2A against Gastrointestinal Nematodes
Elshaima Mohamed Fawzi,aTeresa Cruz Bustos,bMercedes Gómez Samblas,bGloria González-González,bJenifer Solano,b María Elena González-Sánchez,aLuis Miguel De Pablos,bMaría Jesús Corral-Caridad,aMontserrat Cuquerella,aAntonio Osuna,b José María Alundaa
Departmento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spaina
; Institute of Biotechnology, Biochemistry and Molecular Parasitology Group, University of Granada, Campus Fuentenueva, Granada, Spainb
Seven 3-month-old, female, helminth-free lambs were immunized intranasally with three doses (1 mg total) of a recombinant
part of the catalytic region of the serine/threonine phosphatase 2A (PP2Ar) (group 1 [G1]). In addition, four lambs were used as
an adjuvant control group (G2), four as unimmunized, infected controls (G3), and four as unimmunized, uninfected controls
(G4). Fifteen days after the last immunization, lambs from G1, G2, and G3 were challenged with 10,000 larval stage 3 (L3)
organ-isms in a plurispecific nematode infection composed of ca. 40%
Trichostrongylus colubriformis
, 40%
Haemonchus contortus
, and
20%
Teladorsagia circumcincta
. All the lambs were clinically monitored throughout the experiment. Parasitological (fecal egg
output and immunological response), biopathological (packed-cell volume and leukocyte and eosinophil counts), and
zootech-nical (live-weight gain) analyses were conducted. On day 105 of the experiment, all the animals were slaughtered and the adult
worm population in their abomasa examined. Intranasal administration of PP2Ar with bacterial walls as an adjuvant elicited a
strong immune response in the immunized lambs, as evidenced by their humoral immune response. Immunized animals and
animals receiving the adjuvant shed significantly (
P
<
0.001) fewer numbers of parasites’ eggs in their feces. The immunization
significantly reduced the helminth burden in the abomasa by the end of the experiment (
>
68%), protection being provided
against both
Haemonchus
and
Teladorsagia
. Live-weight gain in the immunized lambs was similar to that in the uninfected
con-trols versus the infected or adjuvanted animal groups. Our results suggest that heterologous immunization of ruminants by
in-tranasal administration may be efficacious in the struggle to control gastrointestinal helminths in these livestock.
T
richostrongylidosis is a worldwide parasitic disease affecting
ruminants of all species. Infection by these helminths
pro-vokes digestive disturbances such as loss of appetite, diarrhea,
al-terations in energy and protein metabolism, and anemia,
accom-panied in severe cases by hypoproteinemia and edema. The
relative severity of the clinical signs and the outcome of the disease
depend on the host species, the age of the animals, the parasite
load, and the specific composition of the infection. In extensive
grazing systems, the general rule is a situation of mixed infections.
Control of the disease has been based almost exclusively on the use
of anthelmintics, but their massive and indiscriminate use has led
to the appearance of parasite isolates with resistance to most of the
drugs currently in use (
1
). In certain livestock-raising areas,
resis-tance levels have reached nearly 90% against some of the
anthel-mintics (
2
), implying that the average viable life of new
anthel-mintics is estimated to be around 10 years. Under these
conditions, alternative control methods should be explored,
among them immunoprophylaxis. Discovering a vaccine against
helminth parasites, which affect domestic animals as well as
hu-mans, thus adding economic damage to human suffering,
consti-tutes a major challenge for researchers. So far, vaccination against
gastrointestinal nematodes in ruminants has yielded only limited
success (
3
,
4
).
The success of vaccination systems, measured in terms of the
reduction and viability of the eggs excreted, yields values from
32% to 90% and a reduction in the adult population of up to 78%.
