The Immune Response to Nematode Infection.

Nematode infection is a major threat to the health of humans, domestic animals and wildlife. Nematodes vary in their effect on the host and in the mechanisms underlying immunity but the general features are becoming clear. There is considerable variation among individuals in resistance to infection and much of this variation is due to genetic variation in the immune response. The major histocompatibility complex has a strong influence on resistance to infection but other genes are collectively more important. Resistant individuals produce more IgA, eosinophils, IgE and mast cells than susceptible individuals and this is a consequence of stronger type 2 (Th2) immune responses. A variety of factors promote Th2 responses including genetic background, diet, molecules produced by the parasite and the location of the infection. A variety of cells and molecules including proteins, glycolipids and RNA act in concert to promote responses and to regulate the response. Nematodes themselves also modulate the host response and over 20 parasite-derived immunomodulatory molecules have been identified. Different species of nematodes modulate the immune response in different ways and probably use multiple molecules. The reasons for this are unclear and the interactions among immunomodulators have still to be investigated.

The heritability of Nematodirus battus fecal egg counts.

Although Nematodirus battus is a serious threat to the health and survival of young lambs, there are few options to control this parasite. Bayesian Monte Carlo Markov Chain modelling with a zero-inflated Poisson distribution was used to estimate the heritability of egg counts in both June and July for each of five consecutive cohorts of 200 Scottish Blackface lambs. In one of the 10 analyses, the results failed the diagnostic tests. In seven of the analyses, there was no convincing evidence that the variation in egg counts was heritable. In the 2 years of high infection, the heritability was approximately 0.4 in June but the estimates lacked precision and the 95% highest posterior density credible intervals ranged from just above zero to 0.7. Selective breeding for resistance to N. battus will be difficult because genetically resistant or susceptible lambs cannot be consistently identified by phenotypic markers.

Increased susceptibility to Haemonchus contortus infection by interleukin-5 modulation of eosinophil responses in sheep.

Eosinophils are prominent effector cells in immune responses against gastrointestinal nematode infections in ruminants, but their in vivo role has been hard to establish in large animals. Interleukin-5 is a key cytokine in the induction and stimulation of anti-parasitic eosinophil responses. This study attempted to modulate the eosinophil response in sheep through vaccination with recombinant interleukin-5 (rIL-5) and determine the effect on subsequent Haemonchus contortus infection. Nematode-resistant Canaria Hair Breed (CHB) sheep vaccinated with rIL-5 in Quil-A adjuvant, had lower blood eosinophil counts and higher mean worm burdens than control sheep vaccinated with Quil-A adjuvant alone. In addition, adult worms in IL-5-vaccinated sheep were significantly longer with higher eggs in utero in female worms, supporting an active role of eosinophils against adult parasites in CHB sheep. These results confirm that eosinophils can play a direct role in effective control of H contortus infection in sheep and offer a new approach to study immune responses in ruminants.

Specific humoral response of hosts with variable schistosomiasis susceptibility.

The schistosome blood flukes are some of the largest global causes of parasitic morbidity. Further study of the specific antibody response during schistosomiasis may yield the vaccines and diagnostics needed to combat this disease. Therefore, for the purposes of antigen discovery, sera and antibody-secreting cell (ASC) probes from semi-permissive rats and sera from susceptible mice were used to screen a schistosome protein microarray. Following Schistosoma japonicum infection, rats had reduced pathology, increased antibody responses and broader antigen recognition profiles compared with mice. With successive infections, rat global serological reactivity and the number of recognized antigens increased. The local antibody response in rat skin and lung, measured with ASC probes, increased after parasite migration and contributed antigen-specific antibodies to the multivalent serological response. In addition, the temporal variation of anti-parasite serum antibodies after infection and reinfection followed patterns that appear related to the antigen driving the response. Among the 29 antigens differentially recognized by the infected hosts were numerous known vaccine candidates, drug targets and several S. japonicum homologs of human schistosomiasis resistance markers-the tegument allergen-like proteins. From this set, we prioritized eight proteins that may prove to be novel schistosome vaccine and diagnostic antigens.

Dynamics of IL-4 and IL-13 expression in the airways of sheep following allergen challenge.

