International Journal for Parasitology xxx (2014) xxx–xxx

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Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths Elshaima M. Fawzi, María Elena González-Sánchez, María Jesús Corral, Montserrat Cuquerella, José M. Alunda ⇑ Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain

a r t i c l e

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Article history: Received 11 December 2013 Received in revised form 26 February 2014 Accepted 27 February 2014 Available online xxxx Keywords: Hc23 Haemonchus contortus Vaccination Lambs Native protein Immunochromatography Haemonchosis

a b s t r a c t A somatic protein from adult Haemonchus contortus (Hc23), the most abundant component in a low molecular weight fraction with known immunizing effect against experimental haemonchosis, has been purified by immunochromatography. The immunoprophylactic value of Hc23 was tested in groups of 5– 6 months old Assaf lambs using Al(OH)3 or Escherichia coli lipopolysaccharide + inactivated Propionibacterium acnes as adjuvant and the results compared with uninfected control, uninfected and challenged or infected and challenged lambs. Immunization with Hc23 in either adjuvant elicited significant reductions in fecal egg counts after challenge with 15,000 L3s (70.67%–85.64%, respectively) and reduced (67.1% and 86%) abomasal worm counts (45 days post-challenge). Immunized lambs displayed higher peripheral eosinophil counts, were less anaemic and had weight gains than challenged controls. The results suggest that the Hc23 antigen can induce a partially protective response against haemonchosis in lambs. Ó 2014 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.

1. Introduction The abomasal nematode Haemonchus contortus is a highly pathogenic blood feeding parasite of small ruminants, especially lambs. The L4 and adult stages of the parasite attach to the abomasal mucosa of the host, resulting in anemia which can be severe enough to cause death. Haemonchus contortus infections have a wide range of clinical courses from acute, often fatal, especially for young lambs, to chronic forms, particularly in older lambs and sheep. Mortality ranging from 30–50% has been reported in lambs and kids in acute cases (Aumont et al., 1997; Baker, 1997). Production losses in the chronic subclinical infection include weight loss, reduced weight gain, reproductive inefficiency (decreased fertility), reduction of wool growth and quality, and decline in the milk yield. Globally, haemonchosis is by far the most important nematode infection of small ruminants (Waller and Chandrawathani, 2005) and represents 15% of all gastrointestinal diseases of these species worldwide (http://www.fao.org). Control of the infection relies mainly on the use of anthelmintic drugs, although high levels of anthelmintic resistance (AR) have been described in the major parasites affecting ruminants (Echevarria ⇑ Corresponding author. Tel.: +34 913943701; fax: +34 913943908. E-mail address: [email protected] (J.M. Alunda).

et al., 1996; Waller, 1997; Kaplan, 2004). The appearance of AR has become a common phenomenon, including multiple drug resistance against the three major classes of anthelmintic drugs (benzimidazoles, imidothiazoles and macrocyclic lactones) that compromise successful chemotherapy (Jackson and Coop, 2000; Kaminsky, 2003; Coles et al., 2006; Wrigley et al., 2006). Among possible alternative strategies to control H. contortus, vaccine development is the most investigated method (Miller and Horohov, 2006). To date no commercial vaccines are available for any gastro-intestinal nematode (GIN). Several types of proteins have been used to immunize lambs or kid goats against GIN, using both hidden and exposed antigens (Ags). Hidden Ags are mainly enzymes from the gut of the parasite and are not recognized during infection. Vaccination induces high levels of antibodies (Abs) which probably neutralize these enzymes. Natural Ags are recognized during the infection and include excretory/secretory (ES), surface and somatic Ags and can be effective against both blood and non-blood feeding nematodes (Newton and Meeusen, 2003). An advantage of the so-called natural antigens is the possibility of natural boosting whilst being continuously infected on pasture. Variable results have been obtained in vaccination trials with Haemonchus contortus but some Ags have achieved notable protection levels. Thus Schallig and van Leeuwen (1997) found protection between 75% and 85% with an enriched fraction of ES Ags containing two proteins (15 and 24 KDa). Newton and Munn (1999)

http://dx.doi.org/10.1016/j.ijpara.2014.02.009 0020-7519/Ó 2014 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.

