June-Der Lee

Larvicidal Constituents of Zingiber officinale (Ginger) against Anisakis simplex

In this study, we investigated the anthelmintic activity of [10]-shogaol, [6]-shogaol, [10]-gingerol and [6]-gingerol, compounds isolated from the roots of Zingiber officinale L., Zingiberaceae (ginger), against Anisakis simplex. The above compounds kill or reduce spontaneous movement in A. simplex larvae. The maximum lethal efficacy of [10]-shogaol and [10]-gingerol was approximately 80% and 100%, respectively. We further examined the time course of compound-induced loss of mobility in A. simplex. The results showed that various concentrations of [10]-shogaol, [6]-shogaol, [10]-gingerol and [6]-gingerol have maximum effects on loss of spontaneous movement from 24 to 72 h. In addition, the time course of mortality and the percentage of loss of spontaneous movements were ascertained to determine the minimum effective doses of [10]-gingerol and [10]-shogaol. [10]-Gingerol exhibited a larger maximum larvicidal effect and greater loss of spontaneous movement than [10]-shogaol and albendazole. In addition, these constituents of Zingiber officinale showed effects against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and peroxyl radicals. These constituents of Zingiber officinale are responsible for its larvicidal activity against A. simplex.

Anthelmintic constituents from ginger (Zingiber officinale) against Hymenolepis nana

This study investigated the anthelmintic activity of gingerenone A, [6]-dehydrogingerdione, [4]-shogaol, 5-hydroxy-[6]-gingerol, [6]-shogaol, [6]-gingerol, [10]-shogaol, [10]-gingerol, hexahydrocurcumin, 3R,5S-[6]-gingerdiol and 3S,5S-[6]-gingerdiol, a constituent isolate from the roots of ginger, for the parasite Hymenolepis nana. The cestocidal activity or ability to halt spontaneous parasite movement (oscillation/peristalsis) in H. nana of above constituents was reached from 24 to 72h in a time- and dose-dependent manner, respectively. The [10]-shogaol and [10]-gingero1 have maximum lethal efficacy and loss of spontaneous movement than the others at 24-72h. In addition, worms treated with 1 and 10μM [10]-gingero1, more than 30% had spontaneous movement of oscillation at 72h but [10]-shogaol at 72h only about 15-20% of oscillation. This showing that [10]-gingero1 had less loss of spontaneous movement efficacy than [10]-shogaol. After exposure to 200μM [10]-shogaol, 100% of H. nana had died at 12h rather than died at 24h for [10]-gingerol, showing that [10]-gingero1 had less lethal efficacy than [10]-shogaol. In addition, these constituents of ginger showed effects against peroxyl radical under cestocidal activity. In order to evaluate the cestocidal activity and cytokine production caused by gingers extract R0 in the H. nana infected mice, we carried out in vivo examination about H. nana infected mice BALB/c mice were inoculated orally with 500 eggs. After post-inoculation, R0 (1g/kg/day) was administered orally for 10 days. The R0 exhibited cestocidal activity in vivo of significantly reduced worms number and cytokines production by in vitro Con A-stimulated spleen cells showed that INF-γ and IL-2 were significantly increases by R0. IL-4, IL-5, IL-6, IL-10 and IL-13 were significantly decreases and Murine KC and IL-12 were not significantly changes by R0. Together, these findings first suggest that these constituents of ginger might be used as cestocidal agents against H. nana.

Role of T cell subpopulations in mice infected with Angiostrongylus cantonensis

When C57BL/6 mice were infected with Angiostrongylus cantonensis, the percentage of T helper (CD4+) cells and T supressor (CD8+) cells in peripheral blood increased weekly until the third and seventh week respectively, and then gradually decreased. C57BL/6 mice were depleted of CD4+ and CD8+ T cells by in vivo injection of anti-CD4 and anti-CD8 monoclonal antibodies, respectively, and then infected with A. cantonensis. There were significantly more and less worms recovered in the mice depleted of CD4+ and CD8+ T cells respectively than in undepleted mice. Discrete subpopulations of T cells from mice exposed to A. cantonensis for 3 weeks or 7 weeks were adoptively transferred to syngeneic recipients which were then given a challenge infection. Protection was mediated by a CD4+ T cell population present in mice after 3 weeks of infection but was not demonstrable with cells taken 7 weeks after infection. When CD4+ T cells obtained from 3-week infected mice were mixed with 5% CD8+ T cells obtained from mice infected for 7 weeks, no significant transfer of resistance was observed. Thus, immune responses to A. cantonensis in mice were regulated by discrete subpopulations of T lymphocytes.

