. mansoni and S. japonicum retroelements: non-long terminal repeat retrotransposons (nLTRs, 76%), long terminal repeat retrotransposons (LTRs, 14%), and Penelope-like elements (PLEs, 10%). Most of these regions (86.4%) contained frameshifts, gaps, and stop-codons. The largest portion of them was homological to nLTRs of the RTE clade (67%). The number of sequences homologous to the members of CR1 lineage was 7 times smaller (9%). Homology with LTRs of Gypsy/Ty3 and BEL clades was revealed in 5% and 9% of cases respectively. We assume that the repetitive elements including retroelement-like sequences described in the current study may serve as the source of clonal variability detected previously in T. szidati and other digenean trematodes. Such genome regions rapidly accumulate mutations and thus may play an important functional role in the life history of the species.

Epidemiology of cercarial stage of trematodes in freshwater snails from Chiang Mai province,Thailand

Objective:To investigate the epidemiological situation of cercarial trematodes infection in freshwater snails from different water resources in Chiang Mai province,Thailand.Methods:The snail specimens were collected from 13 districts of Chiang Mai province during April 2008 to February 2012.The prevalence of cercarial infection in snails was investigated using the crushing method.The drawing was done with the help of a camera lucida for the morphological study.Results:A total of 2479 snail individuals were collected and classified into 7 families,11 genera,and 14 species,Among them,8 snails species were found to be infected with an overall prevalence of 17.27%(428/2479),which infected with nine groups of cercariae;gymnocephalous cercaria,strigea cercaria,megalurous cercaria,monostome cercaria,parapleurolophocercous cercaria(Haplorchis cercaria),pleurolophocercous cercaria,furcocercous cercaria(Transversotrema cercaria),xiphidiocercaria,and virgulate cercaria.The parapleurolophocercous cercaria was found to be the dominant type among the cercarial infection in the snails(64.25%).Conclusions:The various species of snails found in the research location act as the intermediate hosts for the high prevalence of parasitic infection of many species of mammals.This work will provide new information on both the distribution and first intermediate host of trematodes.

Synthesis and cercaricidal activities of a serial of novel self-diffused cercaricides derived from niphensamide

A serial of novel cercaricides that can self-diffuse on the water surface were designed and synthesized according to the special habit of cercariae larvae.These compounds were derived from niphensamide.While the compounds were dropped on the surface of water,the liquor diffused along the air-water interface and formed thin membranes floating on the water surface immediately.The strong cercaricidal activities against the cereariae larvae of Schistosome japonicum of these compounds have been revealed by further experiments.

Larval stages of digenetic trematodes in Melanopsis praemorsa snails from freshwater bodies in Palestine

Objective:To detect the species of larval trematodes(cercariae) in Melanopsis praemorsa snails from 5 different fresh water bodies in Palestine.Methods:A total of 1 880 Melanopsis praemona snails were collected from different fresh water bodies in Palestine from October,2008 to November,2010.Cercariae in Melanopsis praemona snails were obtained by lighting and crushing methods.The behavior of cercariae was observed using a dissecting microscope.Results:Three different species of larval trematodes were identified from Melanopsis praemona snails collected only from Al-Bathan fresh water body,while snails from other water bodies were not infected. These species were microcercous cercaria,xiphidiocercaria and brevifurcate lophocercous cercaria.These cercariae called Cercaria melanopsi palestinia 1,Cercaria melanopsi palestinia H and Cercaria melanopsi palestinia III have not been described before from this snail in Palestine. The infection rate of Melanopsis praemona collected from Al-Bathan fresh water body was 5.7%, while the overall infection rate of snails collected from all fresh water bodies was 4.3%.Details are presented on the morphology and behavior of the cercariae as well as their development within the snail.Conclusions:These results have been recorded for the first time and these cercariae may be of medical and veterinary importance.