Their efficacy is determined not only by the type of antigen used
but also by the adjuvant and the way of administration. Greatest
efficacy has been achieved with vaccines that use irradiated larvae
(
5
), but the logistical problems involved in their production and
administration (
6
) have led to a search for native antigens or
re-combinant ones (
7
). The latter are easier to produce and
distrib-ute, and many of them are proteases, cysteine proteases,
metallo-proteases, aspartyl proteases (PEPs), all proteins from the
helminth intestine, and some with unknown biological functions
(
8–11
). Due to the homology in their sequences, some of these
antigens have proved to be effective against different nematode
species (
3
,
12
,
13
). In addition, most of the adjuvants used are oily
suspensions such as Freund’s adjuvant, Montanide, or Lipovant
(
14–16
). Freund’s adjuvant induces potentially serious, adverse
side effects, granulomes or ulcers at the inoculation site, and
therefore formulations with immunoadjuvant activity have been
sought (
17
). Other adjuvants, including plant saponins (
18
),
nanocapsules such as ISCOMs (
19
,
20
), and lipopolysaccharides
(LPSs) from bacterial walls or bacterial ghosts have been described
(
21
,
22
).
The immunization route is another key factor to be borne in
mind in the development of vaccines (
23
). Most vaccines are
ad-ministered either intramuscularly or subcutaneously, although
the administration of vaccines through the mucosa offers a
num-ber of major advantages, such as easy administration and the
re-duction of adverse side effects. Furthermore, the fact that many
Received23 May 2013Accepted3 June 2013
Published ahead of print12 June 2013
Address correspondence to José María Alunda, jmalunda@ucm.es. Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/CVI.00336-13
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pathogenic agents invade the host via mucosal barriers renders the
activation of immunity in the mucosa an attractive subject for
further study (
24
).
Mucosal immunization techniques have been assayed against
intestinal helminths and the larvae of tissue-dwelling helminths
(
25
). Recently, Solano-Parada et al. (2010) (
20
,
26
) obtained
promising results after intranasal immunization against
experi-mental angiostrongylosis, caused by
Angiostrongylus costaricensis
,
using a recombinant part of the catalytic region of
serine/threo-nine phosphatase 2A (PP2Ar) with bacterial walls as an adjuvant.
Serine/threonine protein phosphatase (PP2A) is an enzyme that
catalyzes the elimination of phosphate groups from the
phosphor-ylated proteins. It has been incriminated in many biochemical and
cellular processes such as cell motility, embryogenesis, and
differ-entiation (
28–30
) and is present in many nematode species (
20
,
31–34
). These characteristics make the catalytic region of PP2 a
good candidate for an antigen to be used in vaccines, especially
against parasites that must undergo morphogenic processes in the
definitive host before reaching maturity.
Our aim has been to explore the potential immunoprotective
value of a recombinant heterologous PP2Ar from
A. costaricensis
against a challenge with a multispecific infection by
trichostron-gylids in ruminants, which was administered intranasally to
acti-vate the mucosa, using the bacterial walls of
Escherichia coli
as an
innocuous adjuvant.
MATERIALS AND METHODS
Lambs and experimental design.Nineteen 3-month-old, female, hel-minth-free lambs (Manchego breed) were obtained from a local pro-ducer, kept in isolation pens in our facilities, and clinically monitored throughout the experiment. They were fed with commercial pelleted food (Superfeed, Spain), hay, and waterad libitum. The conditions were ap-proved by the Madrid Veterinary Faculty Committee for Animal Experi-mentation. The lambs were distributed into 4 stratified groups according to their live weight. They were immunized intranasally either with three doses (1 mg) of recombinant peptide (G1, 7 animals) or with the adjuvant at fortnightly intervals as an adjuvant control (G2, 4 lambs). In addition, 4 lambs were kept as unimmunized, infected control (G3) and 4 as unim-munized, uninfected control (G4). Fifteen days after the last immuniza-tion, lambs from G1, G2, and G3 were challenged with 10,000 larval stage 3 organisms (L3) of a plurispecific nematode infection composed of ca. 40%Trichostrongylus colubriformis, 40%Haemonchus contortus, and 20% Teladorsagia circumcincta. An infective dose was prepared using L3 ob-tained by coproculture (26°C, 10 days, and⬎80% relative humidity) of monospecifically infected lambs kept in our department.H. contortuswas obtained from Merck, Sharp & Dohme, Spain, in 1987 and maintained in our facilities by serial passage in donor lambs.T. colubriformisand Tela-dorsagia circumcinctawere originally supplied by the Moredun Research Institute (Edinburgh, Scotland) and maintained by serial infection in our department. All the animals were weighed at the beginning of the exper-iment and each week thereafter, blood and serum samples being taken at the beginning, after immunization, before the challenge, and at slaughter, as described below (Fig. 1).
