Evaluation of LAMP for Fasciola hepatica detection from faecal samples of experimentally and naturally infected cattle.

Fasciola hepatica causes liver fluke disease in production animals and humans worldwide. Faecal egg counts (FEC) are the most common diagnostic tool for the diagnosis of liver fluke disease. However, FEC has low sensitivity and is often unreliable for the detection of patent infection. In this study, loop-mediated isothermal amplification (LAMP) was optimised and evaluated for the detection of Fasciola hepatica infection, with the aim of increased sensitivity and making it suitable for on-farm application. LAMP was initially conducted under laboratory conditions, optimised to enable visual detection using calcein dye. DNA extraction based on bead-beating was developed to enable on-farm application. LAMP results were compared to FEC and polymerase chain reaction (PCR). Under laboratory conditions, LAMP was conducted using two incubation methods: a conventional PCR thermocycler and a field-deployable LAMP instrument. When compared to a 'rigorous' FEC protocol consisting of multiple counts using a comparatively large volume of faeces and with infection confirmed post-mortem, LAMP was highly sensitive and specific (using silica membrane DNA extraction sensitivity 88 %, specificity 100 %; using sieving and beat-beating DNA extraction sensitivity 98.9 %, specificity 100 %). When applied on-farm, LAMP was compared to conventional FEC, which suggested high sensitivity but low specificity (sensitivity 97 %, specificity 37.5 %). However, further analysis, comparing field LAMP results to laboratory PCR, suggested that the low specificity was likely the outcome of the inability of conventional FEC to detect all true F. hepatica positive samples. Based on the high sensitivity and specificity of LAMP compared to a 'rigorous' FEC protocol and its ability to be used in field settings, the study demonstrates the potential of LAMP for diagnosing F. hepatica infection in agriculture.

Environmental detection of Fasciola hepatica by loop-mediated isothermal amplification.

Fasciola hepatica, commonly referred to as liver flukes, is a substantial zoonotic parasitic disease of humans and livestock globally. While infection is readily controlled by anthelmintics, namely triclabendazole, the heavy reliance on triclabendazole has resulted in drug resistance appearing worldwide. Due to drug resistance, it is imperative to adopt an integrated parasite management program to preserve the efficacy of currently available anthelmintics. A integrated liver fluke management plan would benefit from a simple rapid, field-deployable diagnostic for detection of F. hepatica in environment and the host. Therefore, a rapid DNA test using loop-mediated isothermal amplification was developed and optimised for the detection of F. hepatica from faecal and water samples to enable the detection of parasites both within the host and from the environment. The assay presented here is fast, with amplification in ≤20 min, and highly sensitive, with a detection limit of 5 × 10-4 ng/µL. The workflow presented here provides a time to result of ≤60 min without requiring a commercial kit for the extraction of DNA from faecal and water samples, and pending further validation from field-samples, could potentially be used to enable real-time decision making to mitigate parasite prevalence on a farming property and with no requirement for sample transportation.

Bovine Natural Antibody Relationships to Specific Antibodies and Fasciola hepatica Burdens after Experimental Infection and Vaccination with Glutathione S-Transferase.

Fasciola hepatica is the causative agent of fasciolosis, a significant parasitic disease occurring worldwide. Despite ongoing efforts, there is still no vaccine to control liver fluke infections in livestock. Recently, it has been suggested that natural antibodies (NAbs) can amplify specific antibodies (SpAb) and have a direct killing effect, but it is unknown if this phenomenon occurs during parasitic helminth infection or targeted vaccination. NAbs are antibodies produced by the innate immune system, capable of binding antigens without prior exposure. This study explores the role of bovine NAbs, using the exogenous glycoprotein keyhole limpet hemocyanin (KLH), in response to F. hepatica infection and SpAb production after infection and vaccination. The cattle's NAbs were differently influenced by parasite infection and vaccination, with an increase in KLH-binding IgG and IgM levels after infection and reduced KLH-binding IgM levels following vaccination. Underlying NAbs reacting to KLH showed no correlations to the final fluke burdens after experimental infection or vaccination. However, NAbs reacting to whole-worm extract (WWE) prior to infection were positively correlated to increased fluke burdens within the infected bovine host. Furthermore, after infection, the specific IgG reacting to WWE was positively reflected by the underlying NAb IgG response. Following subcutaneous vaccination with F. hepatica native glutathione S-transferase (GST), there was a non-significant 33% reduction in fluke burden. Vaccinated animals with higher underlying NAbs had a higher induction of vaccine-induced SpAbs, with trends observed between KLH-binding IgM and anti-GST IgG and IgM. Our findings provide a platform to allow further investigation to determine if NAb levels could mirror fluke-SpAb production for exploitation in a combined selective breeding and vaccination program. Additionally, this work suggests that liver fluke could possibly evade the host's immune system by utilising surface-bound IgM NAbs.

