Vaccine 30 (2012) 7199–7204

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Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Field vaccination of sheep with a larval-specific antigen of the gastrointestinal

nematode, Haemonchus contortus, confers significant protection against an
experimental challenge infection
D.P. Piedrafita a,b,∗ , M.J. de Veer a , J. Sherrard a , T. Kraska a , M. Elhay c , E.N. Meeusen a,b
a
Biotechnology Research Laboratories, School of Biomedical Sciences, Monash University, Vic 3800, Australia
b
The ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Vic 3800, Australia
c
Veterinary Medicine Research and Development, Pfizer Animal Health, 45 Poplar Rd, Parkville, Vic 3052, Australia

a r t i c l e i n f o a b s t r a c t

Article history: The availability of effective vaccines would add a valuable tool to the management of gastrointestinal
Received 2 May 2012 nematode infections in livestock. While some experimental vaccines have shown protection in laboratory
Received in revised form trials, few have been tested in the field. In the present study, eight month old sheep kept on pasture were
25 September 2012
treated with anthelmintic 8 weeks before vaccination with a larval surface antigen of the nematode
Accepted 5 October 2012
parasite, Haemonchus contortus, under a commercially acceptable protocol, i.e. 2 immunizations using a
Available online 27 October 2012
commercial adjuvant; they were then given a controlled challenge infection 4 weeks later in indoor pens.
Vaccination of sheep with 4 increasing doses of antigen resulted in significant reductions of 61% and 27%
Keywords:
Helminth vaccine
in cumulative faecal egg counts in the two highest dose groups, and a 69% reduction in worm burden in
Gastrointestinal nematodes the highest dose group. Blood loss, as determined by packed cell volume, was also significantly reduced
Parasites in the highest dose group of sheep. One outlier sheep showed an unusual increase in egg count without
Field trial a concomitant increase in worm burden compared to the control sheep, indicating a vaccine-induced
stress response.
Antigen-specific serum antibody levels steadily increased in sheep while on pasture and decreased
when transported to indoor pens. No difference in antibody levels could be detected between vaccinated
and unvaccinated sheep, but all showed increased antibody levels compared to uninfected control sheep
kept in indoors pens for 2–3 months, suggesting sheep were sensitized to the larval antigen either from
low dose pasture contamination or cross reaction with pasture-related antigens.
The results of these studies confirm the protective properties of the larval surface antigen and its
protective effect when vaccinations are performed in the field.
© 2012 Elsevier Ltd. All rights reserved.

1. Introduction
Haemonchus contortus is the most pathogenic of the gastroin-
testinal nematode parasites of sheep with a worldwide distribution.
As no vaccines are currently on the market against any of the
major helminth parasites affecting livestock, parasite control is
achieved by pasture management and regular anthelmintic treat-
ment. Heavy reliance on anthelmintics has however led to the
continuing development of drug resistance to each successive new
anthelmintic class and control of drug-resistant worms, particu-
larly multiple resistance, has now become a major threat to the
∗ Corresponding author at: School of Applied Sciences & Engineering, Building
2W, Rm: 203, Monash University, Churchill, Vic 3842, Australia.
Tel.: +61 3 5122 6205.
E-mail addresses: David.Piedrafita@monash.edu (D.P. Piedrafita),
els.meeusen@monash.edu (E.N. Meeusen).
viability of sheep farming in many regions in the world [1,2]. The
availability of effective anti-parasite vaccines would add a signif-
icant new weapon to the management and control of nematode
parasite infections in livestock, as well as providing a prototype for
human anti-helminth vaccine development.
As the adult stage of H. contortus ingests blood from the host,
several studies have used the ‘hidden antigen’ approach to tar-
get this process. In particular, vaccination with native, gut-derived
antigens from the parasite has been used very effectively in reduc-
ing parasite burden and pathology [3]. The difficulty in reproducing
these results with recombinant antigens has so far precluded the
commercial exploitation of these discoveries, although continuous
progress is made in this area [4].
Another approach for helminth vaccine development, is to tar-
get the infective larval stage of the parasite against which natural
immunity is directed [5]. By focusing on the local antibody response
generated in the lymph nodes draining the infected tissue (aboma-
sum or true stomach), we were able to identify and isolate a surface