Antigen and adjuvant: recombinant protein.We used a recombinant peptide corresponding to the catalytic region of the serine/threonine
pro-tein phosphatase (PP2A) expressed by a CT2-2 clone corresponding to the PP2Ar catalytic region ofA. costaricensis, produced as described elsewhere by Solano-Parada et al. (20). This fragment had the sequence VVDEFCT NHNIDLILRAHQITAEMVYGGYRIFAGGRLVTIFSAPNYQNMMND GCVMRIKRDLTANFIIFRPVVRRH. We began with the cloneTriplEx-2 -CT2-2, described elsewhere (20), and converted it to pTrip1Ex 2-CT2, which was sequenced with an ABI PrismTM BIGDYE sequencing kit (Ap-plied Biosystems, Foster City, CA, USA) using the forward primer 5=TCC GAGATCTGGACGAGC3=and the reverse primer 5=CCCTATAGTGAG TCGTATTA3=. The CT2-2 sequence was confirmed as corresponding to the catalytic region of genepph-1serine/threonine protein phosphatase (NCBI accession numberCAJ18121.1[23]). The cDNA was cleaved from the pTrip1Ex 2 with the restriction enzymes BamHI and HindIII and subcloned in pQE31 (Qiagen). TheE. colistrain used for the transforma-tion was Rosetta 2(DE3)pLysS (Novagen), which was replaced by tRNAs for 7 codons rarely used inE. coli(AGA, AGG, AUA, CUA, GGA, CCC, and CGG) and enhanced the expression of the eukaryote proteins that contain these codons. The recombinant protein in the form of inclusion bodies was purified from colonies isolated in LB plaques with ampicillin (100g/ml) and chloramphenicol (34g/ml) before being cultured for 12 h in 2⫻YT ampicillin-chloramphenicol broth. Recombinant produc-tion was induced with 0.5 mM IPTG (isopropyl--D
-thiogalactopyrano-side) for 3 h and then centrifuged at 4,000⫻gfor 10 min. The pellet was frozen at⫺20°C for at least 24 h, thawed in ice, and resuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM EDTA, 5 mM-mercaptoethanol, 0.35 mg/ml lysozyme, 8 U/ml Benzo-nase (Novagen), and 0.5% Triton X-100 before being incubated for 30 min at 20°C. This was followed by sonication with 6 cycles of 10 s at 200 to 300 W. The lysate was centrifuged again at 10,000⫻gfor 30 min at 4°C. The pellet obtained was washed three times with phosphate-buffered saline (PBS) and resuspended in sterile distilled water. Inclusion bodies were lyophilized and solubilized in 20 mM sodium phosphate buffer con-taining 8 M urea, 0.5 M Na Cl, 20 mM imidazole, and 1 mM -mercap-toethanol, pH 7. Purification of the purified protein was carried out by affinity chromatography with nickel-agarose, nickel-nitrilotriacetic acid (Ni-NTA) (Qiagen), previously equilibrated with 20 mM sodium phos-phate, 0.5 M NaCl, and 20 mM imidazole, pH 7.4. The sample was loaded, and the column was washed with 20 mM sodium phosphate, 0.5 M NaCl, and increasing concentrations of imidazole, from 10 mM to100 mM. A final elution was performed with 20 mM phosphate buffer with 8 M urea, 0.5 M NaCl, 500 mM imidazole, and 1 mM-mercaptoethanol, pH 7.4 (35). All fractions (uninduced, induced, purified, and nonpurified) were analyzed by 12.5% SDS-PAGE and stained with Coomassie brilliant blue dye (Fig. 2).