BACKGROUND: IL-4 and IL-13 play a critical yet poorly understood role in orchestrating the recruitment and activation of effector cells of the asthmatic response and driving the pathophysiology of allergic asthma. The house dust mite (HDM) sheep asthma model displays many features of the human condition and is an ideal model to further elucidate the involvement of these critical Th2 cytokines. We hypothesized that airway exposure to HDM allergen would induce or elevate the expression profile of IL-4 and IL-13 during the allergic airway response in this large animal model of asthma. METHODS: Bronchoalveolar lavage (BAL) samples were collected from saline- and house dust mite (HDM)- challenged lung lobes of sensitized sheep from 0 to 48 h post-challenge. BAL cytokines (IL-4, IL-13, IL-6, IL-10, TNF-α) were each measured by ELISA. IL-4 and IL-13 expression was assessed in BAL leukocytes by flow cytometry and in airway tissue sections by immunohistology. RESULTS: IL-4 and IL-13 were increased in BAL samples following airway allergen challenge. HDM challenge resulted in a significant increase in BAL IL-4 levels at 4 h compared to saline-challenged airways, while BAL IL-13 levels were elevated at all time-points after allergen challenge. IL-6 levels were maintained following HDM challenge but declined after saline challenge, while HDM administration resulted in an acute elevation in IL-10 at 4 h but no change in TNF-α levels over time. Lymphocytes were the main early source of IL-4, with IL-4 release by alveolar macrophages (AMs) prominent from 24 h post-allergen challenge. IL-13 producing AMs were increased at 4 and 24 h following HDM compared to saline challenge, and tissue staining provided evidence of IL-13 expression in airway epithelium as well as immune cells in airway tissue. CONCLUSION: In a sheep model of allergic asthma, airway inflammation is accompanied by the temporal release of key cytokines following allergen exposure that primarily reflects the Th2-driven nature of the immune response in asthma. The present study demonstrates for the first time the involvement of IL-4 and IL-13 in a relevant large animal model of allergic airways disease.

Effective pulmonary delivery of an aerosolized plasmid DNA vaccine via surface acoustic wave nebulization.

BACKGROUND: Pulmonary-delivered gene therapy promises to mitigate vaccine safety issues and reduce the need for needles and skilled personnel to use them. While plasmid DNA (pDNA) offers a rapid route to vaccine production without side effects or reliance on cold chain storage, its delivery to the lung has proved challenging. Conventional methods, including jet and ultrasonic nebulizers, fail to deliver large biomolecules like pDNA intact due to the shear and cavitational stresses present during nebulization. METHODS: In vitro structural analysis followed by in vivo protein expression studies served in assessing the integrity of the pDNA subjected to surface acoustic wave (SAW) nebulisation. In vivo immunization trials were then carried out in rats using SAW nebulized pDNA (influenza A, human hemagglutinin H1N1) condensate delivered via intratracheal instillation. Finally, in vivo pulmonary vaccinations using pDNA for influenza was nebulized and delivered via a respirator to sheep. RESULTS: The SAW nebulizer was effective at generating pDNA aerosols with sizes optimal for deep lung delivery. Successful gene expression was observed in mouse lung epithelial cells, when SAW-nebulized pDNA was delivered to male Swiss mice via intratracheal instillation. Effective systemic and mucosal antibody responses were found in rats via post-nebulized, condensed fluid instillation. Significantly, we demonstrated the suitability of the SAW nebulizer to administer unprotected pDNA encoding an influenza A virus surface glycoprotein to respirated sheep via aerosolized inhalation. CONCLUSION: Given the difficulty of inducing functional antibody responses for DNA vaccination in large animals, we report here the first instance of successful aerosolized inhalation delivery of a pDNA vaccine in a large animal model relevant to human lung development, structure, physiology, and disease, using a novel, low-power (<1 W) surface acoustic wave (SAW) hand-held nebulizer to produce droplets of pDNA with a size range suitable for delivery to the lower respiratory airways.

The in vitro anthelmintic effects of plumbagin on newly excysted and 4-weeks-old juvenile parasites of Fasciola gigantica.

