High prevalence of fasciolosis and evaluation of drug efficacy against Fasciola hepatica in dairy cattle in the Maffra and Bairnsdale districts of Gippsland, Victoria, Australia.

Liver fluke (Fasciola hepatica) is a common parasite amongst grazing livestock in the south-eastern region of Australia and is responsible for significant production losses in the beef and dairy industries. Gippsland in Victoria is a major region for dairy production but no fluke prevalence data in livestock has been obtained in this region since the late 1970s prior to the introduction of Triclabendazole (TCBZ). TCBZ resistance is also now widespread in cattle in south east Australia. In this study, we evaluated the prevalence and intensity of liver fluke infections in dairy cattle in Gippsland and assessed the efficacy of TCBZ and other drenches against F. hepatica on one farm. We obtained 30 individual faecal samples from each of 15 different farms and, using the liver fluke coproantigen ELISA, tested bulk faecal samples pooled from each farm. Any farm that returned a positive bulk sample had all of the samples tested individually to assess the intra-herd prevalence. One farm in the Maffra district also had a coproantigen reduction test and faecal egg count reduction test to assess the efficacy of TCBZ, Clorsulon (CLOR) and Oxyclozanide (OXY). The coproantigen ELISA proved to be a highly sensitive test for liver fluke with a high correlation (R(2)=0.8849) observed between ELISA data from bulk samples and individual samples, suggesting that future larger scale screening on farms for fasciolosis could use the bulk analysis technique. The ELISA data revealed that animals on six of the 15 farms were infected with F. hepatica and the herd prevalence of the infected herds ranged from 47 to 100% (mean 81%) which exceeds the prevalence value for production losses of 25%. The intensity of fluke infection in cattle varied considerably both within and between herds with a proportion of animals exhibiting a positive control value in the coproantigen ELISA of 50-88%. We also confirmed that TCBZ resistance was present on one farm but that CLOR or OXY can be used to remove the adult stage of the TCBZ-resistant parasites. We conclude that fasciolosis is a significant disease and a likely cause of production losses in dairy cattle in the irrigation zones of Gippsland and that TCBZ resistance is a serious threat to fluke control. We suggest that more work needs to be performed in Gippsland to further define the extent of fasciolosis and drug resistance and to ensure that effective chemical and non-chemical methods of fluke control are incorporated on farms in order to improve animal welfare and reduce financial impacts on producers.

Evidence for high genetic diversity of NAD1 and COX1 mitochondrial haplotypes among triclabendazole resistant and susceptible populations and field isolates of Fasciola hepatica (liver fluke) in Australia.

In recent years, the global incidence of Fasciola hepatica (liver fluke) infections exhibiting resistance to triclabendazole (TCBZ) has increased, resulting in increased economic losses for livestock producers and threatening future control. The development of TCBZ resistance and the worldwide discovery of F. hepatica population diversity has emphasized the need to further understand the genetic structure of drug susceptible and resistant Fasciola populations within Australia. In this study, the genetic diversity of liver flukes was estimated by sequencing mitochondrial DNA (mtDNA) encoding the NAD1 (530 bp) and COX1 (420 bp) genes of 208 liver flukes (F. hepatica) collected from three populations: field isolates obtained from abattoirs from New South Wales (NSW) and Victoria (Vic); three TCBZ-resistant fluke populations from NSW and Victoria; and the well-established TCBZ-susceptible Sunny Corner laboratory isolate. Overall nucleotide diversity for all flukes analysed of 0.00516 and 0.00336 was estimated for the NAD1 and COX1 genes respectively. Eighteen distinct haplotypes were established for the NAD1 gene and six haplotypes for the COX1 gene, resulting in haplotype diversity levels of 0.832 and 0.482, respectively. One field isolate showed a similar low level of haplotype diversity as seen in the Sunny Corner laboratory isolate. Analysis of TCBZ-resistant infrapopulations from 3 individual cattle grazing one property revealed considerable sequence parasite diversity between cattle. Analysis of parasite TCBZ-resistant infrapopulations from sheep and cattle revealed haplotypes unique to each host, but no significant difference between parasite populations. Fst analysis of fluke populations revealed little differentiation between the resistant and field populations. This study has revealed a high level of diversity in field and drug resistant flukes in South-Eastern Australia.