The recombinant protein was sequenced and identified at the Servicio de Proteómica del Centro de Biología Molecular Severo Ochoa (CBMSO) in Madrid, Spain. The relevant band from the SDS-PAGE was excised manually, along with the least possible quantity of gel, and digested auto-maticallyin situwith a robot digester (Bruker) using trypsin according to a protocol described elsewhere (36). The supernatant from the digestion (containing the peptides) was acidified with trifluoroacetic acid (final concentration, 0.1%) and dried in a Speed Vac (Thermo) before being resuspended in 0.1% trifluoroacetic acid with 33% acetonitrile. A 0.5-ml aliquot was placed on an AnchorChip plate (Bruker) using 2.5-dihy-droxybenzoic acid (DHB) as a matrix, to a concentration of 5 g/liter via the “fast evaporation” method. The plate was measured in an Autoflex matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometer (Bruker) equipped with a reflector. The mass spectra thus obtained were used as a peptide fingerprint to identify proteins in the database using search engines available on the Internet (MASCOT or ProFound).
The protein concentration of the PP2Ar solubilized in denaturizing buffer (6 M urea) was determined spectrophotometrically at 595 nm with the Bio-Rad protein assay. After electrophoresis in SDS-PAGE (12%), the separated proteins were transferred to a nitrocellulose membrane (Hy-FIG 1Design of the immunization experiment.
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bond-C Extra; GE) according to the method described by Towbin et al. (1979) (37). Blots were exposed to a pool of sera from immunized mice with the recombinant PP2A (tested at 1:50) for 2 h at 37°C, in order to verify the purification, followed by peroxidase-conjugated polyclonal goat anti-mouse immunoglobulins HRP (DakoCytomation) for 2 h at 37°C. The reaction was developed with 3-3=diaminobenzidine tetrahy-drochloride in 0.1 M Tris-HCl buffer (pH 7.2).
Nontransformed bacteria from the same strain were pelleted and fro-zen at⫺20°C for at least 24 h, thawed in ice, and resuspended in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 10 mM EDTA, 0.35 mg/ml lysozyme, 8 U/ml Benzonase (Novagen), and 0.5% Triton X-100 before being incubated for 30 min at 20°C. The suspension was sonicated with 6 cycles of 10 s at 200 to 300 W. The lysate was centri-fuged at 30,000⫻gfor 30 min at 4°C three times, and the pellet was lyophilized prior to being used as an adjuvant (20).
Sequence alignment.Using the sequence of PP2A fromA. costaricen-sisas the query sequence (NCBI accession numberCAJ18121.1) (23), we conducted a BLAST search against the nonredundant nucleotide data-base. The sequences fromA. costaricensisand those from CrPP2A from Caenorhabditis elegans(NCBI accession number NP_506609), CbPP2A fromCaenorhabditis briggsae(NCBI accession number XP_002637795) (38), TvPP2A fromTrichostrongylus vitrinus(NCBI accession number CAM84505) (34), OdPP2A fromOesophagostomum dentatum(NCBI
ac-cession number AAO85518) (33), TePP2A from Trichinella spiralis (NCBI accession numberABL14203) (39), BmPP2A fromBrugia malayi (NCBI accession number XP_001892306) (31), and AsPP2A fromAscaris suum(NCBI accession numberADY46840) (40) were then aligned and edited using ClustalW with GeneDoc programs.
Blood sampling and parasitological, biopathological, and immuno-logical determinations.During the experiment, the animals were daily observed for clinical monitoring and possible adverse reactions. Individ-ual coprological analyses were carried out with a modified McMaster technique (41). Fecal egg output values were log transformed to normalize the values used for statistical and graphic representations.
