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Vaccination of lambs with the recombinant protein rHc23 elicits significant protection against Haemonchus contortus challenge Elshaima M. Fawzi 1 , María Elena González-Sánchez 1 , María Jesús Corral, José Ma Alunda ∗ , Montserrat Cuquerella Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain
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Article history: Received 13 February 2015 Received in revised form 28 April 2015 Accepted 30 April 2015 Keywords: rHc23 Recombinant antigen Haemonchus contortus Vaccination Lambs DNA Cloning Expression Sheep
a b s t r a c t Gene encoding a somatic protein of Haemonchus contortus (Hc23) known to confer significant protection against experimental haemonchosis has been cloned and expressed in a prokaryotic system. A cDNA library of H. contortus using the vector ZAP II was obtained. Full-length gene was amplified, cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in Ni-NTA column. Recombinant protein (rHc23) had 203 aminoacids and a molecular mass of 24.15 kDa. Recombinant protein (100 g/dose) with aluminum hydroxide as adjuvant was administered to 5–6 months age female Assaf lambs on days −42, −28 and −14. On day 0 animals were infected with 15,000 L3 of H. contortus. Vaccination with rHc23 elicited a significant protection against challenge, with > 80% reductions in both fecal egg counts and average abomasal parasite burdens at the end of the experiment (45 days post challenge) besides lack of variations in packed cell volume. Results support the feasibility of vaccination against lamb haemonchosis with a recombinant product from an exposed antigen. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Helminth infections are common in most domestic animals and are the rule in animal species such as ruminants exploited under extensive management systems in many regions of the world. Haemonchus contortus is a highly pathogenic blood feeding nematode of the abomasum of ruminants. Infections are responsible of weight loss, decrease fertility and wool and milk production in adult animals and 30–50% mortality in lambs and kids (Aumont et al., 1997). Haemonchosis, the infection caused by this parasite, is considered the most important helminth infection of small ruminants (Waller and Chandrawathani, 2005) representing ca. 15% of all gastrointestinal diseases of this livestock (http://www.fao. org). Practical control of haemonchosis has been mainly based on the use of anthelmintics. However, indiscriminate medication, lack of strategic planning, potential refuge populations in wild reservoirs and impracticality of pasture management in many regions of the world besides the biological potential of Haemonchus have
∗ Corresponding author at: Dpto. Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain. Tel.: +34 913943701; fax: +34 913943908. E-mail address: [email protected] (J.M. Alunda). 1 Both authors deserve the same credit as first authors.
resulted in widespread resistance to the major classes of available anthelmintics (Jackson and Coop, 2000; Kaminsky, 2003; Kaplan, 2004). Moreover, there is a growing social concern regarding the presence of residues with pharmacological activity in animal products. Natural or experimental repeated H. contortus infections elicit a protective response in many sheep breeds provided that animals are older than 6–8 months age; therefore vaccination against haemonchosis seems feasible. Immunoprophylactic potential of several “native” antigens of H. contortus (i.e., H11, H-gal-GP, E/S antigens, somatic antigens) has been remarkable in terms of reduction in helminth egg excretion and the abomasal worm burdens (e.g., Schallig and van Leeuwen, 1997; Newton and Munn, 1999; Cachat et al., 2010). Insofar, results obtained with native proteins could not be replicated with the recombinant counterparts (e.g., Schallig and van Leeuwen, 1997; Redmond and Knox, 2004; Yanming et al., 2007). Unfortunately vaccination with native proteins, even with low doses immunizations, would require a permanent source of adult helminths from infected hosts. Recently a purified somatic protein from adult H. contortus (Hc23) has been shown to elicit a substantial protection against experimental challenge (Fawzi et al., 2014). In this manuscript we present the cloning and expression in Escherichia coli of a recombinant form of this protein (rHc23). Vaccination with the recombinant protein adjuvanted
http://dx.doi.org/10.1016/j.vetpar.2015.04.029 0304-4017/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Fawzi, E.M., et al., Vaccination of lambs with the recombinant protein rHc23 elicits significant protection against Haemonchus contortus challenge. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.04.029
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with aluminum hydroxide induced a significant protection of lambs against the experimental challenge with H. contortus. 2. Material and methods 2.1. Parasites The H. contortus strain used was originally supplied by Merck, Sharp and Dohme (Madrid, Spain) and maintained for the last 20 years by serial passage in lambs (Department of Animal Health, UCM). Third stage larvae (L3) for experimental challenges were obtained by fecal culture at 26 ◦ C, baermanization of the feces and partial purification on filter paper (MAFF, 1971). Adult H. contortus for DNA extraction were obtained from the abomasums of monospecifically infected lambs. 2.2. Cloning, expression and purification of Hc23 Adult H. contortus (50–100 mg, both sexes) were employed to obtain total RNA (Chomczynski and Sacchi, 1987). Isolation of messenger RNA (mRNA) was performed with mRNA purification kit (Stratagene) and a H. contortus cDNA library was constructed using lambda ZAP cDNA® Synthesis kit (Stratagene). A DNA fragment encoding an incomplete form of p26/23 from previous experiments (García-Coiradas et al., 2010) was labelled with digoxigenin-dUTP, quantified with DIG DNA Labelling Kit (Roche) and used to screen the obtained cDNA H. contortus library (Sambrook et al., 1989). The gene specific primer (5 TCA GTC TTT CGC GGA CTT G 3) was used in combination with T3 cDNA library vector primer (5 AAT TAA CCC TCA CTA AAG GG 3) to amplify the gene encoding Hc23 in the positive colonies. The resulting fragment was cloned in pGEM-T easy vector (Promega) and the construct was used to transform E. coli XL2-Blue (Stratagene). Recombinant plasmids containing the rHc23 gene sequence were purified using plasmid miniprep