Psoroptes ovis: Identification of vaccine candidates by immunoscreening

Experimental Parasitology 120 (2008) 194–199

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Research brief

Psoroptes ovis: Identification of vaccine candidates by immunoscreening Alasdair J. Nisbet *, Aileen M. Halliday, Lois Parker, W. David Smith, Fiona Kenyon, David P. Knox, John F. Huntley Moredun Research Institute, Parasitology Division, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland, UK

a r t i c l e

i n f o

Article history: Received 30 May 2008 Accepted 19 June 2008 Available online 25 June 2008 Keywords: Psoroptes ovis Astigmata Acari Sheep scab mite Glutathione S-transferase (GST) EC2.5.1.18 Recombinant vaccine Catchin-like protein (CLP)

a b s t r a c t Serum from successful vaccine trials against the sheep scab mite, Psoroptes ovis, was used to immunoscreen a cDNA library constructed from mixed-stage and gender P. ovis to identify potential recombinant vaccine candidates. Immunodominant recombinant proteins recognised by IgG in these sera were selected for further analysis. Two candidates were identified in this way; a catchin-like protein (CLP) and a novel mu class glutathione S-transferase (GST). Both candidates were expressed in bacteria as recombinant proteins, the GST as an active enzyme, and combined with four other recombinant allergens in a multi-component recombinant vaccine. Strong serum IgG responses were induced in sheep against each of the components of the recombinant vaccine, however, the protective efficacy of the vaccine could not be determined because of variability in the establishment of a challenge infection. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction Sheep scab is caused by the mite Psoroptes ovis and is, arguably, the most important ectoparasitic disease of sheep in the UK. The most recent data relating to costs of sheep scab in the UK suggest that the disease costs £8 million per annum. These disease costs include lost performance, preventative measures, and treatment of affected animals, with the principal cost component for sheep scab coming from prevention (Nieuwhof and Bishop, 2005). Since deregulation in 1992, annual dipping of sheep in the UK is no longer compulsory. In 1999, organophosphate sheep dips were withdrawn on grounds of risks to operator health but were relicensed (for Diazinon only) in 2001. The use of synthetic pyrethroid (SP) dips (cypermethrin) has also recently been re-evaluated. As a result of these changes in dipping practice the prevalence of sheep scab has escalated and the disease is now endemic in both hill and lowland sheep in all areas of the British Isles, with an estimated 7000 outbreaks in 2004 (Bisdorff et al., 2006). There have therefore been sustained efforts over the last 10 years to develop a vaccine against P. ovis (for recent review see Nisbet and Huntley, 2006), encouraged by the demonstration of a protective immune response in sheep, where prior infestation with the mite leads to a suppression of lesion development in subsequent infestations (Van den Broek et al., 2000). Partial protection of rab* Corresponding author. Fax: +44 131 445 6111. E-mail address: [email protected] (A.J. Nisbet). 0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.06.008

bits against lesions induced by P. cuniculi (believed to be conspecific with P. ovis) (Uhlírˇ, 1992) and partial protection of cattle against P. ovis (Pruett et al., 1998) has been induced by immunisation with various fractions of the mites. Vaccination of sheep with soluble extracts of P. ovis has resulted in 5-fold reductions in mite numbers and more than 50% reduction in lesion area (Smith et al., 2002). Subsequent progressive purification of the soluble mite extract resulted in the isolation of a fraction which stimulated high titres of mite-specific serum antibodies, inhibited lesion growth to less than a third of that seen in controls and reduced mite numbers by more than 13-fold from 28 days after a challenge infection in vaccinated sheep (Smith and Pettit, 2004). In addition to this ‘‘pragmatic proteomic approach” involving sequential protein fractionation, vaccination, and mite challenge, we have identified a number of potential vaccine candidates by a ‘‘rational genomic approach” to identify homologues of known powerful immunogens and protective antigens from other pest and parasite species (Lee et al., 2002a; Kenyon et al., 2003; Huntley et al., 2004; Nisbet and Huntley, 2006; Nisbet et al., 2006, 2007). Using this latter approach we have identified homologues of the potent house dust mite allergens Der p 1 [cysteine proteinase, (Lee et al., 2002a; Nisbet et al., 2007)], Der p 10 [tropomyosin (Huntley et al., 2004; Nisbet et al., 2006)] and Der p 11 [paramyosin (Huntley et al., 2004; Nisbet et al., 2006)] as immunodominant allergens in natural sheep scab infestations. In this study, we describe the use of immunoscreening with serum from successful pragmatic proteomic vaccine trials to identify

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further vaccine candidates and the application of a multi-component recombinant vaccine derived from both approaches in an attempted protection trial in sheep.