Throughout the experiment, blood samples were obtained by jugular venipuncture in evacuated tubes every 14 days. Packed-cell volume (PCV) and leukocyte and eosinophil counts were determined by standard labo-ratory techniques. Serum-specific antibody response was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, MaxiSorpTM 96-well microplates (Nalge Nunc Intl., Rochester, NY, USA) were coated with 10g/well of PP2Ar. The antigens were diluted in 0.1 M NaHCO3(pH
8.6) to give a concentration of 10g/well. Individual lambs’ sera were diluted 1:100 to 1:1,600 in carbonate buffer at pH 8.6, and the secondary antibody was anti-sheep IgG (whole molecule) peroxidase conjugate (Sigma) diluted 1:10,000. Absorbance was read at 492 nm (Multiskan Spectrum; Thermo Scientific).
On day 105 postchallenge, the animals were slaughtered at a local abattoir (Getafe, Madrid), whereupon the abomasa and small intestines were removed and taken to the laboratory under refrigeration. Individual abomasa were opened, and the mucosas and adult helminths in the con-tent were washed in cold PBS. A 10% aliquot of all the helminths recov-ered was fixed in 5% buffrecov-ered formalin, and the worms present were counted (males and females) under a stereomicroscope (Leitz).
Statistical analysis.Logarithmic transformation of the titers in sera and egg numbers was used for the graph representation and statistical analysis. The Tukey-Kramer multiple-comparisons test was used to esti-mate the significance of the difference between means. The results are indicated as mean values (standard errors of the mean of the different groups at different times for each experiment performed were deter-mined). APvalue of⬍0.001 was considered to be highly significant, and aPvalue of⬍0.01 was considered significant. GraphPad Instat v3.05 (GraphPad Software, Inc., La Jolla, CA, USA) software was used for the statistical analysis.
RESULTS AND DISCUSSION
After analyzing the recombinant protein sequence obtained and
carrying out a multiple alignment using the ClustalW program,
we confirmed the high homology of the sequence with that from
the catalytic region of serine/threonine phosphatase (PP2Ac) in
other species of nematodes, including some species of interest in
human and veterinary medicine, such as
Trichostrongylus vitrinus
,
Oesophagostomum dentatum
,
Trichinella spiralis
,
Brugia malayi
,
and
Ascaris suum
(
Fig. 3
). The catalytic subunits of PP2Ar (36
kDa) and PP2A (65 kDa) constitute the constant structural
sub-FIG 2SDS-PAGE analysis of purified PP2A. Lane 1, total proteins of the transformed bacteria; lane 2, PP2A band after affinity chromatography with nickel-agarose column purification; lane 3, recognition by the immune serum against the PP2Ar. Molecular mass is given in kDa.
FIG 3Multiple alignments (ClustalW2) of the sequence of the catalytic center of PP2A in different nematodes. Black indicates the positions with 100% conservation, while gray represents a decline in conservation. A rectangle marks the sequence of PP2A fromAngiostrongylus costaricensis. Ac,Angiostrongylus costaricensis; Cr,Caenorhabditis elegans; Cb,C. briggsae; Tv,Trichostrongylus vitrinus; Od,Oesophagostomum dentatum; Te,Trichinella spiralis; Bm,Brugia malayi; As,Ascaris suum. The positions with 100% conservation appear in black, while a gray scale indicates the gradient of this conservation.
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units of the enzyme core. Both subunits have only two isoforms,
but the third subunit, known as subunit b, has numerous families,
each of which in turn has multiple isoforms (
28
,
29
,
42
). PP2A is
ubiquitous, structurally conserved, and involved in many cell
pro-cesses (
43
), including the functioning of the cytoskeleton (
28
),
flagellum mobility (
35
), and cell cycle and meiosis (
44
). Götz et al.
(
45
) have also suggested its involvement in embryonic
develop-ment. Furthermore, it participates in the mechanisms of cell
sig-naling (
29
,
46
) and its deregulation gives rise to pathological
pro-cesses such as cancer (
43
,
47–49
).
The intranasal administration of a potential immunogenic
molecule with bacterial cell walls of
E. coli
was very effective under
our conditions since all the lambs in G1 showed significantly high
levels of IgG anti-PP2Ar after day 28 (before the second
vaccina-tion set) until the end of the experiment (
Fig. 4
).
