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Final Report Summary - FLUKVAC (Establishing new concepts and approaches for future vaccine development against parasitic pathogens of cattle)

Fasciolosis is a disease of ruminants caused by the liver fluke Fasciola hepatica, and results in worldwide economic losses of greater than US $3 billion per annum. It is also recognised by the World Health Organisation (WHO) as an important zoonosis; global infections are estimated between 2 and 17 million and 180 million people are at risk of infection. The economic losses in ruminants are associated with the liver damage caused by parasites migrating through the definitive hosts resulting in poor food conversion, impaired fertility and reduced wool and milk production. Parasite populations resistant to the frontline anthelminthic used to treat acute fasciolosis, triclabendazole, are becoming more prevalent leaving farmers with no means of controlling acute infection.
Parasite migration begins following the ingestion of the infective encysted stage, the metacercariae, that excyst within the intestine as newly excysted juveniles (NEJs). The NEJs traverse the intestinal wall into the peritoneal cavity, where they continue to migrate through the liver capsule into the liver parenchyma. Those parasites that reach the liver, normally between 4 and 6 days post-infection, cause extensive tissue damage and haemorrhaging in the liver parenchyma resulting in the hepatic pathogenesis associated with acute fasciolosis. F. hepatica secretes molecules that modulate the host immune response and induce the development of a Th2 response and concomitant inhibition of protective pro-inflammatory responses as the disease progresses to chronicity. This polarisation of the immune responses is sufficiently potent to influence the host’s susceptibility to co-infections with bacterial pathogens.
The development of effective new vaccines against early fasciolosis requires an understanding of how the parasite infects the ruminant host, induces tissue damage and modulates the host immune response particularly in the first days of infection. Studies have shown that as the parasite migrates through the intestinal epithelium clinical signs are not evident, although an immunological response is induced, as illustrated by the large number of immune cells infiltrating into the peritoneal cavity. Since the parasite migrates from the intestine to the liver via the peritoneum we considered that investigation of the peritoneal fluid of infected animals may provide new information of the early immune response that can be exploited for vaccine development. At the same time, the data could also identify host-specific proteins that may act as biomarkers of infection.
Proteomic analysis of the host response to F. hepatica has been carried out on host bile and serum, with the analysis of bile representing the chronic stages of infection when the adult parasites have migrated through the liver to the bile ducts and the serum representing the systemic response. However, to date, the use of proteomics tools for the analysis of peritoneal fluid has only been reported in patients with uremia, endometriosis, ovarian cancer and following cases of peritoneal dialysis, which has facilitated the development of biomarkers for these respective diseases/pathologies. Thus, this study contains the first differential proteomic analysis of peritoneal fluid comparing uninfected and F. hepatica infected sheep.
We investigated the peritoneal fluid of infected sheep during the first 18 days of infection with F. hepatica as a means of monitoring liver damage using a combination of immunological and proteomic analyses. At 18 days post-infection (dpi), histopathology of sheep liver showed white necrotic foci/tracts indicative of F. hepatica migration. Within the peritoneal fluid of infected animals, specific F. hepatica antigen FhCL1 antibodies coincided with an intense innate and adaptive cellular immune response, with increasing numbers of total leukocytes and a marked eosinophilia (49%). Cytokine qPCR analysis revealed IL-10, IL-12, IL-13, IL-23 and TGFβ were elevated but not statistically significant at 18 dpi compared to uninfected sheep indicating that whilst the immune response is developing it has not polarised to the Th2 type associated with acute and chronic fasciolosis. Proteomic analysis of the peritoneal fluid from infected and non-infected sheep identified 324 proteins, with 31 proteins uniquely observed in the infected animals. Components of the liver extracellular matrix, including collagen, periostin and the vascular adhesion protein, VCAM-1, exhibited increased expression associated with early fasciolosis, which may be important in signalling host immune responses to tissue damage.
In conclusion, in early F. hepatica infection cellular infiltration with marked eosinophilia, adaptive immune responses and products of liver pathology are evident in the peritoneum. Although cytokine responses are developing they are mixed and not yet polarised to a Th2-type. We have characterised major biomarkers of parasite-induced liver damage such as periostin and VCAM-1 which could be exploited for diagnosis, and vaccine development aimed at reducing/preventing liver pathology.

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