Please cite this article in press as: Fawzi, E.M., et al. Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.02.009

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achieved over 90% protection with H11 from L4 and adult worms and Smith et al. (2003) observed reductions in egg excretion and worm counts of 93% and 72% with H-gal-GP. Comparable values have been obtained more recently (Cachat et al., 2010). Lower values of protection (50%) have been obtained with cystine proteases (Bakker et al., 2004). Previous experiments showed that the low molecular weight fraction p26/23, obtained from soluble extracts of adult H. contortus, conferred notable protection against experimental haemonchosis in lambs provided that Freund’s adjuvant was employed (Domínguez-Toraño et al., 2000). However, this adjuvant is not acceptable and the protective components of this fraction were not identified with certainty. Our aim was to purify native somatic protein Hc23 from the p26/23 fraction and to determine its protective ability when formulated with aluminum hydroxide (Al (OH)3) or a bacterial immune modulator preparation (lipopolysaccharide of Escherichia coli (LPS) + inactivated Propionibacterium acnes) as adjuvant. 2. Materials and methods 2.1. Parasites The H. contortus strain used, originally supplied by Merck, Sharp and Dohme (Madrid, Spain), has been maintained for the last 20 years in the Department of Animal Health, Universidad Complutense de Madrid (UCM), Spain by serial passage in lambs. L3s were obtained by fecal culture at 26 °C, after baermanization of the feces and partial purification on filter paper (MAFF, 1971). 2.2. Purification of native protein Hc23 Adult H. contortus were obtained from the abomasums of lambs infected with a pure isolate of the parasite. Male and female worms in PBS were subjected to eight cycles of freezing/thawing ( 20 °C/ room temperature), homogenized in a glass-in-glass homogenizer and centrifuged (30,000g, 4 °C, 30 min; Klesius et al., 1984); the supernatant was recovered and stored at 80 °C (adult soluble extract, ASE) and the protein concentration was determined with the Bradford method (Bradford, 1976). Purification of native Hc23 protein from H. contortus was carried out by immunoaffinity chromatography using polyclonal Ab raised against a non-protective recombinant form of p26/23 (rHcp26/23) in rabbits (GarcíaCoiradas et al., 2010) following Fitzgerald et al. (2011). A 3 mL column of protein A agarose was prepared to purify 10–20 mg of Ab. The column was pre-equilibrated with five volumes of 100 mM Tris–HCl pH 8 buffer and the sample was clarified by centrifugation (10 min at 10,000g, 4 °C). The sample was loaded into the column, washed with 100 mM Tris–HCl pH 8 buffer and the bound immunoglobulins (Igs) eluted with 100 mM Glycine pH 3 and neutralized with 1 M Tris–HCl pH 8. The Ig-containing fractions were identified (by absorbance at 280 nm (A280)) and the purified Igs was dialyzed against PBS. The polyclonal Abs were obtained and the concentration was determined (Bradford, 1976). Purified Abs were mixed with buffer (0.2 M ammonium bicarbonate and 0.5 M NaCl pH 8.3) and were coupled to a N-hydroxysuccinimide (NHS) activated Sepharose HP column (GE Healthcare, USA) overnight at 4 °C. Uncoupled Abs were removed with blocking buffer (0.2 M Tris–HCl and 0.5 M NaCl pH 8.3) and the column was washed (0.1 M sodium acetate and 0.5 M NaCl pH 4). Haemonchus contortus ASE was loaded into the column and left overnight at 4 °C. The flow-through was collected and the column was washed with equilibration buffer (PBS) until A280 returned to the baseline. Protein was eluted by 0.1 M glycine-HCl pH 2.25 and neutralized with 2 M Tris–HCl, pH 8.6