Intranasal immunization has been used before both in
labora-tory mouse models (
24
,
50
,
51
) and in domestic animal species
(
52
), while bacterial components have also been used successfully
in immunization (
21
,
22
,
53
,
54
), but the combination of these
methods for nematode vaccination has not been tested except in a
recent experiment using the same protein, PP2Ar, against
A.
cos-taricensis
(
20
) in mice. Immunization provoked lower parasite
burdens and increased levels of interleukin-17 (IL-17) and specific
IgA. Recently, the synthesis of specific IgA has been found to be
dependent on the Th17 response by Peyer’s patches in the
intes-tine (
55
,
56
).
Our results confirmed the validity of this method of
immuni-zation and its potential use in ruminants. Moreover, the higher
antibody levels observed in vaccinated lambs could indicate a
massive liberation of membrane and intracellular antigen
result-ing from the death of the helminthes. The relatively low titers
found in the infected and adjuvanted group may indicate that the
native antigen is not an excreted antigen by the worms.
No clinical signs were observed in any of the challenged
ani-mals throughout the experimental period. The hematological
pa-rameters determined are shown in
Fig. 5
. PCV diminished slightly
in all the groups throughout the experiment (
Fig. 5
, top left),
although the reduction was within the physiological range and
unrelated to the infection status of the lambs. The lack of any
significant variation in the unimmunized, infected lambs (G2)
could be due to the relatively moderate burden of
H. contortus
in
the infective dose. Similarly, leukocyte counts in peripheral blood
did not show any variation (
Fig. 5
, top right). However,
eosino-phils displayed both infection- and immunization-related
behav-ior (
Fig. 5
, bottom). In spite of the wide variations found, the
immunized lambs had significantly higher levels than all the other
groups on days 70 (G2 and G3,
P
⬍
0.05; G1,
P
⬍
0.01), 84 (
P
⬍
0.05), and 97 (G3 and G4,
P
⬍
0.05; G2,
P
⬍
0.01)
postimmuni-zation. A rise in eosinophil counts is considered to be a distinct
characteristic of helminth infection (
57
), and eosinophilia has in
fact been related to the protection of lambs (
58–60
), although this
response is variable (
61
). The absence of clinical signs and notable
hematological variations could be related to the moderate
infec-tive dose administered.
Figure 6
shows the results of the fecal egg output along the
patent period of the infection. Unvaccinated and challenged
groups (groups 2 and 3) showed an increase in egg values along the
experimental period, whereas vaccinated lambs displayed a
pla-teau 8 weeks after challenge onwards. Significantly higher egg
val-ues were found in the nonvaccinated animals. However, as
ex-pected, there was a high variability among individual lambs from
each group. This finding is frequently observed in lambs given a
primary infection with gastrointestinal helminths, both in
natu-rally infected animals with plurispecific infections and in
con-trolled experiments with single-species administration (
9
). It has
been considered the expression of the “responder” and
“nonre-sponder” animal phenotype described in this host parasite system
with
T. colubriformis
(
62
) and
H. contortus
(
63
). In turn, this
in-tragroup variability makes necessary the transformation of egg
values for statistical analysis (
64
). In our experiment, the infective
dose was composed of three species from the genera
Haemonchus
,
Teladorsagia
, and
Trichostrongylus
. The reproductive capacity of
H. contortus
is much higher than that of the other two genera
employed, and therefore, fecal egg output is a poor estimation of
induced protection when plurispecific infections are employed.
Individual coprocultures were carried out only at the end of the
experiment (day 105 postinfection [p.i.]).
H. contortus
third-stage
larvae were the most commonly recovered, and only immunized
lambs showed a significant (
P
⬍
0.05) reduction of
Trichostrongy-lus
L3 obtained from the eggs compared to the adjuvant control
and unimmunized challenged groups (G2 and G3) (not shown).