The effect of plumbagin (PB, 5-hydroxy-2-methyl-1,4-naphthoquinone) against newly excysted juveniles (NEJs) and 4-weeks-old immature parasites of Fasciola gigantica were compared with triclabendazole (TCZ). The anthelmintic efficacy of 1, 10 and 100µg/ml of PB or TCZ following incubation in vitro for 1-24h was compared using a combination of relative motility (RM), survival index (SI) and larval migration inhibition (LMI) assays for parasite viability. The RM and SI values of the PB-treated group decreased at a more rapid rate than the TCZ-treated group. For NEJs, the decreased RM values were first observed at 1h incubation with 1µg/ml PB, and 90% of flukes were killed at 24h. In contrast, in TCZ-treated groups a 10-fold higher concentration of TCZ (10µg/ml) resulted in only 9% dead parasites after 24h incubation. In 4-weeks-old juvenile parasites, PB reduced the RM value at 10µg/ml with 100% of flukes dead after 3h, while TCZ decreased RM values at the concentration of 100µg/ml but with only 5% of flukes killed at 24h. NEJs treated with PB exhibited 88%, 99% and 100% of LMIs at the concentrations of 1, 10 and 100µg/ml, respectively. NEJs incubated with TCZ have an LMI of only 32% at the highest concentration of 100µg/ml. Similarly PB had a significantly greater killing of immature 4weeks juvenile stages than TCZ at all concentrations; however, 4-weeks-old juvenile parasites were more resistant to killing by PB or TCZ at all concentrations when compared to NEJs. Further studies were carried out to investigate the alterations of the parasite tegument by scanning electron microscope (SEM). PB caused similar tegumental alterations in 4-weeks-old juveniles as those observed in TCZ treatment but with greater damage at comparative time points, comprising of swelling, blebbing and rupture of the tegument, loss of spines, and eventual erosion, lesion and desquamation of the total tegument. These data indicate that PB had a greater fasciolicidal effect against immature stages of F. gigantica parasites than TCZ and warrant further studies for use as a potential new anthelmintic against Fasciola infections.

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.

Strongyle egg shedding and egg reappearance periods in horses with pituitary pars intermedia dysfunction.

Pituitary pars intermedia dysfunction (PPID) is the most common endocrine disorder of older horses. Immune dysfunction in horses with PPID could increase susceptibility to infectious diseases, including strongyle infections; however, few data are available. The aim of this study was to determine if horses with PPID had increased strongyle faecal egg counts (FEC) compared with control horses, over a fourteen-week period in Victoria, Australia. Clinical signs and plasma adrenocorticotropic hormone (ACTH) concentrations were used to categorise horses into PPID (n=14) or control (n=31) groups. Faecal samples were collected for FEC determination prior to anthelmintic treatment, and fortnightly post-treatment for each horse. Generalised linear mixed modelling, using a gamma distribution, was used to compare differences between groups in the repeated measures study. The confounding variable of age was controlled for as a fixed effect. Following anthelmintic treatment, mean FEC was greater for the PPID group compared to the control group on day 56 (405 ± 756 eggs per gram [EPG] vs 40 ± 85 EPG, p=0.05) and day 70 (753 ±1598 EPG vs 82 ±141 EPG, p=0.04). There were no differences in mean FEC between groups on days 84 and 98. Cumulative FEC (day 14 to day 98) was significantly greater for the PPID horses than control horses (2118 ± 4016 EPG vs 798 ± 768 EPG, p<0.0001). Group egg reappearance period was shorter for PPID horses (day 56 post-anthelmintic treatment) compared to control horses (day 70) and 30% of the PPID horses reached a FEC threshold of >200 EPG on day 42, compared to 0% of control horses (p=0.02). These results suggest that the rate of a re-established patent infection between groups could be different due to a comprised immune response in PPID horses or differences in the host-parasite relationship regarding encysted stage larvae. However, despite differences between groups, some horses with PPID consistently had no detectable or low FEC (<200 EPG) during the study period. These findings highlight the importance of individual FEC monitoring to determine if anthelmintic treatment is required, in line with sustainable parasite management practices.

The developing schistosome worms elicit distinct immune responses in different tissue regions.

Schistosome parasites follow a complex migration path through various tissues, changing their antigenic profile as they develop. A thorough understanding of the antibody response in each tissue region could help unravel the complex immunology of these developing parasites and aid vaccine design. Here we used a novel strategy for analysing the local antibody responses induced by Schistosoma japonicum infection at each site of infection. Cells from rat lymph nodes draining the sites of larval migration (the skin and lungs), the liver-lymph nodes where adults reside and the spleens were cultured to allow the in vivo-induced antibody-secreting cells to release antibody into the media. The amount and isotype of antibodies secreted in the supernatants differed significantly in the different lymph nodes and spleen, corresponding with the migration path of the schistosome worms. In addition, there were significant differences in binding specificity, as determined by surface labelling, western blots and by screening a glycan array. Through capturing the local antibody response, this study has revealed dramatic differences in the quality and specificity of the immune response at different tissue sites, and highlighted the existence of stage-specific protein and carbohydrate antigens. This will provide a valuable tool for the isolation of novel vaccine targets against the larval stages of schistosomes.

