1 Manuscript vaccination against trichostrongylids
1 2 3
INTRANASAL IMMUNIZATION OF LAMBS WITH SERINE/THREONINE 4
PHOSPHATASE 2 A (PP2A) AGAINST GASTROINTESTINAL NEMATODES 5
6
Elshaima Mohamed Fawzi1, Teresa Cruz Bustos2, Mercedes Gómez Samblas 2 ,
7
Gloria González- González2, Jenifer Solano2, Mª Elena González-Sánchez1, Luis
8
Miguel de Pablos2, Mª Jesús Corral-Caridad1, Montserrat Cuquerella1 , Antonio Osuna2 9
& José Mª Alunda1.
10 11
1 Dpto. de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de
12
Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain. 13
2 Institute of Biotechnology, Biochemistry and Molecular Parasitology Group,
14
University of Granada, Edif. Mecenas, Campus Fuentenueva, 18071 Granada, Spain. 15
16
Author for correspondence: J.M. Alunda, Dpto. de Sanidad Animal, Facultad de 17
Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 18 Madrid, Spain. 19 Tel. +34 91 3943701 20 Fax: +34 91 3943908 21 E-mail: [email protected] 22 23 Abstract 24 25
Seven three-month-old, female, helminth-free lambs were immunized 26
intranasally with three doses (1 mg total) of a recombinant part of the catalytic region of 27
the serine/threonine phosphatase 2 A (PP2Ar) (G1). In addition, four lambs were used 28
as an adjuvant control group (G2), four as unimmunized, infected controls (G3) and 29
four as unimmunized, uninfected controls (G4). Fifteen days after the last 30
immunization, lambs from G1, G2 and G3 were challenged with 10,000 L3 of a 31
plurispecific nematode infection composed of ca. 40% Trichostrongylus colubriformis; 32
40% Haemonchus contortus and 20% Teladorsagia circumcincta. All the lambs were 33
clinically monitored throughout the experiment. Parasitological (fecal egg output and 34
Copyright © 2013, American Society for Microbiology. All Rights Reserved. Clin. Vaccine Immunol. doi:10.1128/CVI.00336-13
CVI Accepts, published online ahead of print on 12 June 2013
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2 immunological response), biopathological (packed-cell volume, leukocyte and
35
eosinophil counts) and zootechnical (live-weight gain) analyses were conducted. On day 36
105 of the experiment all the animals were slaughtered and the adult worm population 37
in their abomasa examined. Intranasal administration of PP2Ar with bacterial walls as 38
adjuvant elicited a strong immune response in the immunized lambs, as evidenced by 39
their humoral immune response. Immunized and animals receiving the adjuvant shed 40
significantly (p<0.001) numbers of parasites’ eggs in their feces. The immunization 41
significantly reduced the helminth burden in the abomasa by the end of the experiment 42
(> 68%), protection being provided against both Haemonchus and Teladorsagia. Live-43
weight gain in the immunized lambs was similar to that in the uninfected controls 44
versus the infected or adjuvanted animals groups. Our results suggest that heterologous 45
immunization of ruminants by intranasal administration may be efficacious in the 46
struggle to control gastrointestinal helminths in these livestock. 47
48
Key words: vaccination; serine/threonine phosphatase 2 A; PP2A; lambs; Haemonchus 49
contortus; Trichostrongylus colubriformis; Teladorsagia circumcincta; helminths.
