To explore the resistance against challenge infection in the two susceptible strains of the offspring mice born to mother mice immunized with schistosomulum cells. Both susceptible female Kunming strain and Balb/c strain of mice to schiostosome were immunized with Schistosomulum japonicum cells for 3 time, respectively. After mating with the corresponding strain of male mice, the offspring mice either born to immunized mothers or to untreated mothers were divided into 4 groups, A1:experiment offspring mice born to immunized Kunming strain of mother mice, B1: control offspring mice born to untreated Kunming strain of mothers, A2: experiment offspring mice born to immunized Balb/c strain of mother mice, B2: control offspring mice born to untreated Balb/c strain of mothers. About 6-8 weeks post neutralization rearing (both strains of mice in weight of 16-20 g), the two strains of offspring mice either born to immunized mothers (A1 and A2 group) or born to untreated normal mothers (B1 and B2 group) were challenged with S. j cercariae and the protection efficiency was evaluated with parasitological and immunological indicators. The worm burdens and liver egg loads collected from above 4 groups were counted and compared; The liver sections were prepared and stained with HE, and the diameters and areas of liver egg granulomas were estimated; The levels of Ig G against soluble juvenile worm antigen (SJWA) in the sera of the progenies collected at different time points were detected by ELISA. Compared with two strains of control groups B1 and B2, both strains of experiment offspring mice (A1 and A2) possessed certain level of protection against challenge infection with either Kunming strain of offspring mice exhibiting 35.6％ reduction in worm burden and 59.4％ reduction of liver egg burden or Balb/c strain of offspring mice showing 29.8％ reduction in worm burden and 47.4％ reduction in liver egg burden. The liver appearances of A1 and A2 mice were relatively normal as compared with that of control mice. At the same time, the mean diamaters and areas of granulomas in the sections of livers from A1 and A2 groups were significantly smaller than that in the sections of livers from B1 and B2 groups. Furthermore, ELISA results showed that the levels of specific Ig G against SJWA in the sera from A1 and A2 group of mice were higher than the Ig G levels in the sera from corresponding strain of control groups. By employing two susceptible strains of mice to S. j infection as models, the experiment results indicated that the mother mice immunized with live schistosomulum cells could deliver the protective immunity to next generation and make them to inherit immune protection against initial acquired challenge of Schistosoma japonicum.

Background Schistosomiasis , one of the neglected tropical diseases, is endemic in more than 70 countries. However, the clinical diagnosis of patients with a low degree of infection is an unsolved technical problem. In areas endemic for schistosomiasis japonica, proctoscopy detection of eggs has been one method used for clinical diagnosis. However, it is often a challenge to find typical live eggs and it is difficult to distinguish live eggs from large numbers of partially degraded and/or completely degraded eggs within colon biopsy tissue. To address this problem, we tested six different morphological and biochemical/molecular markers (ALP; morphological characteristics of egg; CalS (calcified substance); AOS (antioxidase); SDHG (succinic dehydrogenase) and SjR2 mRNA (retrotransposons 2 of S.japonicum genome mRNA)), including four new markers (CalS; AOS; SDHG and SjR2 mRNA.), to determine the viability of S. japonicum eggs deposited in human and mouse colon tissues. Our ultimate aim is to obtain a new method that is more sensitive, practical and accurate to clinically diagnose schistosomiasis. Methods Tissue samples were collected from mice at six different time points during S. japonicum infection with or without treatment with praziquantel (PZQ). Four new biochemical or molecular markers were used for the detection of egg viability from mouse liver and intestinal samples: CalS; AOS; SDHG and SjR2 mRNA. Subsequently, all markers were employed for the detection and analysis of eggs deposited in biopsy materials from patients with suspected schistosomiasis japonica for clinical evaluation. Microscopic examination of the egg morphology, worm burden in vivo and ALP (alkaline phosphatase) levels were used as a reference standard to evaluate the sensitivity and reliability of four new markers detecting egg viability. Results The results of the study showed that the morphology of S. japonicum eggs deposited in tissues of hosts with schistosomiasis, especially cases with chronic schistosomiasis, is complex and egg viability is difficult to judge morphologically, particularly eggs with a fuzzy structure or partially modified eggs. We found that the majority of the viable schistosome eggs determined by four new markers (CalS, AOS, SDHG and SjR2 mRNA) were morphologically difficult to identify. Conclusions Among the markers, the most sensitive and specific method was the detection of SjR2 mRNA and the most simple, rapid and practical method was the detection of SDHG. Therefore, the detection of SDHG is the most practical for clinical application and its use could improve the accuracy in diagnosing active schistosome infection.