Comparative kinetics of serological and coproantigen ELISA and faecal egg count in cattle experimentally infected with Fasciola hepatica and following treatment with triclabendazole.

Three methods of diagnosing Fasciola hepatica (F. hepatica) infection (a coproantigen ELISA, Bio-X Diagnostics, Belgium, Faecal Egg Count (FEC), and a serum IgG ELISA,Bio-X Diagnostics, Belgium) were evaluated in artificially infected cattle, with and without drug treatment. Specifically, the potential value of the coproantigen ELISA in the quantitation of F. hepatica infection was sought. Twelve steers were each infected with 100, 200 or 500 metacercariae (n=4 cattle/group). On day 84, post infection (PI), 2 animals from each group were treated orally with triclabendazole (TCBZ). Faecal and blood samples were collected weekly after infection from all animals, as well as over 5 consecutive days (days 105-109 PI) for the six animals remaining infected to determine the repeatability of these assays. Cattle were killed 126 days PI and the coproantigen, FEC and IgG levels were compared with the number of fluke recovered. Animals first tested positive for infection with the serum ELISA, with 11/12 animals positive on day 28, and IgG responses increased to day 42 PI. The coproantigen ELISA was first positive on day 42 (3/12 animals), with all animals positive by day 56 PI. The first F. hepatica egg was detected on day 49 from an animal infected with 500 metacercariae; however only on one occasion (day 84) did all animals return positive FEC. Within one week of treatment with TCBZ, all six treated animals had returned to negative status by coproantigen ELISA and FEC whereas IgG levels persisted. Weekly variation in both coproantigen level and FEC was evident throughout the trial. Results from the consecutive daily collections varied greatly between days for both methods, with 2-6-fold differences in coproantigen levels and 2-4-fold variation in FEC. Strong correlations were observed between fluke burdens (day 126) and day 125 coproantigen levels (R(2)=0.8718) and FEC (R(2)=0.8368). The coproantigen ELISA was more sensitive than FEC (FEC displayed false negatives) and detected infection earlier. This ELISA showed good correlation to fluke burdens in these cattle and has promise as a test for detecting low fluke burdens.

Improving animal and human health through understanding liver fluke immunology.

Sheep, goats and cattle represent the most numerous and economically important agricultural species worldwide used as sources for milk, fibre and red meat. In addition, in the developing world, these species often represent the sole asset base for small-holder livestock farmers and cattle/buffaloes often provide the majority of draught power for crop production. Production losses caused by helminth diseases of these animals are a major factor in extending the cycle of poverty in developing countries and a major food security issue for developed economies. Fasciola spp. are one of the most important zoonotic diseases with a global economic impact in livestock production systems and a poorly defined but direct effect on human health. Improvements in human and animal health will require a concerted research effort into the development of new accurate and simple diagnostic tests and increased vaccine and drug development against Fasciola infections. Here, the use of definitive natural host breeds with contrasting resistance to Fasciola infections is discussed as a resource to contrast parasite-host interactions and identify parasite immune evasion strategies. Such studies are likely to boost the discovery of new vaccine, drug and diagnostic candidates and provide the foundation for future genetic selection of resistant animals.

Host responses during experimental infection with Fasciola gigantica and Fasciola hepatica in Merino sheep II. Development of a predictive index for Fasciola gigantica worm burden.