0264-410X/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.vaccine.2012.10.019
7200 D.P. Piedrafita et al. / Vaccine 30 (2012) 7199–7204
antigen, specific to the infective larval stage (HcsL3) [6,7]. Anti-
bodies generated against HcsL3were able to mediate larval killing
by eosinophils in an in vitro assay [8] and vaccination with HcsL3
significantly reduced worm burden after challenge in a small exper-
imental pen trial [9].
Translation of experimental vaccine studies to the field is not
always successful as it has to comply with commercial reali-
ties, including fewer vaccinations with licensed adjuvants, and
using animals with varying levels of exposure in the field. In the
present study, we performed a larger dose-response trial with the
HcsL3antigen by vaccinating sheep in the field using a commercially
acceptable adjuvant and vaccination protocol. The results suggest
that sufficient protection against infection may be achievable with
this vaccine regimen under acceptable management practices.
2. Materials and methods
2.1. Preparation and formulation of the vaccine
A surface extract was prepared from exsheathed L3 as described
previously [6] with modifications. Briefly, L3 were exsheathed with
CO2 , viable larvae were separated from the sheaths by active migra-
tion through two 20 ␮m filters, then resuspended in PBS and placed
in a boiling water bath for 15 min. Larvae were pelleted by cen-
trifugation and the supernatant, containing the surface extract,
was depleted of small MW molecules and concentrated in one
step using 50 kDa cut-off Centricon Centrifugal Filter Units (Mil-
lipore). This resulted in one dominant, typically broad 75–90 kDa
band on coomassie-stained PAGE which reacted strongly with
the HcsL3-specific mAb Hc22 (not shown). Comparative analysis
against standard dilutions of bovine serum albumin gave an esti-
mate of 0.2 mg HcsL3recovered from 1 million L3 larvae. However,
as the antigen stains very poorly with coomassie (and not at all with
silver stain) [6], this is likely a serious underestimate and vaccine
doses were therefore expressed as L3-equivalents i.e. the antigen
recovered from 200 K (Group A), 20 K (Group B), 2 K (Group C) or
0.2 K (Group D) processed L3 larvae.
The adjuvant was a commercial preparation of Rehydrogel® LV
Aluminium Hydroxide (Reheis Inc.) in a liquid colloid. The alu-
minium hydroxide was adjusted to a concentration of 1 mg/ml
aluminium in the final vaccination dose and mixed thoroughly with
the antigen on an automated rotator for 1 h at room temperature.
Each vaccine dose was contained in 1 ml solution. Enough vaccine
was prepared for 2 immunization doses, and the second dose was
stored at 4 ◦ C until used. One trial group (control) was given the
same adjuvant preparation without antigen.
2.2. Experimental animals, immunization and challenge protocol
Merino wethers were raised and maintained on pasture at
a Woodend farm (Northern Victoria). At 8 months of age (week
−14), fifty sheep were selected and treated with a long-acting
anthelmintic, Cydectin® , to remove any existing parasites. Faecal
egg counts were taken at 8 and 10 months of age and at 8 weeks
after Cydectin treatment and revealed low counts throughout the
grazing period (<100 eggs per gram) and no egg counts after
treatment. At week −8, sheep were randomized and allocated to
5 experimental groups (n = 10) based on stratified body weight
ranking, bled for pre-vaccination serum and given their first immu-
nization dose by subcutaneous injection in the neck region. Four
weeks later (week −4), they were given their second immuniza-
tion and bled one week later (week −3) for the post-vaccination