kit, cloned in pET-29B and used to transform E. coli BL21(DE3) (Novagen). To assess the expression of Hc23, positive colonies were cultured in LB medium with 0.5–4 mM isopropyl--d-thiogalactopyranoside (IPTG) (Roche). The purification of recombinant His6 tagged Hc23 was carried out in columns of 10 × 1 cm (GE–Healthcare) loaded with Ni-NTA agarose resin (QIAGEN) and the protein (rHc23) was eluted with 100 and 250 mM imidazol. The nucleotide sequence of PCR products and the positive bacterial clones of E. coli XL2-Blue and BL21 were determined by the Department of Genetics (Faculty of Veterinary Science, UCM, Madrid) and Genomics Service (Servicios Generales de Apoyo a la Investigación, Universidad de La Laguna, Tenerife). The analysis of sequence similarity, domain identification and other features deduced from the DNA sequence were carried out with BLAST programs of the National Center for Biotechnology Information (www. ncbi.nlm.nih.gov/BLAST/). The EST datasets of H. contortus and other nematodes were analyzed using NEMBASE (www.nematodes.org/ nematodeESTs/nembase.html). For the design of primers and analyses of DNA sequences or amino acids, the program Gene Runner V3.1 and Primer 3 were used. Cloning, expression and immunization procedure with rHc23 has been protected [ES2453391 B2 (BOPI 14/8/2014); WO 2014/037592 A1 (13/3/2014)]. 2.3. Electrophoresis and peptide identification Analysis of expression of rHc23 by transformed E. coli BL21(DE3) was carried by 12.5% polyacrylamide electrophoresis under denaturing and reducing conditions (SDS-PAGE) and the gels stained with Coomassie Blue R250. Protein concentration was determined with Bradford method (Bradford, 1976). Conditions of 2D electrophoresis were those described by Fawzi et al. (2014). Briefly,
lyophilized samples of rHc23 were subjected to isoelectric focusing (pH 3–11) (1st dimension) and second dimension SDS-PAGE was run on homogeneous 12.5% T and 2.6% C polyacrylamide gels at room temperature, 100 V/gel for 2 h. 2D gels were stained following the colloidal Red Ponceau protocol. Electrophoresis equipment was from BioRad. 2D electrophoresis, MS and Peptide Mass Fingerprinting were carried out by the Proteomics Services of the UCM. Samples were digested (with trypsin and Staphylococcus aureus Endo V8) and homologies of mass maps were checked against Protein Prospector (http://prospector.ucs.edu) and Source Database: NCBI Resources, NIH, Bethesda MD, USA, Matrix Science, MASCOT (http://www.matrixscience.com).
2.4. Vaccination against Haemonchus contortus infection in lambs with rHc23 Female 4–5 months old Assaf lambs were obtained from a local producer (Finca la Mora, Pozuelo del Rey Madrid, Spain). At their arrival at our animal facilities coproscopical analysis showed no helminths eggs and a slight coccidial infection. All animals were treated with Borgal® 24% Sulphadoxine–Trimetoprim (Virbac, Spain) (5 mL/animal, 2 doses with 48 h interval). Lambs were maintained under H. contortus free conditions, fed with commercial pelleted food (Rubio Sanidad y Alimentación Animal, Madrid, Spain), hay and tap water ad libitum. Lambs were divided in a stratified manner (i.e. weight) into three comparable groups of seven lambs. To reduce the number of animals employed (3Rs recommendations) three groups of lambs (unvaccinated + challenged, Group 2; uninfected control group, Group 3; infected + challenged, Group 4) were kept as control groups for this and a parallel experiment (Fawzi et al., 2014). Animals from the present experiment (Group 1) were vaccinated with rHc23 (100 g/ injection) and aluminum hydroxide (Sigma) as adjuvant on days −42, −28 and −14. On day 0 lambs from groups G1, G2 and G4 were challenged with a single dose of 15,000 L3 of H. contortus administered with a bucoesophagic catheter. At the end of the experiment (45 days post challenge), all animals were slaughtered at a local abattoir (Villarejo de Salvanés, Madrid). Experimental design and procedures were approved by the Ethical Committee from the UCM.
2.5. Live weight, blood sampling, parasitological determinations and ELISA Live weight of the animals was monitored 3 times during the experimental period. Blood samples were obtained by jugular venipuncture in evacuated tubes. Packed cell volume (PCV) (%) and eosinophil counts were performed with standard laboratory techniques; serum samples were preserved at −20 ◦ C until used. Individual fecal samples were taken to determine the beginning of the patency and once a week afterwards. Eggs per gram of feces (epg) were determined with a modified McMaster technique (MAFF, 1971). All animals were slaughtered (45 days post challenge), the abomasum removed and adult worms were recovered following the technique described by Slotved et al. (1996). ELISA was carried out following Cuquerella et al. (1991) with rHc23 (1 g/mL) to coat 96-well microtiter plates using 0.05 M carbonate buffer, pH 9.6 (16 h, 4 ◦ C). After blocking (5% BSA in PBS) for 1 h at 37 ◦ C lambs’ sera 1/200 diluted with PBS-Tween were incubated for 1 h at 37 ◦ C. Second antibody (alkaline phosphatase-labeled rabbit anti-sheep IgG, Sigma–Aldrich) (1/32000) was used. Reaction was developed with 4 p-nitrophenil phosphate disodium salt hexahydrate (Sigma) (30 min, 37 ◦ C) and read at 405 nm.
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Fig. 1. (a) Electrophoretic (12.5% SDS-PAGE) analysis of the expression of rHc23 by transformed E. coli BL21with different concentrations of isopropyl--dthiogalactopyranoside (IPTG). Lane 1: molecular mass markers in kDa; lanes 2 and 3 non-induced bacterial cultures; cultures induced for 2 h (lane 4) and 4 h (lane 5) with 0.5 mM IPTG. Representative gel stained with Coomassie Blue. (b) Analysis by SDS-PAGE (12.5%) of the purification of recombinant His-6-tagged Hc23 from transformed solubilized extracts of E. coli BL21. Sonicates from bacterial cultures were loaded to a Ni-NTA column. Lane 1: molecular mass markers (kDa); lane 2: bacterial sonicate; lane 3: bacterial unbound fraction; lane 4: eluate with 100 mM imidazol. Gels were stained with Coomassie Blue. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
2.6. Statistical analysis Live weight gain and epg between groups were compared with repeated measures analysis of variance. Parasite burdens were compared by one way ANOVA and correlations between different parameters (i.e. fecal egg counts and PCV) were calculated. Level of significance was p < 0.05 and figures were done with GraphPad Prism V5 software.
