Fasciola hepatica NP-40 released protein extract (FhNPE) exhibits potent Th1 immunosuppressive properties in vitro and in vivo. However, the protein composition of this active fraction, responsible for Th1 immune modulatory activity, has yet to be resolved. Therefore, FhNPE, a Nonidet P-40 extract, was subjected to a proteomic analysis in order to identify individual protein components. This was performed using an in house F. hepatica EST database following 2D electrophoresis combined with de novo sequencing based mass spectrometry. The identified proteins, a mixture of excretory/secretory and membrane-associated proteins, are associated with stress response and chaperoning, energy metabolism and cytoskeletal components. The immune modulatory properties of these identified protein(s) are discussed and HSP70 from F. hepatica is highlighted as a potential host immune modulator for future study.

Background Fasciola hepatica, along with Fasciola gigantica, is the causative agent of fasciolosis, a foodborne zoonotic disease affecting grazing animals and humans worldwide. Pathology is directly related to the release of parasite proteins that facilitate establishment within the host. The dominant components of these excretory-secretory (ES) products are also the most promising vaccine candidates, the cathepsin L (Cat L) protease family. Methodology/Principal Findings The sub-proteome of Cat L proteases from adult F. hepatica ES products derived from in vitro culture and in vivo from ovine host bile were compared by 2-DE. The individual Cat L proteases were identified by tandem mass spectrometry with the support of an in-house translated liver fluke EST database. The study reveals plasticity within the CL1 clade of Cat L proteases; highlighted by the identification of a novel isoform and CL1 sub-clade, resulting in a new Cat L phylogenetic analysis including representatives from other adult Cat L phylogenetic clades. Additionally, for the first time, mass spectrometry was shown to be sufficiently sensitive to reveal single amino acid polymorphisms in a resolved 2-DE protein spot derived from pooled population samples. Conclusions/Significance We have investigated the sub-proteome at the population level of a vaccine target family using the Cat L proteases from F. hepatica as a case study. We have confirmed that F. hepatica exhibits more plasticity in the expression of the secreted CL1 clade of Cat L proteases at the protein level than previously realised. We recommend that superfamily based vaccine discovery programmes should screen parasite populations from different host populations and, if required, different host species via sub-proteomic assay in order to confirm the relative expression at the protein level prior to the vaccine development phase.

Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. We report the first post-genomic investigation of cellular proteins expressed by embryonic F. hepatica via two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Antioxidant proteins and protein chaperones are prominently expressed by embryonic F. hepatica. Molecular differences between the egg and other characterized F. hepatica lifecycle stages were noted. Furthermore, proteins expressed within liver fluke eggs differ to those isolated from the well-characterized eggs of the human blood flatworm Schistosoma mansoni were revealed. Plasticity in expression of major proteins, particularly a prominently expressed 65kDa protein cluster was seen between natural populations of embryonating F. hepatica eggs suggesting that liver fluke embryogenisis is a plastic process. Immunoblotting revealed that the abundant 65kDa protein cluster is recognised by infection sera from three F. hepatica challenged host species. Mass spectrometry and BLAST analyses demonstrated that the 65kDa antigen shows homology to egg antigens of other flatworm parasites, and is represented in a F. hepatica EST database constructed from adult fluke transcripts. EST clones encoding the egg antigen were re-sequenced, predicting two forms of the protein. Four clones predict a 312 aa polypeptide, three clones encode a putative 110 amino acid extension at the N-terminus which may be involved in protein secretion, although this extension was not expressed by natively extracted proteins. Consistent expression of alpha crystallin domains confirmed the protein to be a member of the alpha crystallin containing small heat shock protein (AC/sHSP) superfamily. AC/sHSPs are ubiquitous in nature, however, this is the first time a member of this protein superfamily has been described from F. hepatica. The antigenic AC/sHSP was named Fh-HSP35alpha based on predictions of molecular weight. Production of recombinant Fh-HSP35alpha reveals considerable mass discrepancy between native and recombinant proteins, although descriptions of other characterized flatworm AC/sHSPs, suggest that the native form is a dimer. Immunoblot analyses confirm that the recombinant protein is recognised by F. hepatica challenged hosts, but does not react with sera from non-infected animals. We discuss the potential of recombinant Fh-HSP35alpha as an egg-based diagnostic marker for liver fluke infection.

