In vitro viability test for the eggs of Echinococcus granulosus: a rapid method

Abstract

In this study an attempt was made to develop an efficient, rapid, simple, and reproducible method for the in vitro viability test of Echinococcus granulosus eggs. The eggs were obtained from an experimentally infected dog and kept at 4°C until use. To prepare the dead or damaged eggs, the eggs were heated in hot water (69-72°C for 10 min), preserved in 70% ethyl alcohol (16 days) or exposed to direct sunlight (18 h). Sodium hypochlorite (0.5-0.7%) was used for the hatching process, and the hatched oncospheres were stained with 0.1% eosin for the viability test. With 0.5% sodium hypochlorite, the hatching rates for viable eggs and eggs killed or damaged by heat (69°C), 70% ethyl alcohol, and direct sunlight were 96%, 97.5%, 91.5%, and 94.6% respectively and there was no significant difference between the hatching rate for viable and dead or damaged eggs (p> 0.05). After staining with 0.1% eosin, the rates of the viable oncospheres hatched from viable eggs and the eggs killed or damaged by heat (69°C), 70% ethyl alcohol, and direct sunlight were 97.5% 3.6%, 7%, and 10.5%, respectively. The difference between the rates of viable oncospheres hatched from viable and dead or damaged eggs was extremely significant (P < 0.0001). With 0.7% sodium hypochlorite, the hatching rates for viable and dead eggs (killed by 72°C for 10 min) were 99.1% and 99.9%, respectively. In this condition, the rate of viable oncospheres was an average of 98.5% for viable eggs and 0.0% for dead ones. The results of this study showed that hatching of eggs by 0.7% sodium hypochlorite and staining of hatched oncospheres by 0.1% eosin are practical methods for the differentiation of viable and nonviable (dead) eggs of Echinococcus granulosus.

A Sensitive, One-Way Sequential Sieving Method to Isolate Helminths’ Eggs and Protozoal Oocysts from Lettuce for Genetic Identification

“…In the case of taeniid eggs, the assessment of their maturity/viability has been achieved by exposing them to sodium hypochlorite and counting the number of oncospheres. However, it has been noted that the SHR test is not reliable to assess the viability of long-time preserved T. hydatigena eggs (Deplazes and Eckert, 1988) or heat-exposed eggs of E. multilocularis [25] or E. granulosus [50]. In the case of E. multilocularis, it is possible to assess the viability of the eggs with an in vivo method as used in this study, which is based in the subcutaneous injection of sodium hypochlorite resistant oncospheres to mice [25].…”

“…However, it has been noted that the SHR test is not reliable to assess the viability of long-time preserved T. hydatigena eggs (Deplazes and Eckert, 1988) or heat-exposed eggs of E . multilocularis [ 25 ] or E. granulosus [ 50 ]. In the case of E .…”

Public health risks associated with food‐borne parasites

“…Immature eggs are destroyed by this procedure while viable oncospheres are protected by a resistant membrane (Lightowlers et al., ). However, the results of the SH‐RT do not correlate with the in vitro activation and development rate of taeniids, including E. multilocularis (Deplazes et al., ; Moazeni and Rakhshandehroo, ; Federer et al., ). In vivo studies yield the most reliable results, provided that the intermediate and definitive hosts are available for the target species or an appropriate surrogate species is selected.…”

Assessing the Contamination of Food and the Environment With Taenia and Echinococcus Eggs and Their Zoonotic Transmission

“…However, the results of the SH-RT did not correlate with the in vitro activation and development rate of T. hydatigena oncospheres after 270 days preservation in water at + 4°C or − 28°C. Furthermore, SH-RT was not reliable to assess the viability of heat-exposed eggs of E. multilocularis [43•] or E. granulosus [103]. Therefore, SH-RT is useful to determine the rate of mature eggs after isolation from gravid worms, and this is needed to standardize infection doses in experimental studies [101].…”

“…Therefore, SH-RT is useful to determine the rate of mature eggs after isolation from gravid worms, and this is needed to standardize infection doses in experimental studies [101]. An in vitro method including treatment with sodium hypochlorite and assessing the permeability of oncospheres to eosin was used to estimate the viability of E. granulosus eggs after exposure to different temperatures [103]. This method could be used in the future for tenacity studies; however, no in vivo validation of the method was performed.…”