PIM_432.

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Parasite Immunology, 2002, 24, 39 46

Mechanisms of immunity to Haemonchus contortus infection in sheep

Blackwell
ORIGINAL
Immunity to H. contortus
Science Ltd
ARTICLE infection

A. BALIC, V. M. BOWLES & E. N. T. MEEUSEN

Centre for Animal Biotechnology, School of Veterinary Science, The University of Melbourne, Victoria, Australia

SUMMARY their tissue niche as observed previously and which is associated

with changes in globular leucocyte population but no mobilization
In two separate experiments, sheep were immunized by nine to
of the local immune system. In contrast, when challenge
12 weekly immunizing infections with 4000 Haemonchus
larvae reach their tissue niche, dramatic changes in the local
contortus third stage larva (L3), drenched with anthelmin-
immune system occur, including a pronounced infiltration of
thics and maintained free of H. contortus infection for a further
eosinophils. These two immune mechanisms may be associated
12 weeks. The anamnestic cellular immune responses in both
with the rapid and delayed rejection of parasite infections in
the abomasal lymph node (ALN) and mucosa of the immun-
immune sheep.
ized sheep were examined 3 and 5 days post challenge with
50 000 H. contortus L3. Sheep in the two experiments clearly
Keywords Haemonchus contortus, gastrointestinal nematode,
segregated out in two distinct groups, one in which challenge
anamnestic, lymphocyte, eosinophil, lymph node
larvae were obviously present in the tissues of all 12 sheep at Journal of Animal Ecology (2001) 70, 000000

3 and 5 days post challenge while no challenge larvae were

detected in tissues of seven of the eight sheep in the other INTRODUCTION
group. In sheep in which no tissue larvae were detected, very
While there have been many studies characterizing the
few changes were noted in either the ALN or mucosa. In con-
cellular and cytokine responses to gastrointestinal nematode
trast, dramatic changes were observed in the cellular profiles
infections in rodent models (1), few of these models have
of the ALN and mucosa after challenge infection in sheep in
examined anamnestic immune responses after challenge
which larvae were observed in the abomasal tissues. In the
infection of previously infected animals. In contrast to
ALN, these changes were characterized by an increase in the
rodent models, resistance to nematode larval establishment
relative percentage of -TCR+ T cells and B cells and an
in ruminants generally requires multiple infections and the
increase in the proportion of CD4+ T cells coexpressing the
mechanism(s) involved may be different from those active
activation markers MHC class II and CD25. In the abomasal
during rejection of primary infections (2).
mucosa, an increase in the number of infiltrating CD4+ and
Previous reports of challenge infections of sheep immune
-TCR+ T cells and B cells was observed by 3 days postinfection
to Haemonchus contortus and Trichostrongylus colubriformis
and these levels were further increased at 5 days postinfection.
have indicated that, when challenged, these sheep show little
This infiltration of the abomasal mucosa by lymphocytes was
increase in lymphocyte recruitment to the gastrointestinal
accompanied by a dramatic increase in the number of infiltrat-
mucosal tissue and the majority of the challenge larvae are
ing eosinophils, which were often in intimate association with
expelled before they reach their tissue niche (rapid expul-
the surface of H. contortus larvae. None of these changes
sion) (3,4). However, in a proportion of immunized sheep,
occurred in the mucosa of the sheep that showed no sign of
expulsion of challenge larvae occurs over a period of days,
challenge larvae in the tissues; however, a transient increase
presumably after the larvae had reached their tissue niche
in T cells in the ALN and a drop in intraepithelial globule
(delayed expulsion) and is associated with infiltration of the
leucocytes were uniquely observed in these sheep at 5 days post
gastrointestinal tissue by lymphocytes (4).
challenge. These results suggest that two different types of
Rapid expulsion of challenge larvae in sheep has been
immune responses can be generated after challenge infection
associated with the presence of mucosal mast cells and espe-
of immunized sheep, one where tissue larvae are excluded from
cially the intraepithelial mucosal mast cell (globule leucocyte)
subpopulation (5). It has been suggested that the presence
Correspondence: Dr Els Meeusen, Centre for Animal Biotechnology, of these cells in the mucosa results in an immediate type 1
School of Veterinary Science, The University of Melbourne, Victoria
3010, Australia (e-mail: address: e.meeusen@unimelb.edu.au).
hypersensitivity reaction to challenge larvae. In contrast,
Received: 26 July 2000 the immune mechanisms responsible for the delayed type
Accepted for publication: 8 October 2001 expulsion of nematode larvae are unknown.