(Subramanian, 2002). Representative fractions (flow-through, washing with PBS and fractions containing Hc23) were analyzed by PAGE under denaturing and reducing conditions (SDS–PAGE 12.5%) using 0.025 M Tris, 0.192 M glycine and 0.1% SDS as electrophoresis buffer. For two dimensional (2D) electrophoresis, lyophilized samples were resuspended in MilliQ water (200 lL), precipitated with 2D-Clean Up (GE Healthcare), centrifuged in acetone and resuspended in strip buffer (see below) up to a final concentration of 50 lg/sample. The 2D electrophoresis was performed using BioRad equipment. For the first dimension, 7 cm immobilized pH gradient (IPG) strips pH 3–11 NL (GE Healthcare) were used. They were hydrated, containing sample, with 7 M urea, 2 M thiourea, 4% (w/v) 3-((3-Cholamidopropyl)dimethylammonio)-1propanesulfonate, CHAPS), 100 mM DeStreak, and 2% pharmalytes at pH 3–11, overnight. Isoelectric focusing (IEF) was performed at 20 °C using the following program: 120 V for 15 min, 500 V for 15 min, 500–1000 V in gradient for 2 h, 1000–500 V in gradient for 2 h, and 5000 V for 2 h. Subsequently, strips were equilibrated for 12 min in reducing solution (6 M urea, 50 mM Tris–HCl at pH 6.8, 30% (v/v) glycerol, 2% (w/v) SDS, and 2% (w/v) DTT) and then for 5 min in alkylating solution (6 M urea, 50 mM Tris-HCl at pH 6.8, 30% (v/v) glycerol, 2% (w/v) SDS and 2.5% (w/v) iodoacetamide). The second-dimension SDS–PAGE was run on homogeneous 12.5% T and 2.6% C polyacrylamide gels. Electrophoresis was carried out at room temperature, 100 V/gel for 2 h. To visualize proteins, 2D: gels were stained following the colloidal Coomassie blue protocol. For western blot assays, 2D gels were transferred to a polyvinylidene difluoride (PVDF) membrane. After washing and blocking, the membrane was incubated with pooled sera from group III (GIII) lambs which had been diluted 1/ 100 for 3 h at 37 °C. The conjugate was horseradish peroxidase (HRP)-labeled donkey anti-sheep IgG (Sigma–Aldrich, USA) diluted 1/1000 (1 h at 37 °C). Color was developed with 4-chloro-1-naphtol (0.5 mg/mL). Molecular weight (MW) markers were from GE Healthcare and Bio-Rad (USA). 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). 2.3. Lambs and experimental design The experimental design and procedures were approved by the Ethical Committee of the UCM. Female 4–5 months old Assaf lambs were obtained from a local producer (Finca La Mora, Pozuelo del Rey, Madrid, Spain). Coproscopical analyses, carried out immediately after their arrival, showed a slight coccidial infection and all animals were treated with BorgalÒ 24% Sulfadoxine-Trimetoprim (Virbac, Spain) (5 mL/ animal i.m., two doses at 48 h intervals). Lambs were maintained under H. contortus-free conditions at the Faculty of Veterinary Medicine, UCM where they were fed commercial pellets (Rubio Sanidad y Alimentación Animal, Madrid, Spain), hay and tap water ad libitum. The lambs (34.8 ± 5.9 kg) were allocated to four groups of seven and one of six, and balanced for weight. Group I (GI) was immunized with Hc23 (three doses of 100 lg of Hc23 + 0.9 mL of Al(OH)3 gel colloidal suspension (13 mg/mL) (Sigma-Aldrich) on days 42, 28 and 14. GII was vaccinated with 100 lg of Hc23 and a bacterial adjuvant (LPS of E.coli + Propionibacterium acnes) (Lab. Calier, Spain) (1 mL/10 kg of live weight (lw)) per dose, on days 2, 0, 7 and 14 of the experiment) following the manufacturer’s recommendations. Immunizing doses were administered by i.m. and s.c. injections in the legs and the groin, respectively. GIII was unvaccinated and uninfected, GIV was unvaccinated but

Please cite this article in press as: Fawzi, E.M., et al. Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.02.009

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challenged and GV was infected (day 56: 10,000 L3s of H. contortus, via the oral route) and challenged; the primary infection of GV was terminated by anthelmintic treatment (Valbazen) on day 21. GI, GII, GIV and GV were challenged on day 0 of the experiment with 15,000 H. contortus L3s. All animals were killed 45 days after challenge and their abomasums removed for worm counting. The animals were weighed at the beginning and end of the trial and on the day of challenge.