3. Results 3.1. Cloning, expression and purification of recombinant Hc23 (rHc23) The recombinant pET-29 b (+) plasmids containing the Hc23 gene sequence (612 bp) isolated from the H. contortus cDNA library was used to transform cultures of E. coli BL21. Optimal overexpression of rHc23 in the system was achieved by the induction of bacterial cultures with 0.5 mM IPTG for 2 h (Fig. 1(a)) (lane 4). Electrophoretic analysis showed that rHc23 was efficiently solubilized with 0.15% sarcosyl and purified with Ni-NTA agarose column with 100 mM imidazol (Fig. 1(b)) (lane 4). MS analysis and peptide mass fingerprinting of rHc23 treated with trypsin, in solution and in gel, and Endo V8 allowed the determination of four peptides in solution, nine in gel and three after Endo V8 digestion. Fig. 2(a)) shows the alignment of the identified peptides with the rHc23 sequence obtained in solution. Alignment of amino acid (aa) sequences identified supported the homology of rHc23 to the purified native Hc23 (Fawzi et al., 2014) besides the His6 histidine tag from the expression and purification system. Recombinant protein (rHc23) was subjected to 2-D electrophoresis. Electropherograms showed a predominant spot of isoelectric point (Ip) of 6.71 and the expected molecular mass
Fig. 2. (a) Red Ponceau 2D electrophoretic analysis of rHc23. Arrows indicate spots (1–4) identified and analyzed by mass spectrometry (MS) and peptide finger printing (FP). MW: molecular mass markers in kDa. (b) Alignment of identified peptides by MS and FP in the 2D-electrophoresis (in bold) and the rHc23 sequence obtained in solution. Comparison with the deduced aminoacid sequence of Hc23 from Haemonchus contortus (Fawzi et al., 2014).
(24.15 kDa) (R1) (Fig. 2(b)). In addition three spots of comparable Ip and mass were detected. The spots obtained (R1, R2, R3 and R4) were analyzed by mass spectrometry (MS) and peptide fingerprinting. Trypsin cleavage of R1 and R2 yielded four similar peptide sequences; R3 yielded four sequences and R4, four sequences. All obtained sequences matched those from Hc23 this confirming that all spots corresponded to the same recombinant protein.
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Table 1 Haemonchus contortus adult burden in the abomasums of the experimental lambs (vaccinated with rHc23 and challenged with 15,000 L3) at the end of the experiment (day 45 post-challenge). Experimental animal no.
Number of female worms
15 16 17 18 19 20 21 Mean ± S.E.M Non vaccinated + challenged lambsa
Number of male worms
10 10 230 0 130 260 0 91.43 ± 43.39 765.71 ± 262.3
10 0 30 0 70 280 0 55.71 ± 38.59 264.29 ± 83.48
Total number of helminths 20 10 260 0 200 540 0 147.14 ± 76.86 1030 ± 333.85
Results are meansstandard error of the mean (S.E.M.). a Abomasal burden in matched non vaccinated + challenged lambs (Fawzi et al., 2014).
a
epg reduction (%)
100 80 60 40 20 0
3
4
5
6
Weeks post Challenge Fig.3. Reduction (%) of epg counts from individual lambs vaccinated with rHc23 and challenged with 15,000 L3 (Group 1) of Haemonchus contortus compared to mean epg value of unvaccinated and challenged animals (Group 2) at weekly intervals during the experiment. Each solid symbol corresponds to an animal (#1–#7); squares: average epg reduction of the group.
b
3.2. Vaccination trial with rHc23 against lamb haemonchosis Lambs vaccinated with rHc23 + aluminium hydroxide (Group 1) exhibited high levels of circulating specific antibodies on the day of challenge (day 0) and these levels were maintained for the duration of the experiment (not shown). The maximum average value of epg found in the Group 1 was 1495 epg. The global reduction (%) in fecal counts was 83.49% for Group 1 lambs compared to Group 2 animals (p = 0.01) (Fig. 3). Uninfected control lambs (Group 3) did not show eggs along the experiment. Table 1 shows the helminths found in the abomasum of experimental animals. Despite individual variations and the size of the groups vaccination with the recombinant protein provoked a significant reduction of helminth burdens of experimental lambs from Group 1 (84.7% reduction) (p = 0.04) when compared to the unvaccinated challenged animals (Group 2). Average number of helminths in vaccinated lambs was 147.14 ± 76.86 whereas unvaccinated and similarly challenged lambs reached 1030 ± 333.85 (Fawzi et al., 2014). Challenge infection of non vaccinated lambs (Group 2) elicited a significant fall of PCV values from week 3 post challenge onwards animals (p < 0.001) (Fig. 4(a)) reaching the lowest value on day 35th pi. However, Group 1 lambs remained within the normal physiological range and displayed peripheral eosinophil counts significantly higher on days 7,21,35 and 44 pi (p < 0.01–0.001) (Fig. 4(b)). Liveweight determination was limited but vaccinated lambs gained (p < 0.001) on average 5.5 kg/animal at the end of the experiment whereas non vaccinated and challenged animals (Group 2) lost on average 1.3 kg/animal.
Fig. 4. Packed cell volume (PCV) values (a) and peripheral eosinophil counts (b) of the lambs vaccinated with rHc23 (Group 1) at weekly intervals during the experiment. Values are means ± standard error (SEM). Grey line represents the mean values from non-vaccinated + challenged lambs (Group 2). *Statistically significant differences (p < 0.05– p < 0.001).
4. Discussion H. contortus Hc23 gene encodes a protein of 203 amino acids (aa) with a predicted molecular mass of ca. 23 kDa. The cloned and expressed recombinant product (rHc23) (Fig.2a) is homologous to the native Hc23 purified from adult H. contortus on the basis of the (aa) sequence determined by Edman’s degradation (AGLFAHHPPPECGLPPFVND) (García-Coiradas et al., 2009) and the peptides identified by Fawzi et al. (2014). The protein sequence of H. contortus showed 99% identity to the deduced sequence of CBG 17,730 (GenBank Accession number CDJ 88,397.1) and the protein of unknown function containing the DUF148 domain (CDJ 92,660.1), sequence frequently found in nematodes e.g. Loa, Caenorhabditis and Ascaris suum (Fig. 5). No significant homology with S/E proteins reported by Schallig et al. (1997) was observed considering
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Fig. 5. Sequence alignment of rHc23 and some related proteins from nematodes. Shaded areas correspond to aminoacidic identities. A: Hc23 from Haemonchus contortus; B: hypothetic protein CAEBREN 05254 (Caenorhabditis brenneri); C: protein CBR-ORA-1 (C. briggsae); D: antigen ORA-1 (Onchocerca).