With the development of the information technology, the open classroom teaching is increasingly welcomed by students. The exploration and practice of teaching methods in medical parasitology for medical students, including small classes, microlectures, the high-quality digital pictures of parasite specimens, the reform for parasitological experimental exam and discussion classes, will improve the education level for them.This attempt benefits the exploration of innovative training patterns for medical talents in our country.

Objective To establish a molecular pathology method which can be used for clinical pathological diagnosis on the species identification of helminthes in paraffin-embedded tissue sections.Methods The specimens known and from the clinical variety of worms and host tissues were collected and made.The DNA was extracted from the white thin slices,HE stained slices and immunohistochemical staining slices of the specimens with modified proteinase K digestion,respectively.The primers were designed and synthesized for detecting the specific gene fragments of Paragonimus,Sparganum mansoni and cysticercus cellulosae of Taenia solium and conducting PCR amplification.PCR products were analyzed by gel electrophoresis for observing the target gene strip.The sensitivity and specificity of detecting and identifying minimum tissue amount of the three species of worms were analyzed.Results The specific target gene fragments were amplified from the primers of the genes of three species of worms detected.The results of PCR amplification of the target gene fragments showed that the sensitivity of extracting the worm DNA with modified proteinase K digestion was significantly better than that of commercial kits extraction method.A smallest area and thickness of the worm organizations which could submit DNA with modified proteinase K digestion in Paragonimus,Sparganum mansoni,and cysticercus cellulosae were 1.50 mm2 and 10 μm,4.87 mm and 5 μm,5.80 mm and 5 μm,respectively.The results of 10 clinical specimens detected with the above methods were all very ideal.Conclusions This study successfully obtains a simple method of PCR identifying the species of helminthes by using DNA extracted from paraffin-embedded tissue sections,and it provides a means of molecular pathology for clinical pathological differential diagnosis of these three kinds of worms.

OBJECTIVE:To screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum, not cercaria, tegument of Schistosoma japonicum.METHODS:A 12 phage-display peptide library was screened with the S. japonicum schistosomula and cercariae as the target cells for biopanning by degrees, 15 positive clones were picked randomly and deduced by DNA sequencing. According the sequencing result, ELISA test, elution recovery test and immunohistochemical staining were performed to determine the specificity of the phages to the tegument. To further examine its binding properties, the positive peptide conjugated to RhB and recombinant pEGFP-C2 plasmid were similarly synthesized.RESULTS:After 3 rounds of biopanning, the phage recovery rate increased from 3.50 x 10(-5)% to 3.20 x 10(-2)%, indicating that the phage library was successfully enriched in the tegument of schistosomula. The analyzed sequences were identical with 3 peptide sequence of ZL6, ZL4 and ZL1. ELISA showed that the P/N value of MppZL4, MppZL6 and MppZL binding the schistosomulum membrane protein was 6.72, 3.65 and 2.22, while 1.58, 5.15 and 1.20 of binding the membrane protein of cercariae, respectively. Elution recovery test showed that the elution recovery rate of MppZL4 [(4.60 +/- 0.27) x 10(-2)%] was much higher than that of MppZL6 [(2.10 +/- 0.23) x 10(-3)%], MppZL1 [(1.20 +/- 0.28) x 10(-3)%] and M13KE [(1.30 +/- 0.60) x 10(-7)%] (P<0.01). Immunohistochemical staining showed that MppZL4 specifically bound to the tegument of schistosomula with a positive rate of 83.0% (83/100). Fluorescent microscopy revealed that the synthesized RhB-ZL4 bound to the tegument of schistosomula. The ZL4/pEGFP-C2 plasmid was introduced into juvenile S. japonicum and expressed in the parasite.CONCLUSION:The peptide of ZL4 specifically binds to the schistosomulum tegument but not to that of cercaria.

Schistosoma japonicum adults are pre-embedded in a double-layer agar and made the block, then dehydrated with alcohol, isobutyl alcohol and n-butyl alcohol. Various staining procedures can be conducted after conventional sectioning and dewaxing. Complete longitudinal serial sections of the pre-embedded worms can be obtained, and the desired sections can be easily located accurately.

