Anti-encystment and amoebicidal activity of Lonicera japonica Thunb. and its major constituent Chlorogenic acid in vitro

Objective:To examine the acanthamoebicidal effects of ethyl acetate,aqueous and butanol fractions of dried flower buds of Lonicera japonica(L.japonica) Thunb.(Flos Lonicerae) in vitro.Methods:Acanthamoeba triangularis isolates were obtained from environmental water samples and identified by PCR.They were exposed to ethyl acetate,water and butanol fractions of L.japonica Thunb.at concentrations ranging from 0.5 mg/m L to 1.5 mg/m L.The extracts were evaluated for growth inhibition at 24,48 and 72 h,respectively.Chlorogenic acid at a concentration of 1 mg/mL was examined for inhibition of encystment.Results:Ethyl acetate fraction at a concentration of 1.5 mg/mL evoked a significant reduction of trophozoite viability by 48.9% after 24 h,49.2% after 48 h and 33.7% after 72 h Chlorogenic acid,the major active constituent of L.japonica Thunb.at the concentration of 1 mg/mL reduced the cysts/trophozoite ratio by 100% after 24 h,84.0% after 48 h and 72.3% after 72 h.This phenolic compound at concentration of 1 mg/mL concurrent with 0.6% hydrogen peroxide inhibited hydrogen peroxide-induced encystment by 92.8% at 72 h.Conclusions:Results obtained from this study show that ethyl acetate fraction at 1.5 mg/m L is the most potent fraction of L.japonica Thunb.and its major constituent chlorogenic acid showed the remarkable inhibition of encystment at a concentration of 1 mg/mL.

IN VIVO AND IN VITRO ENCYSTMENT OF ECHINOCHASMUS LILIPUTANUS CERCARIAE AND BIOLOGICAL ACTIVITY OF THE METACERCARIAE

Aim To explore if Echinochasmus liliputanus cercariae can develop into metacercariae both invivo and in vitro and biological activity of the metacercariae,then to determine the effects of silver ni-trate on eereariae encystation in vitro.Methods Cereariae of Echinochasmus liliputanus from Bellamyaaeruginosa snails treated with or without silver nitrate were used to infected goldfish,the second inter-mediate host,or to incubated in many different solutions for 24 h to record the encystation rates.Themetacercariae formed both in vivo and in vitro were then used to infected New Zealand rabbits to testtheir infectivity to its definitive hosts or to exeyst in 0.1% sodium deoxycholate excystation medium at37℃ for 1 h.Results In vivo encystment of cercariae occurred in the gills of goldfish.However,thecercariae were also able to encyst in vitro in Locke’s solution,NaCl solution,artificial gastric juice orhuman gastric juice with eneystation rates of 74.28 %,44.94 %,8.37 % and 10.79 %.0.7×-1.2×Locke’s or 0.7%-1.2% NaCl solution was shown to be appropriate for in vitro encystment to occurwithin 24 hour,however,full- strength Locke’s solution was shown to be optimal.The one- day- oldencysted metacereariae formed in vivo showed 88.53 % exeystation when treated in 0.1% sodium de-oxycholate exeystation medium at 37℃ for 1h.The metacercariae formed in vitro,however,showed88.60% and 84.95% excystation for normal and abnormal ones respectively.While abnormal cysts atroom temperature usually die within 10 days,about 70% normal cyst,both in vivo and in vitro,canstill excyst after stored in 0.5×Locke’s at 4℃ for 3 mouths.Cysts formed in vivo and in vitro were e-qually infective to rabbits.1 uM silver nitrate had a dramatic effect on the cercariae encysting in vitro.When treated with silver nitrate,the cereariae encystation rates decreased to 16.25% in Locke’s solu-tion and 6.69% in NaCl solution,however,the encystment was largely restored wben the cercariaewere washed to remove Ag+.Conclusion The finding of E.liliputanus cercariae encysting in vitro,especially in human gastric juice,might be helpful in elucidating mechanisms of the definitive hosts in-fected by the cercariae directly.The encystment of the cercariae in vitro could be inhibited when thecercariae were treated with 1 uM silver nitrate.As silver nitrate binds to the papillae,especially to theciliated papillae,on the cercarial surface,it is suggested that papillar chemoreceptors may be involved inencystment of the cercariae.