molecular mass and number of aa, or N-terminal sequence. Moreover the sequence of rHc23 showed a 100% identity with the partial ˜ et al. (2000); Garcíaaa sequence identified by Domínguez-Torano Coiradas et al. (2010). Fecal eggs output in H. contortus infections is probably the best in vivo phenotypic marker of resistance or protection (Douch et al., 1996; Bakker et al., 2004). In our case, besides a slight lengthening of the prepatent period after challenge, vaccinated animals showed a reduction over 80% in epg counts. Similarly, vaccination with rHc23 elicited in our experiment a significant reduction (ca. 85%) in the number of adult H. contortus recovered from the abomasums as compared to non vaccinated and challenged animals (Group 2). These levels of reduction in both estimative parameters are in the range of values attained in vaccination trials with native antigens (e.g., Schallig and van Leeuwen, 1997; Newton and Munn, 1999; Knox et al., 2003; Piedrafita et al., 2012; Fawzi et al., 2014) and have never been achieved before with a recombinant product. Moreover vaccinated lambs did not show significant variations in PCV values and in spite of the experimental limitations their lw gain was significantly higher than that found in challenged animals although below (ca. 50%) the gain observed in matched uninfected and unchallenged lambs (Group 3). Considering all results, the expressed recombinant protein (rHc23) apparently elicits a significant protection against experimental H. contortus challenge in lambs with reduction of abomasal helminth burden (ca. 85%), fecal egg counts (over 80%) and lw loss at the end of the experiment. Vaccinated lambs did not show any significant PCV fall after challenge and peripheral eosinophilia was present in partially protected animals. As far as we know this is
the first immunization with a recombinant H. contortus antigen eliciting a significant protection in lambs. Moreover, since Hc23 is an exposed antigen animals could be naturally revaccinated under field conditions. Wide intra-group variations in epg and abomasal burdens were found in the vaccinated group (Group 1). These variations, common in infection + challenge and vaccination trials against Haemonchus and other trichostrongylids (Outteridge, 1993), could be due to the presence of “responders” and “non responders” animals within each group and the size of the experimental groups. The presence of these phenotypes could be a shortcoming of the vaccination with rHc23. However, it has been suggested that a natural antigen can be considered as control method when achieves 60% efficacies in 80% of the flock (Barnes et al., 1995). We are aware of the limitations of our experiment and studies are underway to evaluate the protective response induced by rHc23 with different immunization schedules (e.g. adjuvants, doses of rHc23, calendar of vaccinations) and more stringent conditions (e.g. trickle infection of lambs, different sheep breeds; higher number of animals). However, results obtained point towards the interest of this recombinant H. contortus protein (rHc23) in the non pharmacological control of haemonchosis. Conflict of interest No conflict of interest is present. Acknowledgements Elshaima Fawzi received a pre-doctoral fellowship from the Spanish Ministry of Science and Technology. Research was partially
Please cite this article in press as: Fawzi, E.M., et al., Vaccination of lambs with the recombinant protein rHc23 elicits significant protection against Haemonchus contortus challenge. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.04.029
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funded by the projects AGL2006-10589 and AGL2014-54049-R. The research is a contribution by UCM research group ICPVet (UCM 910993). 2D-PAGE and mass spectrometry identification were carried out in the Proteomics Facility UCM–PCM, a member of ProteoRed-ISCIII network. The authors thank J. Gil Estevão for his help in animal care. We are grateful to Dr. V. Valladares and Dr. E. Carmelo for their help and advice. Valuable suggestions from Dr. P. Girón (Faculty of Statistics, UCM) are acknowledged. References Aumont, G., Pouillot, R., Simon, R., Hostache, G., Varo, H., Barré, N., 1997. Parasitisme digestif des petits ruminants dans les Antilles Francaises. INRA Prod. Anim. 10, 79–89. Bakker, N., Vervelde, L., Kanobana, K., Knox, D.P., Cornelissen, A.W.C.A., de Vries, E., Yatsuda, A.P., 2004. Vaccination against the nematode Haemonchus contortus with a thiol-binding fraction from the excretory/secretory products (ES). Vaccine 22, 618–628. Barnes, E.H., Dobson, R.J., Barger, I.A., 1995. Worm control and anthelmintic resistance: adventures with a model. Parasitol. Today 11, 56–63. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cachat, E., Newlands, G.F.J., Ekoja, S.E., McAllister, H., Smith, W.D., 2010. Attempts to immunize sheep against Haemonchus contortus using a cocktail of recombinant proteases derived from the protective antigen, H-gal-GP. Parasite Immunol. 32, 414–419. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. ˜ Cuquerella, M., Gómez-Munoz, M.T., Alunda, J.M., 1991. Serum IgG response of Manchego lambs to infections with Haemonchus contortus and preliminary characterization of adult antigens. Vet. Parasitol. 38, 131–143. ˜ I.A., Cuquerella, M., Gómez-Munoz, ˜ Domínguez-Torano, M.A.T., Méndez, S., Fernández-Pérez, F.J., Alunda, J.M., 2000. Vaccination of Manchego lambs against Haemonchus contortus with a somatic fraction (p26/23) of adult parasites. Parasite Immunol. 22, 131–138. Douch, P.G., Green, R.S., Morris, C.A., McEewan, J.C., Windon, R.G., 1996. Phenotypic markers for selection of nematode resistant sheep. Int. J. Parasitol. 26, 899–911. Fawzi, E.M., González-Sánchez, M.E., Corral, M.J., Cuquerella, M., Alunda, J.M., 2014. Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths. Int. J. Parasitol. 44, 429–436. García-Coiradas, L., Angulo-Cubillán, F., Méndez, S., Larraga, V., de la Fuente, C., Cuquerella, M., Alunda, J.M., 2009. Isolation and immunolocalization of a putative protective antigen (p26/23) from adult Haemonchus contortus. Parasitol. Res. 104, 363–369.
