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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.

Fasciola hepatica infection in livestock is an increasing threat to the animal production industry and results in severe economic losses. Emergence of fluke drug resistance warrants an efficacious vaccine to combat the liver fluke infections. The tegument of the parasite is a target for antibody‐dependent cell‐ mediated cytotoxicity of juvenile parasites in vitro. Thus, we propose that tegument proteins of F. hepatica could serve as potential vaccine candidates. Proteomic analysis of the adult fluke tegument identified proteins with homology to annexins (Anx) and tetraspanins (TSP) of Schistosoma sp. Further, using bioinformatics analysis, we identified 9 Anx sequences (Anx 1‐9) and 33 TSP sequences (TSP 1‐33) in the F. hepatica genome. Differential transcript levels were observed for Anx 1‐9 and TSP 1‐33 in the life cycle stages of F. hepatica. Anx 1, 2, 3 and 8; and TSP 2, 3, 5 and 8 were predicted to be surface‐expressed based on their homology to known surface‐expressed orthologues from Schistosoma sp and Opisthorchis viverrini. We expressed the full length Anx 1, 2, 3 and 8 proteins and the extra cellular loop 2 of TSP 2, 3, 5 and 8 in Pichia pastoris. Immunolocalisation of the selected annexins and tetraspanins using specific rabbit antisera confirmed the surface expression of each antigen on juvenile flukes, suggesting the potential involvement of these proteins in the host‐ parasite interaction. Vaccination of rats with recombinant Anx 1 and 2; and TSP 2, 3, 5 and 8 followed by F. hepatica challenge infection resulted in the protection levels ranging from 0‐47%. We also tested the rAnx 1 and 2; and rTSP 2, 3, 5 and 8; along with two other tegument proteins CD 59.1 and CUB 2, as combination vaccines in cattle; vaccination with these proteins followed by F. hepatica challenge infection resulted in 0‐39.5% protection, confirming the potential of using certain tegument proteins as vaccine targets in cattle.Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Animal, Plant and Soil Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

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 2: Fig. S1. SDS-PAGE and western blot analysis using anti-histidine antibodies reactive with r6cys A and r6cys B proteins. Lane M: protein marker. Lane 1: reducing conditions. Lane 2: non- reducing conditions. Lane P: multiple-tag as positive control. MW of 6cys A: ~ 70 kDa���MW of 6cys B: ~ 80 kDa.

Additional file 3: Fig. S2. Schematic description of the outline of the in vivo experiments performed in the r6cys A and r6cys B immunization study.

Additional file 7: Fig. S6. Tick hemolymph analysis. A: 48 hemolymph samples were analyzed for the presence of kinetes by light microscopy. The numbers of kinete per sample were computed and used to calculate kinete infectivity rates per animal. The chart represents averages of the kinete infectivity rate in hemolymph collected from ticks feeding on immunized and control animals. Analysis was performed on ticks that dropped on the 3rd, 4th and 5th days after the onset of droppings. P values (> 0.05) indicate there were no significant differences between the two groups. B: Quantitative PCR (qPCR) analysis for the evaluation of B. bovis DNA in hemolymph from the two pooled tick groups derived from the immunized and control animals collected on day 4 and 5, as described in A. There was no significant difference between the two tick groups of tested animals.

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 5: Fig. S4. Antibody titrations performed on sera from the six experimental animals against the r6cys A and r6cys B proteins.

Additional file 7: Fig. S6. Tick hemolymph analysis. A: 48 hemolymph samples were analyzed for the presence of kinetes by light microscopy. The numbers of kinete per sample were computed and used to calculate kinete infectivity rates per animal. The chart represents averages of the kinete infectivity rate in hemolymph collected from ticks feeding on immunized and control animals. Analysis was performed on ticks that dropped on the 3rd, 4th and 5th days after the onset of droppings. P values (> 0.05) indicate there were no significant differences between the two groups. B: Quantitative PCR (qPCR) analysis for the evaluation of B. bovis DNA in hemolymph from the two pooled tick groups derived from the immunized and control animals collected on day 4 and 5, as described in A. There was no significant difference between the two tick groups of tested animals.

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 5: Fig. S4. Antibody titrations performed on sera from the six experimental animals against the r6cys A and r6cys B proteins.

Additional file 3: Fig. S2. Schematic description of the outline of the in vivo experiments performed in the r6cys A and r6cys B immunization study.

Additional file 1: Table S1. Abbreviations used to describe the distinct treatments performed in the in vitro B. bovis sexual induction experiment.

Additional file 6: Fig. S5. Detection of B. bovis DNA by conventional PCR analysis on daily blood samples collected from all animals under experimental study: Immunized animals are C1501, C1506 and C1508. Control animals are C1502, C1505 and C1512. Numbers on top represents days after challenge. Size markers are indicated on the right.

Additional file 6: Fig. S5. Detection of B. bovis DNA by conventional PCR analysis on daily blood samples collected from all animals under experimental study: Immunized animals are C1501, C1506 and C1508. Control animals are C1502, C1505 and C1512. Numbers on top represents days after challenge. Size markers are indicated on the right.

Additional file 2: Fig. S1. SDS-PAGE and western blot analysis using anti-histidine antibodies reactive with r6cys A and r6cys B proteins. Lane M: protein marker. Lane 1: reducing conditions. Lane 2: non- reducing conditions. Lane P: multiple-tag as positive control. MW of 6cys A: ~ 70 kDa���MW of 6cys B: ~ 80 kDa.

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 1: Table S1. Abbreviations used to describe the distinct treatments performed in the in vitro B. bovis sexual induction experiment.

Additional file 10: Fig. S9. Bioinformatics analysis for the prediction of N-glycosylation sites in the 6cysA and 6cysB proteins. XP_001610178.1 represents the 6cys A protein, and XP_001610179.1 represents the 6cys B protein.

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).