KDa

MW

MW KDa 250 150 100

97

66

75

45

50

37

2.4. Parasitological determination, lw gain, blood sampling and ELISA Individual fecal samples were taken from the rectum on days 16, 18 and 21 p.i. and once each week afterwards. Fecal egg counts were determined using a modified McMaster technique (MAFF, 1971) and expressed as eggs per gram of feces (epg). Adult worms were recovered from the abomasal contents following the technique described by Slotved et al. (1996). Briefly, 100 mL of a 2% agar solution (Difco, Bacto-Agar, USA) was mixed with 100 mL from each sample of abomasal contents and transferred to a container filled with 0.9% saline solution, at 37 °C, overnight. Helminths (10%) recovered from the bottom of the container were taken, fixed in 5% formalin and preserved at room temperature until counted. Blood was collected from the jugular into Vacutainer tubes with or without anti-coagulant. Packed cell volume (PCV) (%), leukocyte and eosinophil numbers were determined using standard laboratory techniques. To obtain individual sera, blood was left to clot at room temperature and the sera harvested after centrifugation at 648g for 25 min, divided into aliquots and preserved at 20 °C until tested. ELISA conditions were those described by Cuquerella et al. (1991). ASE was employed at 5 lg/mL to coat 96-well microtiter plates using coating buffer (0.05 M carbonate buffer pH 9.6) for 16 h at 4 °C. BSA (5% in PBS) was used as blocking solution (for 1 h at 37 °C); sera were used at l/200 dilution in PBS-Tween (PBS-T) (1 h at 37 °C) and the conjugate (alkaline phosphataselabeled rabbit anti-sheep IgG, Sigma-Aldrich) was used at l/8000 dilution. Color was developed with 1 mg/mL of 4 p-nitrophenil phosphate disodium salt hexahydrate (Sigma–Aldrich) for 30 min at 37 °C. O.D. at 405 nm was measured with an Opsys MR microplate reader (Dynex Technologies, USA). 2.5. Statistical analysis The data obtained were analyzed with the SPSS statistical package version 15. Repeated measures ANOVA was used for comparisons of epg, PCV and lw gain. Parasite recovery data were compared by one way ANOVA and Bonferroni’s Multiple Comparison Test. Correlations between different parameters (i.e. fecal egg counts and PCV) were calculated. For all tests, P < 0.05 was set as the minimum level of significance. Figures of the vaccination trials were displayed with GraphPad Prism V5 software. 3. Results 3.1. Purification of native Hc23 The gel profiles of the fractions obtained during purification of Hc23 from adult H. contortus extracts (ASE) are shown in Fig. 1. Lane 6 shows the purified protein by immunochromatography with an apparent MW of 23 KDa and a faint band observed ca. 46 KDa. Characterization of the isolated protein yielded a nominal mass (Mr) of 21.61 and an pI of 6.5. Approximate protein yielding was ca. 1.8% of the total ASE. MS analysis and peptide mass fingerprinting of trypsin-treated purified Hc23 allowed the deter-

30 25

20

20.1 1

2

3

4

5

6

7

8

9

10

Fig. 1. Coomassie stained representative electrophoteric analysis (SDS–PAGE) of the purification process of Haemonchus contortus somatic protein Hc23 by immunochromatography. Lanes 1 and 10 show molecular mass markers (MW) in KDa. Lanes 2 and 3 show an analysis of a representative fraction of unbound H. contortus soluble extract (ASE; flow-through). Lanes 3 to 5 show representative fractions of non-specific proteins eluted with PBS (equilibration buffer) applied to the Sepharose column. Lane 6 is an analysis of a representative fraction containing purified Hc23 eluted with 0.1 M glycine-HCl pH 2.25. Lanes 7 to 9 show an analysis of representative fractions after elution with washing buffer (100 mM sodium phosphate, 1.5 M NaCl, pH 7.4).