García-Coiradas, L., Angulo-Cubillán, F., Valladares, B., Martínez, E., de la Fuente, C., Alunda, J.M., Cuquerella, M., 2010. Immunization against lamb haemonchosis with a recombinant somatic antigen of Haemonchus contortus (rHcp26/23). Vet. Med. Int. 2010, 1–8. Jackson, F., Coop, R.L., 2000. The development of anthelmintic in sheep nematodes. Parasitology 120, S95–S107. Kaminsky, R., 2003. Drug resistance in nematodes: a paper tiger or a real problem? Curr. Opin. Infect. Dis. 16, 559–564. Kaplan, R.M., 2004. Drug resistance in nematodes of veterinary importance: a status report. Trends Parasitol. 20, 476–481. Knox, D.P., Redmond, D.L., Newlands, G.F., Skuce, P.J., Pettit, D., Smith, W.D., 2003. The nature and prospects for gut membrane proteins as vaccine candidates for Haemonchus contortus and other ruminant trichostrongyloids. Int. J. Parasitol. 33, 1129–1137. MAFF Ministry of Agriculture, Fisheries and Food, 1971. Manual of Veterinary Parasitological Laboratory Techniques. HMSO, London. Newton, S.E., Munn, E.A., 1999. The development of vaccines against gastrointestinal nematode parasites, particularly Haemonchus contortus. Parasitol. Today 15, 116–122. Outteridge, P.M., 1993. High and low responsiveness to vaccines in farm animals. Immunol. Cell Biol. 71, 355–366. Piedrafita, D.P., de Veer, M.J., Sherrard, J., Kraska, T., Elhay, M., Meeusen, E.N., 2012. Field vaccination of sheep with a larval-specific antigen of the gastrointestinal nematode, Haemonchus contortus, confers significant protection against an experimental challenge infection. Vaccine 30, 7199–7204. Redmond, D.L., Knox, D.P., 2004. Protection studies in sheep using affinity-purified and recombinant cysteine proteinases of adult Haemonchus contortus. Vaccine 22, 4252–4261. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning, a laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory Press, New York, USA, pp. 5. 61-5.72, 5.83, 6. 2-6.62. Schallig, H.D., van Leeuwen, M.A., 1997. Protective immunity to the blood-feeding nematode Haemonchus contortus induced by vaccination with parasite low molecular weight antigens. Parasitology 114, 293–299. Schallig, H.D., van Leeuwen, M.A., Verstrepen, B.E., Cornelissen, A.W., 1997. Molecular characterization and expression of two putative protective excretory secretory proteins of Haemonchus contortus. Mol. Biochem. Parasitol. 88, 203–213. Slotved, H.C., Barnes, E.H., Bjørn, H., Christensen, C.M., Eriksen, L., Roepstorff, A., Nansen, P., 1996. Recovery of Oesophagostomum dentatum from pigs by isolation of parasites migrating from large intestinal contents embedded in agar-gel. Vet. Parasitol. 63, 237–245. Waller, P.J., Chandrawathani, P., 2005. Haemonchus contortus: parasite problem No: 1 from tropics- Polar Circle. Problems and prospects for control based on epidemiology. Trop. Biomed. 22, 131–137. Yanming, S., Ruofeng, Y., Muleke, C.I., Guangwei, Z., Lixin, X., Xiangrui, L., 2007. Vaccination of goats with recombinant galectin antigen induces partial protection against Haemonchus contortus infection. Parasite Immunol. 29, 319–326.
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Vaccination of lambs with the recombinant protein rHc23 elicits significant protection against Haemonchus contortus challenge Elshaima M. Fawzi 1 , María Elena González-Sánchez 1 , María Jesús Corral, José Ma Alunda ∗ , Montserrat Cuquerella Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain
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Article history: Received 13 February 2015 Received in revised form 28 April 2015 Accepted 30 April 2015 Keywords: rHc23 Recombinant antigen Haemonchus contortus Vaccination Lambs DNA Cloning Expression Sheep
a b s t r a c t Gene encoding a somatic protein of Haemonchus contortus (Hc23) known to confer significant protection against experimental haemonchosis has been cloned and expressed in a prokaryotic system. A cDNA library of H. contortus using the vector ZAP II was obtained. Full-length gene was amplified, cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in Ni-NTA column. Recombinant protein (rHc23) had 203 aminoacids and a molecular mass of 24.15 kDa. Recombinant protein (100 g/dose) with aluminum hydroxide as adjuvant was administered to 5–6 months age female Assaf lambs on days −42, −28 and −14. On day 0 animals were infected with 15,000 L3 of H. contortus. Vaccination with rHc23 elicited a significant protection against challenge, with > 80% reductions in both fecal egg counts and average abomasal parasite burdens at the end of the experiment (45 days post challenge) besides lack of variations in packed cell volume. Results support the feasibility of vaccination against lamb haemonchosis with a recombinant product from an exposed antigen. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Helminth infections are common in most domestic animals and are the rule in animal species such as ruminants exploited under extensive management systems in many regions of the world. Haemonchus contortus is a highly pathogenic blood feeding nematode of the abomasum of ruminants. Infections are responsible of weight loss, decrease fertility and wool and milk production in adult animals and 30–50% mortality in lambs and kids (Aumont et al., 1997). Haemonchosis, the infection caused by this parasite, is considered the most important helminth infection of small ruminants (Waller and Chandrawathani, 2005) representing ca. 15% of all gastrointestinal diseases of this livestock (http://www.fao. org). Practical control of haemonchosis has been mainly based on the use of anthelmintics. However, indiscriminate medication, lack of strategic planning, potential refuge populations in wild reservoirs and impracticality of pasture management in many regions of the world besides the biological potential of Haemonchus have
∗ Corresponding author at: Dpto. Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain. Tel.: +34 913943701; fax: +34 913943908. E-mail address: [email protected] (J.M. Alunda). 1 Both authors deserve the same credit as first authors.