50 51
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3 Introduction
52 53
Trichostrongylidosis is a worldwide parasitic disease affecting ruminants of all 54
species. Infection by these helminths provokes digestive disturbances such as loss of 55
appetite, diarrhea, alterations in energy and protein metabolism, and anemia, 56
accompanied in severe cases by hypoproteinemia and edema. The relative severity of 57
the clinical signs and the outcome of the disease depend on the host species, the age of 58
the animals, parasite load and the specific composition of the infection. In extensive 59
grazing systems the general rule is a situation of mixed infections. Control of the 60
disease has been based almost exclusively on the use of anthelmintics, but their massive 61
and indiscriminate use has led to the appearance of parasite isolates with resistance to 62
most of the drugs currently in use (1). In certain livestock-raising areas, resistance levels 63
have reached nearly 90% against some of the anthelmintics (2), implying that the 64
average viable life of new anthelmintics is estimated to be around 10 years. Under these 65
conditions alternative control methods should be explored, among them 66
immunoprophylaxis. Discovering a vaccine against helminth parasites, which affect 67
domestic animals as well as humans, thus adding economic damage to human suffering, 68
constitutes a major challenge for researchers. So far, vaccination against 69
gastrointestinal nematodes in ruminants has only yielded limited success (3, 4). 70
71
The success of vaccination systems, measured in terms of the reduction and 72
viability of the eggs excreted, yields values from 32% to 90% and a reduction in the 73
adult population of up to 78%. Their efficacy is determined not only by the type of 74
antigen used but also by the adjuvant and the way of administration. Greatest efficacy 75
has been achieved with vaccines that use irradiated larvae (5) but the logistical problems 76
involved in their production and administration (6) have led to a search for native 77
antigens or recombinant ones (7). The latter are easier to produce and distribute and 78
many of them are proteases, cystein proteases, metaloproteases, aspartyl proteases 79
(PEPs), all proteins from the helminth intestine, and some with unknown biological 80
functions (8-11). Due to the homology in their sequences some of these antigens have 81
proved to be effective against different nematode species (3, 12, 13). In addition, most 82
of the adjuvants used are oily suspensions such as Freund, Montanide or Lipovant (14-83
16). Freund’s adjuvant induces potentially serious, adverse side-effects, granulomes or 84
ulcers at the inoculation site, and therefore formulations with immunoadjuvant activity 85
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4 have been sought (17). Other adjuvants, including plant saponins (18), nanocapsules 86
such as ISCOMs (19, 20), LPSs from bacterial walls or bacterial ghosts have been 87
described (21, 22). 88
89
The immunization route is another key factor to be born in mind in the 90
development of vaccines (23). Most vaccines are administered either intramuscularly or 91
subcutaneously, although the administration of vaccines through the mucosa offers a 92
number of major advantages, such as easy administration and the reduction of adverse 93
side-effects. Furthermore, the fact that many pathogenic agents invade the host via 94
mucosal barriers renders the activation of immunity in the mucosa an attractive subject 95
for further study (49). 96
97
Mucosal immunization techniques have been assayed against intestinal 98
helminths and the larvae of tissue-dwelling helminths (24). Recently Solano-Parada et 99
al. (2010) (20, 25, 26) got promising results after intranasal immunization against 100
experimental angiostrongylosis, caused by Angiostrongylus costaricensis, using a 101
recombinant part of the catalytic region of serine/threonine phosphatase 2 A (PP2Ar) 102
with bacterial walls as an adjuvant. Serine/threonine protein phosphatase (PP2A) is an 103
enzyme that catalyzes the elimination of phosphate groups from the phosphorylated 104
proteins. It has been incriminated in many biochemical and cellular processes such as 105
cell motility, embryogenesis and differentiation (27-29) and is present in many 106
nematode species (20, 30-33). These characteristics make the catalytic region of PP2 a 107
good candidate for an antigen to be used in vaccines, especially against parasites that 108
must undergo morphogenic processes in the definitive host before reaching maturity. 109
110
Our aim has been to explore the potential immunoprotective value of a 111
recombinant heterologous PP2Ar from A. costaricensis against a challenge with a 112
multispecific infection by trichostrongylids in ruminants, which was administered 113
