SPF golden Syrian hamsters Mesocricetus auratus from the SPF animal facility at the Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences (ICG SB RAS) were used in this study as generally accepted laboratory model animals [7 (link), 10 (link)–13 (link)]. All the procedures were performed aseptically. We applied an appropriate randomization strategy (blocking) to control possible variables, such as potential infection, among the experimental animals. We took into account nuisance variables that could bias the results (a litter and an investigator).
For collecting O. felineus metacercariae, a naturally infected freshwater fish (Leuciscus idus) was net-caught in the Ob River near Novosibirsk (Western Siberia, Russia) by research assistant Viktor Antonov (ICG SB RAS) without the use of chemicals. O. felineus metacercariae were extracted as described previously [7 (link), 16 (link)]. C. sinensis and O. viverrini metacercariae were extracted from naturally infected freshwater fish (Seoul, Republic of Korea, and Khon Kaen, Thailand, respectively) and delivered on ice. After several washes with normal saline, metacercariae were identified under a light microscope. All the procedures with hamsters were performed at the SPF animal facility at the ICG SB RAS.
Sixteen hamsters were distributed into four groups, and animals from three of them were infected with 75 metacercariae (of one of the three liver fluke species separately) by gastric intubation at intervals of 3–5 days to avoid bacterial cross-infection. One group was kept uninfected. One month after the infection, the hamsters were euthanized using carbon dioxide. All the procedures were done aseptically. Bile samples were collected via puncture of the gall bladder and stored at -80°C until use. Colorectal feces were extracted and stored at -80°C until use. Adult worms were carefully extracted from the biliary tract, washed more than 10 times with sterile saline, then soaked for several hours in sterile saline at 37°C, and finally stored at -80°C until analysis.
Although all procedures with hamsters were carried out in the same Animal Facility, we cannot exclude any small differences in the standard protocol for the metacercariae isolation from fish that might affect the microbiome.
For collecting O. felineus metacercariae, a naturally infected freshwater fish (Leuciscus idus) was net-caught in the Ob River near Novosibirsk (Western Siberia, Russia) by research assistant Viktor Antonov (ICG SB RAS) without the use of chemicals. O. felineus metacercariae were extracted as described previously [7 (link), 16 (link)]. C. sinensis and O. viverrini metacercariae were extracted from naturally infected freshwater fish (Seoul, Republic of Korea, and Khon Kaen, Thailand, respectively) and delivered on ice. After several washes with normal saline, metacercariae were identified under a light microscope. All the procedures with hamsters were performed at the SPF animal facility at the ICG SB RAS.
Sixteen hamsters were distributed into four groups, and animals from three of them were infected with 75 metacercariae (of one of the three liver fluke species separately) by gastric intubation at intervals of 3–5 days to avoid bacterial cross-infection. One group was kept uninfected. One month after the infection, the hamsters were euthanized using carbon dioxide. All the procedures were done aseptically. Bile samples were collected via puncture of the gall bladder and stored at -80°C until use. Colorectal feces were extracted and stored at -80°C until use. Adult worms were carefully extracted from the biliary tract, washed more than 10 times with sterile saline, then soaked for several hours in sterile saline at 37°C, and finally stored at -80°C until analysis.
Although all procedures with hamsters were carried out in the same Animal Facility, we cannot exclude any small differences in the standard protocol for the metacercariae isolation from fish that might affect the microbiome.
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