<i>Schistosoma mansoni</i>: Phytochemical Effect on Aquatic Life Cycle

Background: Two aspects need to be considered for schistosomiasis control: morbidity and transmission. In this context, many soluble substances have been tested and Euphorbia milii latex is one of the most promising Brazilian molluscicides. Phytochemical studies involving simulation of the applicability of this latex on all aquatic forms of the S. mansoni life cycle are rare in the literature. The aim of this work was to evaluate the effect of E. milii latex on S. mansoni in the egg, miracidium and different developmental stages in Biomphalaria glabrata. Methods: The laboratory study was designed to simulate the different forms of exposure of the life cycle stages of S. mansoni to the LC50 of E. milii latex;we tested the exposition from four situations of S. mansoni contact with the latex and observed the exposure on different snails’ infection stage too. All snails were analyzed weekly for cercarial shedding and reproductive biology. Results: The results showed that contact of S. mansoni eggs and miracidia with the LC50 of E. milii negatively influenced the development of the parasite life cycle in the intermediate host, with consequent reduction of cercarial shedding. The exposure of infected snails affected the reproductive biology and cercarial shedding in all intra-mollusk development stages of S. mansoni, but the reduction was greater in the first, fourth, fifth, sixth, seventh and eighth weeks of infection. The LC50 of E. milii latex had toxic action on eggs and miracidia, and the number of cercariae shed by snails during the study period declined by about 80%. Conclusions: We can conclude that the use of natural biodegradable compounds containing low concentrations of substances already characterized as having eco-toxico- logical potential can be an important tool to reduce the transmission of Schistosomiasis.

Prevalence of cercarial infections in freshwater snails and morphological and molecular identification and phylogenetic trends of trematodes

Objective:To investigate the prevalence of cercarial infections in freshwater snails from several water sources in Nakhon Nayok,Nonthaburi,and Pathum Thani provinces of Central Thailand,and to reconstruct a phylogenetic tree for improved understanding of the relationships in the cercarial stage.Methods:The snail specimens were collected from 34 total sampling sites and investigated for cercarial infections using the crushing method.The cercarial specimens were classified and used for the phylogenetic tree analysis using the Internal Transcribed Spacer 2(ITS2).Results:A total of 1921 snail specimens were classified into five families and seven species.The results showed that four snail species were identified as intermediate hosts of the larval stages of trematodes,with an overall prevalence of infection of 2.45%(47/1921).The infected snail specimens included five groups of the cercarial type:cercariaeum cercariae,echinostome cercaria,megalurous cercaria,parapleurolophocercous cercaria,and xiphidiocercariae.This is particularly true of xiphidiocercariae,which was found to be the dominant type among cercarial infections in bithyniid snails by approximately 38.00%.With regard to molecular identification,the phylogenetic tree was reconstructed using the neighbor-joining method with 10000 bootstraps and separated the trematodes into three clades:Echinostomatoidea,Microphalloidea and Opisthorchioidea.Conclusions:The study reveals a high prevalence of cercarial infection for each cercarial type and maturation into a definite trematode genus and delineates morphological characteristics and evolutionary trends among each larval trematode in Nakhon Nayok,Nonthaburi and Pathum Thani provinces.In addition,the ITS2 sequence data of cercariae could be used to examine classification of these species at the family level.

Antigen presenting cells may be able to distinguish between normal and radiatedSchistosoma japonicum cercaria:anin vitro observation