mination of six peptides of amino acid sequences coincidental with the deduced sequence of HCC00515 (García-Coiradas et al., 2009) (42–50: K.CYHEQGLTR.D; 51–61: R.DLVETLPTEIR.R; 67–75: K.DALLPPPVR.K; 77–86: K.APEEVQEQFR.K; 77–87: K.APEEVQEQFRK.I; 118–129: K.EYNEFTAHIEDR.H). Analysis of the excised protein band treated with trypsin allowed the identification of four coincidental biomolecules (110–129: K.VLTGDNLKEYNEFTAHIEDR.H; 157–178: K.EKHDIIASLNEQAQEELFQVFK.L; 159–178: K.HDIIASLNEQAQEELFQVFK.L; 159–180: K.HDIIASLNEQAQEELFQVFKLR.H) whereas the digestion with Endo V8 from S. aureus yielded six homologous sequences (46–54: E.QGLTRDLVE.T; 68–79: D.ALLPPPVRKAPE.E; 68–80: D.ALLPPPVRKAPEE.V; 106– 118: E.LAQKVLTGDNLKE.Y; 144–155: E.AKAAYDKIAKLE.K; 158– 167: E.KHDIIASLNE.Q). To rule out the possibility of comigrating proteins being present in the isolated band, 2D-electrophoresis was carried out. Coomassie stained electropherograms showed seven spots with MW comparable with Hc23, the most prominent being N3 and N6 (Fig. 2). Moreover a faint spot (N1) was observed ca. 46 KDa. Individual spots were excised, subjected to trypsin digestion and the peptides identified. Peptide fingerprinting confirmed the homology of all spots with the deduced sequence of Hc23. Peptide sequences obtained overlapped those found in the previous analyses or extended the homology (Spot N0: 32–38: K.DIWKNWK.E, 103–110: K.KMNELAQK.V; spots N3, N4 and N6: 32–38: K.DIWKNWK.E; spots N3 and N4: 65–76: K.ISKDALLPPPVR.K; 89–100: K.IINDKTIPVEEK.H). Comparison of the predicted sequence of Hc23 and actual results obtained by MS and peptide mass fingerprinting of the purified protein by immunochromatography is shown in Fig. 3. The apparent homology of the purified Hc23 and the protein deduced was confirmed by western blotting. Native Hc23 was recognized by pooled sera from immunized lambs with native Hc23 + Al(OH)3 (Fig. 4A, lane 2) (GI), immunized lambs with native Hc23 + bacterial adjuvant (lane 3) and animals infected (10,000 L3s) and challenged (15,000 L3s) (lane 6), whereas no recognition was evident after a primary infection (lane 5). Uninfected control lambs did not show any reactivity (lane 4). 2D western blots with purified Hc23 also showed

Please cite this article in press as: Fawzi, E.M., et al. Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.02.009

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E.M. Fawzi et al. / International Journal for Parasitology xxx (2014) xxx–xxx pH MW

11

3

A

100

1

2

3

4

5

6

MW 66

75

45

N1 50

30

37

N0

N3 N2

N4

20.1

25

20

N5

N7 N6

Fig. 2. Coomassie stained two dimensional electrophoretic analysis of Haemonchus contortus somatic protein Hc23 isolated by immunochromatography. Arrows indicate spots (N0–N7) identified. MW, molecular mass markers in KDa.

B

pH MW

11

3

150 100 75

50

37

Fig. 3. Comparison of the deduced amino acid sequence of the somatic protein Hc23 from Haemonchus contortus and the results obtained by mass spectrometry and peptide mass fingerprinting. Identical residues are shaded gray. Unshaded bold residues have been previously determined by Edman degradation (García-Coiradas et al., 2009).

recognition by pooled sera from lambs vaccinated with native protein and Al(OH)3 (Fig. 4B). The pattern of reactivity matched the findings from 2D-electrophoresis of purified Hc23. In addition, vaccinated lambs reacted at the expected MW region of ASE of H. contortus whereas uninfected animals did not show any recognition (Fig. 4C). 3.2. Vaccination trial of lambs with native Hc23 3.2.1. Fecal egg output Determination of the fecal egg output (epg) of infected + challenged lambs (GV) confirmed the infectivity of the H. contortus isolate employed in the experiment (Fig. 5), the efficacy of the anthelmintic treatment and the immunizing effect of the primary infection. Thus, the prepatent period in this group ranged from days 16 to 18 p.i. during the primary infection whereas after challenge (15,000 L3s) this group showed a lengthening of the prepatent period compared with uninfected + challenged animals (GIV). Notably, lambs immunized with Hc23 (GI and GII) showed a similar pattern (P < 0.05, day 24 post-challenge) as infected and challenged animals; several lambs (#17 and 18, GII) started Haemonchus egg excretion on day 24 post-challenge. Peak epg counts were reached during the fourth week post-challenge in GI (2742 epg) and GII (1600 epg) while the non-infected and challenged group (GIV) reached the maximal values 24 days post-challenge with an average excretion of 10,100 epg. The reduction in fecal egg counts was ca. 70% in GI, 85% in GII and >90% in GV compared with unimmunized challenged lambs. Differences were significant (P < 0.01 – P < 0.05) from day 24 post-challenge onwards. Maximum epg values found in GV and GIV were similar after primary

