Grace Mulcahy

Fasciola hepatica cathepsin L-like proteases: biology, function and potential in the development of first generation liver fluke vaccines.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.

Thioredoxin Peroxidase Secreted by Fasciola hepatica Induces the Alternative Activation of Macrophages

ABSTRACT Alternatively activated macrophages (AAMφ) are primarily associated with the chronic stages of parasitic infections and the development of a polarized Th2 response. We have shown that Fasciola hepatica infection of BALB/c mice induces a polarized Th2 response during both the latent and chronic stage of disease. The activation status of macrophages was analyzed in this model of helminth infection by evaluating the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arg1. AAMφ were recruited to the peritoneum of mice within 24 h of F. hepatica infection and after intraperitoneal injection of parasite excretory-secretory (ES) products. Administration of a recombinant antioxidant thioredoxin peroxidase (TPx), which is contained within the ES products, also induced the recruitment of AAMφ to the peritoneum. In vitro studies showed that this recombinant TPx directly converts RAW 264.7 macrophages to an alternatively activated phenotype characterized by the production of high levels of interleukin-10 (IL-10), prostaglandin E2, corresponding with low levels of IL-12. Our data suggest that the Th2 responses induced by the helminth F. hepatica are mediated through the secretion of molecules, one of which is TPx, that induce the recruitment and alternative activation of macrophages.

Bioinformatic discovery and initial characterisation of nine novel antimicrobial peptide genes in the chicken

AbstractAntimicrobial peptides (AMPs) are essential components of innate immunity in a range of species from Drosophila to humans and are generally thought to act by disrupting the membrane integrity of microbes. In order to discover novel AMPs in the chicken, we have implemented a bioinformatic approach that involves the clustering of more than 420,000 chicken expressed sequence tags (ESTs). Similarity searching of proteins—predicted to be encoded by these EST clusters—for homology to known AMPs has resulted in the in silico identification of full-length sequences for seven novel gallinacins (Gal-4 to Gal-10), a novel cathelicidin and a novel liver-expressed antimicrobial peptide 2 (LEAP-2) in the chicken. Differential gene expression of these novel genes has been demonstrated across a panel of chicken tissues. An evolutionary analysis of the gallinacin family has detected sites—primarily in the mature AMP—that are under positive selection in these molecules. The functional implications of these results are discussed.

Fasciola hepatica infection downregulates Th1 responses in mice

Immune responses induced with helminth parasites have been extensively studied, but there is limited information on those to Fasciola hepatica, especially on the subtype of T cell induced with this parasite. We investigated the local and systemic T cell responses of different strains of mice following oral infection with doses of metacercariae from F. hepatica. Spleen cells from BALB/c and 129Sv/Ev mice given a low‐dose (5 metacercariae) infection exhibited a Th2 response, producing high levels of the cytokines IL‐4 and IL‐5, and low levels of IFN‐γ and IL‐2. In contrast, C57BL/6 mice showed a mixed Th1/Th2 response. A more marked polarization to a Th2 response was observed in BALB/c, 129Sv/Ev exposed to a high‐dose (15 metacercariae) infection and the C57BL/6 mice also exhibited a clear Th2 response. IL‐4 defective (IL‐4−/−) C57BL/6 mice infected with 5 metacercariae produced less IFN‐γ and more IL‐5 compared to their wild‐type C57BL/6 counterparts, suggesting that IL‐4 is important in establishing the Th2 type response in murine fasciolosis. However, the secretion of IFN‐γ and IL‐2 was completely suppressed in the high‐dose infection and this was also observed in IL‐4−/− mice. Thus, liver flukes may secrete molecules that downregulate Th1 responses. T cell responses in the mesenteric (MLN) and hepatic lymph nodes (HLN) were also examined since newly excysted juveniles infect through the intestinal wall of their host before migrating to the hepatic tissue. Cells from both MLN and HLN secreted higher levels of IL‐4 and IL‐5 compared to spleen cells. We also observed a difference in cytokine profiles secreted by the MLN and HLN, which may reflect responses to antigens liberated by newly excysted juveniles and hepatic stage parasites, respectively.