intranasally to activate the mucosa, using the bacterial walls of E.coli as an innocuous 114 adjuvant. 115 116 117 118
Materials and Methods 119
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5 120
Lambs and Experimental Design 121
Nineteen 3-month-old, female, helminth-free lambs (Manchego breed) were 122
obtained from a local producer, kept in isolation pens in our facilities and clinically 123
monitored throughout the experiment. They were fed with commercial pelleted food 124
(Superfeed, Spain), hay and water ad libitum. The conditions were approved by the 125
Madrid Veterinary Faculty Committee for Animal Experimentation. The lambs were 126
distributed into 4 stratified groups according to their live weigh. They were either 127
immunized intranasally with three doses (1 mg) of recombinant peptide (G1: 7 animals) 128
or administered intranasally with the adjuvant at fortnightly intervals as an adjuvant 129
control (G2: 4 lambs). In addition, 4 lambs were kept as unimmunized, infected control 130
(G3) and 4 as unimmunized, uninfected control (G4). Fifteen days after the last 131
immunization, lambs from G1, G2 and G3 were challenged with 10,000 L3 of a 132
plurispecific nematode infection composed of ca. 40% Trichostrongylus colubriformis, 133
40% Haemonchus contortus and 20% Teladorsagia circumcincta. An infective dose 134
was prepared using L3 obtained by coproculture (26°C, 10 days and >80% relative 135
humidity) of monospecifically infected lambs kept in our department. H. contortus was 136
obtained from Merck, Sharp & Dohme, Spain in 1987 and maintained in our facilities 137
by serial passage in donor lambs. T. colubriformis and Te. circumcincta were originally 138
supplied by the Moredun Research Institute, Edinburgh (Scotland) and maintained by 139
serial infection in our department. All the animals were weighed at the beginning of the 140
experiment and each week thereafter, blood and serum samples being taken at the 141
beginning, after immunization, before the challenge and at slaughter, as described below 142
(Figure 1). 143
144
Antigen and adjuvant 145
146
Recombinant Protein
147 148
We used a recombinant peptide corresponding to the catalytic region of the 149
serine/threonine protein phosphatase (PP2A) expressed by a CT2-2 clone corresponding 150
to the PP2Ar catalytic region of A. costaricensis, produced as described elsewhere by 151
Solano-Parada et al. (20). This fragment had the sequence: 152 VVDEFCTNHNIDLILRAHQITAEMVYGGYRIFAGGRLVTIFSAPNYQNMMNDG 153
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6 CVMRIKR DLT ANFIIFRPVV RRH. We began with the clone λ TriplEx- 2- CT2-2, 154
described elsewhere (20), and converted it to pTrip1Ex 2 – CT2, which was sequenced 155
with an ABI PrismTM BIGDYE sequencing kit (Applied Biosystems, Foster, USA) 156
using the forward primer 5´TCCGAGATCTGGACGAGC 3´ and reverse primer 157
5´CCCTATAGTGAGTCGTATTA 3´. The CT2-2 sequence was confirmed as 158
corresponding to the catalytic region of gene pph-1 serine/threonine protein phosphatase 159
(NCBI accession number CAJ18121.1 (23)). The cDNA was cleaved from the 160
pTrip1Ex 2 with the restriction enzymes BamH1 and HindIII and subcloned in pQE31 161
(Qiagen). The E. coli strain used for the transformation was Rosetta 2(DE3)pLysS 162
(Novagen), which was replaced by tRNAs for 7 codons rarely used in E. coli (AGA, 163
AGG, AUA, CUA, GGA, CCC, and CGG) and enhanced the expression of the 164
eukaryote proteins that contain these codons. The recombinant protein in the form of 165
inclusion bodies was purified from colonies isolated in LB plaques with ampicillin (100 166
µg/mL) and chloramphenicol (34 µg/mL) before being cultured for 12 h in 2xYT 167
ampicillin/chloramphenicol broth. Recombinant production was induced with 0.5 mM 168
of IPTG for 3 h and then centrifuged at 4000 x g for 10 min. The pellet was frozen at -169
20ºC for at least 24 h, thawed in ice and resuspended in lysis buffer containing 50 mM 170
Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM EDTA, 5 mM β-mercaptoethanol, 0.35 171
mg/mL lysozyme, 8 U/mL benzonase (Novagen) and 0.5% Triton X-100 before being 172
incubated for 30 min at 20ºC. This was followed by sonication with 6 cycles of 10 sec 173
at 200-300 W. The lysate was centrifuged against at 10,000 x g for 30 min at 4ºC. 174
The pellet obtained was washed three times with PBS and resuspended in sterile 175
distilled water. Inclusion bodies were lyophilized and solubilized in 20 mM sodium 176
phosphate buffer containing 8 M urea, 0.5 M Na Cl, 20 mM imidazole and 1 mM β- 177
mercaptoethanol, pH 7. Purification of the purified protein was carried out by affinity 178
chromatography with nickel-agarose, Ni-NTA (Qiagen), previously equilibrated with 179
20mM sodium phosphate, 0.5 M NaCl and 20 mM imidazole, pH 7.4. Sample was 180
loaded and the column was washed with 20mM sodium phosphate, 0.5 M NaCl and 181
