| BackgroundsAngiostrongylus cantonensis is a neurotrophic and pulmonary parasite which normally lives in the pulmonary artery of permissive hosts (rats). After about a week, the eggs produced by the adult females hatch in the lung parenchyma. The resultant first-stage larvae (L1)are excreted in the host's faeces. Some of these larvae may enter a molluscan intermediate host,where they moult twice to become the third-stage larvae (L3).The L3 in molluscs eaten by rats are carried in the blood to the central nervous system, where they moult twice to become immature(non-egg-laying)adults.On reaching the branches of the pulmonary artery, they grow rapidly, attain sexual maturity, and release eggs.The L1 can be recovered from the faeces of rats approximately 42 days post-infection.In contrast,the non-permissive hosts, such as mice, guinea pigs,rabbits, rhesus monkeys,and humans,infected with this parasite, the worms migrate to the brain, but fail to develop within the heart and lungs to sexual maturity. Angiostrongylus cantonesis from Southeast Asia spread to South Pacific, Africa, India, Philippines and other countries and regions, has more than 3000 cases reported, With the freshwater snails as intermediate hosts in the spread of southern China, the cases of angiostrongyliasis has increased rapidly due to the extension of the natural focus of angiostrongyliasis and the change of human dietary patterns.It has become one of the most potentially dangerous food-borne parasitic diseases in our country.In the permissive and nonpermissive animal hosts for Angiostrongylus cantonensis infection, its structure and vital organs during development, resulting in Angiostrongylus cantonensis not self-penetrating non-host brain, lungs mature? Their parasites during the? These issues has not been reported.Among the host proteolytic enzymes, the plasminogen/plasmin system is composed of an inactive proenzyme, plasminogen, that can be converted to plasmin by either one of two plasminogen activators (PAs)-tissue-type PA (tPA) or urokinase-type PA (uPA). The enzymatic system is involved in the processing of pro-matrix metalloproteinase (pro-MMP) activation. The MMPs have a pathogenic role in several CNS diseases. Among MMPs, the family of gelatinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), has the capacity to metabolize native collagens types IV, V, VII, X, and XI. The activities of tPA, uPA, MMP-2, and MMP-9 can be measured reliably in CSF, and the assessment of these enzymes may be useful for studying the pathogenesis of neurological diseases. Our previous study showed that the tPA and uPA may play a role in eosinophilic meningitis of BALB/c mice. Also, MMP-9 was associated with eosinophilic meningitis. The enzyme was a useful marker for angiostrongyliasis meningitis in BALB/c and ICR mice.In general, a parasite can only develop and reach maturity in a very limited number of hosts. There is still no conclusive evidence on whether the host immune response is positively correlated with the intensity of the PAs/MMPs enzymatic system. In an attempt to understand the differences of proteolytic enzymes and pathological mechanisms in the permissive (rats) and nonpermissive (mice) hosts, to examine the enzymatic activities of PAs and MMP-9, as well as pathological changes in permissive and nonpermissive hosts to Angiostrongylus cantonensis infection.There is no conclusive evidence that the host immune response and matrix metalloproteinase systems strength -related.The secretion of hydrolytic enzymes if the difference is caused by Angiostrongylus cantonensis in the normal host and non-normal host migration outcome different? How their role? Mechanisms play a role in specific? Whether it is related with the parasite's own development? These issues are not clear.Objectives1. The establishment of Angiostrongylus cantonesis infection animal model in rats and mice. Light microscopy and scanning electron microscopic observation of larval structures in rat and mouse.2. Angiostrongylus cantonesis infection in rats and mice for twenty-first days and the larvae were cultured in vitro, the collected condensed excretory-secretory product were used to elevate the activity of protease and underwent spectrum analysis, comparing their differences.3. To elevate the value of LESA on early Angiostrongylus infection animal,s diagnosis.Methods1. The establishment of Angiostrongylus cantonesis infection animal model in rats and mice. Light microscopy and scanning electron microscopic observation of larval structures in rat and mouse1.5g pepsin artificial digestion liquid,37? were used to digest the white jade snail that were infected by the Angiostrongylus, after 2 hours,80 mesh plug mesh filtration to remove tissue debris, to PBS wash filtrate3, static natural precipitation collecting sediments in larvae, in which50 Angiostrongylus cantonesis infection stage larvae of the successful establishment of Angiostrongylus cantonesis infection of rat and mouse animal model. Rat infection model using SD rats, mice and Kunming mice BALB/c mice, model after observation of the two host life condition and infection of symptoms and signs of change; to 21dpi anatomy collection two host brains of larvae, scanning electron microscopy analysis of two kinds of host insect sensory papillae and chemoreceptors differences light microscopic observation, two host brain larval body length, width and pharyngeal gut scale and the analysis of its differences;2. Analysis of Angiostrongylus cantonesis excretory secretory proteases associated with spectrumWhen the Infection animal model is established, rat and mouse (BALB/c)of twenty-first days after infection larvae were cultured in vitro, get the larvae secretion excretions. Protein analysis were performed between the two species of host;3. To elevate the value of LESA on early Angiostrongylus cantonesis infection animal's diagnosis.To collect the larvae of the two species and to prepare