This study reports on the predictive relationship between serological, immunological and pathological responses following experimental inoculation with incremental doses of Fasciola gigantica in sheep. Fifty, 6-month-old, naive Merino wethers were allocated to one of 5 experimental groups, four of which received 50, 125, 225 and 400 metacercariae, respectively, whilst a 5th group acted as non-inoculated control. Strong individual correlations were observed between liver score, GLDH (glutamate dehydrogenase), GGT (gamma glutamyl transferase), CatL5 (cathepsin L5) antibody titre (IgG1, IgA), eosinophilia, and the total worm count or worm biomass. A combination of multiple indicator traits performed significantly better than any single indicator trait alone. The best predictive index accounted for up to 88% of observed worm burden (Wb) if information on inoculation dose was available. Without knowledge of inoculation dose, such as under field conditions, up to 67% of variation in worm burden could be predicted. In contrast, the best single predictor variable (liver damage score) accounted for up to 50% of worm burden, and in the absence of post-slaughter information, serum levels of anti-cathepsin IgA antibody titres accounted for 35% of predicted variation in worm burden. The utility of a predictive index under both field and experimental inoculation conditions is discussed.

Vaccination with a Plasmodium chabaudi adami multivalent DNA vaccine cross-protects A/J mice against challenge with P. c. adami DK and virulent Plasmodium chabaudi chabaudi AS parasites.

A current goal of malaria vaccine research is the development of vaccines that will cross-protect against multiple strains of malaria. In the present study, the breadth of cross-reactivity induced by a 30K multivalent DNA vaccine has been evaluated in susceptible A/J mice (H-2a) against infection with the Plasmodium chabaudi adami DK strain and a virulent parasite subspecies, Plasmodium chabaudi chabaudi AS. Immunized A/J mice were significantly protected against infection with both P. c. adami DK (31-40% reduction in cumulative parasitemia) and P. c. chabaudi AS parasites, where a 30-39% reduction in cumulative parasitemia as well as enhanced survival was observed. The 30K vaccine-induced specific IFN-gamma production by splenocytes in response to native antigens from both P. c. chabaudi AS and P. c. adami DK. Specific antibodies reacting with surface antigens expressed on P. c. adami DS and P. c. chabaudi AS infected red blood cells, and with opsonizing properties, were detected. These results suggest that multivalent vaccines encoding conserved antigens can feasibly induce immune cross-reactivity that span Plasmodium strains and subspecies and can protect hosts of distinct major histocompatibility complex haplotypes.

Discovery and validation of serum biomarkers expressed over the first twelve weeks of Fasciola hepatica infection in sheep.

Serum biomarkers associated with Fasciola hepatica infection of Corriedale sheep were analysed during the first 12 weeks of infection using surface-enhanced laser desorption ionisation time of flight mass spectrometry (SELDI-TOF MS). In the discovery phase of analysis, pooled sera collected at week 0 and at each week p.i. to week 12 were fractionated by anion-exchange chromatography and the protein mass fingerprints obtained in individual fractions were in the M/z range 1.5-150 kDa. A total of 2302 protein clusters (peaks) were identified that varied between time-points following infection with peaks increasing or decreasing in intensity, or showing transient variation in intensity, during the 12 weeks of parasite challenge. In the validation phase, candidate biomarkers in sera of individual sheep at weeks 3 and 9 p.i. were analysed, identifying 100 protein peaks, many of which are small peptides <10 kDa in size: 54% of these peaks were up-regulated in intensity at week 3 or 9 p.i. Twenty-six biomarkers were chosen for further study, ranging in size from 1832 to 89,823 Da: six biomarkers were up-regulated at weeks 3 and 9 p.i., 16 biomarkers were up-regulated only at week 9 p.i. and four biomarkers were down-regulated at week 9 p.i. Two biomarkers up-regulated at week 9 were identified as transferrin (77.2 kDa) and Apolipoprotein A-IV (44.3 kDa), respectively. The results show that the interaction between the host and F. hepatica is complex, with changes in biomarker patterns beginning within 3 weeks of infection and either persisting to weeks 9-12 or showing transient changes during infection. Identification of biomarkers expressed during ovine fasciolosis may provide insights into mechanisms of pathogenesis and immunity to Fasciola and may assist in the rational development and delivery of vaccines.

Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection.

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.