2. Materials and methods 2.1. Isolation of cDNAs encoding catchin-like protein (CLP) and glutathione S-transferase (GST) by immunoscreening Total RNA was extracted from mixed-stage P. ovis under liquid nitrogen by homogenization in TriPureTM reagent (Roche) according to the manufacturer’s protocol. A cDNA library was constructed, from this RNA, in kTriplEx2 by long distance pcr following manufacturer’s instructions for SMARTTM cDNA Library Construction (Clontech), packaged in Gigapack Gold III packaging extract (Stratagene) and amplified in Escherichia coli XL1-Blue strain, (Stratagene). The amplified library titre was 1  1010 pfu ml1, with 96% of the plaques containing recombinant DNA. Two strategies were used to immunoscreen the library. In the first, antibody (IgG) was derived from a pool of sera collected from animals that showed some degree of protection following vaccination with either the water-soluble (S1, 12 sera) or membrane associated (S2, 14 sera) mite extract in a trial described previously (Smith et al., 2002). The resultant positive clones were then subjected to a differential immunoscreen using sera obtained from animals that were strongly protected from mite challenge by vaccination with the S1 or S2 mite antigen with sera from animals that were weakly protected. Those clones that were recognised by IgG in the sera from the strongly protected individuals were selected for further study as they may be involved in the host protective immune response. This screen identified the cDNA encoding catchin-like protein (CLP). In the second immunoscreening strategy, sera, collected immediately pre-challenge from sheep which had been injected on 3 occasions, at 3 week intervals, with a soluble mite extract prepared as described previously (Smith and Pettit, 2004) was used to immunoscreen (detecting IgG) the amplified P. ovis cDNA library. In vaccination trials this mite extract stimulated high titres of mite-specific serum antibodies, inhibited lesion growth to less than a third of that seen in controls and reduced mite numbers by more than 13-fold from 28 days after a challenge infection (Smith and Pettit, 2004). Sera collected from three sheep which had shown high levels of protection in this trial were pooled for use in the immunoscreens. This screen focussed attention on the GST. All pooled sera were pre-absorbed with E. coli (XL1-Blue strain, Stratagene) lysate prepared as described previously (Sambrook et al., 1989) then diluted to a final concentration of 1 in 1000 in Tris buffered saline (25 mM Tris, 150 mM NaCl, and 3 mM KCl) containing 0.1% Tween 20 (TBST) plus normal horse serum (final concentration 5% v/v) before use in immunoscreens. Immunoscreening of the P. ovis cDNA library was performed as described in the protocols supplied with the SMARTTM cDNA Library Construction kit (Clontech). Immunopositives were identified using horse radish peroxidase labelled mouse anti-sheep IgG secondary antibody (Sigma; 1:1000 dilution in TBST, 5% normal horse serum) and developed with 3,30 -diaminobenzidine/urea/H2O2 substrate (Sigma). Positive plaques were purified by successive rounds of screening as described above, amplified by PCR using vector-specific primers and sequenced directly from the PCR products using ET terminator chemistry on a MegaBACE DNA analyser. All sequences were aligned using the CAP3 EST assembly programme (Huang, 1996), employing a minimum sequence overlap length cut-off of 30 bases and an identity cut-off of 98%. Contigs derived from this analysis were compared with those in the GenBank non-redundant

