| Angiostrongylus cantonensis (AC), a metastrongyloid nematode, is the major causative agent of humanEosinophilic meningitis or meningoencephalitis (also known as the angiostrongyliasis cantonensis) in many parts of the world. In general, the human infected with A. cantonensisby eating raw or undercooked food, aquatic products, vegetables, or fruit, which containing with AC infective larvae (three-stage larvae, L3). The A. cantonensis circulated prevalent in tropical and subtropical regions, major reported cases concentrated in the islands of the South Pacific, Southeast Asia, China and some other countries and regions. Until2011, China (including Hong Kong and Taiwan) have been over1000cases of human angiostrongyliasis cantonensis reported. In the recent years, with an expansion of the epidemic focus of the disease, changes in structure of people’s diet, and frequenceexchanges in various civilization and cultural, the incidence of the angiostrongyliasis cantonensis continues to rise in the number of even non-epidemic focus region (such as Europe, North America) sporadic case reported. The angiostrongyliasis cantonensisbrought serious harm to human health, as well as public health and social stability, causing the government attach great importance and widespread concern in society. However, as a new emerging infectious diseases, many of pathogenic biological characteristics and mechanisms of angiostrongyliasis cantonensis are unclear, for the angiostrongyliasis cantonensis the prevention and control of great challenge.In the nematode family, there has been found a variety of menbers have different kinds of chemotaxis activities. As the nematode model organism Caenorhabditis elegans, the chemotaxis activities researcheswhichhadobserved involved neurons about the pathway and the role-playinghave reachedquite high level.Bargmann considers chemotactic neurons in C. elegansare with chemotaxis activities of the external environment perception, feeding, growth and development, apoptosis, activities navigation behavior.After the screening of hundreds of chemicals, Ward summed up the attract agents for C. elegans:(a) cyclic nucleotides:cAMP, cGMP;(b) anion:Cl", Br-, I", etc.;(c) cations:Na+, Li+, K+, Mg2+, etc.;(d) OH-;(e) others:lysine, histidine, cysteine. Tobata-Kudo confirmed that in the concentration of NaCl gradient plate Strongyloides rattiL3the attraction gradient is range from20mm to230mm, which the80mM area particularly adaptable. Masataka Kogafound that S. ratti larvae will inevitably reach the lower intestine and development into adults before passing through the brain through the research about the chemotaxis of larvae chemical, and the in-vitro experiments showed Na+and serum albumin has a attractive chemotactic activity of larvae. Forbes found that low concentrations of NaCl attractedStrongyloides stercoralis infective larvae, while high concentrations of NaCl S. stercoralisshows repellent effect. Adachi et al found C. elegans have a tendency of sodium chloride or isoamyl alcohol, and chemotaxis was enhanced following the temperature increases. Kusaba found that infective larvae of Brugia pahangi in serumor blood of many animals (including mammals, reptiles, fish and invertebrates) had significant chemotactic activities, and chemotaxis index (C.I.) was0.562to0.960(standerized), otherwise in saline control groupC.I. was only0.045(standerized). Liao demonstrated that the tropism of the Caenorhabditis elegans related6kinds of compounds of the explosive was diluted to1000-fold after acetone,2-butanone, nitromethane, cyclohexanone, RDX, methenamine, inflammable and explosive was diluted to1000-fold after H2O2, potassium perchlorate, sulfur and potassium nitrification4related compounds repellent; wherein H2O2diluted100-fold after having repellent effect.In the suitable host beings, AC L3migrate into the brain by crossing the blood-brain barrier after passing through the intestinal wall, and go across the blood-brain barrier againto pulmonary artery and develop to adults. AC L3infecttingto human, mouse or other non-suitable host beings, the larvae migrate into the brain by crossing the blood-brain barrier after passing through the intestinal wall, and stay in the brain. Obviously, getting into the hosts’brain is an indispensable process of the larvae life. What factorsare driving the AC larvae gut-brain-lung tropism migration? It had found that the C. elegans have chemotaxis activities to sodium chloride and isoamyl alcohol. S. stercoralis infective larvaewere show attractive effect in low concentrations of sodium chloride, and repellent effectin high concentrations.No literature or experimental reports on Angiostrongylus cantonensis larvae chemotactic activity in similar areasyet.ObjectivesIn this study, different multiples concentrations of artificial cerebrospinal fluid (1×,3×,5×and10×, respectively) as well as different concentrations (20mM,50mM,100mm and150mM concentration gradient) inorganic salts and glucose were taken on Angiostrongylus cantonensis three-stage larvae in vitro chemotaxis assays, and observed under a stereo microscope on AC L3larvae chemotactic effect. The chemotaxis plateswere separated into solution zone and control zone(triple-distilled water zone). And in accordance with the different solutions, thenumber of AC L3 collected by man and analysedstatistically, and ultimately