2002 Blackwell Science Ltd 39

PIM_432.fm Page 40 Wednesday, January 9, 2002 4:43 PM
A. Balic et al.
In the current study, previously immunized sheep were
challenged with Haemonchus contortus larvae several months
after the immunizing infections and the cellular responses in
the abomasal tissues and lymph nodes were characterized.
The results indicate that two different mechanisms may be
active against larval challenge in immunized sheep.
MATERIALS AND METHODS
Animals and experimental design
Experiment 1
The sheep used in Experiment 1 were Merino weaner lambs
obtained at 3 months of age. While these sheep were poten-
tially exposed to nematode larvae on the pasture, no nema-
tode eggs were detected by faecal examination on arrival.
All sheep were drenched with levamisole (Nilverm, Coopers
Animal Health, North Ryde, NSW, Australia) and housed
indoors for 1 month before commencement of the experi-
ment. All sheep were orally infected with 4000 third stage
larva (L3) H. contortus per week for 12 weeks, drenched
with levamisole, and maintained indoors for 12 weeks. No
nematode eggs were detected in the faeces of any sheep
during this period. Sheep were divided into three groups
of four animals, then challenged orally with phosphate
buffered saline (PBS) (control) or with 50 000 L3 H. contortus
suspended in 50 ml of PBS. Control sheep and sheep
challenged with H. contortus larvae were killed at either 3
or 5 days post challenge (DPC). Sheep were approximately
10 months old at the end of the experiment.
Experiment 2
Eighteen 23-year-old pasture reared Merino wethers were
obtained from a local farm. Faecal examination indicated that
most sheep were infected with Teladorsagia sp. However, no
H. contortus eggs were detected. All sheep were drenched
with levamisole and rested for 1 month. No nematode eggs
were detected during this period by faecal examination. All
sheep were orally infected with 4000 L3 H. contortus per
week for 9 weeks, drenched with levamisole, and maintained
indoors for 12 weeks. Sheep were divided into three groups
of six animals, then challenged orally with PBS (control) or
with 50 000 L3 H. contortus and killed at either 3 or 5 DPC.
Histology
Abomasal specimens were prepared by opening the abo-
masum along the greater curvature, and washing the
contents out with gently running tap water. Fundic folds
were removed with scissors and processed as follows.
Tissue samples were placed in 10% formalin for 5 days and
embedded in paraffin. Sections were cut and stained with
40
Parasite Immunology
haemotoxylin and eosin Y (H&E) to visualize eosinophils
and globule leucocytes.
For immunohistochemistry and lamina propria mast cell
identification, tissue samples were embedded in Optimal
Cutting Temperature (OCT) solution (Tissue Tek, Miles
Inc., Elkhart, IN, USA), and frozen on liquid nitrogen. Tis-
sue blocks were stored at 70C until used. Six-m frozen
tissue section were cut using a CM1900 cryostat (Leica,
Martin Place, Sydney, Australia), transferred onto micro-
scope slides (HD Scientific, Bayswater, Victoria, Australia)
and allowed to dry at room temperature. Mucosal mast cells
were visualized by staining with 1% toluene blue (50% PBS,
50% methanol). Lymphocyte subsets were identified by
immunoperoxidase staining as described previously (6).
Flow cytometry of abomasal lymph node cells
Lymph nodes were carefully excised from the lesser curva-
ture of the abomasum and the cells isolated as described
previously (7) and adjusted to 4 107 cells per ml for flow
cytometry analysis.
50 l of monoclonal antibody (mAb) supernatant was added
to 50 l of cells, in a 96-well V-bottomed plate at 4C for 30 min
with shaking. The cells were then washed three times in FACS
wash buffer and incubated with 50 l of sheep fluorescein-
isothyocyanate (FITC)-antimouse immunoglobulin (Ig) (1 : 80,
Silenus, Hawthorn, Victoria, Australia) (one- and two-colour
analysis) or PE-antimouse Ig (1 : 2000; Silenus) (three-colour
analysis) at 4C for 30 min Cells were again washed and for
single-colour analysis fixed in 3% paraformaldehyde in PBS
at this stage. For two- and three-colour analysis, normal mouse
serum (15% v/v) in FACS wash was added to the wells to block
remaining antibody binding sites for 30 min at 4C. 50 l
of diluted biotinylated mAb was then added to cells at 4C
for 30 min After washing, 50 l PE-streptavidin (1 : 400;
Biosource International, Camarillo, CA, USA) (two-colour
analysis) or FITC-mAb 28-1 (1 : 500) + Tricolour-streptavidin
(1 : 500; Caltag Laboratories, Burlingame, CA, USA) (three-
colour analysis) was added to each well, and incubated as
before. Cells were then washed and fixed in 3% paraformal-
dehyde (Sigma, St Louis, MO, USA) in PBS. For surface
Ig staining (sIg), rabbit antisheep Ig FITC conjugate (1 : 40;
Silenus) was used instead of the rabbit antimouse FITC.
During analysis by flow cytometry (FACscan, Becton
Dickinson, Franklin Lakes, NJ, USA) lymphocytes were gated
and counted, based on their forward scatter versus side scatter
profiles. mAbs specific for MHC class II (MHCII, 281),
CD8 (38 65), CD4 (44 97 + 44 38), WC1 (1919), CD25
(914) and CD45R (2096) were produced in our laboratories.
The mAbs specific for -TCR (86D) and CD21 (CC21) were
kindly provided by Dr Charles Mackay (Basel, Switzerland)
and Dr Chris Howard (Comptom, UK), respectively.
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Volume 24, Number 1, January 2002 Immunity to H. contortus infection