PurposeOwing to the specialist nature of biological experimentation, scientific research staff have been largely neglected from the pro‐environmental initiatives which have inundated other areas of higher education. This dearth of studies is surprising given that scientific research is recognised as a substantial contributor to the environmental impact of tertiary institutes. The present study seeks to utilise the current sustainability literature to identify barriers to sustainability in scientific fieldwork and determines which methods or procedures might increase pro‐environmental behaviours in this technical environment. The resultant information serves to provide a comparison with previously identified barriers to sustainability in the laboratory environment and identifies which environmental initiatives might be successful in both the field and laboratory.Design/methodology/approachThis study gathers qualitative data from a sample of scientific researchers presently conducting field experimentation in the agricultural sciences. A “sustainability in science” questionnaire was developed and distributed to all staff undertaking research at the Institute of Grassland and Environmental Research.FindingsDespite favourable sustainability beliefs and perceptions, almost three‐quarters (71 per cent) of researchers at this institute agreed that they were not conducting their current research activities in the most sustainable way possible. Barriers to sustainability included lack of support, lack of information, lack of training and lack of time. The provision of awards for pro‐environmental behaviours and the application of costs for unsustainable behaviours were the initiatives most likely to encourage research staff to be sustainable in the work environment.Research limitations/implicationsMany agricultural field based research projects manipulate the environment in order to cultivate and develop commercial foodstuffs. Identifying the potential to reduce such waste was an inherent part of the present study. However, identifying the ways in which such environmental manipulation modifies the landscape – whether sustainably or unsustainably – was outwith the scope of the present study and presents an interesting area for future sustainability research.Practical implicationsThe information presented in this paper has immediate practical implication for tertiary bodies and agricultural institutes wishing to adopt more sustainable fieldwork practises.Originality/valueThis is the first study to design a sustainability questionnaire specifically targeting field active research scientists in a tertiary institute.

PurposeThis study aims to identify the current barriers to sustainability in the bioscience laboratory setting and to determine which mechanisms are likely to increase sustainable behaviours in this specialised environment.Design/methodology/approachThe study gathers qualitative data from a sample of laboratory researchers presently conducting experimentation in the biological sciences. A questionnaire, regarding sustainability in the laboratory, was developed and distributed to all bioscience researchers at Aberystwyth University.FindingsAlthough the majority of respondents had favourable attitudes to sustainability, almost three‐quarters (71 per cent) stated that they were not conducting their research in the most sustainable way possible. The factors most likely to hinder sustainable behaviour were lack of support, lack of information and time constraints. However, monetary costs and benefits, closely followed by “other” costs and benefits, were most likely to encourage sustainable behaviour in the laboratory.Research limitations/implicationsThere is a need to extend the present research to other types of biological research, such as field‐based studies. Different biological disciplines may have different consumable requirements and waste streams, thereby changing the barriers to sustainability observed.Practical implicationsThe findings have immediate practical implication for higher education institutions wishing to adopt researcher‐approved mechanisms to reduce the environmental impact of biological laboratory research.Originality/valueThis is the first study to design a sustainability questionnaire which is specific to research scientists and laboratory users. The paper is therefore of immense value to the numerous global higher education institutions with working laboratories which seek to minimise the environmental impact of research.

Three-spined sticklebacks in natural lacustrine populations are often infected with plerocercoids of the indirectly transmitted pseudophyllidean cestode Schistocephalus solidus. Field studies typically show infections to be associated with reduced host condition, gonadogenesis and energy reserves, though infection phenotypes can vary considerably both between and within host populations. Experimental infection studies allow the impact of infections on hosts to be studied under a variety of rearing conditions, and so can be used to determine the environmental component of infection phenotypes. Here, we review recent laboratory studies undertaken by our group, examining the growth and condition of experimentally infected fish reared under conditions that differed in terms of absolute ration, temporal pattern of feeding and level of competition between fish. We compare infection phenotypes generated in our experimental studies with those of fish sampled in field based studies. Experimental studies in which infected fish were reared under competition for limited food resources, or were fed a restricted diet, generated infection phenotypes that most closely resembled those found in the majority of natural populations. When access to food was unrestricted, however, infected fish were able to sustain high growth rates and lay down energy reserves. If experimental studies are to be used to understand the impact of infection under natural conditions, husbandry protocols that closely match field conditions must be designed. We suggest that a full understanding of the impact of parasites on their hosts can only be gained by integrating controlled laboratory experiments with detailed field studies. The stickleback-Schistocephalus system is ideally suited to examining these questions, and we provide several suggestions for future research.

Compensatory growth responses are made by individual fish to restore their original growth trajectory following a period of growth depression. Little is known about whether diseases impact a fish's capacity for growth compensation. In this study we investigate the effect of Schistocephalus solidus, a common cestode parasite of threespine sticklebacks (Gasterosteus aculeatus), on the ability of host fish to undertake growth compensation following short-term food deprivation. Placebo-infected controls completely compensated for a 2-week deprivation period after 3 weeks postdeprivation feeding, but experimentally infected sticklebacks showed only partial compensation and after 6 weeks of refeeding had attained only 80% of the weight of continually fed infected fish. A major factor limiting the compensatory growth response of infected fish was their reduced hyperphagic response during the period of refeeding. Feed deprivation had no effect on ultimate parasite size of infected fish. We discuss the possible mechanisms limiting hyperphagia in infected fish and consider the fitness implications - for parasites and hosts - of the reduced ability of infected fish to undertake compensatory growth responses.