serum. Two weeks after the second immunization (week −2),
they were transported and housed in a large indoor shed at the
Monash University experimental Werribee farm. After two weeks
acclimatization (week 0), they were infected twice with 5000 L3 on
day 0 and day +5. The H. contortus strain, Haecon-5, was used for
infection. This strain was isolated in 2006 from the field by Novartis
Animal Health, and shown to be more pathogenic than the previ-
ously used laboratory strains; it was kindly provided by the late
Paul Presidente. Two sheep died on pasture and one indoors due to
causes unrelated to the trial.
2.3. Parasitological, serological and haematological
measurements
Faecal egg counts (FECs) were assessed between 21 and 59 days
post infection (dpi) according to the modified McMaster method
and expressed as eggs per gram (EPG) faeces. Egg counts were
determined for each collection day, and cumulative mean egg
counts (cEPG) were calculated by adding all EPG values for each
sheep over the whole collection period. Sheep in the control and
high dose groups were killed at 63 dpi for worm recovery and num-
bers of worms counted as described previously [9]. Briefly, the
stomach (abomasum) was removed and cut along the greater cur-
vature. Abomasal contents and washings were collected and made
up to a volume of 2 L with tap water containing 1% formalin. The
solution was vigorously bubbled with air and 10% transferred to
glass trays for manual counting of parasites. All parasites from each
sheep were then aggregated using tweezers, placed on tissue paper
to remove excess fluid and total wet weights determined.
Blood samples were collected on day 0 and at various times after
infection. For determining packed cell volume (PCV), blood was
collected in 10 ml EDTA tubes and spun in a Haematocrit centrifuge.
Serum anti-HcsL3 antibodies were determined by enzyme
linked immunosorbant assay (ELISA) as described previously [9].
Briefly, ELISA plates (Nunc) were coated overnight with L3 surface
extract in 100 ␮l carbonate buffer, pH 9.6, incubated with various
dilutions of sheep sera followed by polyclonal horse-radish perox-
idase (HRP) rabbit anti-sheep Ig (Silenus, USA) and developed with
3 ,3 ,5 ,5 -tetramethyl-benzidine dihydrochloride hydrate (TMB;
Invitrogen, VIC, Australia).
Antigen-specific IgE was determined by ELISA of ammonium
sulphate-treated serum samples as described previously [10].
Briefly, equal volumes of serum and 60% ammonium sulphate solu-
tion were mixed and the sample centrifuged after 30 min in a
microcentrifuge (13,000 rpm for 10 min). The supernatants (diluted
1:1 with 0.1% Tween 20/H2 0) were added to antigen-coated plates
and incubated overnight at 4 ◦ C, followed by washing and incuba-
tion with anti-IgE mAb (clone XB6-YD3). Plates were then washed
and incubated with HRP-conjugated goat anti-mouse IgG (Sigma,
NSW, Australia), washed and developed with TMB-substrate.
Antigen-specific IgG1 was similarly determined in untreated serum
samples using an anti-sheep IgG1 mAb (clone McM2).
2.4. Statistical analysis
Data were analyzed using GraphPad Prism5. Each vaccinated
group was compared for protection against the adjuvant control
group using Student’s t-test with significance set at p < 0.05. Egg and
worm counts were log transformed before analysis. The Spearman’s
rank correlation coefficient was calculated to determine depend-
ence between two variables.
3. Results
3.1. Protection levels after challenge infection of field vaccinated
sheep
Faecal egg counts (FECs) of individual sheep in all groups are
represented in Fig. 1. Uniformly high egg counts were seen in the
 D.P. Piedrafita et al. / Vaccine 30 (2012) 7199–7204 7201