70 Experiment 1 Control
Morphometric analysis
Three DPC
60
Five DPC

abomasal lymph node

To determine the number of lymphocytes, eosinophils or lamina

% positive cells in
propria mast cells in the abomasal mucosa, the number of 50
cells in a 028 mm 028 mm area of the lower two-thirds of 40
the abomasal mucosa were counted for 100 different fields
for each animal at 400 magnification. To assess globule 30
leucocyte numbers, a 028 mm 028 mm area in the upper 20
two-thirds of the abomasal mucosa was counted for 100
10 * *
different fields for each animal at 400 magnification.
0
CD4 CD8 WC1 sIg
Statistical analysis
Results are expressed as group means and SDs. Cell popu- 70 Experiment 2
Control
lations from different groups were compared using a two-tailed 60 Three DPC
Students t-test with Microsoft Excel software. P < 005 was abomasal lymph node Five DPC
% positive cells in
50
considered statistically significant.
40 *
*
RESULTS 30

General observations
Experiment 1
Careful examination of H&E stained paraffin sections of
several different regions of the abomasal fundus failed to
find any nematode larvae in the nematode challenged sheep,
except in the case of Sheep 64, which was killed 5 DPC.
Nave donor sheep were successfully infected with the same
batch of H. contortus L3, indicating that the challenge larvae
were infective but, except for one sheep, did not penetrate
the abomasal tissues of the immunized sheep. With the
exception of Sheep 64, there was no obvious cellular infiltra-
tion of the abomasal tissue in the nematode challenged
sheep compared to control sheep.
Experiment 2
Examination of H&E stained paraffin sections of the abo-
masal fundus indicated that all nematode challenged sheep
had nematode larvae present either within the gastric pits or
on the abomasal mucosal surface. Cellular infiltration of the
abomasal mucosa was observed in all nematode challenged
sheep. Lymphocytes and eosinophils were most commonly
observed at the base of the abomasal mucosa, especially
adjacent to thin walled arterioles.
Total abomasal lymph node weight
Experiment 1
The group mean for the combined weight of the abomasal lymph
nodes was 229 082 g in the control group. There was no signif-
icant change in the abomasal lymph node weights between any
group after challenge (158 07 g 3 DPC, 178 02 g 5 DPC).
20
* *
10 *
0
CD4 CD8 WC1 sIg CD45R
Figure 1 Changes in abomasal lymph node lymphocyte populations,
showing mean and SD in control and H. contortus challenged
groups. *Indicates a significant (P < 005) change in percentage
of a particular lymphocyte population, compared to the control
group, for the marker tested.
Experiment 2
The group average for the combined weight of the abomasal
lymph nodes was 276 027 g in the control group. In
immunized sheep, there was no change in abomasal lymph
node weight at three DPC (332 112 g). However, by 5 DPC,
there was an increase in the abomasal lymph node weight
(551 200 g) which was significant (P < 001; P < 005)
compared to the control and 3 DPC groups, respectively.
Cellular changes in the abomasal lymph node after
challenge infection of immunized sheep
Experiment 1
No differences were observed in the percentage of CD4+
T cells or B cells, as defined by sIg positive cells, in the