25 20

C

2 1

ASE MW 97 66

45 30 20.1 14.4 Fig. 4. One and two dimensional western blot analysis of Hc23 from Haemonchus contortus. (A) Analysis of immune recognition of purified somatic protein Hc23 from Haemonchus contortus by rabbit hyperimmune serum against defective recombinant (r)Hcp26/23 (lane 1); pooled sera from vaccinated lambs with Hc23 + Al(OH)3 (GI) (lane 2); pooled sera from vaccinated lambs with Hc23 + bacterial adjuvant (GII) (lane 3); negative lamb sera (GIII) (lane 4); pooled sera from challenged animals (15,000 L3s) (GIV) (lane 5); pooled sera from infected and challenged lambs (GV) (lane 6). (B) Two dimensional western blot of Hc23 probed with pooled sera from GI lambs. (C) Western blot analysis of immune recognition of H. contortus soluble extract (ASE) by pooled sera from vaccinated lambs with Hc23 + Al(OH)3 (GI) (lane 1); lane 2: negative lamb sera. MW, molecular mass markers in KDa.

Please cite this article in press as: Fawzi, E.M., et al. Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.02.009

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Eggs per gram (epg)

15000 12000 9000 6000 3000 0

-8

-7

-6

-5

-4

-3

-2

-1

0

1

2

**

*

*

4

5

6

3

Weeks post-challenge Fig. 5. Fecal excretion of Haemonchus contortus eggs (eggs per gram of feces, epg) in four groups lambs during the experimental period.d: GI; j: GII; s: GIV; h: GV. Arrow indicates the day of challenge with 15,000 L3s. Values are means ± S.E.M. ⁄Statistically significant difference of GI and GII compared with GIV (P < 0.01).

infections administered in spite of the different infective doses given (10,000 and 15,000 L3, respectively). Lambs from the uninfected + unchallenged control group (GIII) did not show any parasite eggs during the entire experiment.

Packed Cell Volume (PCV) (%)

A

3.2.2. Packed cell volume (PCV) All groups challenged with Haemonchus displayed a significant reduction in PCV during the trial (Fig. 6A), especially in the unimmunized and challenged lambs (GIV). Those given the primary

46 40

*

34

*

*

*

3

5

6

28 22 16 10

-6

-4

-2

0

1

Weeks post-challenge

Eosinophils (10 3/ µL)

B 1.5

*

1.0

*

*

*

* 0.5

0.0

-6

-4

-2

0

1

3

5

6

Weeks post-challenge Fig. 6. Packed cell volume (PCV) values (A) and peripheral eosinophil counts (B) during the experimental period in the lamb groups (d: GI; j: GII; D: GIII; s: GIV; h: GV). Arrow indicates the day of challenge with 15000 L3s. Values are means ± S.E.M. ⁄Statistically significant difference of GI and GII compared with GIII and GIV (eosinophils) and with GIV (packed cell volume) (P < 0.01).

Please cite this article in press as: Fawzi, E.M., et al. Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.02.009

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higher levels from (P < 0.01 P < 0.001).

1.50

*

O.D. 405nm

1.25 1.00

*

*

*

0.75 0.50 0.25 0.00 -8

0

1

3

6

Weeks post-challenge Fig. 7. Specific serum IgG responses of groups of experimental lambs (d: GI; j: GII; D: GIII; s: GIV; h: GV) against Haemonchus contortus adult soluble extract.. The arrow indicates the day of challenge with 15,000 L3s. Values are means ± S.E.M. ⁄ Statistically significant difference of GI and GII compared with GIII and GIV (P < 0.01).

Live weight gain (kg)

12

8

4

0 -8

-4

0

6

Weeks post-challenge

Fig. 8. Live weight gain (kg) of groups of lambs during the experiment. GI: solid bars; GII: white bars; GIII: grey bars; GIV: horizontal striped bars; GV: vertical striped bars. Values are means ± S.E.M.

infection also showed a drop in PCV although this was less pronounced than that observed in the unimmunized and challenged lambs (GIV). A negative correlation was found between epg counts and PCV (r = 0.82). 3.2.3. Eosinophils Peripheral leukocyte counts were not related (P > 0.05) to the immunization and infection schedule during the experiment although all immunized groups and the infected + challenged lambs showed a slight increase (data not shown). More relevant was the peripheral eosinophil pattern found in the experimental groups (Fig. 6B). Eosinophil counts were related both to the Haemonchus challenge administered and the immunization schedule. Unimmunized and challenged lambs (GIV) only exhibited a transient non-significant rise at early prepatency (day 7 post-challenge) returning afterwards to pre-challenge levels and the values found in uninfected control animals (GIII). All other groups exhibited increased levels related to the immunization. Thus, lambs immunized with Hc23 + Al(OH)3 (GI) showed higher eosinophil counts (P < 0.05) before challenge (day 14) and vaccinated (GI, GII) and infected and challenged (GV) animals had significantly