More relevant were the results of helminth burdens and
live-weight gain. Adult burden provides a good estimation of induced
protection and has been extensively used in immunization trials
against gastrointestinal (GI) helminths. As expected, the recovery
of
Trichostrongylus
from the small intestine yielded inconsistent
results, which were excluded from further analysis. The specific
composition of the adult helminths in the abomasa of challenged
lambs varied widely. No absolute establishment rate (ER) could be
determined since all the lambs were slaughtered at the end of the
experimental period, and the possibility of some of them having
expelled part of the infective dose cannot be ruled out. The ER
found for
H. contortus
in the unimmunized challenged animals
was comparable to that found for other isolates (
65
,
66
) and
sim-FIG 4Serum antibody response (log titer) of lambs estimated by ELISA against PP2A throughout the experimental period.ⴙ, vaccinated;, unim-munized and challenged;Œ, adjuvant control group. Values are means⫾ standard errors.
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ilar to results obtained previously with this parasite stock (
67
), and
thus no conclusion could be reached. The most abundant species
in the three infected groups was
Teladorsagia circumcincta
(about
3 times more abundant than
Haemonchus
) in spite of the lower
number of infective larvae administered. One lamb (lamb 4) from
the immunized group (G1) and another from the unvaccinated
challenged group (G2) (lamb 9) did not show any
Haemonchus
in
their abomasa.
Under our conditions, immunization with PP2Ar led to a
re-duction in adult helminth burdens in the abomasum by the end of
the experiment. A protective effect (
P
⬍
0.05) was observed
against the total populations of
H. contortus
(78.33% reduction)
and
Teladorsagia circumcincta
(68.56% reduction) compared to
unimmunized challenged lambs (
Fig. 7
). Notably, these
protec-tion levels were achieved against two species of nematodes with
different feeding behaviors. Only lambs from G1, receiving
bacte-rial walls plus PP2Ar, displayed any significant reduction in the
50 15 0
38 44 50
V
ol
um
e (
%
)
10.2 12.6 15.0
x 10
9/ L
26 32
P
a
cked C
e
ll
V
5.4 7.8
Leukocyt
es
0 14 28 42 49 56 70 84 97
20
Days post Vaccination
0 14 28 42 49 56 70 84 97
3.0
Days post Vaccination
0 70
0.42 0.56 0.70
(10
3/ µ
L
)
0.14 0.28
E
o
si
nophi
ls
(
0 14 28 42 49 56 70 84 97
0.00
Days post Vaccination
FIG 5Variation in physiological parameters determined in the lambs throughout the experiment: packed-cell volume (PCV) (top left); leukocytes (top right); and eosinophils (bottom). Values were determined in peripheral blood. Symbols in top panels:⫹, vaccinated;Œ, unimmunized and challenged;Œ, adjuvant control group;p, unchallenged control animals. Code for bottom panel: black column, immunized; gray column, unimmunized and challenged; hatched column, adjuvant control group; clear gray column, uninfected control animals. For all panels, the arrow shows the day of challenge. Values are means⫾standard deviations.
FIG 6Fecal egg output (means⫾standard errors) throughout the patent period. Individual egg counts were log transformed. **, significant differences (P⬍0.01); ***, highly significant differences (P⬍0.001). White, vaccinated group (G1); gray, unimmunized and challenged group (G3); dark gray, adju-vant control group (G2).
FIG 7Adult helminth burden (means⫾standard deviations) from the abo-masa of experimental lambs. Numbers of adult worms from all the lambs were determined at the end of the experiment. Light gray, vaccinated animals; gray, nonvaccinated, nonadjuvanted animals; dark gray, adjuvanted animals. Hae-monchus,Haemonchus contortus;Teladorsagia,Teladorsagia circumcincta; F, female; M, male; ns, not significant. Values are means⫾standard deviations.
