A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance.

Fasciola hepatica infection is responsible for substantial economic losses in livestock worldwide and poses a threat to human health in endemic areas. The mainstay of control in livestock and the only drug licenced for use in humans is triclabendazole (TCBZ). TCBZ resistance has been reported on every continent and threatens effective control of fasciolosis in many parts of the world. To date, understanding the genetic mechanisms underlying TCBZ resistance has been limited to studies of candidate genes, based on assumptions of their role in drug action. Taking an alternative approach, we combined a genetic cross with whole-genome sequencing to localise a ~3.2Mbp locus within the 1.2Gbp F. hepatica genome that confers TCBZ resistance. We validated this locus independently using bulk segregant analysis of F. hepatica populations and showed that it is the target of drug selection in the field. We genotyped individual parasites and tracked segregation and reassortment of SNPs to show that TCBZ resistance exhibits Mendelian inheritance and is conferred by a dominant allele. We defined gene content within this locus to pinpoint genes involved in membrane transport, (e.g. ATP-binding cassette family B, ABCB1), transmembrane signalling and signal transduction (e.g. GTP-Ras-adenylyl cyclase and EGF-like protein), DNA/RNA binding and transcriptional regulation (e.g. SANT/Myb-like DNA-binding domain protein) and drug storage and sequestration (e.g. fatty acid binding protein, FABP) as prime candidates for conferring TCBZ resistance. This study constitutes the first experimental cross and genome-wide approach for any heritable trait in F. hepatica and is key to understanding the evolution of drug resistance in Fasciola spp. to inform deployment of efficacious anthelmintic treatments in the field.

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.

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.

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.

Current Status for Controlling the Overlooked Caprine Fasciolosis.

The disease fasciolosis is caused by the liver flukes Fasciola hepatica and F. gigantica, which infect a wide range of mammals and production livestock, including goats. These flatworm parasites are globally distributed and predicted to cost the livestock industry a now conservative USD 3 billion per year in treatment and lowered on-farm productivity. Infection poses a risk to animal welfare and results in lowered fertility rates and reduced production yields of meat, milk and wool. This zoonotic disease is estimated to infect over 600 million animals and up to 2.4 million humans. Current and future control is threatened with the global emergence of flukes resistant to anthelmintics. Drug resistance calls for immediate on-farm parasite management to ensure treatments are effective and re-infection rates are kept low, while a sustainable long-term control method, such as a vaccine, is being developed. Despite the recent expansion of the goat industry, particularly in developing countries, there are limited studies on goat-focused vaccine control studies and the effectiveness of drug treatments. There is a requirement to collate caprine-specific fasciolosis knowledge. This review will present the current status of liver fluke caprine infections and potential control methods for application in goat farming.

Analysis of daily variation in the release of faecal eggs and coproantigen of Fasciola hepatica in naturally infected dairy cattle and the impact on diagnostic test sensitivity.

The liver fluke, Fasciola hepatica (F. hepatica) is a widespread parasite infection in dairy cattle in Victoria, South-eastern Australia. Robust diagnosis of fluke infection is needed in dairy cattle to identify sub-clinical infections which often go unnoticed, causing significant production losses. We tested the coproantigen ELISA (cELISA) and the FlukeFinder faecal egg count kit® on naturally infected cows in a fluke endemic region of Victoria. The aim of the study was to investigate the variation in the release of coproantigens and eggs into faeces over a 5-day period, at the morning (AM) and afternoon (PM) milkings, and to assess the impact of the timing of faecal sample collection on diagnostic test sensitivity. Ten cows were enrolled into the study based on positive F. hepatica faecal egg counts (LFEC) and faecal samples from the ten cows were collected twice daily, at the 7-9 AM and 4-6 PM milking, for five consecutive days. At the conclusion of the sampling period, the cows were euthanized and F. hepatica burden determined at necropsy. A moderate negative correlation between cow age and cELISA optical density (OD) was observed using data from all samples (R -0.63; 95 % CI -0.68 to -0.57). Over the 5-day sampling period, we observed within-animal variation between days for both the cELISA OD (2.6-8.9 fold) and LFEC (5-16 fold), with more variation in values observed in the PM samples for both tests. The correlation with total fluke burden was higher in the AM sampling using both the cELISA and LFEC (R 0.64 and 0.78, respectively). The sensitivity was 100 % for the cELISA using various cut offs from the literature (0.014 OD, 0.030 OD, and 1.3 % or 1.6 % of the positive control). The sensitivity of the FlukeFinder kit® (based on 588 faecal samples and not accounting for lack of independence in the data) was 88 % (95 % CI 85 %-90 %). Seventy one false negatives were recorded from the 588 LFEC tests all of which were observed in the cows with fluke burdens <14 flukes; 42 of the 71 false negative LFECs occurred in one individual cow which had the lowest burden of nine flukes. In dairy cows, the cut-off for production losses due to fasciolosis is estimated at> 10 fluke. Both the cELISA and the LFEC identified all cows that had burdens equal to or greater than this cut-off. Five of the ten cows also exhibited relatively high paramphistome egg counts.