Objective： To observe the discrepancies of responses induced by Schistosoma japonicum （S. japonicum） normal cercaria antigen （NCA） and ultraviolet （UV） -radiation-attenuated cercaria antigen （UVACA） in an in vitro system. Methods： S. japonicum cercariae were collected and UVACA and NCA were prepared. Mouse macro- phage model cells （RAW 264.7） were treated with medium, NCA （40 μg/mL） or UVACA （40 μg/mL） in the presence or absence of recombinant mouse interferon gamma （rmIFN-γ; 4 ng/mL） for 48 h. Cell surface staining and flow cytometry were used to assess the major histocompatibility complex （MHC） Ⅱ expression, and data were expressed as mean fluorescence intensities （MFI）. Interleukin （IL） -10, IL-6 and prostaglandin E2 （PGE2） in cell culture supernatant were evaluated by commercial enzyme-linked immunosorbent assays. Results： NCA significantly suppressed IFN-7-induced MHC Ⅱ expression on RAW 264.7 cells. In the presence of 1FN-7, NCA significantly promoted IL-6, IL-10 and PGE2 secretion from RAW 264.7 cells. In the presence of IFN-γ, UVACA significantly promoted IL-10 but not IL-6 and PGE2 secretion from RAW 264.7 cells and showed no effect on IFN-γ-induced MHC Ⅱ expression. Compared with UVACA, NCA significantly suppressed IFN-γ-induced MHC Ⅱ expression and significantly promoted IL-6, PGE2 and IL-10 secretion from RAW 264.7 cells. Conclusion： RAW 264.7 cells respond differently to NCA and UVACA. NCA can significantly suppress IFN-γ-induced MHC Ⅱ expression and significantly promote IL-6, IL-10 and PGE2 secretion from RAW 264.7 cells compared with UVACA.

Effect of temperature on the Bulinus globosus-Schistosoma haematobium system

Background:Given that increase in temperature may alter host-parasite relationships,the anticipated rise in temperature due to global warming might change transmission patterns of certain diseases.However,the extent to which this will happen is not well understood.Methods:Using a host-parasite system involving Bulinus globosus and Schistosoma haematobium,we assessed the effect of temperature on snail fecundity,growth,survival and parasite development under laboratory conditions.Results:Our results show that temperature may have a non-linear effect on snail fecundity and snail growth.Snails maintained at 15.5℃ and 36.0℃ did not produce egg masses while those maintained at 25.8℃ laid 344 and 105 more egg masses than snails at 31.0℃ and 21.2℃,respectively.Attainment of patency led to a reduction in egg mass production among the snails.However,the reduction in fecundity for snails maintained at 21.2℃ occurred before snails started shedding cercariae.Parasite development was accelerated at high temperatures with snails maintained at 31.0℃ reaching patency after three weeks.Furthermore,snail growth rate was highest at 25.8℃ while it was inhibited at 15.5℃ and reduced at 31.0℃.Increase in temperature increased snail mortality rates.Snails maintained at 36.0℃ had the shortest survival time while those maintained at 15.5℃ had the longest survival time.Conclusions:We concluded that temperature influences fecunxdity,growth,survival and parasite development in the snail and thus dictates the time it takes the parasite to complete the life cycle.This has implications on transmission of schistosomiasis in the context of global warming.

Types of trematodes infecting freshwater snails found in irrigation canals in the East Nile locality,Khartoum,Sudan

Background:The planorbid freshwater snails of the two genera,Biomphalaria and Bulinus-have been vigorously studied due to the role they play as intermediate hosts of schistosomiasis.In Sudan specifically,most studies have focused on the chemical and ecological control of the two genera,but few studies have looked at their biological control.This study explored the coexistence of other species of freshwater snails and the two genera along with their trematode infections in relation to a number of environmental factors in the East Nile locality,Khartoum state,Sudan.Methods:Freshwater snails from irrigation canals(abueshreens)were sampled monthly from January 2004 to December 2005.The snails were examined for trematode infections by cercarial emergence immediately after collection and then weekly for an additional four weeks to allow for the maturation of prepatent infections.Vegetation cover in the study sites as well as the physicochemical characteristics of the water,including temperature,were also recorded.Results:A total of 10,493 snails,representing seven species,were collected.The most abundant species was Biomphalaria pfeifferi,representing 48.6%of the sample.Overall,14.1%of the snails were found to be shedding some type of cercariae.Five species were found to have infections;among these the Bulinus truncatus species was found to be the most heavily infected,with an overall prevalence of 46.2%.Double infections were recorded in only two B.truncatus snails and one Cleopatra bulimoides snail.Twenty different morphotypes of cercariae were recorded,seven of which appeared not to conform to previously described cercariae from Africa.Xiphidiocercariae type 1 was the most common type of cercariae recovered,accounting for 44.3% of all infections.The density of snails tended to be lower during the summer months than the winter months,except for M.tuberculata snails,which were not affected by seasonal changes.Conclusion:The findings of this study indicate that besides schistosomes,other larval trematodes are found,and some use the same intermediate hosts as the schistosomes.Further studies should be conducted to determine whether some of these trematodes could be manipulated for the biological control of schistosomiasis.