day

7

post-challenge

onwards

3.2.4. Serum antibody response (ELISA) Fig. 7 shows the specific serum peripheral IgG response of lambs against H. contortus ASE. Immunized lambs (GI, GII) had increased levels on the day of challenge (day 0), particularly the animals immunized with Hc23 + Al (OH)3 (GI). Primary infection (GV) elicited a very low Ab response. After challenge differences found in the Ab responses were related both to the infection course and the adjuvant employed with Hc23. Animals adjuvanted with Al (OH)3 reached their maximum Ab levels on day 7 post-challenge and maintained this level until the end of the experiment. However, lambs immunized with the bacterial preparation (GII) increased specific IgG levels until the third week post-challenge whereas the infected + challenged lambs (GV) showed a slow and moderate increase in their anti-ASE IgG response. As expected, both uninfected control animals (GIII) and uninfected + challenged lambs (GIV) did not show any significant IgG-specific response. Ab responses of vaccinated animals were significantly higher than those found in control and uninfected + challenged lambs on days 0 (P < 0.01) and days 7, 21 and 42 post-challenge (P < 0.001). 3.2.5. Lw gain All lamb groups increased their average lw before challenge (Fig. 8). Lowest lw gain was found in the lambs subjected to the primary infection with 10,000 L3s (GV) whereas all other animal groups showed comparable weight gains. However, after challenge (15,000 L3s) the lw pattern was very variable. Uninfected control lambs (GIII) showed the highest gain at the end of the experimental period (week 6 post-challenge) (10 kg), this value being significantly different (P < 0.001) from those found in all other animals. By their part unvaccinated + challenged animals (GIV) exhibited a significant weight loss ( 1.3 kg) (P < 0.001) compared with the other experimental groups. Both vaccinated lamb groups (GI, GII) displayed significantly (P < 0.001) higher weight gains (6.5 and 5 kg, respectively) than infected + challenged animals (GV). This group, in spite of showing a clear recovery after the primary infection was administered, did not reach the values found in the immunized animals (3.2 kg lw gain). 3.2.6. Abomasal worm burdens Adult H. contortus burdens determined at the end of the experiment are shown at Table 1. Abomasal helminth burden was correlated (r = 0.86) to the last fecal egg counts (not shown). Wide variations were observed between animal groups and within each group. However, uninfected + challenged lambs (GIV) had the highest values (1030 ± 338.85 adults/animal) and no helminths were recovered from the abomasums of uninfected control animals (GIII). The highest reduction was found in infected + challenged animals (89.15% reduction) although vaccinated animals also showed lower values with both Al(OH)3 (GI: 67.17% reduction) (P > 0.05) and the bacterial (GII: 86.78% reduction) (P < 0.05) adjuvants. No clear differences in worm size were found among the experimental groups (not shown) and the female/male ratio of adult H. contortus ranged from 1: 0.62 (GI) to 1:0.34 (GIV) although the differences were not significant. 4. Discussion Several hidden Ags from H. contortus have been shown to induce protection against experimental or natural challenge with the nematode (e.g. Newton and Meeusen, 2003; Lejambre et al., 2008). However, information on the effect of purified isolated ‘‘exposed’’ Ags is comparatively scarce (e.g. Schallig and van

Please cite this article in press as: Fawzi, E.M., et al. Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.02.009

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E.M. Fawzi et al. / International Journal for Parasitology xxx (2014) xxx–xxx Table 1 Haemonchus contortus adult burden in the abomasums of the experimental lambs at the end of the experiment (day 45 post-challenge). Lamb Groups

Experimental animal No.

Number of female worms

Number of male worms

Total number of helminths

GI (a)

1 2 3 4 5 6 7 Mean ± S.E.M. 8 9 10 11 12 13 14 Mean ± S.E.M. 21 22 23 24 25 26 27 Mean ± S.E.M. 28 29 30 31 32 33 34 Mean ± S.E.M.

