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 of Loop-Mediated Isothermal Amplification Technologies for the Detection of Honey Bee Pathogens.

Approximately one-third of the typical human Western diet depends upon pollination for production, and honey bees (Apis mellifera) are the primary pollinators of numerous food crops, including fruits, nuts, vegetables, and oilseeds. Regional large scale losses of managed honey bee populations have increased significantly during the last decade. In particular, asymptomatic infection of honey bees with viruses and bacterial pathogens are quite common, and co-pathogenic interaction with other pathogens have led to more severe and frequent colony losses. Other multiple environmental stress factors, including agrochemical exposure, lack of quality forage, and reduced habitat, have all contributed to the considerable negative impact upon bee health. The ability to accurately diagnose diseases early could likely lead to better management and treatment strategies. While many molecular diagnostic tests such as real-time PCR and MALDI-TOF mass spectrometry have been developed to detect honey bee pathogens, they are not field-deployable and thus cannot support local apiary husbandry decision-making for disease control. Here we review the field-deployable technology termed loop-mediated isothermal amplification (LAMP) and its application to diagnose honey bee infections.

Characterization of a profilin-like protein from Fasciola hepatica.

Fasciola hepatica is the causative agent of fasciolosis, an important disease of humans and livestock around the world. There is an urgent requirement for novel treatments for F. hepatica due to increasing reports of drug resistance appearing around the world. The outer body covering of F. hepatica is referred to as the tegument membrane which is of crucial importance for the modulation of the host response and parasite survival; therefore, tegument proteins may represent novel drug or vaccine targets. Previous studies have identified a profilin-like protein in the tegument of F. hepatica. Profilin is a regulatory component of the actin cytoskeleton in all eukaryotic cells, and in some protozoan parasites, profilin has been shown to drive a potent IL-12 response. This study characterized the identified profilin form F. hepatica (termed FhProfilin) for the first time. Recombinant expression of FhProfilin resulted in a protein approximately 14 kDa in size which was determined to be dimeric like other profilins isolated from a range of eukaryotic organisms. FhProfilin was shown to bind poly-L-proline (pLp) and sequester actin monomers which is characteristic of the profilin family; however, there was no binding of FhProfilin to phosphatidylinositol lipids. Despite FhProfilin being a component of the tegument, it was shown not to generate an immune response in experimentally infected sheep or cattle.

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.

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.