Sequence analysis in partial genes of five isolates of Angiostrongylus cantonensis from Taiwan and biological comparison in infectivity and pathogenicity between two strains.

Angiostrongylus cantonensis is the most common infectious agent causing eosinophilic meningitis and is present in Taiwan, Thailand and the Pacific islands. Clinical symptoms vary within different endemic regions, and their severity is probably dependent on the number of ingested parasites and the diversity among strains. The experimentally definitive host is the rat, and non-permissive hosts are certain mammals such as humans and mice. In this study, the partial gene sequences of two A. cantonensis strains isolated from five different regions in Taiwan were selected and molecularly analyzed. The internal transcribed spacer gene and cytochrome-c oxidase subunit I gene sequences of the Hualien (H) strain of A. cantonensis differed from those of the Pingtung (P) strain and the other three strains by 19% and 11%, respectively. We analyzed the infectivity, fecundity, and development of the H and P strain in rats and host pathogenicity in mice inoculated with both strains. The number of the emerged first-stage larvae, adult recovery, and average length of adults in Sprague-Dawley rats significantly differed between rats inoculated with the H and P strain. Young adult recovery, average length of young adults, eosinophil counts in the cerebrospinal fluid (CSF), glutathione peroxidase concentration, levels of reactive oxygen species as well as malondialdehyde concentration in the CSF, and the survival of mice significantly differed between BALB/c mice inoculated with the H and P strain. The H strain of A. cantonensis had lower infectivity, delayed fecundity, and poor development in rats, and caused milder pathology and lower mortality in mice than the P strain. These data clearly indicate that the H strain of A. cantonensis is a pathogenically distinct strain with lower infectivity to its definitive host, and causing mild pathogenic symptoms to its non-permissive host.

Haplorchis infections in intermediate hosts from a clonorchiasis endemic area in Meinung, Taiwan, Republic of China.

Snails and freshwater fish were examined from four ponds in the Meinung township in which Clonorchis sinensis was known to be endemic 18 years ago. No metacercariae were found in 478 Tilapia nilotica, whereas of 451 Ctenopharyngodon idellus examined, 16.2%, 3.3% and 0.9% were found to be infected with Haplorchis pumilio, H. taichui and Clonorchis sinensis, respectively. In addition, there were some unidentified metacercariae in 12.0% of Ctenopharyngodon idellus examined. Overall, no positive correlation between infection rates and sizes of infected fish was shown. Six species of snails were collected in this survey and two frequently-occurring snails, Melanoides tuberculata and Thiara granifera were commonly infected with H. pumilio. Reasons for the prevalence of Haplorchis species and the absence of Clonorchis sinensis in fish and snail hosts in a previously reported endemic area for human clonorchiasis are discussed.

A Survey on the Intestinal Parasites of the School Children in Kaohsiung County

The present study on the prevalence of intestinal parasites infection, from September to December 1999, was conducted among school children in Taoyuan Hsiang, Kaohsiung county. The investigated areas included three (Jiannshan, Shingjong and Taoyuan) primary schools. The overall infection rate in 305 children determined by Merthiolate-Iodine-Formaldehyde Concentration method of stools was 17%. Four confirmed species of helminthes (Ascaris lumbricoides, Hookworm, Trichuris trichiura and Hymenolepis nana) and three species of protozoa (Giardia lamblia, Entamoeba coli and Blastocystis hominis) were detected. Males and females had the infection rates of 24% and 11%. The infection rates of aboriginal and non-aboriginal children were 17% and 14%, respectively. Grade 1 and Grade 6 had the highest infection rate (21%). Following tape perianal examination of 302 children, the overall infection rate of Enterobius vermicularis was 25%. Males and females had the infection rates of 24% and 26%. The infection rates of aboriginal and non-aboriginal children were 27% and 11%, respectively. Grade 1 had the highest infection rate (37%). Based on these data, the infection rate of intestinal parasites among rural primary school children in Kaohsiung county remains high.

The susceptibility of five stains mice to infections with Angiostrongylus cantonensis.

The susceptibility to the infection of Angiostrongylus cantonensis was studied in five inbred stains of mice including four H-2 haplotypes. Three weeks after infection, C57 BL/10J (H-2b) and C57BL/6J (H-2b) stains of mice showed the lowest, CBA (H-2k) and A/J (H-2a) stains showed intermediate, and the BALB/cJ (H-2d) mice showed the greatest susceptibility to infection. BALB/cJ mice also displayed the most body weight loss, the highest number of worms and showed the least number of days of survival. The s susceptibility of C57BL/6J (H-2b) and C57BL/10J (H-2b) strains of mice sharing same H-2 haplotype was the same despite their different genetic backgrounds. These data suggested that the H-2 gene might influence the susceptibility of mice to A. cantonensis.