increasing comcentrations of imidazole, from 10mM to100mM. Final elution was 182
performed with 20mM phosphate buffer with 8 M urea, 0.5 M NaCl, 500 mM imidazole 183
and 1 mM β- mercaptoethanol, pH 7.4. All fractions (uninduced, induced, purified and 184
non-purified) were analyzed by by 12.5% SDS-PAGE (Laemmli, 1970) (42) and stained 185
with Coomassie Brilliant Blue dye (Figure 2). 186 187
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7 The recombinant protein was sequenced and identified at the Servicio de 188
Proteómica del Centro de Biología Molecular Severo Ochoa (CBMSO) in Madrid, 189
Spain. The relevant band from the SDS-PAGE was excised manually, along with the 190
least possible quantity of gel, and digested automatically in situ with a robot digester 191
(Bruker) using trypsin according to a protocol described elsewhere (34). The 192
supernatant from the digestion (containing the peptides) was acidified with 193
trifluoroacetic acid (0.1% final concentration) and dried in a Speed Vac (Thermo) 194
before being resuspended in 0.1% trifluoroacetic acid with 33% acetonitrile. A 0.5-mL 195
aliquot was placed on an anchor-chip plate (Bruker) using 2.5-dihydroxybenzoic acid 196
(DHB) as a matrix, to a concentration of 5 g/liter via the “fast evaporation” method. The 197
plate was measured in an Autoflex matrix assisted laser desorption ionization–time of 198
flight (MALDI-TOF) mass spectrometer (Bruker) equipped with a reflector. The mass 199
spectra thus obtained were used as a peptide fingerprint to identify proteins in the 200
database using search engines available on the internet (Mascot, Profound). 201
Protein concentration of the PP2Ar solubilized in denaturizing buffer 6 M urea, was 202
determined spectrophotometrically at 595 nm with Bio-Rad Protein Assay).After 203
electrophoresis in SDS PAGE (12%) the separated proteins were transferred to 204
nitrocellulose membrane (Hybond™-c extra GE) according to the method described by 205
Towbin et al. (1979) (35). Blots were exposed to a sera pool from immunized mice 206
with the recombinant PP2A (tested at 1:50) for 2 h at 37ºC, in order to verify the of the 207
purification , followed by peroxidase conjugated (polyclonal goat anti-mouse 208
immunoglobulins HRP (DakoCytomation) for 2 h at 37ºC. The reaction was developed 209
with 3-3´diaminobenzidine tetrahydrochloride in 0.1M Tris-HCl buffer (pH 7.2). 210
Nontransformed bacteria from the same strain were pelleted and frozen at -211
20ºC for at least 24 h, thawed in ice and resuspended in lysis buffer containing 50 mM 212
Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM EDTA, 0.35 mg/mL lysozyme, 8 U/mL 213
benzonase (Novagen) and 0.5% Triton X-100 before being incubated for 30 min at 214
20ºC. Suspension was sonicated with 6 cycles of 10 sec at 200-300 W. The lysate was 215
centrifuged at 30,000 xg for 30 min at 4ºC, three times, and the pellet lyophilized prior 216
being used as adjuvant (20). 217 218 219 Sequence alignment 220
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8 Using the sequence of PP2A from A. costaricensis as the query sequence (NCBI 221
accession number CAJ18121.1) (23), we conducted a BLAST search against the non-222
redundant nucleotide database. The sequences from A. costaricensis, CrPP2A from 223
Caenorhabditis elegans (NCBI accession number NP_506609), CbPP2A from
224
Caenorhabditis briggsae (NCBI accession number XP_002637795) (36), TvPP2A from
225
Trichostrongylus vitrinus (NCBI accession number CAM84505) (33), OdPP2A from
226
Oesophagostomum dentatum (NCBI accession number AAO85518) (32), TePP2A from
227
Trichinella spiralis (NCBI accession number ABL14203) (37), BmPP2A from Brugia
228
malayi (NCBI accession number XP_001892306) (30) and AsPP2A from Ascaris suum
229
(NCBI accession number ADY46840) (38) were then aligned and edited using 230
ClustalW with GeneDoc programs. 231
232
Blood sampling, parasitological, biopathological, and immunological 233
determinations 234
Along the experiment the animals were daily observed for clinical monitoring 235
and possible adverse reactions. Individual coprological analyses were carried out with a 236
modified McMaster technique (39). Fecal egg output values were log transformed to 237
normalize values used for statistical and graphic representations. 238
Throughout the experiment blood samples were obtained by jugular 239
venipuncture in evacuated tubes every 14 days. Packed-cell volume (PCV), leukocyte 240
and eosinophil counts were determined by standard laboratory techniques. Serum-241
specific antibody response was determined by ELISA. Briefly, MaxiSorpTM 96-well 242
microplates (Nalge Nunc Intl.,Rochester, NY, USA) were coated with 10 μg/well of 243
PP2Ar. The antigens were diluted in 0.1M NaHCO3 (pH 8.6) to give a concentration of
244
10 μg /well. Individual lambs’ sera were diluted 1 : 100 to 1: 1600 in carbonate buffer at 245
pH 8.6 , the secondary antibody was anti sheep IgG (whole molecule) peroxidase 246
