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Angiostrongylus cantonensis is an important zoonotic parasite causing eosinophilic meningitis and eosinophilic meningoencephalitis in humans. In this study, the protein expression profiles of the infective third- and pathogenic fifth-stage larvae (L3 and L5) of this parasite were compared by proteomic techniques. Isolated protein samples were separated by two-dimensional gel electrophoresis (2-DE), stained with silver nitrate, and analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Proteins from L5 were mainly at pH 5-7 and with molecular weight (MW) 40-100 kDa, whereas those from L3 were at pH 5-6 and with 5-35 kDa. Of 100 protein spots identified, 33 were from L3 whereas 67 from L5 and 63 had known identities, whereas 37 were hypothetical proteins. There were 15 spots of stress proteins, and HSP60 was the most frequently found heat stress proteins in L5. More binding and protein transport-related proteins were found in L5 including peptidylprolyl isomerase (cyclophilin)-like 2, serum albumin, preproalbumin precursor, and dilute class unconventional myosin. L3 had a higher expression of cytoskeleton and membrane proteins than L5. In addition, four protein spots were identified in the sera of the rat host by Western blot analysis. The present proteomic study revealed different protein expression profiles in L3 and L5 of A. cantonensis. These changes may reflect the development of L3 from the poikilothermic snails to L5 in the homoeothemic rats. This information may be useful for the finding of stage-specific proteins and biomarker for diagnosis of angiostrongyliasis.

The kinetics of changes in the eotaxin concentration in the serum and cerebrospinal fluid (CSF) of BALB/c mice after infection with Angiostrongylus cantonensis and the correlation between the concentration of eotaxin and worm recovery were investigated. The mean concentration of eotaxin in serum of infected mice gradually increased from 46.3+/-6.5 pg/ml at week 0 to 104.9+/-44.8 pg/ml at week 3 after infection, while the mean eotaxin level in the CSF of infected mice rapidly increased from 18.7+/-2.1 pg/ml to 193.2+/-23.6 pg/ml 1 week after infection and then increased further to 507.8+/-167.9 pg/ml at week 3. The concentrations of eotaxin in the CSF of infected mice each week after infection were all significantly higher than those in serum ( P0.0001). In parallel with the increase in eotaxin in the CSF, infected mice showed gradual increases in CSF eosinophilia and a reduction in intracranial worm recovery. The concentration of eotaxin in CSF was higher in infected mice with more worms in the brain, except when the number of worms in the brain was30. In addition, when the worm counts in the brains of infected mice were30, eotaxin concentrations in the CSF were positively correlated with worm counts in the brain ( P0.001). Thus, the release of eotaxin in the CSF of mice infected with A. cantonensis observed in this study was time dependent and worm-load dependent, and in parallel with the increase in eotaxin in the CSF, and gradual decreases in worm counts in the brains of infected mice.

Although there have been some advances in the cryopreservation of Angiostrongylus cantonensis, the degrees of viability and infectivity of the cryopreserved developmental stages have not been high. A two-step freezing protocol using a programmable freezer was determined to be effective in improving the infectivity of the cryopreserved third-stage larvae of this parasite. After washing steps and suspension in 10% (v/v) dimethylsulfoxide and equilibrium at room temperature the larvae were placed into the freezer. The temperature was lowered first at 0.8 degrees C/min from room temperature to -40 degrees C and then at 10 degrees C/min to -70 degrees C. The samples were plunged into liquid nitrogen. After storage in liquid nitrogen for 7-15 days the larvae were thawed rapidly in 37 degrees C water and 27.6% were found to show vigorous "S-shape" movement without significant changes in appearance. These larvae (50/rodent) could develop to the fifth stage in mice (42.6%) and establish patent infection in rats (40.4%). Moreover, there was no significant difference in the recovery rates of cryopreserved worms and their unfrozen counterparts. These findings indicate that steady precooling conditions may decrease damage with regard to the infectivity of cryptopreserved third-stage larvae of A. cantonensis.

BALB/cByJ mice were divided into zinc-deficient (ZD), high zinc-supplemented (ZH), adequately zinc-replete (ZA) and normal control groups by daily dietary control. The body weight in ZD and ZH mice became significantly less than that of normal control mice from 4 weeks (P