Fasciola hepatica Control Practices on a Sample of Dairy Farms in Victoria, Australia.

In Australia, little is known about the strategies used by farmers to control Fasciola hepatica (F. hepatica) infection in dairy cattle. Triclabendazole-resistant F. hepatica have recently been found on several dairy and beef properties in Australia. It is difficult to draw conclusions about how widespread resistance is in Australian dairy cattle because we have little information about flukicide usage, drug resistance testing, and alternative flukicide usage on-farm. The study objectives were to determine how dairy farmers are currently controlling F. hepatica and to identify knowledge gaps where F. hepatica control strategies need to be communicated to farmers to improve management. The survey was distributed online or by hard copy and 36 dairy farmers completed the survey. There were 34 questions including closed, open-ended, multicheck box, demographic, and text questions. Descriptive statistics were used to quantify each response. The survey results showed high use of clorsulon, limited rotation of flukicides, and limited use of diagnostic tests to inform treatment options and timing. There was poor adherence to best management practice in determining the dose of flukicides administered to cattle, with farmers often relying on estimating body weights or average body weights, suggesting that underdosing of animals is likely to be prevalent. Most respondents in this study did not isolate and quarantine treated and newly returned or purchased animals before joining them with the main herd. The research identified four knowledge gaps where communication needs to be enhanced to improve control of F. hepatica: diagnostic testing to inform flukicide use, rotation of flukicide actives, flukicide administration, and increased testing of replacement animals.

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.

Towards understanding the liver fluke transmission dynamics on farms: Detection of liver fluke transmitting snail and liver fluke-specific environmental DNA in water samples from an irrigated dairy farm in Southeast Australia.

Livestock production around the world is impacted by liver fluke (Fasciola spp.) infection resulting in serious economic losses to the beef, dairy and sheep industries with significant losses of about $90 million per annum in Australia. Triclabendazole (TCBZ) is the most effective anthelmintic treatment available to control liver fluke infections; however, the widespread emergence of TCBZ resistance in livestock threatens liver fluke control. Alternative control measures to lower exposure of livestock to liver fluke infection would help to preserve the usefulness of current anthelmintic treatments. Environmental DNA (eDNA) based identification of liver fluke and the intermediate snail host in the water bodies is a robust method to assess the risk of liver fluke infection on farms. In this study, we used a multiplex quantitative PCR assay of water samples to detect and quantify eDNA of Fasciola hepatica (F. hepatica) and Austropeplea tomentosa (A. tomentosa), a crucial intermediate snail host for liver fluke transmission in South-east Australia. Water samples were collected from an irrigation channel for a period of 7 months in 2016 (February, March, May, September, October, November and December) at a dairy farm located at Maffra, Victoria, South-east Australia. Using an effective eDNA extraction method, the multiplex qPCR assay allows for the independent but simultaneous detection of eDNA released from liver fluke life stages and snails using specific primers and a probe targeting the ITS-2 region of the liver fluke and snail, respectively, with minimal inhibition from contaminants in field collected water samples. The sensitivity of this assay to detect eDNA of liver fluke and snails was observed to be 14 fg and 50 fg, respectively, in the presence of field collected water samples. Differential levels of liver fluke and snail specific eDNA in water were observed at the time points analysed in this study. The successful detection of eDNA specific to liver fluke and snails from the field collected water samples provides a precedent for the use of this method as a monitoring tool to determine the prevalence of liver fluke and liver fluke-transmitting snails in irrigation regions. Further, this method has the enormous potential to allow an assessment of the liver fluke transmission zones on farms and to inform the application of effective control strategies.