Control Group A Group B

30000 30000 30000

20000 20000 20000

EPG

EPG
EPG

10000 10000 10000

0 0 0
0 20 40 60 80 0 20 40 60 80 0 20 40 60 80
DPI DPI DPI

cumulative FEC
Group C Group D
80000

30000 30000
60000 *

20000 20000 40000

**
EPG

EPG

10000 10000 20000

0 0 0
0 20 40 60 80 0 20 40 60 80

D
-1
DPI

ro
DPI

A
t
on
C
Group

Fig. 1. Individual faecal egg counts at various days post infection (DPI) of sheep injected with adjuvant alone (control) and sheep vaccinated with HcsL3 antigen recovered
from 200 K L3 (Group A), 20 K L3 (Group B), 2 K L3 (Group C) or 0.2 K L3 (Group D). Bottom right histogram represents the mean cumulative faecal egg counts (cFEC) of
the different groups (mean + SEM). The outlier sheep in group A had a cFEC of 124640 and was excluded from the analysis (Group A-1). Y axis: eggs per gram faeces (EPG).
*Significantly different from control at p < 0.05. **Significantly different from control at p < 0.001.

adjuvant control group with most sheep peaking between 10,000
and 20,000 EPG. FEC of all the vaccinated sheep became more vari-
able with lower antigen dosage, suggesting individual differences
in response to the vaccine. Eight of the nine sheep in the highest
antigen dose group (Group A) had egg counts that peaked below the
control sheep, however 1 sheep in this group showed an unusual
high FEC, peaking well above any of the 10 control sheep at 35,000
EPG. Calculation of the cumulative egg counts for each individual
sheep revealed that values for this sheep fell significantly above
the 95% confidence interval of the control group, and this outlier
was excluded from group A for most statistical analysis (designated
Group A-1). Without this outlier, the cEPG of the different vacci-
nated groups showed a clear dose-dependent protection against
infection compared to the adjuvant control group, with a highly
significant (p < 0.0001) 61% reduction in cEPG in the highest vac-
cine group A-1 (43% reduction, p = 0.04 with the outlier included),
and a smaller but still significant (p = 0.035) 27% reduction in the
second highest dosage group (Group B).
Adult worms were recovered from the abomasums of the adju-
vant control sheep and the highest antigen vaccine dose group A
(Fig. 2), and showed a significant 54% (p = 0.04) and 69% (p = 0.004)
reduction in total worms present in the vaccinated group, with or
without the outlier, respectively. Interestingly, while the outlier
sheep in the vaccine group had much more eggs than the control
sheep (Fig. 1), it did not have a higher worm burden than control
sheep, suggesting that the same number of worms present in this
sheep were induced to increase egg production as a consequence
of vaccination. Total wet worm weights recovered from the vaccine
group showed similar 56% (p = 0.02) and 70% (p = 0.003) reductions,
with or without the outlier, respectively. There was no significant
difference in average worm weight between the groups.
The correlation between worm count and cFEC was highly sig-
nificant (p = 0.0006) across the two groups, with an rs value of 0.72.
3.2. Blood pathology
PCV values began to decline in all groups from 4 weeks post
infection compared to their pre-infection values, reaching their
lowest point at 7 weeks before uniformly increasing (Fig. 3). When
they reached their lowest value at 7 weeks, mean PCV levels in the
vaccinated groups showed a dose-dependent improvement over
the controls, with PCV values in the highest dose vaccine group
(without outlier) reaching significance (p = 0.039). The outlier sheep
in this group had a PCV value of 12 which was lower than any of the
other sheep in the trial and significantly below the 95% confidence
limit of the control group.
PCV values across the two groups were negatively corre-
lated with worm counts (rs = −0.5; p = 0.044) and cFEC (rs = −0.7;
p = 0.002).
3.3. Antibody responses during vaccination and challenge
Total serum antibody levels against the vaccine antigen showed
some unexpected patterns, with no increase in specific antibody
after vaccination or challenge above the control group (Fig. 4, top
panel). In fact, the adjuvant control group showed the highest anti-
body levels overall, and the levels increased while on pasture (from
week −8 to week −3) while they decreased when transferred to
indoor pens (from week −2 to week +1), regardless of the challenge
infection.
To examine in more detail the antibody response, isotype ELISAs
were performed at a range of dilutions for the control and high anti-
gen dose group A (Fig. 4, lower panel). In addition, a separate control
group of 8 sheep that had been kept in indoor pens, worm free
for 2–3 months was included (Penned group). The Penned group of
sheep uniformly showed very low antibody titres against the vac-
cine antigen, indicating that the high antibody levels in the trial
7202 D.P. Piedrafita et al. / Vaccine 30 (2012) 7199–7204

Total worm counts Total worm weight (mg)

4000 20

3000 15

2000 10

1000 5
* *
0 0
ol

ol
-1

-1
A

A
tr

tr
on

on
up

up
C

C
ro

ro
G

G
Fig. 2. Individual worm counts and total worm weights recovered 63 days after infection of sheep injected with adjuvant alone (control) or vaccinated with HcsL3 antigen
recovered from 200 K H. contortus L3 (Group A). The outlier sheep in group A had a worm count and total weight of 3170 and 12.4 g respectively, and was deleted from the
analysis (Group A-1). Horizontal lines represent mean ± SEM. *Significantly different from control at p < 0.01.