resulted in widespread resistance to the major classes of available anthelmintics (Jackson and Coop, 2000; Kaminsky, 2003; Kaplan, 2004). Moreover, there is a growing social concern regarding the presence of residues with pharmacological activity in animal products. Natural or experimental repeated H. contortus infections elicit a protective response in many sheep breeds provided that animals are older than 6–8 months age; therefore vaccination against haemonchosis seems feasible. Immunoprophylactic potential of several “native” antigens of H. contortus (i.e., H11, H-gal-GP, E/S antigens, somatic antigens) has been remarkable in terms of reduction in helminth egg excretion and the abomasal worm burdens (e.g., Schallig and van Leeuwen, 1997; Newton and Munn, 1999; Cachat et al., 2010). Insofar, results obtained with native proteins could not be replicated with the recombinant counterparts (e.g., Schallig and van Leeuwen, 1997; Redmond and Knox, 2004; Yanming et al., 2007). Unfortunately vaccination with native proteins, even with low doses immunizations, would require a permanent source of adult helminths from infected hosts. Recently a purified somatic protein from adult H. contortus (Hc23) has been shown to elicit a substantial protection against experimental challenge (Fawzi et al., 2014). In this manuscript we present the cloning and expression in Escherichia coli of a recombinant form of this protein (rHc23). Vaccination with the recombinant protein adjuvanted
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with aluminum hydroxide induced a significant protection of lambs against the experimental challenge with H. contortus. 2. Material and methods 2.1. Parasites The H. contortus strain used was originally supplied by Merck, Sharp and Dohme (Madrid, Spain) and maintained for the last 20 years by serial passage in lambs (Department of Animal Health, UCM). Third stage larvae (L3) for experimental challenges were obtained by fecal culture at 26 ◦ C, baermanization of the feces and partial purification on filter paper (MAFF, 1971). Adult H. contortus for DNA extraction were obtained from the abomasums of monospecifically infected lambs. 2.2. Cloning, expression and purification of Hc23 Adult H. contortus (50–100 mg, both sexes) were employed to obtain total RNA (Chomczynski and Sacchi, 1987). Isolation of messenger RNA (mRNA) was performed with mRNA purification kit (Stratagene) and a H. contortus cDNA library was constructed using lambda ZAP cDNA® Synthesis kit (Stratagene). A DNA fragment encoding an incomplete form of p26/23 from previous experiments (García-Coiradas et al., 2010) was labelled with digoxigenin-dUTP, quantified with DIG DNA Labelling Kit (Roche) and used to screen the obtained cDNA H. contortus library (Sambrook et al., 1989). The gene specific primer (5 TCA GTC TTT CGC GGA CTT G 3) was used in combination with T3 cDNA library vector primer (5 AAT TAA CCC TCA CTA AAG GG 3) to amplify the gene encoding Hc23 in the positive colonies. The resulting fragment was cloned in pGEM-T easy vector (Promega) and the construct was used to transform E. coli XL2-Blue (Stratagene). Recombinant plasmids containing the rHc23 gene sequence were purified using plasmid miniprep kit, cloned in pET-29B and used to transform E. coli BL21(DE3) (Novagen). To assess the expression of Hc23, positive colonies were cultured in LB medium with 0.5–4 mM isopropyl--d-thiogalactopyranoside (IPTG) (Roche). The purification of recombinant His6 tagged Hc23 was carried out in columns of 10 × 1 cm (GE–Healthcare) loaded with Ni-NTA agarose resin (QIAGEN) and the protein (rHc23) was eluted with 100 and 250 mM imidazol. The nucleotide sequence of PCR products and the positive bacterial clones of E. coli XL2-Blue and BL21 were determined by the Department of Genetics (Faculty of Veterinary Science, UCM, Madrid) and Genomics Service (Servicios Generales de Apoyo a la Investigación, Universidad de La Laguna, Tenerife). The analysis of sequence similarity, domain identification and other features deduced from the DNA sequence were carried out with BLAST programs of the National Center for Biotechnology Information (www. ncbi.nlm.nih.gov/BLAST/). The EST datasets of H. contortus and other nematodes were analyzed using NEMBASE (www.nematodes.org/ nematodeESTs/nembase.html). For the design of primers and analyses of DNA sequences or amino acids, the program Gene Runner V3.1 and Primer 3 were used. Cloning, expression and immunization procedure with rHc23 has been protected [ES2453391 B2 (BOPI 14/8/2014); WO 2014/037592 A1 (13/3/2014)]. 2.3. Electrophoresis and peptide identification Analysis of expression of rHc23 by transformed E. coli BL21(DE3) was carried by 12.5% polyacrylamide electrophoresis under denaturing and reducing conditions (SDS-PAGE) and the gels stained with Coomassie Blue R250. Protein concentration was determined with Bradford method (Bradford, 1976). Conditions of 2D electrophoresis were those described by Fawzi et al. (2014). Briefly,
lyophilized samples of rHc23 were subjected to isoelectric focusing (pH 3–11) (1st dimension) and second dimension SDS-PAGE was run on homogeneous 12.5% T and 2.6% C polyacrylamide gels at room temperature, 100 V/gel for 2 h. 2D gels were stained following the colloidal Red Ponceau protocol. Electrophoresis equipment was from BioRad. 2D electrophoresis, MS and Peptide Mass Fingerprinting were carried out by the Proteomics Services of the UCM. Samples were digested (with trypsin and Staphylococcus aureus Endo V8) and homologies of mass maps were checked against Protein Prospector (http://prospector.ucs.edu) and Source Database: NCBI Resources, NIH, Bethesda MD, USA, Matrix Science, MASCOT (http://www.matrixscience.com).
2.4. Vaccination against Haemonchus contortus infection in lambs with rHc23 Female 4–5 months old Assaf lambs were obtained from a local producer (Finca la Mora, Pozuelo del Rey Madrid, Spain). At their arrival at our animal facilities coproscopical analysis showed no helminths eggs and a slight coccidial infection. All animals were treated with Borgal® 24% Sulphadoxine–Trimetoprim (Virbac, Spain) (5 mL/animal, 2 doses with 48 h interval). Lambs were maintained under H. contortus free conditions, fed with commercial pelleted food (Rubio Sanidad y Alimentación Animal, Madrid, Spain), hay and tap water ad libitum. Lambs were divided in a stratified manner (i.e. weight) into three comparable groups of seven lambs. To reduce the number of animals employed (3Rs recommendations) three groups of lambs (unvaccinated + challenged, Group 2; uninfected control group, Group 3; infected + challenged, Group 4) were kept as control groups for this and a parallel experiment (Fawzi et al., 2014). Animals from the present experiment (Group 1) were vaccinated with rHc23 (100 g/ injection) and aluminum hydroxide (Sigma) as adjuvant on days −42, −28 and −14. On day 0 lambs from groups G1, G2 and G4 were challenged with a single dose of 15,000 L3 of H. contortus administered with a bucoesophagic catheter. At the end of the experiment (45 days post challenge), all animals were slaughtered at a local abattoir (Villarejo de Salvanés, Madrid). Experimental design and procedures were approved by the Ethical Committee from the UCM.
