| Schistosomiasis is one of the most serious parasitic diseases in human and livestock, and widely distributed water-borne disease in the world from the report of World Health Organization. In our country, there is only Schistosoma japonicum endemic. By the end of 2015, four districts and counties (Qixia, Luhe, Pukou, Jiangning) of Nanjing and three counties (Dantu, Yangzhong, Runzhou) of Zhenjiang in Jiangsu Province were still in the level of schistosomiasis transmission control, for the rest of the city had achieved the standard of schistosomiasis transmission interrupted. As the unique intermediate host of schistosome, infected snails (Oncomelania hupensis) in upstream Yangtze river could spread with water, which could lead a threat of schistosomiasis epidemic in Jiangsu province.The conventional methods with serological and etiological examination in schistosomiasis test were not suitable for low endemic areas gradually. In order to explore the endemic status in schistosomiasis transmission control regions and establish a new rapid and accurate method for schistosomiasis detection, the studies were carried out as follow.1. Investigation of Oncomelania hupensis snail and human and livestock schistosomiasisTwo typical area in Qixia district from 2013 to 2014 and Shuiyang river in Gaochun district of Nanjing in 2015 were investigated in our study. The density survey of infection Oncomelania hupensis and schistosomiasis examination in human and livestock were carried out, combined with an analysis of Jiangsu provincial report of schistosomiasis control (2004-2014). Method Mechanical and environmental sampling for snail detection, DDIA for sera screen and fecal examination for human schistosomiasis, stool hatching method with plastic cup for livestock were used in our survey with the national standard for control and elimination of schistosomiasis (GB15976-2015). Results There was no infected snail found in Qixia district and Shuiyang river in Gaochun district. Serological detection with DDIA and hatching fecal microscopic examination using nylon silk bags, the positive rate of serological test was less than 1% in two selected villiages in Qixia, and 2.48% positive in three villages in Gaochun. There is no infected sample found with fecal egg hatching. Furthermore, livestock found no infected. Conclusions The distribution area of snail had decreased year by year in Jiangsu province, the density of live snail maintained in a relative stable low epidemic situation. Schistosomiasis showed a low prevalence and there was no acute schistosomiasis case in recent years. The positive rates of schistosomiasis with serum test was higher than 1% along Shuiyang river in Gaochun, suggested that the regions with higher risk, it is necessary to strengthen the monitoring of the epidemic.2. Establishment of Schistosomiasis detection method based on the different expression peptides in serumTo investigate the serum differential expression peptides in the sera of rabbit with acute infection, and to establish the methods with different peptide peaks by ClinProTools. Methods After serum collection, serum peptide enrichment with magnetic beads, MALDI-TOF/TOF mass spectrometry detection and ClinProTools analysis, serum peptide diagnosis method (ClinProTools, CPT) was established. Results In experimental rabbit model, Mr 1787 protein has significantly increased in the rabbit at the 6th day post-infection (P<0.05). On the 9th day after infection, Mr 2834 protein was high significantly increased (P<0.01), Mr 3483 increased (P<0.05) and Mr 4018 decreased (P<0.05) significantly. In 12 days after infection, Mr 3151 proteins have significantly decreased (P<0.05), and Mr 4018 was reduced significantly (P<0.01), Mr 3530 was increased (P<0.05), the trend continued until the infection after the 30 days. The peptides identification with TOF-TOF, Mr 1787 was predicted as alpha enolase, and Mr 3530 was heat shock protein 90 or 47 fragment, suggested that the two proteins maybe valuable markers. The detection method was established with serum peptides peaks in 5 weeks and health control using ClinProTools software, the blind test with serum in rabbit at 1,2,3,4w post-infection showed 30%,55%,75%,80% positive, and ELISA results were 0,0,20% and 50%. Toxoplasma gondii infection rabbit were also tested for specificity. Conclusions The sensitivity of schistosomiasis detection method based on the different expressed serum peptide was superior to the conventional ELISA method. It was useful for early detection of schistosomiasis and screening of diagnostic value of serum peptide/protein markers.3.Application of ClinProTool method with serum different expression peptides3.1 Application of ClinProTool method in experimental miceMouse is a widely used animal model in the experiment of schistosoma japonicum infection. It is of great significance to develop a rapid, accurate and