60 650 10 290 30 70 250 194.29 ± 86.51 0 170 10 40 220 150 10 85.71 ± 34.56 20 1010 1240 390 2010 400 290 765.71 ± 262.3 70 0 380 70 0 0 20 77.14 ± 51.85

40 450 30 110 0 30 190 121.43 ± 59.93 10 60 0 50 90 80 0 41.43 ± 14.38 30 430 610 170 420 80 110 264.29 ± 83.48 10 0 120 30 0 0 30 27.14 ± 16.28

100 1100 40 400 30 100 440 315.71 ± 145.44 10 230 10 90 310 230 10 127.14 ± 48.09c 50 1440 1850 560 2430 480 400 1030 ± 333.85b,d 80 0 500 100 0 0 50 104.29 ± 67.75c

GII(b)

GIV(c)

GV(d)

Superscript letters indicate a statistically significant difference (P < 0.05) between the group and the animal group designated by that superscript letter (GI, a; GII, b; GIV, c; GV, d)

Leeuwen, 1997; Piedrafita et al., 2012) in spite of their potential benefits, including natural re-vaccination under field conditions. A somatic fraction (p26/23) isolated from adult H. contortus was shown to elicit significant protection in 3.5-5-month-old lambs (Domínguez-Toraño et al., 2000). The most abundant protein present was isolated by affinity chromatography followed by SDS–PAGE, blotting and excision from the membranes (GarcíaCoiradas et al., 2009). This method permitted the purification of Hc23 although it was time consuming and the yield was very low. The immunoaffinity technique described here allowed purification of Hc23 from the H. contortus ASE in a single step and with higher yield. Sequencing and peptide mass fingerprinting by MS confirmed it as Hc23 (García-Coiradas et al., 2009). SDS–PAGE and western blots also showed a faint band around 46 KDa as described previously (Domínguez-Toraño et al., 2000; GarcíaCoiradas et al., 2009). 2D-electrophoresis also showed a spot in this MW region and sequence homology with Hc23 could indicate dimerization of the protein. It is worth mentioning that 2D analysis showed several forms of Hc23 with close pI and molecular mass values. It is possible that they represent isoforms of the protein. Peptide mass fingerprinting and 2D western blots supported their homology to Hc23. No identical or homologous protein to Hc23 has been found in databases of isolated proteins. However, the obtained sequence was almost identical to nucleotide base hypothetical proteins from H. contortus (CDJ92660.1 and CDJ88397.1: NCBI-Blast; U6P8378 and U6PFC7: Uniprot) and showed a 58% homology with the hypothetical protein NECAME_07527 from Necator americanus. In all cases the function of these DUF148 domain-containing proteins is unknown. Immunization of lambs with purified Hc23 induced significant protection against experimental haemonchosis using Al (OH)3 or a commercially available bacterial adjuvant (E. coli LPS + inacti-

vated Propionibacterium), taking into consideration fecal egg counts, anemia and weight gain. The reduction in egg counts (7085%) was similar to that reported previously (e.g. Schallig and van Leeuwen, 1997; Domínguez-Toraño et al., 2000) and was associated with higher eosinophil counts in peripheral blood. Similarly, vaccination with Hc23, particularly with the bacterial adjuvant, provoked reductions in abomasal worm counts (67–86%) comparable to those obtained with other Ags (e.eg. Smith et al., 2003; Cachat et al., 2010). Lambs vaccinated with Hc23 showed higher lw gains although the short duration of the experiment probably limits the value of this parameter. We are aware of the limitations of this pilot trial given that the experimental design excluded the possibility of undesired re-infection of lambs. Moreover both infection and challenge with H. contortus were administered in a single dose. Therefore we cannot anticipate that comparable levels of protection will be reached under natural conditions and experiments are planned to test it. In conclusion we have developed a better method for purifying a protective antigen (Hc23) from adult H. contortus, although the nature of the protective mechanism has yet to be determined. Much work is needed (e.g. refinement of vaccination dosing and schedule; role of adjuvants; field trials with large groups) but immunization of lambs with Hc23 could be a significant step towards alternative control of haemonchosis. Acknowledgements Elshaima M. Fawzi received a predoctoral fellowship from the Spanish Ministry of Science and Technology. Research was partially funded by the project AGL2006-10589. The research is a contribution from the Universidad Complutense de Madrid, Spain (UCM) research group 910993 ICPVet. 2D-PAGE and mass spectrometry

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Please cite this article in press as: Fawzi, E.M., et al. Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths. Int. J. Parasitol. (2014), http://dx.doi.org/10.1016/j.ijpara.2014.02.009