Do algae blooms dilute the risk of trematode infections in threespine sticklebacks？

Human-induced growth of macro-algae is often assumed to increase trematode infections in fishes by increasing the abtmdanee and condition of the parasite＇s intermediate host - snails - as this can boost the release of trematode larvae, cercariae, from the intermediate hosts. However, macro-algae can also impose barriers to the transmission of cercariae and reduce infections. We investigated whether an increased growth of filamentous algae affects the transmission of Diplostomum pseudospathaceum cercariae to the threespine stickleback Gasterosteus aculeatus, a common fish in eutrophied shallow waters. We exposed stickle- backs to trematode cercariae in the absence and presence of artificial filamentous algae, and recorded effects on the proportion of sticklebacks infected and the number of encysted metacercariae per fish. No significant effect of artificial algae on cercariae transmission was detected. However, the body size and the sex of the sticklebacks were strongly correlated with the number of encysted metacercariae per infected fish, with females and larger individuals being more infected. We discuss different factors that could have caused the difference in parasite transmission, including sex-related differences in body size and behaviour of sticklebacks .

Effects of vertical position on trematode parasitism in larval anurans

Spatial distributions of animals can affect interactions with their natural enemies,such as parasites,and thus have important implications for host-parasite dynamics.While spatial variation in infection risk has been explored in many systems at the landscape scale,less attention has been paid to spatial structure at smaller scales.Here,we explore a hypothesized relationship between a common spatial variable,vertical position,and risk of parasite infection in a model aquatic system,larval frogs(Rana)and trematode(Digenea)parasites.Vertical position is relevant to this system given evidence that the densities of snail first intermediate hosts,tadpole second intermediate hosts,and trematode infective stages can vary with depth.To test the effects of depth on infection risk of larval frogs by trematodes,we performed two enclosure experiments,one in the laboratory and one in the field,in which larval frogs in cages just below the water surface or near the bottom of the water column were exposed to parasites.Compared with near-surface cages,mean infection load(number of cysts)in tadpoles in near-bottom cages was 83%higher after 48-h exposures in the laboratory and 730%higher after 10-day exposures in the field.Our findings thus indicate that infection risk depends on depth,which may have adaptive significance,as tadpoles have previously been shown to change vertical position in response to parasite presence.These results motivate future work examining vertical variation in infection risk and may have broader implications for host-parasite dynamics and evolution of host and parasite behavior.

A novel and effective hyperthermia method for Schistosomiasis japonica prevention and treatment

The emerging resistance to schistosoma has been paid much attention and is in urgent need for a novel strategy to control the prevalent parasitic zoonosis. In this study, the efficiency of hyperthermia therapy was investigated by the skin hyperthermia device. The survival rate of cercariae decreased from 68.15 % （37 ℃, 5 min） to 0 （49℃, 10 min） with the thermal dosages increased, which proved the preventing effect of hyperthermia therapy （P 〈 0.05）. Therapeutic effects were assessed in Schistosoma japonicum-infected BALB/c mice. When the cercarial contact region of the skin was treated at 45-49℃ for 5 min within 8 h of infection, worm reduction rate （WRR） reached 74 %-83 % （P 〈 0.01）. The sensitivity of adult schistosoma to heat was also investigated using microwave intraperitoneal hyperthermia （thermal dosages 42-43℃, 20 min）. The WRR, hepatic shift rates and egg reduction rates were 23.7 %, 40 % and 30 %, respectively, comparing with 80.2 %, 59.6 % and 53.9 % of praziquantel （PZQ）-treated group. Encouraging results have been obtained that hyperthermia can effectively kill schistosomula, especially with the appearance of cercarial dermatitis, while PZQ lacks efficacy against the cercariae. Thus, hyperthermia therapy would show significant benefit in preventing and treatment of schistosoma, especially in the early stage.