2.5. Live weight, blood sampling, parasitological determinations and ELISA Live weight of the animals was monitored 3 times during the experimental period. Blood samples were obtained by jugular venipuncture in evacuated tubes. Packed cell volume (PCV) (%) and eosinophil counts were performed with standard laboratory techniques; serum samples were preserved at −20 ◦ C until used. Individual fecal samples were taken to determine the beginning of the patency and once a week afterwards. Eggs per gram of feces (epg) were determined with a modified McMaster technique (MAFF, 1971). All animals were slaughtered (45 days post challenge), the abomasum removed and adult worms were recovered following the technique described by Slotved et al. (1996). ELISA was carried out following Cuquerella et al. (1991) with rHc23 (1 g/mL) to coat 96-well microtiter plates using 0.05 M carbonate buffer, pH 9.6 (16 h, 4 ◦ C). After blocking (5% BSA in PBS) for 1 h at 37 ◦ C lambs’ sera 1/200 diluted with PBS-Tween were incubated for 1 h at 37 ◦ C. Second antibody (alkaline phosphatase-labeled rabbit anti-sheep IgG, Sigma–Aldrich) (1/32000) was used. Reaction was developed with 4 p-nitrophenil phosphate disodium salt hexahydrate (Sigma) (30 min, 37 ◦ C) and read at 405 nm.
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Fig. 1. (a) Electrophoretic (12.5% SDS-PAGE) analysis of the expression of rHc23 by transformed E. coli BL21with different concentrations of isopropyl--dthiogalactopyranoside (IPTG). Lane 1: molecular mass markers in kDa; lanes 2 and 3 non-induced bacterial cultures; cultures induced for 2 h (lane 4) and 4 h (lane 5) with 0.5 mM IPTG. Representative gel stained with Coomassie Blue. (b) Analysis by SDS-PAGE (12.5%) of the purification of recombinant His-6-tagged Hc23 from transformed solubilized extracts of E. coli BL21. Sonicates from bacterial cultures were loaded to a Ni-NTA column. Lane 1: molecular mass markers (kDa); lane 2: bacterial sonicate; lane 3: bacterial unbound fraction; lane 4: eluate with 100 mM imidazol. Gels were stained with Coomassie Blue. (For interpretation of the references to color in text, the reader is referred to the web version of this article.)
2.6. Statistical analysis Live weight gain and epg between groups were compared with repeated measures analysis of variance. Parasite burdens were compared by one way ANOVA and correlations between different parameters (i.e. fecal egg counts and PCV) were calculated. Level of significance was p < 0.05 and figures were done with GraphPad Prism V5 software.
3. Results 3.1. Cloning, expression and purification of recombinant Hc23 (rHc23) The recombinant pET-29 b (+) plasmids containing the Hc23 gene sequence (612 bp) isolated from the H. contortus cDNA library was used to transform cultures of E. coli BL21. Optimal overexpression of rHc23 in the system was achieved by the induction of bacterial cultures with 0.5 mM IPTG for 2 h (Fig. 1(a)) (lane 4). Electrophoretic analysis showed that rHc23 was efficiently solubilized with 0.15% sarcosyl and purified with Ni-NTA agarose column with 100 mM imidazol (Fig. 1(b)) (lane 4). MS analysis and peptide mass fingerprinting of rHc23 treated with trypsin, in solution and in gel, and Endo V8 allowed the determination of four peptides in solution, nine in gel and three after Endo V8 digestion. Fig. 2(a)) shows the alignment of the identified peptides with the rHc23 sequence obtained in solution. Alignment of amino acid (aa) sequences identified supported the homology of rHc23 to the purified native Hc23 (Fawzi et al., 2014) besides the His6 histidine tag from the expression and purification system. Recombinant protein (rHc23) was subjected to 2-D electrophoresis. Electropherograms showed a predominant spot of isoelectric point (Ip) of 6.71 and the expected molecular mass
Fig. 2. (a) Red Ponceau 2D electrophoretic analysis of rHc23. Arrows indicate spots (1–4) identified and analyzed by mass spectrometry (MS) and peptide finger printing (FP). MW: molecular mass markers in kDa. (b) Alignment of identified peptides by MS and FP in the 2D-electrophoresis (in bold) and the rHc23 sequence obtained in solution. Comparison with the deduced aminoacid sequence of Hc23 from Haemonchus contortus (Fawzi et al., 2014).
(24.15 kDa) (R1) (Fig. 2(b)). In addition three spots of comparable Ip and mass were detected. The spots obtained (R1, R2, R3 and R4) were analyzed by mass spectrometry (MS) and peptide fingerprinting. Trypsin cleavage of R1 and R2 yielded four similar peptide sequences; R3 yielded four sequences and R4, four sequences. All obtained sequences matched those from Hc23 this confirming that all spots corresponded to the same recombinant protein.
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Table 1 Haemonchus contortus adult burden in the abomasums of the experimental lambs (vaccinated with rHc23 and challenged with 15,000 L3) at the end of the experiment (day 45 post-challenge). Experimental animal no.
Number of female worms
15 16 17 18 19 20 21 Mean ± S.E.M Non vaccinated + challenged lambsa
Number of male worms
10 10 230 0 130 260 0 91.43 ± 43.39 765.71 ± 262.3
10 0 30 0 70 280 0 55.71 ± 38.59 264.29 ± 83.48
Total number of helminths 20 10 260 0 200 540 0 147.14 ± 76.86 1030 ± 333.85
Results are meansstandard error of the mean (S.E.M.). a Abomasal burden in matched non vaccinated + challenged lambs (Fawzi et al., 2014).
a
epg reduction (%)
100 80 60 40 20 0
3
4
5
6
Weeks post Challenge Fig.3. Reduction (%) of epg counts from individual lambs vaccinated with rHc23 and challenged with 15,000 L3 (Group 1) of Haemonchus contortus compared to mean epg value of unvaccinated and challenged animals (Group 2) at weekly intervals during the experiment. Each solid symbol corresponds to an animal (#1–#7); squares: average epg reduction of the group.