Identification of two allelic forms of ovine CD4 exhibiting a Ser183/Pro183 polymorphism in the coding sequence of domain 3.

The ovine CD4 cDNA sequence from four sheep sources (Australian Merino, Indonesian Thin Tail, Canadian cross bred, Prealpes du sud) predicts a protein of 455 residues with position 130 in the V2 domain exhibiting a W instead of C suggesting that, like the white whale, dog and cat sequences, sheep CD4 contains only two disulphide bonds. The sequence shows 73% amino acid identity and 83% nucleotide identity to a CD4 sequence from the white whale and significant identity to a partial sequence (314 residues) of bovine CD4 (87% amino acid identity, 93% nucleotide identity). Phylogenetic analysis showed that the ovine CD4 sequence forms a clade with the pig, white whale, dolphin, dog and cat CD4. Two forms of ovine CD4 were identified which differ by a single base pair (T/C) in their cDNA sequence at position 622. This polymorphism is also present in sheep genomic DNA in Hardy-Weinberg equilibrium, suggesting that at least two alleles of CD4 exist in the ovine genome with no selection for a particular allele. This polymorphism changes the first codon position of amino acid 183 and results in a Pro/Ser substitution in the N-terminal region of domain 3 of the CD4 protein.

Induction of strain-transcending immunity against Plasmodium chabaudi adami malaria with a multiepitope DNA vaccine.

A major goal of current malaria vaccine programs is to develop multivalent vaccines that will protect humans against the many heterologous malaria strains that circulate in endemic areas. We describe a multiepitope DNA vaccine, derived from a genomic Plasmodium chabaudi adami DS DNA expression library of 30,000 plasmids, which induces strain-transcending immunity in mice against challenge with P. c. adami DK. Segregation of this library and DNA sequence analysis identified vaccine subpools encoding open reading frames (ORFs)/peptides of >9 amino acids [aa] (the V9+ pool, 303 plasmids) and >50 aa (V50+ pool, 56 plasmids), respectively. The V9+ and V50+ plasmid vaccine subpools significantly cross-protected mice against heterologous P. c. adami DK challenge, and protection correlated with the induction of both specific gamma interferon production by splenic cells and opsonizing antibodies. Bioinformatic analysis showed that 22 of the V50+ ORFs were polypeptides conserved among three or more Plasmodium spp., 13 of which are predicted hypothetical proteins. Twenty-nine of these ORFs are orthologues of predicted Plasmodium falciparum sequences known to be expressed in the blood stage, suggesting that this vaccine pool encodes multiple blood-stage antigens. The results have implications for malaria vaccine design by providing proof-of-principle that significant strain-transcending immunity can be induced using multiepitope blood-stage DNA vaccines and suggest that both cellular responses and opsonizing antibodies are necessary for optimal protection against P. c. adami.

A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria.

The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethal Plasmodium chabaudi adami DS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.

Induction of specific T-cell responses, opsonizing antibodies, and protection against Plasmodium chabaudi adami infection in mice vaccinated with genomic expression libraries expressed in targeted and secretory DNA vectors.

It has been proposed that a multivalent malaria vaccine is necessary to mimic the naturally acquired resistance to this disease observed in humans. A major experimental challenge is to identify the optimal components to be used in such a multivalent vaccine. Expression library immunization (ELI) is a method for screening genomes of a pathogen to identify novel combinations of vaccine sequences. Here we describe immune responses associated with, and the protective efficacy of, genomic Plasmodium chabaudi adami DS expression libraries constructed in VR1020 (secretory), monocyte chemotactic protein-3 (chemoattractant), and cytotoxic T lymphocyte antigen 4 (lymph node-targeting) DNA vaccine vectors. With splenocytes from vaccinated mice, specific T-cell responses, as well as gamma interferon and interleukin-4 production, were observed after stimulation with P. chabaudi adami-infected erythrocytes, demonstrating the specificity of genomic library vaccination for two of the three libraries constructed. Sera obtained from mice vaccinated with genomic libraries promoted the opsonization of P. chabaudi adami-infected erythrocytes by murine macrophages in vitro, further demonstrating the induction of malaria-specific immune responses following ELI. Over three vaccine trials using biolistic delivery of the three libraries, protection after lethal challenge with P. chabaudi adami DS ranged from 33 to 50%. These results show that protective epitopes or antigens are expressed within the libraries and that ELI induces responses specific to P. chabaudi adami malaria. This study further demonstrates that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.