Solid sand particle addition can enhance the production of resting cysts in dinoflagellates

Resting cysts are an important part of the life cycle for many harmful algal bloom-forming dinoflagellates, and play vital roles in the recurrence and geographical spread of harmful algal blooms. Numerous factors have been suggested to regulate the formation of resting cysts, although only a few have been proven to be significant. Cyst formation can be induced by adverse environmental conditions such as drastic changes in temperature, light, salinity, and nutrient levels, and by biological interactions. In this study, we evaluated the ability of an artificial factor(fine sand particles) to enhance the formation of resting cysts. Fine sand particles were added to cultures of dinoflagellates that are known to produce cysts. The addition of fine sand particles significantly increased both the production rate and final yield of cysts in cultures of S crippsiella trochoidea, Biecheleria brevisulcata, and Levanderina fissa(= Gymnodinium fissum, Gyrodinium instriatum, Gyr odinium uncatenum). The largest increase in the final yield(107-fold) of cysts as a result of sand addition was in S. trochoidea. However, addition of fine sand particles did not induce cyst formation, or barely af fected cyst formation, in A kashiwo san guinea, Cochlodinium polykrikoides and Pheopolykrikos hartmannii, which are also known to be cyst-producing species. We speculated that addition of sand significantly increased the chances of cell collision, which triggered cyst formation. However, further research is required to test this idea. Importantly, our findings indicate that the addition of fine sand particles is a useful method to obtain a large quantity of cysts in a short time for laboratory studies or tests; for example, if a cyst viability test is being used to assess the eff ectiveness of ships' ballast water treatment.

Eukaryotic cell encystation and cancer cell dormancy:is a greater devil veiled in the details of a lesser evil?

Cancer cell dormancy is the main cause of cancer recurrence and failure of therapy as dormant cells evade not only the anticancer drugs but also the host immune system. These dormant cells veil themselves from detection by imaging and/or using biomarkers, which imposes an additional problem in targeting such cells. A similar form of hibernation process known as encystation is studied in detail for pathogenic unicellular eukaryotic microorganisms. By examination using microarray gene expression profiles, immunocytochemistry tools, and siRNAs during the process of encystation, understanding the covert features of cancer cell dormancy as proposed could be possible. This knowledge can be extended to dormant cancer cells to uncover the mechanisms that underlie this ghost, yet dangerous state of human cancers. We propose a strategy to induce dormancy and exit this state by application of knowledge gained from the encystation induction and retrieval processes in pathogenic eukaryotic microorganisms. Given that early detection and characterization of dormant malignant tumor cells is important as a general strategy to monitor and prevent the development of overt metastatic disease, this homology may enable the design of therapies that could either awake the dormant cell from dormancy to make it available for therapies or prolong such a phase to make cancer appear as a chronic disease

Incidence and histopathology of encysted progenetic metacercaria of Clinostomum complanatum(Digenea:Clinostomidae) in Channa punctatus and its development in experimental host

Objective:To study the incidence of encysted progenetic metacercariae of Clinostomum complanatum(C. complanatum) in Channa punctatus(C. punctatus), associated histopathology and the experimental infection to laboratory chicken to obtain ovigerous adult worms.Methods:Live C. punctatus were brought from local fish market of Aligarh, India, dissected and examined on a monthly basis for the presence of C. complanatumcysts. For histochemistry, infected tissue sections with attached cysts were processed for haematoxylene and eosin staining. Cysts were aseptically fed to 4 day old leghorn chicken to obtain adult worms. Mechanically excysted metacercaria and the ovigerous adult worms were stained in carmine to prepare permanent slides.Results:One year survey for the infection of encysted progenetic metacercaria of C.complanatuminC. punctatusrevealed the prevalence, intensity and abundance of 24.7%, 2.27and 0.608, respectively. Histopathology showed heavy infiltration of immune cells at the site of cyst attachment and some tissue damage was also evident. Following feeding to experimental chicken, about 41.07% of the encysted metacercariae were able to excyst and migrate back to bucco-pharyngeal region where they tenaciously attached and fed on blood, and transformed into ovigerous adult worms from 62 hours onwards of post infection.Conclusions:The parasite is potentially pathogenic to the host, and the availability of a suitable intermediate host can be a contributing factor for the occurrence of C. complanatummetacercaria either in the excysted or encysted form, indicating loose host specificity and zoonotic potential.