the larval excretory-secretory antigen (LESA)and to perpare adult worm antigen preparation (AWA). The two antigens were used to detect the differences among the fectious mice and humans and the normal mice and humans at different time points of IgG and IgM change trend, statistics analysiswere carried on to clear LESA, sensitivity and specificity.4. SPSS17.0 software package for each set of data statistics?RESULTS1.The establishment of Angiostrongylus cantonesis infection animal model in rats and mice and the observation of the associated structures1.1 Observation of the host after infectionAngiostrongylus cantonensis infection models built after observation:mouse models about since 13dpi began to emerge, shedding hair wet, kyphosis, hemiplegia, trembling and looping, signs, symptoms persist. At about 16dpi mice more apparent exacerbation of symptoms and the onset of death in mice. Rat model of infection during the period of observation only found a depilatory symptoms, other in a mouse model of body appears the onset of symptoms is not obvious.1.2 observation of larvae associated structure1.2.1 Electron microscopic observation21dpi,s anatomy collection of larvae were observed through scanning electron microscope to clear the body structure characteristics:(1) body head presence of sensory papillae and chemoreceptors, by scanning electron microscopic observation of the mouth structure and peripheral sensory papillae, chemoreceptors found two host larva in brain development complete, normal and abnormal host host larva in brain (two mouse) by the observation and statistical analysis of the above structure found no statistical differences (P> 0.05); (2) was observed under scanning electron microscope of two kinds of host brain worm tail structure display:SD rats as well as in two kinds of mice tail structure is complete, the tail fold obvious female, anal and vulva opening and male copulatory bursa were clearly visible, statistical analysis showed no difference (P> 0.05). (3) the larval head other structures were observed: two infected host larva in brain head structure development is already mature and Angiostrongylus cantonensis adult head scanning electron microscopic structure similarity;1.2.2 Observation of light microscopic21dpi,anatomy collection of larvae were observed by light microscopic.observation of structures such as the larval body length body, wide and pharyngeal intestine proportion and statistical analysis indicated:the host larva in brain in body length, width and the pharyngeal intestine is proportional to the difference has statistical significance (P< 0.001), observation of rat brain in larval growth rate is in brain of mice with high degree of larval development. Two in the mouse brain and larval body length, width and pharyngeal gut proportions were compared and found no statistically significant difference (P>0.05), while the pharyngeal length of difference (P< 0.05).2. Analysis of Angiostrongylus cantonesis excretory secretory proteases associated with spectrum2.1 Rat and mouse brain 21dpi larva protein mass spectrometry is roughly the same as the main strip, esp in mice is much more than rats esp in the main strip.there is a obvious difference between the two species in different strips.2.2Western blotting and gelatin zymography were used to make a further comparison of two kinds of secretory proteins in the difference on the function,using rats and mice ESP respectively corresponding with the infected rats serum and serum reaction, both results are obvious reaction occurred strip, but esp in Mr35000 and Mr32 OOOis more obvious in mice than in rat.2.3Further zymography analysis suggest that esp in M-40000-Mr35000 has a wide hydrolysis degree in mice than in rat. After treated by EDTA samples in the Mr40000-Mr35000 hydrolysis region has waned,this phenomenon is more obervious in mice.3. elevation of the value of LESA on early Angiostrongylus infection animal,s diagnosis.The collected secreted proteins were used to coat antigen ELISA plate, and the plate were then used to have a reaction with mouse serum. Detection of different time points of IgG and IgM trends show:fifth days after infection antibody levels reached to peak and peak sustainable to tenth days, after the peak the line shows fluctuations; LESA infection of group IgG within group comparisons revealed tenth days IgG level was higher than fifteenth days and twenty-first days, and the difference was statistically significant; two antigen and Angiostrongylus cantonesis patients seropositive rate detection display:AWA detection sensitivity is slightly higher than LESA; At the same time AWA with other parasite antigen has a cross reactivity, and LESA cross-reactivity is not obvious, which patients of acute schistosomiasis in serum, sparganosis patient serum, Strongyloides stercoralis patients serum showed no positive reaction which show a description that LESA can improve the detection of specific as a antigen detection methods on early diagnosis of Angiostrongylus cantonensis infection.Conclusions1. Scanning electron microscopic observation of the larva structural features in the two species showed no difference; Light microscopic observation of the larva structural features that associated with development in the two species showed significant difference; Fail to have a fully development maybe the reason that stop the larva from wearing the brain again.2.'The ecretory protein of the larva in brain of the two species were performed silver staining bands, Western blotting and gelatin zymography analysisprocesses which confirmed the existence of differences,such differences may be the cause of larvae can't fully developed and can't even wearing the brain again.3. Compared with AWA, LESA has a high sensitivity and specificity which suggest that LESA has a explicit value in the early diagnosis of Angiostrongylus cantonensis infection. |
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