abomasal lymph node between any of the three groups
(Figure 1).
At 3 DPC, there was a slight decrease in the percentage
of CD8+ T cells (90 23 to 55 07), concomitant with a
significant (P < 005) increase in the percentage of T cells
(32 09 to 55 09) compared to control sheep (Figure 1).

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A. Balic et al. Parasite Immunology

Table 1 CD4+ T cell subpopulations present in the abomasal lymph nodes of control and nematode challenged sheep

Experiment 1 Experiment 2

Control 3 DPC 5 DPC Control 3 DPC 5 DPC

MHC class II 353 105a 383 150a 366 172a 364 76a 424 105a 610 65b
CD25 221 28a 226 41a 257 73a 254 34a 263 84a,b 329 28b
-selectin 610 76a 458 221a 598 95a 644 60a 645 79a 598 16a
CD45R 337 144a 239 72a 170 41a 93 73a 202 107a,b 216 53b

Values are expressed as mean percentage ( SD) of the CD4+ population. a,bValues without a letter in common are significantly different (P < 005).

These values returned to the levels observed in the control

group by 5 DPC.
No differences were observed in the group mean percent-
ages of CD4+ T cells coexpressing MHC class II (MHCII),
CD25, -selectin or CD45R surface markers (Table 1).

Experiment 2
In contrast to Experiment 1, significant differences in the
mean percentage of lymphocyte populations in the local
abomasal lymph nodes were observed between the three
groups. In particular, there was a significant (P < 005)
increase in the percentage of T cells at 3 and 5 DPC
compared to control sheep. The percentage of T cells was
not significantly different between the two challenged groups
(Figure 1). In addition, the mean percentage of B cells, as
defined by sIg and CD21 expression, was significantly
(P < 005) increased at 5 DPC in comparison to control and
3 DPC groups (Figure 1).
While no changes were observed between control and
challenge groups in the mean percentage of CD4+ T cells in
the abomasal lymph node (Figure 1), significant changes
were observed in the phenotypes of the CD4+ T cell popu-
lations as determined by two-colour analysis (Table 1). In
particular, the percentage of CD4+ T cells, coexpressing
MHCII was significantly (P < 001) increased by 5 DPC
compared to control sheep (Table 1). In addition, the CD4+
MHCII+ T cell subpopulation was located in the blast
cell population, as determined by their forward and side
scatter profiles, whereas the CD4+ MHCII T cell subpopu-
lation was located in the small lymphocyte population (data
not shown). There was also a significant (P < 005) increase
in the percentage of CD4+ T cells coexpressing CD45R.
However, three-colour analysis indicated that the CD4+
CD45R+ and the CD4+ MHCII+ populations were mutually
exclusive (Figure 2a). In all sheep, the CD4+ MHCII+ T cell Figure 2 Three colour flow cytometry analysis of CD4+ abomasal
lymph node cells. (a) Expression of CD45R and MHC class II
population expressed significantly higher levels of CD2
by abomasal lymph node CD4 T cells. (b) Expression levels of
(257 25 peak staining intensity) compared to the CD4+ the CD2 marker by abomasal CD4+ MHCII+ (right peak) and
MHCII subpopulation (1037 212 peak staining intensity) CD4+ MHCII (left peak) T cells. Data are representative of all
(Figure 2b). animals tested.

