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Fascioliasis (or fasciolosis) is a socioeconomically important parasitic disease caused by liver flukes of the genus Fasciola. Flukicide resistance has exposed the need for new drugs and/or a vaccine for liver fluke control. A rapidly improving 'molecular toolbox' for liver fluke encompasses quality genomic/transcriptomic datasets and an RNA interference platform that facilitates functional genomics approaches to drug/vaccine target validation. The exploitation of these resources is undermined by the absence of effective culture/maintenance systems that would support in vitro studies on juvenile fluke development/biology. Here we report markedly improved in vitro maintenance methods for Fasciola hepatica that achieved 65% survival of juvenile fluke after 6 months in standard cell culture medium supplemented with 50% chicken serum. We discovered that this long-term maintenance was dependent upon fluke growth, which was supported by increased proliferation of cells resembling the "neoblast" stem cells described in other flatworms. Growth led to dramatic morphological changes in juveniles, including the development of the digestive tract, reproductive organs and the tegument, towards more adult-like forms. The inhibition of DNA synthesis prevented neoblast-like cell proliferation and inhibited growth/development. Supporting our assertion that we have triggered the development of juveniles towards adult-like fluke, mass spectrometric analyses showed that growing fluke have an excretory/secretory protein profile that is distinct from that of newly-excysted juveniles and more closely resembles that of ex vivo immature and adult fluke. Further, in vitro maintained fluke displayed a transition in their movement from the probing behaviour associated with migrating stage worms to a slower wave-like motility seen in adults. Our ability to stimulate neoblast-like cell proliferation and growth in F. hepatica underpins the first simple platform for their long-term in vitro study, complementing the recent expansion in liver fluke resources and facilitating in vitro target validation studies of the developmental biology of liver fluke.

Additional file 8: Fig. S7. Comparisons of anti-r6cys A and r6cys B antibodies in tick hemolymph and sera from immunized and control animals by iELISA. A: Bovine antibodies were detected in the sera of all immunized animals, 84 dpi, and in the first and second tick hemolymph groups derived from immunized vs control animals. B: Antibody isotype analysis performed on antibodies against r6cys A protein present in hemolymph of ticks compared with immunized animals (84 dpi).

Additional file 9: Fig. S8. Analysis of the effect of the immunization of cattle with r6cys A and r6cys B proteins on the development of B. bovis in ticks. A. Comparisons of tick body (left chart) and egg weights (right chart) among ticks and eggs derived from immunized and control groups. No significant difference among the groups was found (P > 0.05). B. qPCR analysis performed on DNA extracted from temperature-stimulated and non-stimulated larvae derived from the two tick groups (vaccinated or control cattle). No significant difference among the groups was found (P > 0.05).

Additional file 8: Fig. S7. Comparisons of anti-r6cys A and r6cys B antibodies in tick hemolymph and sera from immunized and control animals by iELISA. A: Bovine antibodies were detected in the sera of all immunized animals, 84 dpi, and in the first and second tick hemolymph groups derived from immunized vs control animals. B: Antibody isotype analysis performed on antibodies against r6cys A protein present in hemolymph of ticks compared with immunized animals (84 dpi).

Additional file 4: Fig. S3. Schematic representation of the experimental design of larva analysis derived from the two tick groups, immunized and control. Larvae induced under two temperature conditions, 36 ��� [Induced] and 26 ��� [Non induced] were analyzed.

Additional file 4: Fig. S3. Schematic representation of the experimental design of larva analysis derived from the two tick groups, immunized and control. Larvae induced under two temperature conditions, 36 ��� [Induced] and 26 ��� [Non induced] were analyzed.

Additional file 9: Fig. S8. Analysis of the effect of the immunization of cattle with r6cys A and r6cys B proteins on the development of B. bovis in ticks. A. Comparisons of tick body (left chart) and egg weights (right chart) among ticks and eggs derived from immunized and control groups. No significant difference among the groups was found (P > 0.05). B. qPCR analysis performed on DNA extracted from temperature-stimulated and non-stimulated larvae derived from the two tick groups (vaccinated or control cattle). No significant difference among the groups was found (P > 0.05).