Observation of developing cercaria of Schistosoma japonicum on ultrastructural level and remarks at its morphology of mature stage

Objective To study the developing cercaria of Schistosoma Japonicum (S. japonicum) on ultrastructural level by transmission electron microscope (TEM) for analyzing the morphological dynamic changes of the tegument, glands and musculature. Methods Artificial infected Oncomelania hupensis were dissected under dissecting microscope and the daughter sporocysts picked up for studying the germinal cells stage (S1). The other embryonic cercaria were selected according to the modified parameter of Chen and Bier (1972). The specimens were prepared by conventional procedure of the laboratory of TEM and were observed by Hitachi H600. Results Besides the germinal cell stage (S1), this is the first chronological study on the morphological development of S. japonicum cercaria from S2 S5 concerning the tegument and its elements (glycocalyx, sensory papilla, basal lamina and spine), head and acetabular gland and musculature of the body and tail. This article discusses the ultrastructural morphological differences from prior authors and emphasizes on the postacetalar gland and the pattern of tail musculature of the matured stage cercaria. Conclusions According to the cell division and differentiation, the process of development may be divided into 5 stages: S1, the single germinal cell stage; S2, the germinal ball stage; major multiplication and minor differentiation; S3, tail budding embryonic stage, both active in multiplying and differentiating; S4, the pre matured stage, major differentiation and minor multiplication; and S5, the fully matured stage (completing the differentiation).

Environmental determinants of distribution of freshwater snails and trematode infection in the Omo Gibe River Basin,southwest Ethiopia

Background:Determination of infection rates of snail populations is one of the basic tools for epidemiological studies of snail borne diseases.In this study,we opted to determine the trematode infection of freshwater snails in the Omo-Gibe River Basin,southwest Ethiopia.Methods:We collected snail samples from 130 observation sites in lakes,wetlands,rivers,reservoirs and irrigation canals surveyed during the dry season(March to May)in 2016.The snail samples were examined for trematode infections by cercarial shedding immediately after collection.Habitat conditions,water quality,human water contact practices and other human activities were assessed at each survey site.A redundancy analysis(RDA)was used to examine the relationship between cercarial infection and environmental variables.The statistical significance of eigenvalues and cercariae-environment correlations generated by the RDA were tested using Monte Carlo permutations at 499 permutations.Results:A total of 3107 snails belonging to five species were collected.The most abundant species was Biomphalario pfeifferi,representing 66%of the total collection.Overall,109(3.6%)of the snails were found infected with trematodes(cercariae).Biompholario pfeifferi was found to be the most highly infected,accounting 85%of all infected snails.A total of eight morphologically different types of cercariae were recorded,which included:Echinostoma cercariae,brevifurcate apharyngeate distome cercariae,amphistome cercariae,brevifurcate apharyngeate monostome cercariae,xiphidiocercariae,longifurcate pharyngeate distome cercariae,strigea cercariae and unidentified cercariae.Brevifurcate apharyngeate distome cercariae,and Echinostoma cercariae were the most abundant cercariae,accounting for 36 and 34%of all infection,respectively.The mean concentration of water conductivity and 5 days biological oxygen demand were higher in irrigation canals and lake sampling points.Human activities such as open field defecation,urination,livestock grazing,farming,and swimming were highly correlated with trematode infection.Conclusions:The abundance,occurrence and infection rates of snail species were largely influenced by water physicochemical quality,sanitation and water contact behaviour of the inhabitants.Human activities,such as open field defecation and urination,livestock grazing,farming,and swimming were important predictors of the abundance of cercariae.Therefore,awareness creation should be implemented for proper containment of excreta(urine and faeces)and reducing human and animal contacts with surface waters to reduce snail-borne disease transmission.