Livestock infection by the parasitic fluke Fasciola hepatica causes major economic losses worldwide. The excretory-secretory (ES) products produced by F. hepatica are key players in understanding the host-parasite interaction and offer targets for chemo- and immunotherapy. For the first time, subproteomics has been used to compare ES products produced by adult F. hepatica in vivo, within ovine host bile, with classical ex host in vitro ES methods. Only cathepsin L proteases from F. hepatica were identified in our ovine host bile preparations. Several host proteins were also identified including albumin and enolase with host trypsin inhibitor complex identified as a potential biomarker for F. hepatica infection. Time course in vitro analysis confirmed cathepsin L proteases as the major constituents of the in vitro ES proteome. In addition, detoxification proteins (glutathione transferase and fatty acid-binding protein), actin, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase were all identified in vitro. Western blotting of in vitro and in vivo ES proteins showed only cathepsin L proteases were recognized by serum pooled from F. hepatica-infected animals. Other liver fluke proteins released during in vitro culture may be released into the host bile environment via natural shedding of the adult fluke tegument. These proteins may not have been detected during our in vivo analysis because of an increased bile turnover rate and may not be recognized by pooled liver fluke infection sera as they are only produced in adults. This study highlights the difficulties identifying authentic ES proteins ex host, and further confirms the potential of the cathepsin L proteases as therapy candidates.

Livestock infection by the parasitic fluke Fasciola hepat- ica causes major economic losses worldwide. The excre- tory-secretory (ES) products produced by F. hepatica are key players in understanding the host-parasite interaction and offer targets for chemo- and immunotherapy. For the first time, subproteomics has been used to compare ES products produced by adult F. hepatica in vivo, within ovine host bile, with classical ex host in vitro ES methods. Only cathepsin L proteases from F. hepatica were identi- fied in our ovine host bile preparations. Several host pro- teins were also identified including albumin and enolase with host trypsin inhibitor complex identified as a poten- tial biomarker for F. hepatica infection. Time course in vitro analysis confirmed cathepsin L proteases as the major constituents of the in vitro ES proteome. In addi- tion, detoxification proteins (glutathione transferase and fatty acid-binding protein), actin, and the glycolytic en- zymes enolase and glyceraldehyde-3-phosphate dehy- drogenase were all identified in vitro. Western blotting of in vitro and in vivo ES proteins showed only cathepsin L proteases were recognized by serum pooled from F. he- patica-infected animals. Other liver fluke proteins re- leased during in vitro culture may be released into the host bile environment via natural shedding of the adult fluke tegument. These proteins may not have been de- tected during our in vivo analysis because of an increased bile turnover rate and may not be recognized by pooled liver fluke infection sera as they are only produced in adults. This study highlights the difficulties identifying au- thentic ES proteins ex host, and further confirms the po- tential of the cathepsin L proteases as therapy candidates. Molecular & Cellular Proteomics 6:963-972, 2007. The parasitic fluke Fasciola hepatica infects humans and ruminant livestock worldwide. An estimated 2.4 million people are infected with Fasciola species, and a further 180 million are at risk (1). In addition, F. hepatica causes an estimated loss of $3 billion worldwide per annum through livestock mortality, especially in sheep, and by decreased productivity via reduction of milk and meat yields in cattle (2). Presently the rate of fluke infection in parts of northwest Europe has been estimated as greater than 30%. In the absence of commercial vaccines, the benzimidazole derivative triclabendazole (TCBZ;1 Fasinex� ) is the drug most extensively used against Fasciola. Unlike other fasciolicides, TCBZ shows activity against both juvenile flukes, which are responsible for the damage to the liver of acute fasciolosis, and the mature flukes, which cause the debilitation of chronic fasciolosis. Recently resistance to TCBZ has been reported in several countries suggesting that chemotherapeutic control of this infection may soon be compromised (3-5). The most common diagnostic field method used to detect Fasciola infection requires counting fluke eggs in fecal sam- ples. However, egg counts are time-consuming and expen- sive and have limited ability to diagnose early acute stages of infection. New immunological diagnostic methods with com- mercially protected antigen identification are available in the form of a rapid on-site field test, DriDotTM (Biotechnology Ireland) based on latex agglutination and a companion ELISA for laboratory veterinary applications. Further diagnostics- based research is required to freely validate current tests and to provide alternative molecular approaches for liver fluke populations.