summed up the chemotaxis tendency in different multiple concentration artificial cerebrospinal fluid and different concentrations of inorganic salts and glucose in generally investigated.Methods1. Angiostrongylus cantonensis infection host model:takingthe cleared green leafy vegetables in an enamel square plate.put the infective white jade snails(Achatina fulica) with AC L3on the vegetables.lt isdemanded that small amount of distilled waterindwell in plate, and the water level is appropriate to immersion vegetables. Stamps to prevent snailsfrom climbing outand fromreducing AC larvae harvest quanlity. The snails movein plate at room temperature for2to4hours. And thancollectall liquid, mucus, and distilled water wached vegetable surfaces, centrifuge2500r/min for5minutes, the supernatant is threw away; precipitate was washed with distilled water, centrifuge2500r/min for5minutes, twice. Take sediment separation of three-stage larvae under stereo microscope for counting and packing. The larvae are washedby triple-distilled water twice. Every100A. cantonensisinfective larvae infected every single rat,and Angiostrongylus cantonensis infection model and AC life cycle in laboratory are successfully established.2. Tend embodiment of the experiment:take centrifugated and resuspended AC L320～30μl set chemokines to the center of the petri dish, and hatch at the darkness at room temperature after8hours.And than countthe larvae in the test area and the control area of the stereo microscope (buffer larvae are not included). To avoid operator error, set three chemokines plate simultaneouslyforevery single solution of every single concentration. In order to facilitate endoscopic accurate count, at the bottom of the plate place a small squaredpaper. Larvae on each plate are counted three times, and take the mean for statistical analysis.3. Chemotaxis index (C.I.) calculated:The C.I. is a commonly used metrics which descriptes the trend level of the nematodes in the activity under influence of the external environmental factors.C.I.∈(0,1], compared with triple-distilled water, the solution is attractiveto AC L3; C.I.∈[-1,0), compared to the triple-distilled water, the solution isrepellentto AC L3; if the C.I.=0, AC L3are in random activitiesbetweenthe solution and the distilled water nonspecificly. C.I. and statistical comparisons are based on the following three aspects:1) of different concentrations multiples artificial cerebrospinal fluid tends activities are relatively;2) the same concentration of inorganic salts compared with glucose tends activities;3) of the same inorganic salts and glucose in different concentrations of the solvent tends activities;4) tend to distinguish between ions activities comparison.4. The experimental data are the mean±standard deviation (X±SD). SPSS19.0statistical analysis software package used for each set of data of medical statistics. From the chemotaxis assayexperiment of different multiple-fold concentrations of ACSFtreated the AC L3, originaldata of the solution group and of distilled water group firstly test of homogeneity of variance, if homogeneity of variance (P>0.05), usedt-test paired analysis; if heterogeneity of variance (P≤0.05), used the Wilcoxon signed-rank test for analysis. From the experiment of Angiostrongylus cantonensis infective larvae in different concentrations of the seperated solvent composition chemotaxis assay, original data of solution group and of distilled water group firstlyused the test of homogeneity of variance, if homogeneity of variance (P>0.05), used a t-test paired analysis; if heterogeneity of variance (P≤0.05), used the Wilcoxon signed-rank test analysis.The significance level for P=0.05. Results1.5×ACSF solution is AC L3’s attractant.2. Distinction by concentration:at the range of1.0cm,20mM KC1,Na2HPO4, NaHCO3, MgCl2solution are AC L3’s attractants;50mM NaCl solution is AC L3’sattractant;100mM KCl solution is AC L3’s repellents, the same concentration of CaCl2solution is AC L3’s attractant;150mM NaCl solution is AC L3’s repellent, the concentration of KC1, NaHCO3, MgCl2solution are AC L3’s attractants.3. Distinguish by solvent:at the range of1.0cm, KCl solution at20mM,150mM concentration shows the attraction effect while repellent effect at100mM; NaCl solution at50mM shows the attraction effect while repellent effect at150mM; Na2HPO4solution at20mM shows the attraction effect; NaHCO3solution at20mM and150mM shows the attraction effect; CaCl2solution at100mM shows the attraction effect; MgCl2solution shows the attraction effect at20mM,150mM.4. Distinguish by ions:at the range of1.0cm,solution of K+cation in the concentration of20mM and150mM shows the attraction effect, while repellent effect at100mM concentration; solution of Na+cation in40mM,50mM shows the attraction effect; Cl-anion solution in200mM shows the attraction effect.Conclusion1. The ACSF solution on the the Angiostrongylus cantonensis three-stage larvae has certain chemokines, which involves the concentration of ACSF solution.2. NaCl, Na2HPO4, KCl and other inorganic salts on the the Angiostrongylus cantonensis three-stage larvae chemotaxis, which involves concentration of the inorganic salts.3. Na+cation on the the Angiostrongylus cantonensis three-stage larvae have certain chemokines, which involves concentration of Na+cation. |
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