Evaluation of the immune responses induced by four targeted DNA vaccines encoding the juvenile liver fluke antigen, cathepsin B in a mouse model.

BACKGROUND: Liver fluke can infect cattle and sheep, and is also emerging as a human pathogen in developing countries. Cathepsin B (Cat B2) is a major cysteine protease secreted by the juvenile flukes. To enhance the immune responses of Cat B2, the cDNA sequence was fused with four different DNA vaccine vectors. The induced cellular and antibody responses were compared in vaccinated mice. METHODS: The following recombinant DNA vaccine constructs were constructed: empty vector VR1012 as negative control, cytoplasmic construct pVR1012 Cat B2, secretory construct pVR1020 Cat B2, chemokine-fused construct pMCP3 Cat B2 and lymph node targeting construct pCTLA-4 Cat B2. Plasmids were constructed using standard procedures, and positive constructs screened and selected using restriction digestion analysis followed by sequence analysis. The constructs were then tested in Cos-7 cells for in vitro expression, which was analysed using immunoblotting. Subsequently, female BALB/c mice were immunised with DNA constructs as vaccines. Elicited antibody responses were measured using ELISA. The ratio between IgG1 and IgG2a antibody responses was estimated among different vaccine groups. IgG antibody avidity assay was performed and the relative avidity index was calculated. The induced cytokine production from splenocytes of vaccinated animals was estimated using ELISPOT. RESULTS: DNA vaccine constructs carrying Cat B2 were expressed in Cos-7 cell lines and encoded protein was recognised using western blotting using rat anti- cathepsin B antibody. DNA vaccines elicited high Cat B2- specific IgG, IgG1, IgE and also modest IgG2a antibody responses. Cat B2 specific IL-4 T cell responses were also observed in Cat B2 vaccinated mice. The comparison of immunogenic potential in each of these constructs was demonstrated as enhanced antibody responses on the lymph-node targeting vector pCTLA-4 Cat B2, the high antibody avidity of chemo-attractant pMCP3 Cat B2 and stronger T cellular responses of non-secretory DNA vaccine pVR1012 Cat B2 in vaccinated animals. CONCLUSION: This study showed that the targeting DNA vaccine strategies enhanced specific immune responses to juvenile fluke Cat B2. The results of our current study have demonstrated that a gene-based vaccine as an immunotherapeutic approach to combat Fasciola infection may be feasible.

Evaluation of the Role of Galectins in Parasite Immunity.

Galectin-11 (LGALS-11) and galectin-14 (LGALS-14) are ruminant specific galectins, first reported in sheep. Although their roles in parasite immunity are still being elucidated, it appears that they influence protection against parasites. In gastrointestinal infections with the nematode Haemonchus contortus, both galectin-11 and galectin-14 appear to be protective. However, in a chronic infection of liver fluke, Fasciola hepatica, these galectins may aid parasite survival. To unravel the structural, functional, and ligand profile of galectin-11 and galectin-14, recombinant production of these proteins is vital. Here we present the recombinant production of soluble galectin-11 and galectin-14 from domestic sheep for in vitro and structural biology studies. These methods include parasite cultivation and infection, galectin staining of host and parasite tissue, surface staining of parasites with recombinant galectins, pull-down assays to identify endogenous galectin binding proteins, and in vitro assays to monitor the effect of galectins on parasite development.

Quantifying the sources of variation in eosinophilia among Scottish blackface lambs with mixed, predominantly Teladorsagia circumcincta nematode infection.

Eosinophils play a key role in defence against gastrointestinal nematodes. There is considerable variation among animals in the intensity of eosinophilia following nematode infection. However, the statistical distribution of eosinophils among animals has still to be determined. A better description of the variation among animals could provide biological insight and determine the most appropriate way to analyse the effect of eosinophils. We estimated blood eosinophil numbers in a flock of Scottish Blackface sheep that were naturally exposed to mixed, predominantly Teladorsagia circumcincta infection. Three of the four eosinophil counts were better described by a gamma distribution than by a lognormal distribution. The scale and shape parameters of the gamma distribution varied over time. Eosinophil counts differed among animals kept on separate fields before weaning and between singletons and twins but were not significantly different between years and genders. Eosinophil counts also differed among offspring from different sires and dams. The parameters of the gamma distribution were used to enable a power analysis. Large numbers of animals were required to reliably detect even large differences between two groups. These results indicate that methods appropriate for gamma distributions, such as generalized linear mixed models, will provide more reliable inferences than traditional methods of analysis and experimental design.