Evaluation of Immunogenicity and Efficacy of Fasciola hepatica Tetraspanin 2 (TSP2) Fused to E. coli Heat-Labile Enterotoxin B Subunit LTB Adjuvant Following Intranasal Vaccination of Cattle.

Fasciolosis, caused by the liver flukes Fasciola hepatica and F. gigantica, is an economically important and globally distributed zoonotic disease. Liver fluke infections in livestock cause significant losses in production and are of particular concern to regions where drug resistance is emerging. Antigens of the F. hepatica surface tegument represent promising vaccine candidates for controlling this disease. Tetraspanins are integral tegumental antigens that have shown partial protection as vaccine candidates against other trematode species. The Escherichia coli heat-labile enterotoxin's B subunit (LTB) is a potent mucosal adjuvant capable of inducing an immune response to fused antigens. This study investigates the potential of F. hepatica tetraspanin 2 extracellular loop 2 (rFhTSP2) as a protective vaccine antigen and determines if fusion of FhTSP2 to LTB can enhance protection in cattle. Cattle were immunised subcutaneously with rFhTSP2 mixed in the Freund's adjuvant and intranasally with rLTB-FhTSP2 in saline, accounting for equal molar ratios of tetraspanin in both groups. Vaccination with rFhTSP2 stimulated a strong specific serum IgG response, whereas there was no significant serum IgG response following rLTB-FhTSP2 intranasal vaccination. There was no substantial antigen specific serum IgA generated in all groups across the trial. Contrastingly, after the fluke challenge, a rise in antigen specific saliva IgA was observed in both vaccination groups on Day 42, with the rLTB-FhTSP2 vaccination group showing significant mucosal IgA production at Day 84. However, neither vaccine group showed a significant reduction of fluke burden nor faecal egg output. These results suggest that intranasal vaccination with rLTB-FhTSP2 does elicit a humoral mucosal response but further work is needed to evaluate if mucosal delivery of liver fluke antigens fused to LTB is a viable vaccine strategy.

Molecular characterisation and vaccine efficacy of two novel developmentally regulated surface tegument proteins of Fasciola hepatica.

The surface tegument of Fasciola hepatica is a crucial tissue due to its key role at the host-parasite interface. We characterised three novel proteins, termed Fhteg1, Fhteg5 and Fhteg8, that are found in the tegument membrane fraction of adult F. hepatica. Bioinformatic analysis of proteomic datasets identified Fhteg5 and Fhteg8 as tegumental glycoproteins and revealed that Fhteg1, Fhteg5 and Fhteg8 are associated with exosomes of adult F. hepatica. Fhteg1, Fhteg5 and Fhteg8 appear to be related to uncharacterised sequences in F. gigantica, Fasciolopsis buski, Echinostoma caproni, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma japonicum and S. mansoni, although F. hepatica appears to have expanded this family. Fhteg1 and Fhteg5 were characterised in detail. The Fhteg1 and Fhteg5 gene transcripts each demonstrate significant upregulation in juvenile fluke 2-4 days post-excystment, with transcript levels maintained during development over 3 weeks in vitro. RNAseq data showed that both Fhtegs are expressed in the adult life stage, although the transcript levels were about 8 fold lower than those in juveniles (3 week post infection). Using immunocytochemistry, Fhteg1 and Fhteg5 were each shown to be expressed in cells adjacent to the muscle layer as well as on the surface of 1 week old juveniles, whilst Fhteg5 was also present in cells at the base of the pharynx. RNAi mediated knockdown of Fhteg1 and Fhteg5 transcripts in 4-10 day old juveniles had no effect on parasite survival, movement or growth in vitro. Although no IgG responses were observed for Fhteg1 or Fhteg5 during infection in sheep and cattle, both proteins elicited a low IgG response in a proportion of infected rats. Rats vaccinated with Fhteg1 and Fhteg5 showed good IgG responses to both proteins and a mean 48.2 % reduction in worm burden following parasite challenge. Although vaccination of cattle with both proteins induced a range of IgG responses, no protection was observed against parasite challenge. This is the first study to provide insights into the molecular properties of two novel, developmentally regulated surface tegument proteins in F. hepatica.