血吸虫(schistosoma)又叫作裂体吸虫,是寄生在宿主静脉内的一种扁形动物,借助钉螺这一中间宿主,感染人体后导致相关疾病(图1).血吸虫病(schistosomiasis)是一种严重危害人类健康的寄生虫病,在我国流行已有2000多年的历史[1],高发区域主要集中在我国长江中下流地区以及以南的12个省[2].尽管这些区域内已经实施了相关防控措施,部分区域钉螺灭螺面积达到80%以上,然而仍有5个省(市、区)的110个县、区未达到疫情控制标准,共有血吸虫病人62416人,其中约1/3为晚期血吸虫病患者[3].为了更好更全面的做好晚期血吸虫病的救助工作,及时、准确的诊断显得尤为重要.

OBJECTIVE:To diagnose 10 cases of clinically suspected cases of sparganosis mansoni by pathogen identification.METHODS:In the period from August 2009 to August 2011, 10 biopsy specimens were obtained from 10 patients of four hospitals to identify the pathogen. Among the 10 cases, 4 cases showed abdominal subcutaneous mass, 3 showed eyelid swelling, 1 displayed brain lesions, 1 showed pulmonary mass, and 1 showed pleural effusion. There was one parasite each from three patients with eyelid swelling, and one patient with abdominal subcutaneous mass, which were observed by naked eye and microscope morphologically and histologically. Specimens from other six cases were examined by microscope after paraffin embedding, sectioning, and HE staining. For further identification, the parasite biopsy tissue specimens were detected by immunohistochemistry with Sparganum mansoni-immunized rabbit serum as the primary antibody.RESULTS:Three intact worms, from three patients with eyelid swelling, showed typical S. mansoni morphological characteristics. One residue parasite from the abdominal subcutaneous mass showed network structures and full of calcareous corpuscles in the body under microscope same as that of S. mansoni. The histological structure in three of the six sections showed typically the body wall with folds, which was dense, thick and deeply eosine stained, part of the tegument outside was covered by micro-hairs. In the worm body there was net-like loose structure and calcareous corpuscles without cavity. The structure of the other three worm sections was atypical. The six worm sections were positive by immunohistochemical detection.CONCLUSION:The 10 clinically suspected cases are diagnosed as sparganosis mansoni.

AIM:To understand the influence of astragalus on the development of Schistosoma japonicum(Sj) and immune response of mice infected with Sj.METHODS:Kunming strain of mice were naturally and succes-sively administrated astragalus for 50 d to observe its influence on Sj-infected mice and detect the level of antibody against Sj adoult worm antigens(SjAWA) and level of Sj soluble egg antigens(SjSEA),IL-6,and IL-10 immune response to hosts infected by Sj.RESULTS:The results showed that compared with infected controls,after admin-istration with low-dose astragalus(0.25 g/d),high-dose astragalus(0.5 g/d),low-dose astragalus + praziquantel and praziquantel group,respectively,the reduction rates of worm were 29.2%,30.3%,43.2% and 35.8%,re-spectively,and the body lengths of females reduced 10.49%,10.57%,33.61% and 22.76% and that of males 8.40%,15.33%,32.50% and 28.52% and liver eggs reduced 31.1%,36.0%,47.4% and 46.9%.The lev-els of antibody against SEA,IL-6 and a pro-inflammatory cytokine in the four administrated groups but for infected group were significantly reduced(either P 0.05 or P 0.01),but the level of IL-10,an anti-inflammatory cyto-kine was risen(P 0.05).CONCLUSION:Astragalus plays a role in anti-growth and development of Sj and im-mune response to antibody against eggs,down-regulates proinflammatory factor and up-regulates antiinflammatory factor.