Variation in gut bacterial composition is associated with Haemonchus contortus parasite infection of sheep.

BACKGROUND: One of the greatest impediments to global small ruminant production is infection with the gastrointestinal parasite, Haemonchus contortus. In recent years there has been considerable interest in the gut microbiota and its impact on health. Relatively little is known about interactions between the gut microbiota and gastrointestinal tract pathogens in sheep. Thus, this study was undertaken to investigate the link between the faecal microbiota of sheep, as a sample representing the gastrointestinal microbiota, and infection with H. contortus. RESULTS: Sheep (n = 28) were experimentally inoculated with 14,000 H. contortus infective larvae. Faecal samples were collected 4 weeks prior to and 4 weeks after infection. Microbial analyses were conducted using automated ribosomal intergenic spacer analysis (ARISA) and 16S rRNA gene sequencing. A comparison of pre-infection microbiota to post-infection microbiota was conducted. A high parasite burden associated with a relatively large change in community composition, including significant (p ≤ 0.001) differences in the relative abundances of Firmicutes and Bacteroidetes following infection. In comparison, low parasite burden associated with a smaller change in community composition, with the relative abundances of the most abundant phyla remaining stable. Interestingly, differences were observed in pre-infection faecal microbiota in sheep that went on to develop a high burden of H. contortus infection (n = 5) to sheep that developed a low burden of infection (n = 5). Differences observed at the community level and also at the taxa level, where significant (p ≤ 0.001) in relative abundance of Bacteroidetes (higher in high parasite burden sheep) and Firmicutes (lower in high parasite burden sheep). CONCLUSIONS: This study reveals associations between faecal microbiota and high or low H. contortus infection in sheep. Further investigation is warranted to investigate causality and the impact of microbiome manipulation.

The oligomeric assembly of galectin-11 is critical for anti-parasitic activity in sheep (Ovis aries).

Galectins are a family of glycan-binding molecules with a characteristic affinity for ß-D-glycosides that mediate a variety of important cellular functions, including immune and inflammatory responses. Galectin-11 (LGALS-11) has been recently identified as a mediator induced specifically in animals against gastrointestinal nematodes and can interfere with parasite growth and development. Here, we report that at least two natural genetic variants of LGALS-11 exist in sheep, and demonstrate fundamental differences in anti-parasitic activity, correlated with their ability to dimerise. This study improves our understanding of the role of galectins in the host immune and inflammatory responses against parasitic nematodes and provides a basis for genetic studies toward selective breeding of animals for resistance to parasites.

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.

Development and application of a fecal antigen diagnostic sandwich ELISA for estimating prevalence of Fasciola gigantica in cattle in central Java, Indonesia.

The purpose of this study was to compare the sensitivity and specificity of an ELISA test to detect Fasciola gigantica antigens (coproantigens) in bovine feces, with fecal egg counting and an ELISA for detecting anti-F. gigantica antibodies in serum. Monoclonal antibodies to cathepsin L were generated and used to capture this antigen in feces of infected cattle. Blood, feces, and livers were collected from 150 cattle at an abattoir in Jakarta, Indonesia, for anti-Fasciola antibodies, coproantigen detection, and F. gigantica egg and worm counts. Fluke recovery varied from 1 to 426 per host, with a mean of 32 flukes. The results showed that the sensitivity and specificity of coproantigen detecting ELISA (95 and 91%, respectively) was better than the anti-F. gigantica antibody ELISA (91 and 88%, respectively) and to fecal egg counting (87 and 100%, respectively). The coproantigen ELISA was able to detect 100% of the cattle with >15 flukes. A survey of 305 cattle in central Java over a 10-mo period validated this test in the field, demonstrating a high prevalence of fascioliasis and establishing the test as a useful diagnostic method to determine patent F. gigantica infections in cattle.

The current status of anthelmintic resistance in a temperate region of Australia; implications for small ruminant farm management.