42 2002 Blackwell Science Ltd, Parasite Immunology, 24, 39 46

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Volume 24, Number 1, January 2002 Immunity to H. contortus infection

80 Experiment 1 Control for the large standard deviation with these markers on day
70
Three DPC 5 post challenge.
Five DPC
60
Cells per mm2

Experiment 2
50 In general, more lymphocytes were present in the control
40 sheep of Experiment 2 compared to control sheep in Experi-
30 ment 1, probably reflecting the increased age and antigen
exposure of these sheep as observed previously (3).
20
Both CD4 + and -TCR + T cell numbers in the abo-
10 masal mucosa were significantly increased at 3 and 5
0 DPC compared to control sheep (Figure 3). In contrast,
CD4 CD8 WC1 CD45R
no increase in the number of CD8 + or WC1 + T cell
populations was observed after larval challenge. Differ-
600
* Experiment 2 Control ences in the mean number of CD4+ and T cells
Three DPC
between the 3 and 5 DPC groups were not significant
500 Five DPC
(Figure 3).
* The mean number of CD45R+ B cells was significantly
Cells per mm2

400
increased at 3 and 5 DPC compared to control sheep and,
300 in this case, the increase in the number of CD45R+ B cells
*
200 at 5 DPC was significantly higher compared to 3 DPC
*
* (Figure 3).
100 *

0 Granulocyte populations present in the abomasal

CD4 CD8 WC1 CD45R
Figure 3 Changes in abomasal mucosa lymphocyte populations,
showing mean and SD in control and H. contortus challenged
groups. *Indicates a significant (P < 005) change in the number
of lymphocytes in the abomasal mucosa, compared to the control
group, for the marker tested.
Leucocyte populations present in the abomasal mucosa
after challenge
Experiment 1
No significant differences in the mean number of leucocytes
were noted amongst control or challenged groups for the
CD4+, CD8+, -TCR and WC1+ T cell populations or the
CD45R+ B-cell population (Figure 3). Sheep 64, the only
sheep in this experiment with detectable tissue larvae, had
by far the highest numbers of CD45R+, WC1+ and +
lymphocytes in the abomasal mucosa of all sheep, accounting
mucosa after challenge
Experiment 1
No significant differences were observed in the number of
eosinophils between any group (Table 2). However, Sheep 64
was observed to have the highest number of eosinophils
(148 cells per mm2) compared to other nematode challenged
sheep in this experiment (no other sheep had greater then
22 cells per mm2).
There was a significant decrease in the number of globule
leucocytes in sheep 5 DPC compared to control sheep, with
only Sheep 64 showing detectable (02 cells per mm2) numbers
for the areas counted. No change was observed in the number
of MMCs between any group (Table 2).
Experiment 2
In contrast to Experiment 1, there was a dramatic increase
in the number of eosinophils in the abomasal mucosa at

Table 2 Granulocyte populations present in the abomasal mucosa of control and nematode challenged sheep

Experiment 1 Experiment 2

Control 3 DPC 5 DPC Control 3 DPC 5 DPC

Eosinophils 10 09a 09 08a 44 70a 28 41a 344 133b 632 425b

Mast cells 318 148a 201 234a 244 63a 202 136a 288 92a 239 36a
Globule leucocytes 09 06a 04 03a 002 01b 25 23a 26 50a 14 24a

Values are expressed as the mean number ( SD) of cells per mm2. a,bValues without a letter in common are significantly different (P < 005).