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database, using the Basic Local Alignment Search Tool (BLASTn and BLASTx) programme (Altschul et al., 1990) from the National Center for Biotechnology Information and also using the Washington University Basic Local Alignment Search Tool Version 2.0 (WUBLAST2) programme to search the Invertebrate EST database available through the European Bioinformatics Institute. 2.2. Expression of recombinant GST and CLP cDNAs encoding both the novel mu class GST and the CLP, identified by immunoscreening as described above, were amplified by PCR using a heat-denatured, phage lysate preparation of cDNA library (GST) or purified plasmid containing the putative fulllength open reading frame (ORF) as template (CLP). Primer sequences were:GSTpETF 50 -AATTCGGATCCGATGGACACTAAACCGGT GCTTGG-30 GSTpETR 50 -CCGCAAGCTTAGCGTATTGACCATTCCACAA TGCT-30 CatchpETF 50 -AATTCGGATCCGATGAGTGATATTGACTCTTC– 30 CatchpETR 50 -CCGCAAGCTTACTTACCTGTACTTTTCTTCC-30 (BamH1 and HindIII restriction sites underlined, putative initiation codon in italics). PCR conditions were as follows: primer concentrations (final concentration in 50 ll reaction) 0.2 lM; reaction buffer, polymerase and dNTPs all as described in manufacturer’s instructions for BD AdvantageTM 2 (BD Biosciences). Cycling parameters: 95 °C for 1 min followed by 30 cycles of 95 °C for 15 s, 55 °C (GST) or 52 °C (CLP) for 1 min, 70 °C for 1 min, and a final extension of 68 °C for 10 min. The PCR products were subcloned into the expression vector pET-22b(+) (Novagen) downstream of a periplasmic secretion signal and containing a C-terminal His-tag, cultivated in E. coli JM109 (Promega) competent cells and plasmid was extracted using WizardÒ Plus SV Miniprep kits (Promega). After sequencing to confirm that the constructs were in frame, the plasmids were used to transform E. coli BL21-CodonPlusÒ (DE3)-RIL competent cells (Stratagene). Recombinant protein expression was induced in the presence of 1 mM isopropyl-b-Dthiogalactopyranoside (IPTG). Soluble, recombinant GST (rPoGST) was purified from cell lysates by nickel column affinity chromatography using HisTrapTM HP columns (GE Healthcare) according to the manufacturer’s instructions. Recombinant CLP (rPoCLP) was expressed in the insoluble fraction and was solubilised in 8 M urea. Purification of the rPoCLP was accomplished by electroelution from NuPAGEÒ Bis–Tris 4–12% gels after electrophoresis under reducing conditions and staining with SimplyBlueTM according to the manufacturer’s instructions (Invitrogen). The identity of the purified recombinant proteins was established by matrix-assisted laser desorption/ionization (MALDI-ToF-ToF) after destaining and reductive alkylation using DTT and iodoacetamide. Trypsin digests of the proteins were analysed on an Ultraflex II MALDI-ToF-ToF mass spectrometer (Bruker Daltonics), scanning the 600–5000 Da region in reflectron mode producing monoisotopic resolution. The spectra generated were mass calibrated using known standards and the peaks deisotoped. Masses obtained were then database-searched with the MASCOT search engine using the Swiss-Prot and local databases using a 50 ppm mass tolerance window. Significant matches from the Peptide Mass Fingerprint data were confirmed by MS/MS analysis using the search criteria detailed above and an MS/MS tolerance window of 0.5 Da. 2.3. rPoGST activity assay Following purification, rPoGST was dialysed against phosphate buffered saline (PBS, pH 7.2) for 24 h at 4 °C and the protein concentration of the resulting dialysed solution was determined using the BCATM Protein Assay Kit (Pierce) in accordance with the manufacturer’s instructions, with bovine serum albumin (BSA) standards diluted in PBS. The specific activity of the rPoGST was determined using the substrate 1-chloro-2,4-dinitrobenzene (CDNB), essentially

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as previously described (Lee et al., 2002b). GST activity was measured spectrophotometrically at 340 nm, using a microplate adaptation of the assay. Activities, expressed as lmol min1 mg1, were calculated as described in Lee et al. (2002b). 2.4. Expression of recombinant tropomyosin, paramyosin, Pso o 1 and Der p 14 Bacterially expressed recombinant P. ovis tropomyosin (rPoTRO) and a truncated form of paramyosin (trPoPAR) were produced and purified as detailed in Nisbet et al. (2006). Recombinant Pso o 1 was expressed in Pichia pastoris and purified as described in Nisbet et al. (2007). A truncated form (amino acids 1–224) of the house dust mite allergen Der p 14 (trDerp14) was produced in E. coli BL21-CodonPlusÒ (DE3)-RIL competent cells and solubilised in 8 M urea prior to purification using HisTrapTM HP columns (GE Healthcare), with 8 M urea incorporated into each binding and elution buffer, according to the manufacturer’s instructions. The plasmid containing the ORF for trDerp14 in expression vector pET-22b(+) (Novagen) was a kind gift from Prof. Wayne R. Thomas, Telethon Institute for Child Health Research, West Perth, Australia. Following purification, urea and imidazole were removed from the trDerp14 sample by dialysis as described above for the rPoGST. 2.5. Immune response of sheep to recombinant antigens The six recombinant immunogens (rPoGST, rPoCLP, rPoTRO, trPoPAR, Pso o 1 and trDerp14) were each diluted to 120 lg ml1 with PBS then combined to give a final volume of 1.5 ml containing 30 lg of each protein. This preparation was then mixed with 0.5 ml QuilA (Superfos Biosector, Denmark) so that, in a 2 ml final volume, each sheep received 5 mg of the adjuvant and 30 lg of each recombinant protein (where appropriate) at each immunization. Control immunogen was prepared identically except that PBS was substituted for antigen. Groups of 10 sheep (Suffolk cross, 1–2 years of age) were injected intramuscularly with 2 ml of recombinant or control immunogen. Three injections were administered 3 weeks apart. All sheep were challenged with P. ovis at the same time as the third immunization by the skin transfer method (Smith et al., 2002). Assess-