Immune responses of chronically infected adult cattle to Fasciola hepatica

Eight adult cows, with an existing chronic Fasciola hepatica infection, were experimentally infected with 1300 metacercariae of F. hepatica, given as trickle infections, over two separate 10-day periods. Two fluke-naive heifers were similarly treated. Analysis of parasite-specific immunoglobulin isotypes IgM, IgG1, IgG2 and IgA showed IgG1 to be the dominant isotype in both chronically infected and previously naive animals. Lymphocyte proliferation assays demonstrated (a) an association between lymphocyte response and mature fluke burden in the chronically infected cattle and (b) no association between lymphocyte response and mature or immature fluke burden in naive heifers. There was no production of gamma-interferon (IFN gamma) by lymphocytes responding to adult fluke antigen. At post-mortem examination the burden of immature flukes in chronically infected and previously naive heifers was similar. This suggests that chronically infected animals may be tolerant to a moderate superinfection and that the prevailing immune mechanism in operation may be a non-protective response generated by the Th2 lymphocyte subset.

Correlation of specific antibody titre and avidity with protection in cattle immunized against Fasciola hepatica.

Cattle produce specific serum antibody mainly of the IgG1 isotype in response to infection with the liver fluke, Fasciola hepatica. In these animals a positive correlation between fluke-specific serum IgG1 levels and fluke-burden in non-immunized infected animals was observed. In contrast, immunization of cattle with a combination of the fluke-derived antigens cathepsin L2 (CL2) and fluke haemoglobin (FHb) in Freunds complete/incomplete adjuvant (FCA/FLA) induced a specific antibody response involving IgG2, as well as IgG1. These immunized animals also exhibited very high (72%) levels of protection against a subsequent challenge infection. When the vaccine was administered in FIA alone the specific antibody response, while still involving IgG1 and IgG2, was of lower magnitude (10-fold and 100-fold, respectively) and no significant reduction in fluke burden was observed following challenge. Nevertheless, in these animals, a strong IgG2 response was associated with low fluke burdens. These results provide further evidence of the non-protective nature of specific immune responses in cattle following F. hepatica infection, and demonstrate that vaccination can induce a qualitatively different, and protective, response.

Parasite vaccines — a reality?

Over the last decade, the anti-parasitics market has been the fastest growing sector of the overall

Experimental Fasciola hepatica Infection Alters Responses to Tests Used for Diagnosis of Bovine Tuberculosis

18 billion animal health market. While drugs for the treatment of parasites of livestock still dominate this sector and will continue to be developed or re-formulated, because of consumer demands for chemical-free food and of concerns regarding the environment and animal welfare there is a growing interest in the development of safe and effective vaccines. There is also a call for vaccines in the lucrative

Helminth vaccines: from mining genomic information for vaccine targets to systems used for protein expression

3 billion-plus companion animal market. These demands for vaccines will add a greater impetus to an area that has seen tremendous success in the last 15 years. A number of anti-parasite vaccines have been developed, e.g. the recombinant 45w and EG95 oncosphere proteins against Taenia ovis and Echinococcus granulosis, respectively, and the Bm86 vaccine against Boophilus microplus. In addition, the cathepsin L vaccines against the liver fluke, Fasciola hepatica, and the H11 vaccine against Haemonchus contortus are progressing well. There are also many additional vaccine candidates for H. contortus and for other nematodes such as Ostertagia and Trichostrongylus spp. that may ultimately lead to broad-spectrum gastrointestinal worm vaccines. Live or attenuated-live vaccines are available for the control of avian coccidiosis, toxplasmosis in sheep and anaplasmosis in cattle, although molecular vaccines against protozoans are still proving elusive. The wealth of information in genomics, proteomics and immunology that has been forthcoming together will new methods of vaccine production and delivery should see many new vaccines reach the marketplace in the near future.

Fasciola hepatica is associated with the failure to detect bovine tuberculosis in dairy cattle

ABSTRACT Fasciola hepatica is a prevalent helminth parasite of livestock. Infection results in polarization of the hosts immune response and generation of type 2 helper (Th2) immune responses, which are known to be inhibitory to Th1 responses. Bovine tuberculosis (BTB) is a bacterial disease of economic and zoonotic importance. Control polices for this disease rely on extensive annual testing and a test-and-slaughter policy. The correct diagnosis of BTB relies on cell-mediated immune responses. We established a model of coinfection of F. hepatica and Mycobacterium bovis BCG to examine the impact of helminth infection on correct diagnosis. We found the predictive capacity of tests to be compromised in coinfected animals and that F. hepatica infection altered macrophage function. Interleukin-4 and gamma interferon expression in whole-blood lymphocytes restimulated in vitro with M. bovis antigen was also altered in coinfected animals. These results raise the question of whether F. hepatica infection can affect the predictive capacity of tests for the diagnosis of BTB and possibly also influence susceptibility to BTB and other bacterial diseases. Further studies on the interplay between helminth infection and BTB are warranted.