Widespread anthelmintic resistance in small ruminants is a constraint on the profitability of the meat/wool industry. Limited published data is available on the prevalence and efficacy of anthelmintics, particularly in Australia where parasites affecting ruminant systems vary greatly between geographic regions. This paper reports on the anthelmintic resistance status in a temperate region of Victoria, Australia, a major sheep producing state largely affected by Trichostrongylus species and Teladorsagia circumcincta. The prevalence of anthelmintic resistance to any product was high (71%), with farms reporting varying levels of drug efficacies (21-100%). Resistance to older chemical groups (i.e. fenbendazole and levamisole) and single active macrocyclic lactone treatments was higher than newer chemical groups and combination treatments. This report provides clarity on anthelmintic resistance in the temperate region of Victoria and more importantly suggests that more comprehensive, regional specific anthelmintic resistance studies are required to understand the real level of chemical resistance threatening the effective control of worms.

Protective efficacy of liver fluke DNA vaccines: A systematic review and meta-analysis: Guiding novel vaccine development.

The immunogenicity and efficacy of Fasciola DNA vaccines have not yet been comprehensively summarised in the form of a systematic review and meta-analysis. Though multiple vaccine studies with respect to Fasciola vaccines exist, the variance in the experimental parameters has made comparison difficult. We conducted a bibliographic database search in Scopus, PubMed, Science Direct, Cochrane Library, EMBASE and Web of Science databases, limited to publications from 1998 to 2017. The key words: Liver fluke, Fasciola hepatica, Fasciola gigantica, DNA vaccination, and immunogenicity were used in combination to form search strings. A total of 4760 studies were identified after initial screening, of which 14 qualified for systematic review and 7 for meta-analysis. The mean Odds Ratio (OR) for all studies was 0.565 (95% confidence interval (CI) of 0.293 to 1.087), which means the percentage of protection in terms of decreased fluke burden in animals vaccinated with DNA vaccines was 43.5%. A moderate protective efficacy was observed for cysteine protease and phosphoglycerate kinase vaccine antigen candidates (pooled OR and 95% CI, [0.542; 0.179-1.721] and [0.616; 0.219-1.735], respectively). Vaccine effectiveness was observed in individual studies and cohorts; however, the overall pooled efficacy for all vaccine candidates was found to be non-significant. Despite multiple individual studies showing promising results for various DNA vaccine candidates against fascioliasis, the pooled studies showed the non-significant effect of the vaccine formulations against fluke burden, and displayed minimal protective efficacy against Fasciola infection. Though promising results are observed in isolated studies, further animal trials with standardised experimental parameters are required to develop new vaccine candidates effective against Fasciola.

Proteomic identification of galectin-11 and -14 ligands from Fasciola hepatica.

Fasciola hepatica is a globally distributed zoonotic trematode that causes fasciolosis in livestock, wildlife, ruminants and humans. Fasciolosis causes a significant economic impact on the agricultural sector and affects human health. Due to the increasing prevalence of triclabendazole resistance in F. hepatica, alternative treatment methods are required. Many protein antigens have been trialled as vaccine candidates with low success, however, the tegument of F. hepatica is highly glycosylated and the parasite-derived glycoconjugate molecules have been identified as an important mediator in host-parasite interactions and as prime targets for the host immune system. Galectin-11 (LGALS-11) and galectin-14 (LGALS-14) are two ruminant-specific glycan-binding proteins, showing upregulation in the bile duct of sheep infected with F. hepatica, which are believed to mediate host-parasite interaction and innate immunity against internal parasites. For the first known time, this study presents the ligand profile of whole worm and tegument extracts of F. hepatica that interacted with immobilised LGALS-11 and LGALS-14. LGALS-14 interacted with a total of 255 F. hepatica proteins. The protein which had the greatest interaction was identified as an uncharacterised protein which contained a C-type lectin domain. Many of the other proteins identified were previously trialled vaccine candidates including glutathione S-transferase, paramyosin, cathepsin L, cathepsin B, fatty acid binding protein and leucine aminopeptidase. In comparison to LGALS-14, LGALS-11 interacted with only 49 F. hepatica proteins and it appears to have a much smaller number of binding partners in F. hepatica. This is, to our knowledge, the first time host-specific lectins have been used for the enrichment of F. hepatica glycoproteins and this study has identified a number of glycoproteins that play critical roles in host-parasite interactions which have the potential to be novel vaccine candidates.