2002 Blackwell Science Ltd, Parasite Immunology, 24, 39 46 43

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A. Balic et al.
both 3 and 5 DPC (Table 2). While eosinophils were located
most commonly in the lamina propria, they were also
observed within the overlaying mucus layer, often in intimate
association with L4 larvae (not shown).
No significant difference was observed in the number
of globule leucocytes or MMCs between any group (Table 2).
DISCUSSION
In a previous study, in which naive sheep were given a
primary infection of 50 000 Haemonchus contortus L3,
activation of the local lymph node and recruitment of
lymphocytes to the abomasal mucosal tissue was observed
(7). In the current study, similar changes were also noted in
previously immunized sheep given a challenge infection of
50 000 L3 H. contortus, although this response was only
observed in sheep which had larvae present in the abomasal
tissue. However, differences were noted in the kinetics of
lymphocyte recruitment to the abomasal tissue between
primary infected and previously immunized and challenged
sheep. During primary infection of sheep, increased recruit-
ment of lymphocytes (mainly CD4+ and -TCR+ T cells,
and B cells) to the abomasal tissue did not occur until 5 days
postinfection, while significant recruitment of these
lymphocytes was observed earlier at 3 DPC in previously
infected sheep.
In addition, while there was an increase in the percentage
of activated CD4+ T cells (MHC II+) in the local lymph
node in both the primary infected and previously immun-
ized and challenged sheep, significant increases in the
percentage of T cells and B cells and an increase in
CD4+ CD25+ T cells in the abomasal lymph nodes were only
observed in the previously immunized and challenged sheep.
These data indicate that the anamnestic immune response of
sheep to infection with H. contortus is characterized by an
increased rate of lymphocyte recruitment to the abomasal
mucosa and quantitative and qualitative changes in the
lymphocyte population activated in the local lymph node
compared to primary infected sheep.
In the current study, there was no significant change in
the percentage of CD4 T cells in the abomasal lymph node
of any sheep after nematode challenge. However, in sheep
which had larvae present in the abomasal tissue there was
a significant increase in the proportion of CD4 T cells co-
expressing the activation markers CD25 and MHCII by
5 DPC. The activated CD4+ MHCII+ T cell population also
had higher levels of CD2 expression. Bimodal distribution
of CD2 staining on human CD4+ T cells has been previ-
ously described and the CD4+ CD2high phenotype has been
identified as an activated T cell phenotype (8). In addition,
the CD4+ MHCII+ T cell population was found to comprise
large blast cells, which is indicative of an activated state.
44
Parasite Immunology
These data indicate that the anamnestic immune response to
challenge infection with H. contortus is characterized by the
specific activation of the CD4 T cell population, if larvae
reach their tissue niche.
Transfer of efferent gastric lymph from resistant to naive
sheep has been demonstrated to confer partial resistance to
challenge infection with H. contortus (9). This transfer of
resistance was only evident with efferent lymph cells col-
lected between 3 and 5 DPC of the donor sheep (9). While
the phenotype of the cell population responsible for the
transfer of resistance was not defined, it is likely to be the
activated CD4 T cell population observed in the abomasal
lymph nodes in the current study, as CD4 T cells have been
shown to be required for the expression of resistance to H.
contortus (10).
A memory response was also observed in the abomasal
mucosa of sheep in which larvae were observed in the gas-
tric pits (Experiment 2). This anamnestic immune response
to larval invasion was characterized at 3 and 5 DPC by an
increased infiltration of CD4 and T cells. WC1 T cells
were rarely observed and, similar to the CD8 T cell popula-
tion, did not increase in nematode challenged sheep, indicat-
ing that the increase in the T cell population was due to
increased numbers of a -TCR+ WC1 CD8 T cell subpopu-
lation. Challenge infection of sheep immune to Trichos-
trongylus colubriformis with L3 larvae also results in an
increase in the number of CD4 and -TCR+ WC1 T cells
infiltrating the small intestine lamina propria by 5 DPC (4).
The similarity in the kinetics of the lymphocyte recruitment
and the subpopulations involved in these two infections
suggests that the ovine immune system responds with a
stereotypic anamnestic immune response to gastrointestinal