The protective efficacy of MSP4/5 against lethal Plasmodium chabaudi adami challenge is dependent on the type of DNA vaccine vector and vaccination protocol.

The enhancement of immunogenicity of malarial DNA vaccines is important if they are to have practical application in protecting against blood-stage malaria. Here we describe three different DNA vaccine vector types used in conjunction with the blood-stage merozoite surface protein 4/5 (MSP4/5), the murine homologue of Plasmodium falciparum MSP4 and MSP5, in an attempt to enhance survival against lethal Plasmodium chabaudi adami DS blood-stage challenge. MSP4/5 was inserted into VR1020 (secretory), monocyte-chemotactic protein-3 (MCP-3) (chemoattractant), and cytotoxic T-lymphocyte antigen 4 (CTLA4) (lymph node targeting) vectors. Mice were immunized intradermally via gene-gun, IM injection, or boosting with recombinant MSP4/5 protein. Antibody responses after boosting were predominantly of the IgG1 and IgE isotypes, with low avidity antibodies produced in DNA primed groups. Despite antibody responses comparable to recombinant protein immunization, boosting mice primed with antigens encoded by MCP-3 and CTLA4 vectors did not enhance survival compared to vector control groups. Gene-gun vaccination using VR1020/MSP4/5 followed by recombinant MSP4/5 boosting, or gene-gun DNA vaccination alone using MCP-3/MSP4/5, resulted in enhanced survival compared to empty vector control mice. The results suggest that the enhancement of survival against lethal blood-stage malaria challenge after utilizing MSP4/5 DNA vaccination is therefore highly dependent on the route and type of vaccine vector employed.

Characterisation of humoral immune responses in dogs vaccinated with irradiated Ancylostoma caninum.

Infection with Ancylostoma caninum, an intestinal hookworm of dogs, can cause debilitating and potentially life-threatening disease. In the current study, protective immunity to hookworm infection was induced in dogs following vaccination with irradiation-attenuated third-stage larvae (L3) with significant reductions in both worm (P<0.03) and faecal egg counts (P<0.0004) following a challenge infection. Vaccination with irradiated L3 and challenge with infective L3 stimulated a dominant antibody response to antigens of less than 20 kDa in an excretory/secretory extract from adult parasites. Immunoscreening of an adult A. caninum cDNA library with antisera from the vaccine trial identified a number of clones. The three clones with the strongest immunoreactivity proved to be identical and encoded a peptide with similarity to the N-terminal domain of the tissue matrix metalloproteinase inhibitor (TIMP)-2 mammalian tissue metalloproteinase inhibitor family.

Antibody-dependent cell-mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates.

Passive intraperitoneal transfer of sera from Fasciola hepatica-infected sheep, cattle or rats can protect naive rats from F. hepatica infection, suggesting a parasite killing mechanism within the peritoneal cavity that is dependent on the presence of parasite-specific antibody. We investigated antibody-dependent cell-mediated cytotoxicity by resident peritoneal lavage cell populations, containing large numbers of monocytes/macrophages, as a potential host resistance mechanism by which juvenile flukes could be killed within the peritoneal cavity of naive rats. Comparative studies were conducted using cell populations containing large numbers of monocytes/macrophages from sheep. The results demonstrate that monocyte/macrophage-rich lavage cell populations from rat and sheep differ substantially in their ability to generate nitric oxide. Only resident rat peritoneal lavage cells were able to mediate antibody-dependent cell-mediated cytotoxicity against newly excysted juvenile liver fluke. The mechanism of cytotoxicity was dependent on, and directly proportional to, the production of nitric oxide and required attachment of effector cells to the newly excysted juvenile liver fluke tegument, which occurred following the addition of sera from F. hepatica-infected animals. This is the first report demonstrating a mechanism of cell-mediated cytotoxicity to newly excysted juvenile liver fluke.