ment of disease was made by lesion area measurements as described previously (Smith et al., 2002). Sera, collected from sheep in both the immunised and control groups 2 weeks after the second injection of immunogen (or adjuvant control) were used to probe Western blots of the recombinant antigens as follows: 1.5 lg of mixed recombinant immunogens (250 ng each protein) were electrophoresed on NuPAGEÒ (Invitrogen) Bis–Tris 4–12% gels under reducing conditions employing NuPAGEÒ MES SDS running buffer and proteins were transferred to a nitrocellulose membrane according to the manufacturer’s instructions (Invitrogen). After transfer, the membranes were washed briefly in Tris-buffered saline (TBS; 25 mM Tris, 137 mM NaCl, and 2.7 mM KCl) containing Tween 20 (0.1%) (TBST) then incubated in TBST containing 5% Marvel non-fat milk powder (Premier International Foods Ltd.) for 4 h at room temperature (RT) on an orbital shaker (100 cycles/ min) to block non-specific protein adsorption. Blotted proteins were then incubated in sheep sera diluted 1:1000 in TBST 5% Marvel for 1 h at RT with constant rocking. A control blot was performed by omitting the test sera during this primary incubation step. The blots were washed (10 min/wash) three times in TBST, before being incubated in mouse monoclonal antibody raised against ovine IgG, horse radish peroxidase (HRP) conjugate (Sigma) diluted 1:500 in TBST, 5% Marvel. Following incubation at RT for 1 h, the blots were washed a further three times in TBST and peroxidase activity was revealed using 3,30 -diaminobenzidine (DAB) as a substrate. 3. Results 3.1. Isolation of cDNAs encoding CLP and GST by immunoscreening The initial immunoscreen, using sera from a vaccine trial described by Smith et al. (2002), identified 48 positive plaques which were reduced down to 6 ‘‘strong” positives by the differential immunoscreen outlined above. Of these, 5 encoded the CLP (Accession No. AM991141) and the remaining positive clone encoded an open reading frame of 152 amino acids which showed no clear homology to a specific protein sequence in the databases. The putative ORF of CLP was 747 bp in length, encoding a predicted protein of 28.5 kDa. The predicted protein possessed 30% amino

Fig. 1. Pairwise sequence alignment of P. ovis CLP (PoCLP) with catchin from the Mediterranean mussel, Mytilus galloprovincialis Accession No. Q9U0S5. The sequences showed 30% identity and 46% similarity. The vertical lines indicate residues conserved between the two sequences, highly similar residues by a colon and less similar residues by a full-stop.