Identification of novel diagnostic serum biomarkers for Chagas' disease in asymptomatic subjects by mass spectrometric profiling.

More than 10 million people are thought to be infected with Trypanosoma cruzi, primarily in the Americas. The clinical manifestations of Chagas' disease (CD) are variable, but most subjects remain asymptomatic for decades. Only 15 to 30% eventually develop terminal complications. All current diagnostic tests have limitations. New approaches are needed for blood bank screening as well as for improved diagnosis and prognosis. Sera from subjects with asymptomatic CD (n = 131) were compared to those from uninfected controls (n = 164) and subjects with other parasitic diseases (n = 140), using protein array mass spectrometry. To identify biomarkers associated with CD, sera were fractionated by anion-exchange chromatography and bound to two commercial ProteinChip array chemistries: WCX2 and IMAC3. Multiple candidate biomarkers were found in CD sera (3 to 75.4 kDa). Algorithms employing 3 to 5 of these biomarkers achieved up to 100% sensitivity and 98% specificity for CD. The biomarkers most useful for diagnosis were identified and validated. These included MIP1 alpha, C3a anaphylatoxin, and unusually truncated forms of fibronectin, apolipoprotein A1 (ApoA1), and C3. An antipeptide antiserum against the 28.9-kDa C terminus of the fibronectin fragment achieved good specificity (90%) for CD in a Western blot format. We identified full-length ApoA1 (28.1 kDa), the major structural and functional protein component of high-density lipoprotein (HDL), as an important negative biomarker for CD, and relatively little full-length ApoA1 was detected in CD sera. This work provides proof of principle that both platform-dependent (i.e., mass spectrometry-based) and platform-independent (i.e., Western blot) tests can be generated using high-throughput mass profiling.

A novel achiral seco-cyclopropylpyrido[e]indolone (CPyI) analog of CC-1065 and the duocarmycins: synthesis, DNA interactions, in vivo anticancer and anti-parasitic evaluation.

The synthesis of an achiral seco-hydroxy-aza-CBI-TMI analog (8) of the duocarmycins is reported. Its specificity for the DNA minor groove of AT-rich sequences and covalent bonding to adenine-N3 was ascertained by a thermal cleavage assay. Compound 8 was found to be cytotoxic in the nanomolar range against murine and human cancer cells. It was further demonstrated that compound 8 was active against murine melanoma (B16-F0) grown in C57BL/6 mice. Compound 8 was also shown to inhibit the growth of the protozoan parasites Leishmania donovani, Leishmania mexicana, Trypanosoma brucei, and Plasmodium falciparum in culture.

Determination of the prevalence and intensity of Fasciola hepatica infection in dairy cattle from six irrigation regions of Victoria, South-eastern Australia, further identifying significant triclabendazole resistance on three properties.