conjugate (Sigma) diluted 1 : 10,000. Absorbance was read at 492 nm (Multiskan 247
Spectrum, Thermo Scientific). 248
On day 105 post challenge the animals were slaughtered at a local abattoir 249
(Getafe, Madrid), whereupon the abomasa and small intestines were removed and taken 250
to the laboratory under refrigeration. Individual abomasa were opened and the mucosa 251
and adult helminths in the content washed in cold phosphate buffer saline. A 10% 252
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9 aliquot of all the helminths recovered was fixed in 5% buffered formalin and the worms 253
present counted (males and females) under stereomicroscope (Leitz). 254
255
Statistical Analysis 256
257
Logarithmic transformation of the sera titers and eggs numbers was used for the 258
graph representation and statistical analysis. Tukey–Kramer multiple comparisons test 259
was used to estimate the significance of the difference between means. The results are 260
indicated as mean values (standard errors of the mean of the different groups at different 261
times for each experiment performed were determined). P <0.001 was considered to be 262
highly significant (***) and P<0.01, significant (**). GraphPad Instat v3.05 (GraphPad 263
Software, Inc, La Jolla, USA) software was used for the statistical analysis. 264
265 266
RESULTS AND DISCUSSION 267
268
After analyzing the recombinant-protein sequence obtained and carrying out a 269
multiple alignment using the ClustalW program we confirmed the high homology of the 270
sequence with that from the catalytic region of serin threonin phosphatase (PP2Ac) in 271
other species of nematodes, including some species of interest in human and veterinary 272
medicine, such as Trichostrongylus vitrinus, Oesophagostomum dentatum, Trichinella 273
spiralis, Brugia malayi and Ascaris suum (Fig. 3). The catalytic subunits of PP2Ar of 36
274
kDa and PP2A of 65 kDa constitute the constant structural subunits of the enzyme core. 275
Both subunits have only two isoforms but the third subunit, known as subunit “b”, has 276
numerous families, each of which in turn has multiple isoforms (27, 28, 40). PP2A is 277
ubiquitous, structurally conserved (5) and involved in many cell processes (41), 278
including the functioning of the cytoskeleton (27), flagellum mobility (42) and cell 279
cycle and meiosis (43). Götz et al. (44) has also suggested its involvement in embryonic 280
development. Furthermore, it participates in the mechanisms of cell signaling (28, 45) 281
and its deregulation gives rise to pathological processes such as cancer (41, 46-48). 282
283
The intranasal administration of a potential immunogenic molecule with bacterial walls 284
of E.coli was very effective under our conditions since all the lambs in G1 showed 285
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10 significantly high levels of IgG anti-PP2Ar after day 28 (before the second vaccination 286
set ) until the end of the experiment (Fig. 4). 287
Intranasal immunization has been used before both in laboratory mouse models 288
(49-51) and in domestic animal species (52), whilst bacterial components have also 289
been used successfully in immunization (21, 22, 53, 54), but the combination of these 290
methods for nematode vaccination has not been tested except in a recent experiment 291
using the same protein, PP2Ar, against A.costaricencis (20) in mice. Immunization 292
provoked lower parasite burdens and increased levels of IL 17 and specific IgA. 293
Recently it has been demonstrated the dependence in the synthesis of specific IgA from 294
the Th17 response by Peyer's patches in the intestine (Hirota et al., 2013) (55, 56). 295
Our results confirmed the validity of this method of immunization and its 296
potential use in ruminants. Moreover, the higher antibody levels observed in vaccinated 297
lambs could indicate a massive liberation of membrane and intracellular antigen 298
resulting from the death of the helminthes. By its part, the relatively low titers found in 299
the infected and adjuvanted group may indicate that the native antigen is not an excreted 300
antigen by the worms. 301
No clinical signs were observed in any of the challenged animals throughout the 302
experimental period. The hematological parameters determined are shown in Figure 5. 303
PCV diminished slightly in all the groups throughout the experiment (Fig. 5a) although 304
the reduction was within the physiological range and unrelated to the infection status of 305
the lambs. The lack of any significant variation in the unimmunized, infected lambs 306
(G2) could be due to the relatively moderate burden of H. contortus in the infective 307
dose. Similarly, leukocyte counts in peripheral blood did not show any variation (Fig. 308
5b). However, eosinophils displayed both infection and immunization-related behavior 309