A novel ex vivo immunoproteomic approach characterising Fasciola hepatica tegumental antigens identified using immune antibody from resistant sheep.

A more thorough understanding of the immunological interactions between Fasciola spp. and their hosts is required if we are to develop new immunotherapies to control fasciolosis. Deeper knowledge of the antigens that are the target of the acquired immune responses of definitive hosts against both Fasciola hepatica and Fasciola gigantica will potentially identify candidate vaccine antigens. Indonesian Thin Tail sheep express a high level of acquired immunity to infection by F. gigantica within 4weeks of infection and antibodies in Indonesian Thin Tail sera can promote antibody-dependent cell-mediated cytotoxicity against the surface tegument of juvenile F. gigantica in vitro. Given the high protein sequence similarity between F. hepatica and F. gigantica, we hypothesised that antibody from F. gigantica-infected sheep could be used to identify the orthologous proteins in the tegument of F. hepatica. Purified IgG from the sera of F. gigantica-infected Indonesian Thin Tail sheep collected pre-infection and 4weeks p.i. were incubated with live adult F. hepatica ex vivo and the immunosloughate (immunoprecipitate) formed was isolated and analysed via liquid chromatography-electrospray ionisation-tandem mass spectrometry to identify proteins involved in the immune response. A total of 38 proteins were identified at a significantly higher abundance in the immunosloughate using week 4 IgG, including eight predicted membrane proteins, 20 secreted proteins, nine proteins predicted to be associated with either the lysosomes, the cytoplasm or the cytoskeleton and one protein with an unknown cellular localization. Three of the membrane proteins are transporters including a multidrug resistance protein, an amino acid permease and a glucose transporter. Interestingly, a total of 21 of the 38 proteins matched with proteins recently reported to be associated with the proposed small exosome-like extracellular vesicles of adult F. hepatica, suggesting that the Indonesian Thin Tail week 4 IgG is either recognising individual proteins released from extracellular vesicles or is immunoprecipitating intact exosome-like extracellular vesicles. Five extracellular vesicle membrane proteins were identified including two proteins predicted to be associated with vesicle transport/ exocytosis (VPS4, vacuolar protein sorting-associated protein 4b and the Niemann-Pick C1 protein). RNAseq analysis of the developmental transcription of the 38 immunosloughate proteins showed that the sequences are expressed over a wide abundance range with 21/38 transcripts expressed at a relatively high level from metacercariae to the adult life cycle stage. A notable feature of the immunosloughates was the absence of cytosolic proteins which have been reported to be secreted markers for damage to adult flukes incubated in vitro, suggesting that the proteins observed are not inadvertent contaminants leaking from damaged flukes ex vivo. The identification of tegument protein antigens shared between F. gigantica and F. hepatica is beneficial in terms of the possible development of a dual purpose vaccine effective against both fluke species.

Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro.

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.

Liver fluke vaccines in ruminants: strategies, progress and future opportunities.