Fasciola hepatica (liver fluke) is a widespread parasite infection of livestock in Victoria, South-eastern Australia, where high rainfall and a mild climate is suitable for the main intermediate host Austropeplea tomentosa. The aims of this study were to quantify the prevalence and intensity of F. hepatica in dairy cattle in the irrigated dairy regions of Victoria and determine if triclabendazole resistance was present in infected herds. Cattle in 83 herds from the following six irrigation regions were tested for F. hepatica: Macalister Irrigation District (MID), Upper Murray (UM), Murray Valley (MV), Central Goulburn (CG), Torrumbarry (TIA) and Loddon Valley (LV). Twenty cattle from each herd were tested using the F. hepatica faecal egg count (FEC) as well as the coproantigen ELISA (cELISA). The mean individual animal true prevalence of F. hepatica across all regions was 39 % (95 % credible interval [CrI] 27%-51%) by FEC and 39 % (95 % CrI 27%-50%) by cELISA with the highest true prevalence (75-80 %) found in the MID. Our results show that 46 % of the herds that took part in this study were likely to experience fluke-associated production losses, based on observations that herd productivity is impaired when the true within-herd prevalence is > 25 %. Using the FEC and cELISA reduction tests, triclabendazole resistance was assessed on 3 herds in total (2 from the 83 in the study; and 1 separate herd that did not take part in the prevalence study) and resistance was confirmed in all 3 herds. This study has confirmed that F. hepatica is endemic in several dairy regions in Victoria: triclabendazole resistance may be contributing to the high prevalence in some herds. From our analysis, we estimate that the state-wide economic loss associated with fasciolosis is in the order of AUD 129 million (range AUD 38-193 million) per year or about AUD 50,000 (range AUD 15,000-75,000) per herd per year.

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.

Vaccination against fasciolosis by a multivalent vaccine of stage-specific antigens.

Liver flukes produce cathepsin B and cathepsin L in their excretory-secretory material. These proteases are proposed to be key virulence factors for parasite infection, and are therefore targets for vaccination. Cathepsin B is predominately released in the juvenile stage of the life cycle, while different cathepsin L's are released throughout the cycle. Three proteases (cathepsin L5, cathepsin L1g and cathepsin B) were expressed in yeast from cDNA clones isolated from adult, metacercariae and newly excysted juvenile flukes respectively. Each was used singly or in combination to vaccinate rats that were subsequently challenged with Fasciola hepatica metercercariae. Each protein induced an immune response, and all groups vaccinated with recombinant protein yielded significantly fewer and smaller flukes than the control group. Maximal protection of 83% was seen in the group vaccinated with cathepsin B and cathepsin L5 in combination.

Chemical attenuation of Plasmodium berghei sporozoites induces sterile immunity in mice.

Radiation and genetic attenuation of Plasmodium sporozoites are two approaches for whole-organism vaccines that protect against malaria. We evaluated chemical attenuation of sporozoites as an alternative vaccine strategy. Sporozoites were treated with the DNA sequence-specific alkylating agent centanamycin, a compound that significantly affects blood stage parasitemia and transmission of murine malaria and also inhibits Plasmodium falciparum growth in vitro. Here we show that treatment of Plasmodium berghei sporozoites with centanamycin impaired parasite function both in vitro and in vivo. The infection of hepatocytes by sporozoites in vitro was significantly reduced, and treated parasites showed arrested liver stage development. Inoculation of mice with sporozoites that were treated in vitro with centanamycin failed to produce blood stage infections. Furthermore, BALB/c and C57BL/6 mice vaccinated with treated sporozoites were protected against subsequent challenge with wild-type sporozoites. Our findings demonstrate that chemically attenuated sporozoites could be a viable alternative for the production of an effective liver stage vaccine for malaria.

The silencing of cysteine proteases in Fasciola hepatica newly excysted juveniles using RNA interference reduces gut penetration.

Probing protein function in parasitic flatworms is hampered by the difficulties associated with the development of transgenic approaches. Although RNA interference (RNAi) in schistosomes shows much promise, it has not been reported in other trematodes. Here, we show the successful silencing of the cysteine proteases cathepsin B and L in the infective stage of Fasciola hepatica newly excysted juveniles (NEJs). Silencing resulted in marked reductions in target transcript levels and significant diminution in the encoded proteins in the gut. RNAi of either enzyme in NEJs induced transient, abnormal locomotory phenotypes, and significantly reduced penetration of the rat intestinal wall.

Development of a multiplex quantitative PCR assay for detection and quantification of DNA from Fasciola hepatica and the intermediate snail host, Austropeplea tomentosa, in water samples.