(Fig. 5c). In spite of the wide variations found, the immunized lambs had significantly 310
higher levels than all the other groups on days 70 (G2, G3: p<0.05; G1: p<0.01), 84 311
(p<0.05) and 97 post immunization (G3, G4: p<0.05; G2: p<0.01). A rise in eosinophil 312
counts is considered to be a distinct characteristic of helminth infection (57) and 313
eosinophilia has in fact been related to the protection of lambs (58-60), although this 314
response is variable (61). The absence of clinical signs and notable haematological 315
variations could be related to the moderate infective dose administered. 316
Figure 6 shows the results of the fecal egg output along the patent period of the 317
infection. Unvaccinated and challenged groups (Groups 2 and 3) showed an increase in 318
eggs values along the experimental period whereas vaccinated lambs displayed a 319
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11 plateau 8 weeks after challenge onwards. Significantly higher eggs values were found in 320
the non vaccinated animals. However, as expected, there was a high variability among 321
individual lambs from each group. This finding is frequently observed in lambs given a 322
primary infection with gastrointestinal helminths, both in naturally infected animals 323
with plurispecific infections as well as in controlled experiments with single-species 324
administration (9, 28, 54). It has been considered the expression of the “responder” and 325
“non-responder” animal phenotype described in this host parasite system with T. 326
colubriformis (62) and H. contortus (63). In turn, this intragroup variability makes
327
necessary the transformation of eggs values for statistical analysis (64) (Mukaratirwa 328
and Khumalo, 2010). In our experiment the infective dose was composed of three 329
species from the genera Haemonchus, Teladorsagia and Trichostrongylus. The 330
reproductive capacity of H. contortus is much higher than that of the other two genera 331
employed and, therefore, faecal egg output is a poor estimation of induced protection 332
when plurispecific infections are employed. Individual coprocultures were only carried 333
out at the end of the experiment (day105 pi). H. contortus third-stage larvae were the 334
most commonly recovered and only immunized lambs showed a significant (p<0.05) 335
reduction of Trichostrongylus L3 obtained from the eggs compared to the adjuvant 336
control and unimmunized challenged groups (G2 and G3) (not shown). 337
More relevant were the results of helminth burdens and live-weight gain. Adult 338
burden provides a good estimation of induced protection and has been extensively used 339
in immunization trials against GI helminths. As expected, the recovery of 340
Trichostrongylus from the small intestine yielded inconsistent results, which were
341
excluded from further analysis. The specific composition of the adult helminths in the 342
abomasa of challenged lambs varied widely. No absolute establishment rate (ER) could 343
be determined since all the lambs were slaughtered at the end of the experimental period 344
and the possibility of some of them having expelled part of the infective dose cannot be 345
ruled out. The ER found for H. contortus in the unimmunized challenged animals was 346
comparable to that found for other isolates (65, 66) and similar to results obtained 347
previously with this parasite stock (67), and thus no conclusion could be reached. The 348
more abundant species in the three infected groups was Te. circumcincta (about 3 times 349
more abundant than Haemonchus) in spite of the lower number of infective larvae 350
administered. One lamb (#4) from the immunized group (G1) and another from the 351
unvaccinated challenged group (G2) (#9) did not show any Haemonchus in their 352 abomasa. 353
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12 Under our conditions, immunization with PP2Ar led to a reduction in adult 354
helminth burdens in the abomasum by the end of the experiment. A protective effect 355
(p<0.05) was observed against the total populations of H. contortus (78.33% reduction) 356
and Te. circumcincta (68.56% reduction) as compared to unimmunized challenged 357
lambs (Figure 7). Notably, these protection levels were achieved against two species of 358
nematodes with different feeding behaviors. Only lambs from group G1 receiving 359
bacterial walls plus PP2Ar displayed any significant reduction in the adult burden of 360
Haemonchus and Teladorsagia. However the administration of the adjuvant apparently
361
elicited some degree of protection against challenge since the parasite burdens in this 362
group (G3) although not significant showed consistent lower counts that unimmunized 363
challenged lambs (G2). The immunomodulatory and immunostimulant properties of 364
bacterial walls have been described (21, 22, 53). In addition, exposure to 365
