Metacercariae

Adult parasites from each of five isolates were used for genome sequencing: (1) FhepLivSP, from the laboratory maintained Shrewsbury isolate (Ridgeway Research, UK); (2) FhepLivS1, a clonal line derived from the Shrewsbury population; (3) and (4) FhepLivR1 and R3, clonal lines derived from two isolates recovered from sheep in Northern England naturally infected with F. hepatica; and (5) FhepLivR2, a clonal line derived from a F. hepatica population from naturally infected sheep in South West Wales. For RNA sequencing, the following were used: (1) metacercariae and newly excysted juveniles (NEJ) at 1, 3 and 24 h post excystment from a North American isolate (Baldwin Aquatics Inc., Monmouth, OR, USA). Twenty-one–day-old juvenile flukes were recovered from mice infected with the same isolate; (2) an adult parasite recovered from the bile ducts of cattle naturally infected with F. hepatica in Uruguay. All animal work was conducted with ethical approval from the Universities of Liverpool (UK) and McGill (Canada).

Adult O. viverrini were collected from experimentally infected hamsters (Mesocricetus auratas) maintained at the animal facility of the Khon Kaen University Faculty of Medicine. Protocols approved by the Khon Kaen University Animal Ethics Committee were used for all animal research conducted in this study. Briefly, metacercariae of O. viverrini were collected from naturally infected cyprinoid fish by pepsin digestion. Metacercariae (100 per hamster) were administered intragastrically to hamsters. Hamsters were euthanazed 6 weeks after inoculation, and adult worms were flushed with saline from the bile ducts [69 (link)]. Worms were washed extensively with sterile phosphate-buffered saline (pH 7.2), after which they were snap frozen and stored in liquid nitrogen or employed immediately as the source of fluke RNA.

Illumina HiSeq reads were trimmed to Q≥30 and adaptors removed using
Fastx_toolkit (version 0.0.13). RNAseq libraries were mapped to the putatively
annotated F. hepatica MAKER gene models (9 ) using TopHat2 (17 )
and read counts extracted using htseq-count. Based on these counts normalized
transcript abundance was calculated as transcripts per million (TPM), with
subsequent analysis carried out on those genes with a normalized count of at
least two TPM. Comparison of the number of genes transcribed by each time point
were visualized using an Upset Plot (18 (link))
(supplemental Fig. S2). Comparative analysis with the
transcriptomic responses of juvenile 21 day old and adult parasites was carried
out on RNAseq data generated from samples isolated from rats and bovine
infected with F. hepatica as detailed by Cwiklinski et
al. (9 ).
Network analysis of the 17901 genes expressed within the first 24 h
post-excystment was carried using a Network graph constructed using BioLayout
Express^3D (19 (link)) with a
Pearson correlation threshold of r ≥ 0.97. The graph comprised of 13,559
nodes connected by 765,001 edges, which was clustered using the Markov
clustering algorithm (MCL 2.2.6), resulting in 857 clusters with at least 4
nodes that were temporally expressed by the different lifecycle stages;
metacercariae, NEJ 1 h, 3 h, and 24 h post-excystment. Hierarchical clustering
was also carried out on those genes that displayed at least a 2-fold difference
in expression among any of the four lifecycle stages, represented by 6009 genes
with a baseline cut-off of 2 TPM, graphically represented using heatmaps
generated using the R program, pheatmap. Gene model annotation was carried out
using Uniprot, Gene Ontology and Interpro in silico tools
(9 ) and the KEGG Automatic Annotation
Server (KAAS; (20 (link)). Metabolic pathway
analysis was carried by normalizing the global patterns of expression at the
KEGG module level (21 (link), 22 (link)); graphically represented using heatmaps
generated using the R program, pheatmap.

Adult worms of F. hepatica were collected from the livers of naturally infected cattle acquired from the local abattoir. The worms were washed three times in wash medium (RPMI 1640 medium [Gibco] supplemented with Penicillin-Streptomycin-Amphotericin B [Antibiotic Antimycotic 100×, Gibco] and 10 mM HEPES, pH 7.3) and then transferred to complete culture medium (RPMI 1640 medium supplemented with 2 mM L-glutamine supplemented with 5% calf serum, 55 mM glucose, 30 mM HEPES, penicillin [100 U/ml], streptomycin [100 μg/ml], and gentamicin [25 μg/ml], pH 7.3). After 4-h incubation at 37 °C, the medium with freshly laid eggs was collected. Metacercariae were obtained from Ridgeway Research Ltd. NEJ material was prepared by excystation of metacercariae (19 (link)) and harvested after 24 h cultivation. For protein extraction, the worm tissue in PBS containing protease inhibitor cocktail (539131, Calbiochem) was mechanically disrupted by metal beads (Qiagen TissueLyser II), following by ultrasonication. The lysate was centrifuged at 16,000g at 4 °C for 10 min, and the supernatant was stored at −80 °C. Medium containing the E/S products of adult flukes was collected after cultivation for 4 h at 37 °C in serum-free medium, filtered through an Ultrafree-MC 0.22 mm filter (Millipore), concentrated, and stored at −80 °C.

In order to obtain metacercariae experimentally, we attempted to infect laboratory-reared snails [Biomphalaria glabrata (Say, 1818)] and fish (Poecilia reticulata Peters, 1859). These species were used as experimental hosts due to their availability in the laboratory and previous knowledge on the involvement of snails and fish as second intermediate hosts of echinostomes. The behavior of cercariae in the presence of these potential hosts was observed under a stereomicroscope. After 24hs of exposure to cercariae, the snails and fish were necropsied. We also searched for metacercariae in samples of insects, fishes, snails, and tadpoles collected in the same water bodies where snails were found infected.
Metacercariae found in tadpoles collected in the stream where snails were found infected were used for an experimental infection study. We suspected they could be of the same species based on the number of excretory corpuscles. Aiming to obtain adult parasites for taxonomic identification, a sub-sample of 50 metacercariae was orally administered to one specimen of a dexamethasone-immunosuppressed (50 mg/kg) male Swiss mouse. The infected mouse was maintained on a 12/12h light–dark cycle and allowed access to food and water ad libitum. Coproparasitological examinations by the sedimentation technique were conducted daily, starting from seven days post-infection. The mouse was euthanized via barbituric overdose (sodium pentobarbital, injected intraperitoneally) and necropsied for the search of adult parasites 14 days post-infection.

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.