The development of a vaccine for Fasciola spp. in livestock is a challenge and would be advanced by harnessing our knowledge of acquired immune mechanisms expressed by resistant livestock against fluke infection. Antibody-dependent cell-mediated cytotoxicity directed to the surface tegument of juvenile/immature flukes is a host immune effector mechanism, suggesting that antigens on the surface of young flukes may represent prime candidates for a fluke vaccine. A Type 1 immune response shortly after fluke infection is associated with resistance to infection in resistant sheep, indicating that vaccine formulations should attempt to induce Type 1 responses to enhance vaccine efficacy. In cattle or sheep, an optimal fluke vaccine would need to reduce mean fluke burdens in a herd below the threshold of 30-54 flukes to ensure sustainable production benefits. Fluke infection intensity data suggest that vaccine efficacy of approximately 80% is required to reduce fluke burdens below this threshold in most countries. With the increased global prevalence of triclabendazole-resistant Fasciolahepatica, it may be commercially feasible in the short term to introduce a fluke vaccine of reasonable efficacy that will provide economic benefits for producers in regions where chemical control of new drug-resistant fluke infections is not viable. Commercial partnerships will be needed to fast-track new candidate vaccines using acceptable adjuvants in relevant production animals, obviating the need to evaluate vaccine antigens in rodent models.

Autoantibodies to iron-binding proteins in pigs infested with Sarcoptes scabiei.

Despite the availability of effective treatments, Sarcoptes scabiei remains a major health problem in the pig industry. Unsuccessful control of the disease is often due to the lack of reliable detection methods, with current tests relying on skin scrapings and crude antigen ELISAs. A previous analysis of antigens in pig skin scrapings reported that anti-transferrin antibodies were present in S. scabiei infected animals and that this finding might be considered as a useful diagnostic tool. This paper confirms IgG autoantibodies against transferrin, including the first report of IgM autoantibodies, in both naturally and experimentally infected pigs using ELISA and dot blot assays. Autoantibodies were also detected in pigs to ferritin and to a lesser extent lactoferrin. Immunoblotting confirmed the presence of IgG and IgM autoantibodies in mange positive pigs, as well as IgM antibodies to transferrin and albumin in mange negative pigs. These findings suggest the presence of natural autoantibodies to transferrin and albumin in pigs. The development of the IgG autoimmune response may either be a host mechanism for limiting iron to the mite via antibody mediated clearance, the result of host exposure to mite iron-binding homologues or because of a mite-induced antigenic change to host transferrin. Further investigation into the formation of these autoantibodies may provide insights into the importance of iron in scabies infections and the development and perseverance of S. scabiei infections in pigs. The specificity and sensitivity of the anti-transferrin response reinforces its potential in the diagnosis of scabies in pigs.

First insight into CD59-like molecules of adult Fasciola hepatica.

The present study focussed on investigating CD59-like molecules of Fasciola hepatica. A cDNA encoding a CD59-like protein (termed FhCD59-1) identified previously in the membrane fraction of the F. hepatica tegument was isolated. This homologue was shown to encode a predicted open reading frame (ORF) of 122 amino acids (aa) orthologous to human CD59 with a 25 aa signal peptide, a mature protein containing 10 cysteines and a conserved CD59/Ly-6 family motif "CCXXXXCN". An analysis of cDNAs from two different adult specimens of F. hepatica revealed seven variable types of FhCD59-1 sequences, designated FhCD59-1.1 to FhCD59-1.7, which had 94.3-99.7% amino acid sequence identity upon pairwise comparison. Molecular modeling of FhCD59-1.1 with human CD59 confirmed the presence of the three-finger protein domain found in the CD59 family and predicted three disulphide bonds in the F. hepatica sequence. The interrogation of F. hepatica databases identified two additional sequences, designated FhCD59-2 and FhCD59-3, which had only 23.4-29.5% amino acid identity to FhCD59-1.1. Orthologues of the inferred CD59 protein sequences of F. hepatica were also identified in other flatworms, including Fasciola gigantica, Fascioloides magna, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, Clonorchis sinensis, Opisthorchis viverrini, Taenia solium, Echinococcus granulosus and the free living Schmidtea mediterannea. The results revealed a considerable degree of sequence complexity in the CD59-like sequence families in F. hepatica and flatworms. Phylogenetic analysis of CD59-like aa sequences from F. hepatica and flatworms showed that FhCD59-2 clustered with the known surface-associated protein SmCD59-2 of S. mansoni. Relatively well-supported clades specific to schistosomes, fasciolids and opisthorchiids were identified. The qPCR analysis of gene transcription showed that the relative expression of these 3 FhCD59-like sequences varied by 11-47-fold during fluke maturation, from the newly excysted juvenile (NEJ) to the adult stage. These findings suggest that different FhCD59-like sequences play distinct roles during the development of F. hepatica.