lipopolysaccharides (LPSs) from bacterial walls induces the production of nitrogen 366
species and reactive oxygen as well as pro-inflammatory cytokines by macrophages 367
(68). Since the need for stimulation of mucosal response to induce protection in 368
helminth infections has been suggested, it is possible that the bacterial walls elicited a 369
unspecific activation of mucosal immunity in our experiment. Some genes associated 370
with the early inflammatory response including those encoding toll-like receptors 371
(TLR2, 4 and 9) or involved with free radical production (DUOX1 and NOS2 A) are 372
more abundantly expressed in lambs that are resistant to H. contortus and 373
Trichostrongylus colubriformis infections (69). Further experimentation is needed but
374
this activation could be the responsible of the apparent reduction of helminth burdens in 375
non vaccinated lambs receiving the adjuvant (G3). 376
Live-weight gain in lambs is an important zootechnical parameter of the 377
“resistant” and “resilient” status of animals. Fig. 8 shows the live-weight gain in the 378
four groups of lambs. Overall, analysis showed that the immunized and uninfected 379
control lambs maintained similar weight gains throughout the experiment. Nevertheless, 380
infection led to weight loss in unimmunized lambs 6 weeks p.i. compared to the 381
uninfected controls (p<0.05). At 8 weeks p.i. the immunized lambs (G1) showed no 382
difference from the control group (G4) whereas the unimmunized ones (G2) were 383
significantly lighter (p<0.05). This finding is corroborated by the fact that no significant 384
differences were found during week 0 of infection and preinfection (week -8) (not 385 shown). 386
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13 Vaccination of ruminants against GI helminths, particularly trichostrongylids, 387
has proved to be an elusive issue for decades (4).Thus, in spite of numerous attempts 388
with both “hidden” and “exposed” antigens (12, 70, 71) there is no commercially 389
available vaccine against Haemonchus. Partial protection induced by native forms of 390
parasite proteins has not yielded comparable results with recombinant products. Failures 391
have been related to the presence of non-responder sheep, inappropriate glycosilation or 392
folding of recombinant proteins, besides our poor knowledge of the relevant 393
mechanisms of sheep immune system (71). With regard to other important gastric 394
helminths infecting small ruminants, both in terms of pathology and economics, the 395
identification of native protective antigens is probably lacking (72). At present, whereas 396
conventional immunization procedures work for Ostertagia in cattle, they are unable to 397
elicit protective responses against the closely related genera Teladorsagia and 398
Trichostrongylus in sheep and goats (73).
399
Our results showed that PP2Ar administered intranasally with E.coli walls can 400
elicit a partially protective response against H. contortus and Te. circumcincta in lambs, 401
reflected in the similar weight gain of immunized animals and the notable reduction in 402
the plurispecific helminth burden in the abomasum (over 68% at least). This protection, 403
achieved with a recombinant heterologous protein, is related to mucosal immunization 404
with the adjuvant and the antigen (PP2Ar) and is expressed against two different species 405
of gastrointestinal nematodes. Anthelmintic resistance is a widespread phenomenon (2, 406
74-76) and the immune protection elicited would reduce the need to medicate animals in 407
risk areas and seasons. As such, effector mechanisms, in particular the role played by 408
the bacterial adjuvant employed, should be explored but our results with the intranasal 409
route and the possibility of heterologous immunization against plurispecific helminth 410
infections are encouraging. 411
412 413
Acknowledgements 414
This research was financed by a Spanish Ministry of Science and Technology 415
grant (AGL2011-26098) and the “Programa de Ayudas para la Transferencia de 416
Resultados de Investigación” of the OTRI, University of Granada, Spain. The authors 417
thank B. Rojas for his technical assistance. They also thank Dr. J. Trout of the Scientific 418
Translation Service of the University of Granada for revising and editing their English 419 text. 420
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14 The patent protecting these results is licensed exclusively to Bioorganic
421
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19 Legends to figures:
663 664 665
Figure 1: Experimental design of the immunization experiment. 666
667
Figure 2: SDS-PAGE analysis of purified PP2A. Lane 1, total proteins of the 668
transformed bacteria; lane 2: PP2A band after affinity chromatography with nickel-669
agarose column purification; lane 3: Recognition by the immune serum against the 670
PP2Ar. Molecular weight in kDa.