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Psoroptes ovis A 100

Sarcoptes scabiei mu 66

Dermatophagoides pteronyssinus 87

Psoroptes ovis B

83

Haemaphysalis longicornis 60 100

100

Boophilus microplus

Mu

Rhipicephalus appendiculatus 91

100

Fasciola hepatica Schistosoma mansoni Onchocerca volvulus

100

56

Pi

Dirofilaria immitis 100 100

Chicken

Alpha

Rat GSTA2 100 100

Drosophila melanogaster GST 2 Musca domestica GST1

Sigma

Blatella germanica Bla g 5 56

Rat Kappa 100

Human omega 100 100

Omega

Lucilia cuprina Drosophila melanogaster GST1

Theta

Musca domestica GST2

100

Psoroptes ovis C 69

Kappa

Mouse omega

98

Sarcoptes scabiei delta 100

Human Zeta 1

Delta Zeta

Wheat Proteus

Beta

Fig. 2. Relationships between selected GSTs. Neighbour-joining analysis was performed using representative sequences from each GST class. The neighbour joining tree was bootstrapped 1000 times using Clustal X (Jeanmougin et al., 1998) and the resulting tree viewed with TREEVIEW. Branches with bootstrap values of less than 50% were collapsed. The P. ovis GST sequence discovered by immunoscreening, ‘‘Psoroptes ovis A” (Accession No. AM991140) is boxed. Those P. ovis GST sequences previously described in Lee et al. (2002b) and Kenyon et al. (2003) are indicated as Psoroptes ovis GST B and C, respectively. GST classes of the representative sequences are shown on the right of the diagram. Psoroptes ovis A (AM991140), Psoroptes ovis B (AAF19264), Psoroptes ovis C (BQ834940), Dermatophagoides pteronyssinus (AAX37326), Sarcoptes scabiei mu (AAO15607), Sarcoptes scabiei delta (AAV65948), Boophilus microplus (AAD15991), Haemaphysalis longicornis (AAQ74441), Rhipicephalus appendiculatus (AAQ74442), Onchocerca volvulus (X77393), Fasciola hepatica (P31671), Schistosoma mansoni (AAA29888), Blattella germanica Bla g 5 (O18598), Rat Kappa (P24473), Mouse omega (AAB70110), Human omega (AAF73376), Musca domestica GST1, Musca domestica GST2 (AAA03434, P46431), Lucilia cuprina (P42860), Drosophila melanogaster GST1 (P20432), Drosophila melanogaster GST2 (P41043), Dirofilaria immitis (P46426), Chicken (Q08393), Rat GSTA2 (NP_058709), Human Zeta1 (AAB96392), Wheat (AAD09190), Proteus (P15214).

acid identity and 46% similarity over 249 amino acid residues to a myosin-like protein, catchin, from the bivalve Mytilus galloprovincialis [Fig. 1. (Accession No. Q9U0S5)]. The cDNA sequences of 25 of the 55 immunopositives identified by screening the library with sera from a subsequent vaccine trial (Smith and Pettit, 2004) formed a single contig encoding a novel mu class GST (Accession No. AM991140). The predicted protein possessed highest (61%) amino acid identity to mu-class GSTs of Dermatophagoides pteronyssinus (Accession No. AAX37326) and Sarcoptes scabiei type hominis [AAO15607, (Pettersson et al., 2005)] and 54% amino acid identity to a muclass GST previously identified from P. ovis [AAF19264 (Lee et al., 2002b)] (Fig. 2). The GST is predicted to be intracellular and secretion is unlikely given a signal peptide probability of only 0.006 [using Hidden Markov models] and an S-mean score of only 0.248 for amino acids 1–26 (cleavage site ‘‘C” score of 0.525 between aa 26 and 27) when neural networks analysis was performed using the programme SignalP 3.0 (Dyrløv Bendtsen et al., 2004). Expression of the soluble form of this GST was achieved in a bacterial system and the recombinant protein, rPoGST, was shown to be enzymatically active (Fig. 3).

3.2. Immune response of sheep to recombinant antigens Each of the recombinant proteins was recognised by the IgG component of sera from immunised sheep, but not adjuvant control sheep two weeks after the second immunisation (1 week before the third immunisation and challenge infestation) (Fig. 4). The sera used in the Western blot were all from sheep which subsequently developed lesions following infestation with P. ovis in the challenge infestation, demonstrating that a good immunological response had been generated. However, infestation of the sheep in the trial was poor and variable. Only 7 of the 20 sheep in the trial became infested during the course of the experiment. Of these sheep, only 2 were in the control group and 5 were in the group treated with recombinant proteins. The high variability of the lesion masses in the infested sheep from the control group made statistical analysis of the data redundant (data not shown). 4. Discussion Here, the presence and immunogenicity of two recombinant vaccine candidates for P. ovis has been demonstrated by immunoscreening of a mite cDNA expression library with serum from

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GST activity (μmol/min/mg)

A

8

0.40

5

4

3

2

1

0.30 0.25 62

0.20 0.15

49

0.10

38

trPoPAR rPso o 1 rPoTRO rPoCLP rPoGST trDerp14

0.05 28 2.E-04

4.E-04

6.E-04

8.E-04

1.E-03 17*

Substrate concentration (mol)

GST activity (μmol/min/mg)

6

0.35

0.00 0.E+00

B

7

0.35 **BC

0.3

Control sheep

Vaccinated sheep

0.25 Fig. 4. Immune response of sheep to six recombinant antigens Sera from immunised (lanes 1–5) sheep or adjuvant control (lanes 6 and 7) sheep which subsequently developed lesions following infestation with Psoroptes ovis were used to probe a Western blot of the recombinant vaccine mixture. Sera were taken 2 weeks after the second immunisation with the immunogens (or adjuvant control). * Molecular mass (kDa), **blank control, no primary serum.