Liver fluke (Fasciola hepatica) infection is an increasing threat to livestock production resulting in serious economic losses to the beef, dairy and sheep industries in Australia and globally. Triclabendazole (TCBZ) is the main drug used to control liver fluke infections in Australia and the widespread emergence of TCBZ resistance in cattle and sheep threatens liver fluke control. Alternative control measures to lower exposure of livestock to fluke infection would be useful to help preserve the usefulness of current chemical flukicides. Environmental DNA (eDNA) sampling methodology and associated molecular techniques are suited to rapidly assess the presence of pathogens on farms. In the present study, we developed a water sampling method in combination with a multiplex quantitative PCR assay to detect and quantify DNA of F. hepatica and Austropeplea tomentosa (A. tomentosa), a crucial intermediate snail host for liver fluke transmission in South-east Australia. The multiplex qPCR assay allows for the independent detection of F. hepatica and A. tomentosa DNA using specific primers and a probe targeting the ITS-2 region of the liver fluke or snail. The method allows the highly specific and sensitive (minimal DNA detection levels to 14-50 fg) detection of F. hepatica or A. tomentosa. The method allows the detection of both liver fluke and snail eDNA in water samples. The effective quantification of liver fluke and snail eDNA in water samples using this assay could potentially allow researchers to both identify and monitor F. hepatica transmission zones on farming properties in South-east Australia which will better inform control strategies, with potential application of the assay worldwide.

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.

The A/T-specific DNA alkylating agent adozelesin inhibits Plasmodium falciparum growth in vitro and protects mice against Plasmodium chabaudi adami infection.

There is an urgent need for new anti-malarial drugs to combat the resurgence of resistance to current therapies. To exploit the A/T richness of malaria DNA as a potential target for anti-malarial drugs we tested an A/T-specific DNA synthesis inhibitor, adozelesin, for activity against Plasmodium falciparum in vitro and Plasmodium chabaudi adami in mice. Adozelesin is a DNA alkylating agent that exhibits specificity for the motif A/T, A/T and A. In P. falciparum 3D7 cultures, adozelesin acts as a powerful inhibitor of parasite growth (IC(50) of 70 pM) and is equally potent at killing the drug-resistant strains FCR3 and 7G8. Using a real-time PCR assay, we show that treatment with adozelesin in vitro results in damage of P. falciparum genomic DNA. In synchronized cultures, adozelesin exhibits a concentration-dependent effect on parasitemia and on the development of parasites through the asexual cycle. In asynchronous cultures, parasites arrest at all stages of the asexual cycle suggesting that adozelesin exerts other anti-parasitic effects in addition to inhibiting DNA replication. These anti-parasite effects are irreversible since cultures exposed to adozelesin for more than 6h fail to recover upon removal of the drug. Furthermore, adozelesin is very effective at suppressing malaria infection in vivo; growth of P. c. adami DK in mice was highly impaired by a single injection of adozelesin (25 microg/kg) at 4 days post-infection. These results demonstrate that adozelesin irreversibly blocks parasite growth in vitro and suppresses parasite infection in vivo, suggesting that A/T-specific DNA damaging agents represent a new class of compounds with potential as anti-malarials.

Current Threat of Triclabendazole Resistance in Fasciola hepatica.

Triclabendazole (TCBZ) is the only chemical that kills early immature and adult Fasciola hepatica (liver fluke) but widespread resistance to the drug greatly compromises fluke control in livestock and humans. The mode of action of TCBZ and mechanism(s) underlying parasite resistance to the drug are not known. Due to the high prevalence of TCBZ resistance (TCBZ-R), effective management of drug resistance is now critical for sustainable livestock production. Here, we discuss the current status of TCBZ-R in F. hepatica, the global distribution of resistance observed in livestock, the possible mechanism(s) of drug action, the proposed mechanisms and genetic basis of resistance, and the prospects for future control of liver fluke infections using an integrated parasite management (IPM) approach.