sheep were due to their grazing on pasture. The antigen-specific
IgG1 and IgE isotype responses showed a similar pattern to the total
antibody response, with the vaccine group showing equal or lower
antibody titres compared to the control group. The same pattern
was observed with IgA and IgG2 (not shown).
4. Discussion
Vaccination against gastrointestinal nematode (GIN) infections
is a highly desirable goal in livestock research. H. contortus is one
of the most pathogenic of the GIN in sheep and is often the major
parasite in hot and humid climate conditions, making it a good can-
didate for a commercial vaccine. The complexity of the organism
and its different life cycle stages has so far precluded the develop-
ment of a classical H. contortus vaccine based on boosting of natural
immunity. It has been well established that the molecular and anti-
genic structures of nematode larvae changes with each moulting
[11,12] and it is therefore critical that the identification of poten-
tial protective antigens is focused on the right parasite stage. The
L3 surface antigen (HcsL3) used in the present vaccine prepara-
tion was previously shown to confer protection in a limited pen
trial after 3 intradermal immunizations. The current study reports
a more extensive vaccine trial, with vaccinations performed in the
field and only 2 vaccine doses administered subcutaneously to com-
ply with a desirable product profile. Under these conditions, highly
significant protection against an experimental challenge infection
was achieved in a dose-dependent manner. Importantly, PCV val-
ues as a measure of blood loss and infection-induced pathology,
while reduced in all infected groups, were significantly improved
in the protected sheep compared to the controls indicating that ani-
mals will be healthier and likely more able to generate increased
immunity following renewed exposure. As the antigen is the target
of natural immunity [7,8], it would be expected that immune resis-
tance will be further boosted by natural exposure to contaminated
pasture.
One of the sheep in the highly protected group showed an
unusual high egg count and an extremely low PCV value. This
was clearly an outlier as all other sheep in this group showed sig-
nificant protection compared to the control. Both extreme values
in this sheep were also significantly outside control levels while
worm numbers were normal, suggesting they may be related to a
vaccine-induced stress response. Previous studies have also shown

Fig. 3. Packed cell volume (PCV) of trial sheep after infection. Panel A: Percentage change in PCV at various weeks after infection compared to day 0 PCV values, of sheep
injected with adjuvant alone (control) and sheep vaccinated with HcsL3 antigen recovered from 200 K L3 (Group A), 20 K L3 (Group B), 2 K L3 (Group C) or 0.2 K L3 (Group
D). Panel B: the mean PCV (+SEM) of the different groups at week 7 post infection. The outlier sheep in group A had a PCV of 12 and was excluded from the analysis (Group
A-1). *Significantly different from the control group at p < 0.05.
 D.P. Piedrafita et al. / Vaccine 30 (2012) 7199–7204 7203

Total Ig 1/500
0.7
Control

0.6 Group A
Group B
0.5 Group C
Group D
0.4

0.3

-8 -6 -4 -2 0 2
Weeks before/after infection

IgG1 week -8 IgE week -8

1.0 0.6
Control Control
0.8 Group A Group A
Penned 0.4 Penned
0.6

0.4
0.2
0.2

0.0 0.0
102 103 104 105 106 107 1 10 100 1000

IgG1 week -3 IgE week -3

1.0 0.8
Control Control
0.8 Group A Group A
0.6
Penned Penned
0.6
0.4
0.4
0.2
0.2