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adult burden of
Haemonchus
and
Teladorsagia
. However, the
ad-ministration of the adjuvant apparently elicited some degree of
protection against challenge since the parasite burdens in this
group (G3), although not significant, showed consistently lower
counts than unimmunized challenged lambs (G2). The
immuno-modulatory and immunostimulant properties of bacterial walls
have been described (
21
,
22
,
53
). In addition, exposure to
lipo-polysaccharides (LPSs) from bacterial walls induces the
produc-tion of nitrogen species and reactive oxygen as well as
proinflam-matory cytokines by macrophages (
68
). Since the need for
stimulation of mucosal response to induce protection in helminth
infections has been suggested, it is possible that the bacterial walls
elicited an unspecific activation of mucosal immunity in our
ex-periment. Some genes associated with the early inflammatory
re-sponse, including those encoding toll-like receptors (TLR2, -4,
and -9) or involved with free radical production (DUOX1 and
NOS2 A), are more abundantly expressed in lambs that are
resis-tant to
H. contortus
and
Trichostrongylus colubriformis
infections
(
69
). Further experimentation is needed, but this activation could
be responsible for the apparent reduction of helminth burdens in
nonvaccinated lambs receiving the adjuvant (G3).
Live-weight gain in lambs is an important zootechnical
param-eter of the “resistant” or “resilient” status of animals.
Figure 8
shows the live-weight gain in the four groups of lambs. Overall,
analysis showed that the immunized and uninfected control
lambs maintained similar weight gains throughout the
experi-ment. Nevertheless, infection led to weight loss in unimmunized
lambs 6 weeks p.i. compared to the uninfected controls (
P
⬍
0.05).
At 8 weeks p.i., the immunized lambs (G1) showed no difference
from the control group (G4) whereas the unimmunized ones (G2)
were significantly lighter (
P
⬍
0.05). This finding is corroborated
by the fact that no significant differences were found during week
0 of infection and preinfection (week
⫺
8) (not shown).
Vaccination of ruminants against GI helminths, particularly
trichostrongylids, has proved to be an elusive issue for decades (
4
).
Thus, in spite of numerous attempts with both “hidden” and
“ex-posed” antigens (
12
,
70
,
71
), there is no commercially available
vaccine against
Haemonchus
. Partial protection induced by native
forms of parasite proteins has not yielded results comparable to
those of recombinant products. Failures have been related to the
presence of nonresponder sheep and inappropriate glycosylation
or folding of recombinant proteins, besides our poor knowledge
of the relevant mechanisms of the sheep immune system (
71
).
With regard to other important gastric helminths infecting small
ruminants, both in terms of pathology and economics, the
iden-tification of native protective antigens is probably lacking (
72
). At
present, whereas conventional immunization procedures work
for
Ostertagia
in cattle, they are unable to elicit protective
re-sponses against the closely related genera
Teladorsagia
and
Tricho-strongylus
in sheep and goats (
73
).
Our results showed that PP2Ar administered intranasally with
E. coli
walls can elicit a partially protective response against
H.
contortus
and
Teladorsagia circumcincta
in lambs, reflected in the
similar weight gains of immunized animals and the notable
reduc-tion in the plurispecific helminth burden in the abomasum (over
68% at least). This protection, achieved with a recombinant
het-erologous protein, is related to mucosal immunization with the
adjuvant and the antigen (PP2Ar) and is expressed against two
different species of gastrointestinal nematodes. Anthelmintic
re-sistance is a widespread phenomenon (
2
,
74–76
), and the immune
protection elicited would reduce the need to medicate animals in
risk areas and seasons. As such, effector mechanisms, in particular
the role played by the bacterial adjuvant employed, should be
explored, but our results with the intranasal route and the
possi-bility of heterologous immunization against plurispecific
hel-minth infections are encouraging.
ACKNOWLEDGMENTS
This research was financed by a Spanish Ministry of Science and Technol-ogy grant (AGL2011-26098) and the “Programa de Ayudas para la Trans-ferencia de Resultados de Investigación” of the OTRI, University of Granada, Spain.
We thank B. Rojas for her technical assistance. We also thank J. Trout of the Scientific Translation Service of the University of Granada for re-vising and editing the English text.
The patent protecting these results is licensed exclusively to Bioor-ganic Research and Services SA.
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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 “(200
g/kg).”
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