simple method for the diagnosis of schistosomiasis. Methods After serum collection, serum peptide enrichment with magnetic beads, serum peptide detection method (CPT) was established with differential peptides peaks between 5w post-infection and health control by MALDI-TOF/TOF mass spectrometry detection and ClinProTools. Results Compared with the traditional ELISA method, CPT method detected positive (5%,3/60) in the first week post-infection, and 35%(21/60),75%(45/60),87.93% (51/58) and 98.15%(53/54) showed in 2nd,3rd,4th,5th week respectively, with a gradual upward trend. The ELISA method was only detected after 3 weeks post-infection, the infection rate was 65%(39/60), and 77.59%(45/58),94.44%(51/54) in 4,5 weeks, all of which were lower than the CPT method.70%(7/10) positive mice were detected at 3-4w using the parasitic dissection, which lower than CPT method at the same time. Different infectiosity mice were blind test with CPT method, all the mice infected with 14,18,22 cercariae were predicted to be infected, the accuracy of blind test was 40%(4/10),50%(5/10) and 80%(8/10) in 4,6 and 10 cercariae group of mice. Conclusions Sucessfully diagnosis with ClinProTools method was at least 2-3 weeks in advance than that with ELISA detection, and reduced the workload than the parasitic detection. It could be used for experimental mice test at low infection status, which was expected to provide a new method in "sentinel mice" detection.3.2 Application of ClinProTool in sentinel miceIt is useful to use "sentinel mouse" to monitor the schistosomiasis endemic in the field of Yangtze river in Jiangsu. Parasitology examination for adult and liver egg granuloma is the primary method in sentinel mice diagnosis, but time-consuming and labor intensive. There is an urgent need of new method for the rapid detection in schistosomiasis. Methods Serum peptide fingerprint of the "sentinel mice" were aquired, and the detection method of CPT was used for the blind test.660 sentinel mice from field test in 2015,5 cases of positive and 50 negative "sentinel mice" (2009-2014) after the adult worm examination were verified with CPT method. Results:Five positive sentinel mice were detected with CPT correctly,660 sentinel mice (including 60 from Qixia and Gaochun district,600 from Nanjing, Zhenjiang and Yangzhou) were validated as uninfected, which were consistent with the result of dissection examination. Conclusions The ClinProTool method with serum different expression peptides was earlier than the conventional method for the detection, which was helpful for early survelliance in sentinel mice.3.3 Application of ClinProTool in human advanced schistosomiasis patients identificationThe diagnosis and treatment of patients with advanced schistosomiasis is a key point in schistosomiasis prevention and control. Identification of advanced schistosomiasis and acute schistosomiasis is important for the assessment in schistosomiasis survelliance. Methods sera samples were collected from advanced schistosomiasis, newly development advanced human schistosomiasis(NDAS), cured human and healthy controls, the serum protein fingerprint were acquired and CPT method was established based on differences of serum peptides. Results The groups of advanced schistosomiasis, new development advanced schistosomiasis and cured cases had the same peptides spectrum without significant difference. Compared with the healthy control group, NDAS in Mr 2662,2991,3241,5337 and 5906 were down regulated significantly, whereas Mr 2082 and 4282 were increased significantly. Comparision with the group of cure and the healthy control group, Mr 2662,4209,5337 and 5906 showed down-regulated and Mr 2082 showed up-regulated significantly. Compared with the control group, the newly advanced group was significantly reduced in Mr 3241. It suggested that Mr 3241 and 4282 could be used as the indication of the newly advanced schistosomiasis patients, all the peptides peaks identified with TOF-TOF. The two kinds of peptides were immunoglobulin heavy chain variable region. The groups of advanced, newly advanced schistosomiasis, cured patients and health control were all correctly diagnosed.73.3%(11/15) samples were verified as unknown group,86.2%(25/29) samples from patients with ELISA positive and stool examination negative were verified as advanced patients from epidemiological survey in Qianxia and Gaochun district. Conclusions The advanced and current schistosomiasis could be distinguished with ClinProTools method using different peptide indirectly, but the latter may include previous infection or re-infection, which may validated as advanced schistosomiasis patients. ClinProTools method with serum different peptide combined with conventional parasite examination could be used for preliminary screening in schistosomiasis epidemiology investigation. |
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