Peptides, bound to the tegument of live Schistosoma japonicum schistosomula, were differentially screened by phage display in vitro using three rounds of reverse absorption and bio-panning. Three M13 phage peptides were isolated and identified by determination of their recovery rate, immunohistochemical localization, immunoblot analysis, and their anti-schistosomal effects in vivo and in vitro. Of the three, M13 phage peptide ZL4 (MppZL4, YSGLQDSSLRLR, 1.4 kDa, pI 8.8) bound to the tegument of mechanically transformed schistosomula and to other developmental stages of S. japonicum from the mammalian host. By contrast, MppZL4 did not bind to the surface of cercariae. To further examine its binding properties, MppZL4 was conjugated to Rhodamine B (RhB-YSGLQDSSLRLR, RhB-ZL4) and a peptide control (RhB-AIPYFSGILQWR, RhB-12P) was similarly synthesized. The binding capacities of RhB-ZL4 to the surface membrane of S. japonicum schistosomula in vitro and of S. japonicum adult worms in vivo were examined and revealed specificity for binding. When examined for anti-parasite activity, both MppZL4 and RhB-ZL4 exhibited a potent schistosomicidal effect in vitro. Further MppZL4 also affected the growth and development of schistosomula in vivo. These findings extend previous studies showing that phage display techniques can recover polypeptides that bind specifically to living schistosomes and, moreover, that these bound peptides have the potential to inhibit key physiological processes in these parasites. Our findings suggest further that ectogenic polypeptides, which can bind to the tegument of S. japonicum, might be adapted as vectors to deliver experimental probes and/or pharmacologically relevant compounds to the schistosome tegument, including drugs and immunological mediators.

In order to pack the recombinant adenovirus vector carrying SV40LT gene and to study the effect of the recombinant adenovirus on transfecting schistosomulum cells,The constructed recombinant adenovirus plasmid was re-transformed into competent cells Stbl2,and identified by restriction enzyme digestion.The right recombinant adenovirus plasmid was transfected into 293A cells using Lipofectine 2000.The recombinant adenovirus was amplified,and then identified by polymerase chain reaction(PCR).The effect of the recombinant adenovirus transfecting schistosomulum cells was detected by RT-PCR and immunohistochemistry.The recombinant adenovirus containing SV40LT gene was successfully packed,which effectively infected schistosomulum cells.The expression of SV40LT gene in schistosomulum cells was detected by RT-PCR and immunohistochemistry.The recombinant adenovirus containing SV40LT gene could transfect schistosomulum cells to provide a basis for Schistosoma japonicum cell immortalization.

OBJECTIVETo screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum tegument of Schistosoma japonicum.METHODSA 12 phage-display peptide library was screened with the S. japonicum schistosomula as the target cells for biopanning by degrees, positive clones picked randomly were deduced by DNA sequencing. According the sequence seeing result, immunohistochemical staining was performed to determine the specificity of the phages to the tegument. To test their targeting efficacy, the interested phage clones were infused back to the mice infected with S. japonicum, mice were sacrificed 2.5 hours later, and the phage distribution in the liver and the tegument of schistosomula was appraised, respectively.RESULTSAfter 3 rounds of biopanning, the phage recovery rate increased from 0.77 x 10(-8) to 0.75 x 10(-5), indicating that the phage library was successfully enriched in the tegument of schistosomula. Seventy-five percent (15/20) of the analyzed sequences were identical with a sequence of QHPRIRKOOOOO. The immunohistochemical stainings showed this sequence specifically binding to the tegument. In vivo titering displayed that this sequence selectively targeted the tegument.CONCLUSIONThe peptide of QHPRIRKOOOOO specifically binds to the schistosomulum tegument.

Although draft genome sequences of two of the major human schistosomes, Schistosoma japonicum and Schistosoma mansoni are available, the structures and characteristics of most genes and the influence of exogenous genes on the metabolism of schistosomes remain uncharacterized. Furthermore, which functional genomics approaches will be tractable for schistosomes are not yet apparent. Here, the vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped pantropic retroviral vector pBABE-puro was modified to incorporate the human telomerase reverse transcriptase gene (hTERT) as a reporter, under the control of the retroviral long terminal repeat (LTR). Pseudotyped virions were employed to transduce S. japonicum to investigate the utility of retrovirus-mediated transgenesis of S. japonicum and the activity of human telomerase reverse transcriptase as a reporter transgene in schistosomes. Schistosomules perfused from experimentally infected rabbits were cultured for 6 days after exposure to the virions after which genomic DNAs from virus exposed and control worms were extracted. Analysis of RNA from transduced parasites and immunohistochemistry of thin parasite sections revealed expression of hTERT in the transduced worms. Expression of hTERT was also confirmed by immunoblot analysis. These findings indicated that S. japonicum could be effectively transduced by VSVG-pseudotyped retrovirus carrying the hTERT gene. Given the potential of hTERT to aid in derivation of immortalized cells, these findings suggest that this pantropic retroviral approach can be employed to transduce cells from specific tissues and organs of schistosomes to investigate the influence of transgene hTERT on growth and proliferation of schistosome cells.