671
Figure 3: Multiple alignments (ClustalW2) of the sequence of the catalytic center of 672
PP2A in different nematodes. Black indicates the positions with 100% conservation 673
while grey represents a decline in conservation. A rectangle marks the sequence of 674
PP2A from Angiostrongylus costaricensis Ac: Angiostrongylus costaricensis; Cr: 675
Caenorhabditis elegans; Cb: C. briggsae; Tv: Trichostrongylus vitrinus; Od:
676
Oesophagostomum dentatum; Te: Trichinella spiralis; Bm: Brugia malayi; As: Ascaris
677
suum. The positions with 100% conservation appear in black while a grey scale
678
indicates the gradient of this conservation 679
Figure 4: Serum antibody response (log titer) of lambs estimated by ELISA against 680
PP2A throughout the experimental period. + : vaccinated; ● : unimmunized and 681
challenged; ▲: adjuvant control group. Values are means ± standard error. 682
Figure 5: Variation in physiological parameters determined in the lambs throughout the 683
experiment: (a) packed-cell volume (PCV); (b) leukocytes; and (c) eosinophils. Values 684
were determined in peripheral blood. Symbols in Figures A and B: + : vaccinated; O 685
: unimmunized and challenged; ▲: adjuvant control group; : unchallenged 686
control animals. Symbols Figure C: black column: immunized; grey column: 687
unimmunized and challenged; hatched column: adjuvant control group; clear grey 688
column: uninfected control animals. Arrow shows day of challenge. Values are means ± 689
standard deviation 690
Figure 6: Fecal egg output (means ± standard error) along the patent period. Individual 691
egg counts were log transformed. **: significant (p<0.01) and ***: highly significant 692
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20 (p<0.001) differences. White color: vaccinated (G1); Gray color: unimmunized and 693
challenged (G3); Dark Gray color : adjuvant control Group (G2). 694
695
Figure 7: Adult helminth burden (means ± standard deviation) from the abomasa of 696
experimental lambs. Adult worms from all the lambs were determined at the end of the 697
experiment. Light Gray color vaccinated animals; Gray color non vaccinated non 698
adjuvanted animals; Dark Gray color adjuvanted animals Hc: Haemonchus contortus; 699
Telc: Teladorsagia circumcincta. F: female; M: male. Values are means ± standard 700
deviation. 701
Figure 8: Live-weight gain in lambs throughout the experimental period. Values are 702
means ± standard deviation. Vac: vaccinated; Inf: infected control group; Adj: treated 703
with the adjuvant and challenged; Control: non vaccinated non infected group. Dark- 704
grey column: before the infection; Broadly hatched column: half of the experiment; 705
Light-grey column: at end of experiment. 706 707 708 709 710 711 712 713 714 715 716
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Intranasal Immunization of Lambs with Serine/Threonine Phosphatase
2A against Gastrointestinal Nematodes
Elshaima Mohamed Fawzi, Teresa Cruz Bustos, Mercedes Gómez Samblas, Gloria González-González, Jenifer Solano, María Elena González-Sánchez, Luis Miguel De Pablos, María Jesús Corral-Caridad, Montserrat Cuquerella, Antonio Osuna, José María Alunda
Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain; Institute of Biotechnology, Biochemistry and Molecular Parasitology Group, University of Granada, Campus Fuentenueva, Granada, Spain
Volume 20, no. 9, p. 1352–1359, 2013. Page 1353, column 1, line 35: “(1 mg)” should read “(200g/kg).”
Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/CVI.00315-14