Angiostrongylus cantonensis (Nematode: Metastrongiloidea): In Vitro Cultivation of Infective Third-Stage Larvae to Fourth-Stage Larvae

The present study to attempt to cultivate Angiostrongylus cantonensis from third-stage larvae (AcL3) to fourth-stage larvae (AcL4) in vitro in defined complete culture medium that contained with Minimum Essential Medium Eagle (MEM), supplemented amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA) and 20% fetal calf serum (FCS) was successful. When AcL3 were cultured in the defined complete culture medium at 37°C in a 5% CO2 atmosphere, the larvae began to develop to AcL4 after 30 days of cultivation, and were enclosed within the sheaths of the third molts of the life cycle. Under these conditions, the larvae developed uniformly and reached to the fourth-stage 36 days. The morphology of AcL3 develop to AcL4 were recording and analyzing. Then comparison of A. cantonensis larval morphology and development between in vitro cultivation in defined complete culture medium and in vivo cultivation in infective BALB/c mice. The larvae that had been cultivated in vitro were smaller than AcL4 of infective BALB/c mice. However the AcL3 that were cultured using defined incomplete culture medium (MEM plus 20% FCS with AA+AM, FA, CA, AA+AM+FA, FA+CA, CA+AA+AM or not) did not adequately survive and develop. Accordingly, the inference is made that only the defined complete medium enable AcL3 develop to AcL4 in vitro. Some nematodes have been successfully cultured into mature worms but only a few researches have been made to cultivate A. cantonensis in vitro. The present study is the first to have succeeded in developing AcL3 to AcL4 by in vitro cultivation. Finally, the results of in vitro cultivation studies herein contribute to improving media for the effective development and growth of A. cantonensis. The gap in the A. cantonensis life cycle when the larvae are cultivated in vitro from third-stage larvae to fourth-stage larvae can thus be solved.

Eosinophilic meningitis risk associated with raw Ampullarium canaliculatus snails consumption

In Taiwan, Angiostrongylus cantonensis infection has been reported in foreign laborers who had consumed raw Ampullarium canaliculatus snails. This study analyzed three foreign laborers who had contracted enzyme‐linked immunosorbent assay‐confirmed A cantonensis infection while working in Taiwan. All three workers had consumed either roasted snails or raw snails flavored with seasoning while drinking wine. This study investigated possible risk factors for A cantonensis, including naturally occurring A cantonensis in A canaliculatus snails, viability of third‐stage A cantonensis larvae in raw seasoned snails and in roasted snails, infectivity of larvae, and effects of alcohol while consuming snails. Positive infection rates in snails from five different irrigation canals in south Taiwan ranged from 12.3% to 29.4% and the average number of motile larvae per infected snail ranged from 36 to 65. The number of motile and coiled larvae in snail meat after 120 minutes seasoning was 93 (27.7%) and 233 (69.3%), respectively. After 20 minutes of roasting, most larvae in the snail meat were dead. The infectivities of motile and coiled larvae from snail meat after 60 minutes seasoning were 53.2% and 33.2%, respectively, and those from snail meat after 5 minutes roasting were 33.2% and 7.0%, respectively. Eating Taiwan A canaliculatus snails raw is extremely risky given their high infection rates and infection intensities. Even after 120 minutes seasoning or after 20 minutes roasting, snail meat should be considered unsafe for human consumption. Finally, experimental rodent studies indicated that consuming alcohol while ingesting larvae does not significantly reduced infectivity.

Humoral immune responses in mice infected with gamma-irradiated third-stage larvae of Angiostrongylus cantonensis

Humoral immune responses in C57BL/6 strain mice infected with 1.0 kGy gamma-irradiated third-stage larvae (L3) and with non-gamma-irradiated L3 of Angiostrongylus cantonensis were assessed in this study. The young-adult worm (L5) antigen-specific antibodies in sera of both groups of mice elevated gradually and reached a peak in the third week after infection. The IgM antibodies of mice 1-week after infection with gamma-irradiated L3 and with non-gamma-irradiated L3 recognized several L5 antigens of the same molecular weight. However, only partially identical profiles were observed in the reaction of L5 antigens with IgM and IgG antibodies from mice 3 weeks after infection with gamma-irradiated L3 and with non-gamma-irradiated L3 respectively by western blot analysis. With regard to antibody-dependent cell-mediated cytotoxicity, antibodies from 3-week infected mice significantly increased the adherence of mouse eosinophils and neutrophils to L3 of A. cantonensis. The immunoresponses of splenic cells from mice in the third week of infection to sheep red blood cells were suppressed because the number of plaque forming cells obviously decreased more than the uninfected control.