Fasciola: kinetics and quality of humoral responses to fatty acid binding protein and cathepsin l following delivery as DNA vaccines in mice.

The humoral response to DNA vaccination of mice with two important Fasciola antigens has been investigated. Both F. gigantica fatty acid binding protein (FABP) and F. hepatica cathepsin L5 (FhCatL5) were shown to be expressed in COS 7 cells and induced a humoral response when delivered as secretory constructs in mice. FABP induced an IgGl dominant response, with significant IgE, IgG2a, and IgG2b responses also present, indicating a mixed Th1/Th2 response. The total Ig response peaked at 1:24,500 and antibody titers were sustained for at least 32 weeks. In contrast, the delivery of FABP as a nonsecreted construct did not result in the induction of a measurable humoral response. FhCatL5 was delivered as a secretory construct, with secretion mediated by the native F. hepatica signal sequence, which was shown to operate in COS 7 cells. The humoral response peaked at 1:2000 at week 8 and was sustained for at least 20 weeks. Antibody isotype analysis demonstrated a Th2-like response, which was qualitatively different from that obtained for FABP with an IgE dominant response, and lower titers to IgG1 and IgG3. The results demonstrate that Fasciola antigens can be delivered as DNA vaccines, but that the quality of the response varies between antigens and is influenced by the method of vaccine delivery.

Juvenile Fasciola hepatica are resistant to killing in vitro by free radicals compared with larvae of Schistosoma mansoni.

Free radicals have previously been shown to kill the immature stages of the trematode, Schistosoma mansoni but their effect on newly excysted juvenile (NEJ) flukes of Fasciola hepatica has not been established. Using acetaldehyde and xanthine oxidase to chemically generate reactive oxygen intermediates (ROI), up to 61% of NEJ were killed but only when exposed to high levels of ROI. At low concentrations of acetaldehyde and xanthine oxidase as sources of reactive oxygen intermediates, only 6-29% of NEJ were killed compared with 70-92% of schistosomula. Incubation with lipopolysaccharide (LPS)-stimulated rat peritoneal lavage cells (PLCs) killed only 7-15% of NEJ whereas 78-87% of schistosomula were killed under the same conditions by a mechanism dependent on the production of reactive nitrogen intermediates. Relative to immature and adult parasites, NEJ expressed 2.5-20-fold lower levels of superoxide dismutase and glutathione S-transferase but no catalase activity was detected. Incubation of NEJ with inhibitors of peroxidases and glutathione metabolism increased the mean killing of NEJ by LPS-stimulated rat PLCs to 40-75%. These results demonstrate that, in comparison to schistosomula of S. mansoni, NEJ of F. hepatica are relatively resistant to killing by free radicals and this resistance could, in part, be due to the activity of oxidant scavenger enzymes of NEJ.

Expression library immunization protects mice against a challenge with virulent rodent malaria.

Although several candidate vaccine antigens have been developed for malaria, there is as yet no effective single vaccine available. There is a growing consensus that the ultimate malaria vaccine will be multivalent, requiring the identification of a suitable cocktail of antigens. However, evaluation of the multitude of potential malaria vaccine antigens in suitable combinations is a daunting task. Here we describe the validation of expression library immunization (ELI) as a tool for the discovery of sequences protective against malaria infection. A genomic Plasmodium chabaudi expression library was constructed comprising ten separate pools of 3000 plasmids. Over three vaccine trials using biolistic delivery of pools composed of 616 to 30,000 plasmids we report up to 63% protection of mice from a challenge with P. chabaudi adami DS, a highly virulent strain. Overall, ELI protected 36% of vaccinated mice against virulent challenge compared with only 3.2% survival of control mice. These results demonstrate that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.

A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica.

The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.