0.0 0.0
102 103 104 105 106 107 1 10 100 1000

IgG week +1 IgE week +1

0.8 0.3
Control Control
Group A Group A
0.6
Penned 0.2 Penned

0.4

0.1
0.2

0.0 0.0
102 103 104 105 106 107 1 10 100 1000

Fig. 4. Antibody profiles of sheep before vaccination (week −8), 1 week after vaccination (week −3), at day of challenge (week 0) and 1 week after challenge (week +1). Sheep
were injected with adjuvant alone (control) or vaccinated with HcsL3 antigen recovered from 200 K L3 (Group A), 20 K L3 (Group B), 2 K L3 (Group C) or 0.2 K L3 (Group D).
A separate group of 8 sheep kept in pens indoors for 2–3 months were included as negative controls (Penned). Top panel: Total antigen-specific Ig (mean ± SEM) at serum
dilution of 1/500. Bottom Panel: Mean (± SEM) antigen-specific IgG1 and IgE levels at different serum dilutions (X-axis). Y-axis: Absorbance values.
7204 D.P. Piedrafita et al. / Vaccine 30 (2012) 7199–7204
significant enhancing effects of protective immunity on worm mat-
uration and fertility in filarial parasites [13].
No increase in antigen-specific antibodies could be detected in
the trial sheep after vaccination or challenge. In the previous pen
trial, only low levels of antibodies were detected after 3 immuniza-
tions with alum adjuvant with no boosting after challenge [9]. As
only 2 vaccinations were administered in the present trial, this may
have resulted in even lower antibody levels, undetectable under
present conditions. In addition, as the antigen is only present on
the L3 larvae and this stage moults into an L4 within 1–2 days
after infection, the antigenic stimulus provided by the 10,000 L3
administered as the challenge infection may be too low to result
in detectable boosting of the antibody response. Surprisingly, high
antigen-specific antibody levels were observed in the control sheep
kept on pasture, and these levels seemed to reduce when housed
indoors in pens. This was confirmed when sera obtained from
long-term penned sheep were tested and gave completely negative
results. The trial took place during the winter season (June–July)
and only low numbers of GIN eggs were observed in the flock dur-
ing the trial period. Either this low level of pasture contamination
may be sufficient to raise antibody levels, or antibodies to other
components on the pasture may cross-react with the larval antigen.
Pre-existing levels of specific antibodies against a vaccine antigen
were also observed during experimental vaccination in an endemic
area with a secreted larval hookworm antigen [14]. In this case,
hypersensitivity reactions after vaccination were associated with
high pre-existing serum IgE levels and precluded the further appli-
cation of this vaccine. No adverse reactions were observed with the
HcsL3 antigen in the present study, possibly due to the fact that it
is a surface antigen and not secreted, and/or does not induce high
IgE levels.
The present study confirms the protective efficacy of the larval-
specific surface antigen, and demonstrates that vaccination with
the native antigen can induce sufficient protection against H. con-
tortus infection when vaccination is performed on the farm using a
commercially acceptable adjuvant and vaccination protocol. The
Merino sheep used in this study are a highly susceptible breed
and unvaccinated animals carried large infections resulting in sig-
nificant blood pathology which was ameliorated in the high dose
vaccinated sheep. The present experiment was conducted in sheep
at 8–9 months of age. Lambs under 9 months of age are known
to acquire natural immunity slowly and may be more difficult
to protect effectively with the current vaccine. It is therefore
likely that anthelmintic treatment or appropriate pasture rota-
tions will be required at weaning to protect lambs during the first
grazing season. As wool sheep are kept for several years, subse-
quent vaccinations may help protect these susceptible sheep from
future worm infections and reduce the amount of anthelmintic
treatments required over their lifetime. This may reduce cost
to the farmer and help delay the occurrence of drug resistant
worms.
In the present study, sheep were exposed to varying levels
of natural infection during their first 8 months on pasture. They
were then treated with a long-acting anthelmintic to reduce any
potential effect of varying worm burden on immune responses to
vaccination and to achieve a uniform baseline for assessing pro-
tection against an experimental challenge infection. Vaccinations
were performed during the winter months (June–July) when low
pasture infections are expected. This was confirmed by egg counts
of non-experimental sheep of the same flock, which did not require
anthelmintic treatment during this period. Future vaccination trials
could therefore be conducted without anthelmintic treatment. As
the larval antigen is a natural immunogen during infection, it will
be interesting to see if natural boosting due to low level infections
in the absence of drug treatment may further increase vaccine effi-
cacy in the field. To further evaluate the vaccine, vaccinated sheep
should also be subjected to natural challenge in the field, which
will require a much larger field trial in areas with high infection
prevalence.
While the antigen appears to be a complex glycoprotein that
may be difficult to produce as a synthetic or recombinant product,
H. contortus is a highly prolific egg producer and the preparation
of the antigen from the L3 was achieved with a simple two step
procedure. Sufficient native antigen may therefore be obtained for
vaccination of small sheep and goat holdings. In addition, new tech-
nologies such as phage display and glycan/peptide arrays may be
able to generate antigen mimics that can be produced in commer-
cial quantities.
Acknowledgment
This work was supported by an Australian Research Council
Linkage grant with Pfizer VMRD and the ARC Centre of Excellence
in Structural and Functional Microbial Genomics.
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