b
3.2. Vaccination trial with rHc23 against lamb haemonchosis Lambs vaccinated with rHc23 + aluminium hydroxide (Group 1) exhibited high levels of circulating specific antibodies on the day of challenge (day 0) and these levels were maintained for the duration of the experiment (not shown). The maximum average value of epg found in the Group 1 was 1495 epg. The global reduction (%) in fecal counts was 83.49% for Group 1 lambs compared to Group 2 animals (p = 0.01) (Fig. 3). Uninfected control lambs (Group 3) did not show eggs along the experiment. Table 1 shows the helminths found in the abomasum of experimental animals. Despite individual variations and the size of the groups vaccination with the recombinant protein provoked a significant reduction of helminth burdens of experimental lambs from Group 1 (84.7% reduction) (p = 0.04) when compared to the unvaccinated challenged animals (Group 2). Average number of helminths in vaccinated lambs was 147.14 ± 76.86 whereas unvaccinated and similarly challenged lambs reached 1030 ± 333.85 (Fawzi et al., 2014). Challenge infection of non vaccinated lambs (Group 2) elicited a significant fall of PCV values from week 3 post challenge onwards animals (p < 0.001) (Fig. 4(a)) reaching the lowest value on day 35th pi. However, Group 1 lambs remained within the normal physiological range and displayed peripheral eosinophil counts significantly higher on days 7,21,35 and 44 pi (p < 0.01–0.001) (Fig. 4(b)). Liveweight determination was limited but vaccinated lambs gained (p < 0.001) on average 5.5 kg/animal at the end of the experiment whereas non vaccinated and challenged animals (Group 2) lost on average 1.3 kg/animal.
Fig. 4. Packed cell volume (PCV) values (a) and peripheral eosinophil counts (b) of the lambs vaccinated with rHc23 (Group 1) at weekly intervals during the experiment. Values are means ± standard error (SEM). Grey line represents the mean values from non-vaccinated + challenged lambs (Group 2). *Statistically significant differences (p < 0.05– p < 0.001).
4. Discussion H. contortus Hc23 gene encodes a protein of 203 amino acids (aa) with a predicted molecular mass of ca. 23 kDa. The cloned and expressed recombinant product (rHc23) (Fig.2a) is homologous to the native Hc23 purified from adult H. contortus on the basis of the (aa) sequence determined by Edman’s degradation (AGLFAHHPPPECGLPPFVND) (García-Coiradas et al., 2009) and the peptides identified by Fawzi et al. (2014). The protein sequence of H. contortus showed 99% identity to the deduced sequence of CBG 17,730 (GenBank Accession number CDJ 88,397.1) and the protein of unknown function containing the DUF148 domain (CDJ 92,660.1), sequence frequently found in nematodes e.g. Loa, Caenorhabditis and Ascaris suum (Fig. 5). No significant homology with S/E proteins reported by Schallig et al. (1997) was observed considering
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Fig. 5. Sequence alignment of rHc23 and some related proteins from nematodes. Shaded areas correspond to aminoacidic identities. A: Hc23 from Haemonchus contortus; B: hypothetic protein CAEBREN 05254 (Caenorhabditis brenneri); C: protein CBR-ORA-1 (C. briggsae); D: antigen ORA-1 (Onchocerca).
molecular mass and number of aa, or N-terminal sequence. Moreover the sequence of rHc23 showed a 100% identity with the partial ˜ et al. (2000); Garcíaaa sequence identified by Domínguez-Torano Coiradas et al. (2010). Fecal eggs output in H. contortus infections is probably the best in vivo phenotypic marker of resistance or protection (Douch et al., 1996; Bakker et al., 2004). In our case, besides a slight lengthening of the prepatent period after challenge, vaccinated animals showed a reduction over 80% in epg counts. Similarly, vaccination with rHc23 elicited in our experiment a significant reduction (ca. 85%) in the number of adult H. contortus recovered from the abomasums as compared to non vaccinated and challenged animals (Group 2). These levels of reduction in both estimative parameters are in the range of values attained in vaccination trials with native antigens (e.g., Schallig and van Leeuwen, 1997; Newton and Munn, 1999; Knox et al., 2003; Piedrafita et al., 2012; Fawzi et al., 2014) and have never been achieved before with a recombinant product. Moreover vaccinated lambs did not show significant variations in PCV values and in spite of the experimental limitations their lw gain was significantly higher than that found in challenged animals although below (ca. 50%) the gain observed in matched uninfected and unchallenged lambs (Group 3). Considering all results, the expressed recombinant protein (rHc23) apparently elicits a significant protection against experimental H. contortus challenge in lambs with reduction of abomasal helminth burden (ca. 85%), fecal egg counts (over 80%) and lw loss at the end of the experiment. Vaccinated lambs did not show any significant PCV fall after challenge and peripheral eosinophilia was present in partially protected animals. As far as we know this is
the first immunization with a recombinant H. contortus antigen eliciting a significant protection in lambs. Moreover, since Hc23 is an exposed antigen animals could be naturally revaccinated under field conditions. Wide intra-group variations in epg and abomasal burdens were found in the vaccinated group (Group 1). These variations, common in infection + challenge and vaccination trials against Haemonchus and other trichostrongylids (Outteridge, 1993), could be due to the presence of “responders” and “non responders” animals within each group and the size of the experimental groups. The presence of these phenotypes could be a shortcoming of the vaccination with rHc23. However, it has been suggested that a natural antigen can be considered as control method when achieves 60% efficacies in 80% of the flock (Barnes et al., 1995). We are aware of the limitations of our experiment and studies are underway to evaluate the protective response induced by rHc23 with different immunization schedules (e.g. adjuvants, doses of rHc23, calendar of vaccinations) and more stringent conditions (e.g. trickle infection of lambs, different sheep breeds; higher number of animals). However, results obtained point towards the interest of this recombinant H. contortus protein (rHc23) in the non pharmacological control of haemonchosis. Conflict of interest No conflict of interest is present. Acknowledgements Elshaima Fawzi received a pre-doctoral fellowship from the Spanish Ministry of Science and Technology. Research was partially
Please cite this article in press as: Fawzi, E.M., et al., Vaccination of lambs with the recombinant protein rHc23 elicits significant protection against Haemonchus contortus challenge. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.04.029
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Please cite this article in press as: Fawzi, E.M., et al., Vaccination of lambs with the recombinant protein rHc23 elicits significant protection against Haemonchus contortus challenge. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.04.029