0.2 0.15 0.1 0.05 0 0

1

2

5

10

Duration of heat inactivation (min) Fig. 3. Enzymatic activity of rPoGST. (A) Purified rPoGST (2.6 lg per well) was incubated with the substrate 1-chloro-2,4-dinitrobenzene (CDNB) at various concentrations in the presence of 2 mM reduced glutathione and the increase in absorbance at 340 nm was measured for 20 min at room temperature. Specific activity was calculated as described previously (Lee et al. 2002b), after adjustment of the data to account for spontaneous hydrolysis of the substrate. Each data point shown is the mean of three replicates in a single run of the experiment (±SEM). (B) Purified rPoGST in PBS was incubated at 72 °C for 0, 1, 2, 5, and 10 min, then used in a GST activity assay as described above, in the presence of 1 mM substrate. Each data point shown is the mean of three replicates in a single run of the experiment (±SEM).

animals which possessed high titres of mite-specific antibodies. These sera were derived from animals which had shown inhibited lesion growth and reduced mite numbers in prior vaccination experiments in which the sheep had been injected with fractionated mite extracts (Smith et al., 2002; Smith and Pettit, 2004). The potential for use of these antigens as practical vaccine components could not be assessed in the current experiment as a result of poor ‘‘take” of the experimental infection in the protection trial. This reflects the difficulties involved in using the sheep scab/ovine host model in experimental infections and highlights the need for a more reliable and less expensive testing system. Psoroptes cuniculi, which is believed to be conspecific with P. ovis (Zahler et al., 1998) infests rabbits, however, in psoroptic otoacariasis of rabbits Psoroptes mites ingest whole blood, which is not observed in Psoroptes infestations of sheep, so the relevance of rabbit models for in vivo testing of vaccine candidates may be questionable. Another, possibly better, alternative would be to use the recently developed in vitro feeding system for Psoroptes mites (Thind and Ford, 2007) to test the direct effects of serum antibodies on mite survival and fecundity. Differential immunoscreening of a P. ovis cDNA library with sera from ‘‘strongly” or ‘‘weakly” protected sheep from a vaccine trial

described in Smith et al (2002) identified a number of clones with 35% sequence identity (at amino acid level) with catchin, a molluscan myosin-like protein. While myosin-like proteins have made excellent vaccine candidate molecules for a number of helminth parasites [e.g. Sm62-IrV-5, a myosin from Schistosoma mansoni (Soisson et al., 1992), paramyosin from S. mansoni (Pearce et al., 1988) and S. japonicum (Chen et al., 2000)], they are known to be highly immunogenic and therefore myosin is often encountered in immunoscreens using sera raised against parasite extracts so the relevance of the catchin homologue to protection may not be clear-cut, particularly as it was not identified in the second immunoscreen conducted with sera from the vaccine trial described by Smith and Pettit (2004). Antigen used as vaccine in this trial was a refined version of that used by Smith et al (2002). Here, we show that 45% of the immunopositives identified by screening the cDNA library with immune sera from the vaccine trial described by Smith and Pettit (2004) encoded a novel mu class GST. Recombinant versions of glutathione S-transferases have provided high levels of protection against other species of parasite [e.g. Fasciola gigantica and Schistosoma mansoni in a mouse model (Preyavichyapugdee et al., 2008); Necator americanus in a hamster model (Xiao et al., 2008)] and GSTs in mites have also been shown to provoke the pathophysiology associated with mite allergies and severe complications of mite-related diseases [e.g. crusted scabies (Dougall et al., 2005)]. The vaccine potential of mite GSTs, and other allergens, may therefore lie in diverting the immune response from pathology to protection. Current research is therefore aimed at elucidating the timing and mechanisms of natural immunity against P. ovis for the optimal application of allergens and/or other immunogens as vaccines for sheep scab. Acknowledgments The authors gratefully acknowledge funding by the Department for Environment Food and Rural Affairs, UK Research Contract OD0544 and by the Scottish Government Rural and Environment Research and Analysis Directorate (RERAD).

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