Objective To examine the effectiveness of different chemical dyes at staining Schistosoma japonicum eggs in order to provide a method of staining that can easily distinguish eggs and fecal residue under a microscope.This may have considerable significance for the automated identification of schistosome eggs.Methods At different concentrations and temperatures,S.japonicum eggs in liquid feces were stained with the following chemical dyes:AO-EB,rhodamine,neutral red,acid fuchsin,toluidine blue,trypan blue,Coomassie brilliant blue,and malachite green.Then,the effects of various staining methods were compared with smear microscopy of eggs and fecal residue to provide a primary index to assess contrast.Results Of the eight staining methods,AO-EB staining and neutral red staining resulted in the greatest difference between eggs and fecal residue.These two methods allowed eggs to easily be identified under the microscope.The detection rate for AO-EB and neutral red staining was 67.5% and 65.0%,respectively.These methods were clearly superior to modified Kato staining,which had a detection rate of 40.0%.Conclusion AO-EB staining and neutral red staining helped to increase the rate of schistosome egg detection.

Objective To observe the difference between single administration of astragalus and combined administration of astragalus,pumpkin seeds and arecas on the treatment of schistosomiasis.Methods 9 groups [infected control group,administration of compound 1～10 d after infection,administration of compound 8～17 d after infection,administration of compound 15～24 d after infection,administration of compound 28～37 d after infection,administration of astragalus 1～10 d after infection,administration of pumpkin seeds 1～10 d after infection,administration of pumpkin seeds and arecas 1～10 d after infection,administration of Praziquantel(PZQ) 36 d after infection at the dose of 300 mg /kg·d] were set up to compare the effect differences of the traditional Chinese medicine compounds at different infective stages.Results The worm reduce rates of the groups administrated compounds 1～10 days,8～ 17 days and 15～24 days after infection were 36.21%,26.74% and 39.04 %(P0.05),respectively,and the liver egg reduce rates were 58.6%,32.2% and 47.7%(P0.05),respectively,as compared with control group.The node densities on liver surfaces from treated groups were less than that from control mice.However,as compared the group administrated compounds 28～37 days after infection,administration of astragalus 1～10 d after infection and the group administrated single pumpkin seeds 1 ～10 days after infection as well as the group administrated pumpkin seeds + arecas,there was no difference in worm reduce rate and egg reduce rate when compared with control group.Conclusion The combination of astragalus,pumpkin seeds and arecas could produce a good effect of antischistosomulum,which was better than only use astragalus,or pumpkin seeds or arecas.

Objective To observe the differences in growth rates of Schistosoma japonicum(Sj)at different points in development.Methods Worms were recovered from Kunming mice infected with Sj cercariae on days 0,8,12,16,20,24,and 28 post-infection,respectively.Pathological Image Processing System Motic BA400 and 3.2 software were utilized to observe morphology and measure worm area,perimeter,length,and width.Results Worms at different points in development grew at distinctive rates.Worms on days 8 to 12 had the fastest development,followed by worms on days 12 to 24,0 to 8,and 24 to 28.Even at the same point in time,there were obvious distinctions among the worms,especially after day 12.Conclusion The growth rate of Sj worms differs at different points in the worms' development.The growth rate of 8 to 12-day old worms was the fastest.

In order to observe the influence of immunization route on the immune protective effects induced by primary juvenile worm cells(pJCs),Kunming mice were inoculated intravenously or subcutaneously with pJCs from 12 day-old Schistosoma japonicum respectively.All mice were immunized three times at 2 weeks interval and were challenged each with 30±2 cercariae by abdominal skin penetration 4 weeks after the last booster.Six weeks of post-infection,mice were sacrificed by aether inhalation,and the numbers of worms and hepatic eggs were counted.Specific anti-pJCs levels in the mice sera collected one day before the second,third immunization and challenge infection were tested with ELISA.Mice immunized with pJCs given i.v.or s.c.exhibited high level of specific antibody and protection effects,but given i.v.was significantly higher than given s.c.in anti-pJCs level(P0.05)and showed significant reduction in worm burden(48.53%),liver eggs per gram(LEPG)load(54.54%),worm average weight(29.62%)and egg granuloma size(36.8%).The juvenile worm cells of S.japonicum could induce partial protective immunity against S.japonicum in mice and the protective immunity by i.v.was better than that by s.c..These results indicated intravenous route was a feasible and efficacious route for juvenile worm cell immunization.