nematode infection when larvae reach their predilection site.
In addition to the increase in T cells observed in nematode
infected sheep, the mean number of B cells (CD45R+) in the
abomasal mucosa, increased significantly at 3 and 5 DPC.
Previous studies have shown that the antibodies produced
by these activated B cells recognize a restricted set of L3 and
L4 specific antigens (11,12).
Eosinophil recruitment into the abomasal tissue was
more rapid and of greater magnitude, after nematode chal-
lenge, in previously infected sheep compared to primary
infected sheep (7). This observation has also been made in
experimental infections of sheep with Oesophagostomun
columbianum (13). In addition to these quantitative changes,
qualitative changes in eosinophils function in previously
infected compared to primary infected sheep were also
observed. Thus, although there was eosinophil recruitment
in some larval infected animals after primary infection,
there was no observable association of eosinophils and
developing larvae. In contrast, after larval challenge of pre-
viously exposed sheep, intimate association of eosinophils
2002 Blackwell Science Ltd, Parasite Immunology, 24, 39 46
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Volume 24, Number 1, January 2002
and larvae was frequently observed. Eosinophils were
readily observed in close association with L4 larvae within
both the abomasal mucosa and the overlaying mucus layer.
Further studies aimed at examining more closely the inter-
action between eosinophils and parasites at earlier time
points suggest that larval damage and killing by eosinophils
can occur in these sheep (14) and may contribute to the
delayed rejection of challenge larvae observed in some
experimental systems (4,15).
With the exception of one sheep, no challenge larvae were
observed in the abomasal tissues of the remaining seven
challenged sheep in the first experiment. In these seven
sheep, there was also a complete absence of lymphocyte and
eosinophil recruitment to the mucosal tissue and no lym-
phocyte activation in the local draining lymph nodes. While
it could have been possible that the challenge larvae in this
experiment were not viable or infective, this was discounted
for the following reasons: first, the batch of larvae used for
the challenge infection was shown to be > 90% viable and
used successfully to infect donor sheep; second, one of the
eight challenged sheep in this experiment did show a clear
presence of larvae in the tissues, resulting in tissue reactions
similar to those found in the second experiments where all
sheep had tissue larvae; and finally, the challenge infections
did induce significant changes in the seven challenged sheep
without tissue larvae compared to sham-infected controls,
including an increase of -T cells in the draining lymph
nodes on day 3 and a decrease in globular leucocytes in
tissues on day 5. Previous studies have shown that rapid
expulsion of challenge larvae can occur before they reach
their tissue niche and have implicated mast cells and, in par-
ticular, globule leucocytes as possible mediators of this
rapid rejection/immune exclusion response (5,16). While
the number of mast cells/globular leucocytes observed in the
present study was much lower than that observed in previ-
ous hyper immunization studies (5,16), a significant change
in globule leucocyte numbers after challenge only occurred
in sheep without tissue larvae. Although this change occurred
late after challenge (5 DPC), it supports involvement of
this cell population in a rapid rejection/immune exclusion
response. As recognized previously (17,18), absolute num-
bers of mast cells may not be a sufficient indicator of their
activity and other factors such as the cytokine environment
and the presence of specific IgE, may significantly influence
the strength of the mast cell response. The expression of
various cytokine genes during challenge infection will be
presented in a future study. At present, it is not clear why
these two different immune responses to challenge infection
occurred in the two experiments examined in this study. An
obvious difference between the two experiments is the age of
the sheep (10 months versus 2 years) and it is possible that
younger sheep may be more prone to an immune exclusion
2002 Blackwell Science Ltd, Parasite Immunology, 24, 39 46
Immunity to H. contortus infection
response. However, recent studies have shown that 2-year-
old sheep can also exclude challenge larvae from their
tissue niche if the time between sensitization and challenge
is shortened by a few weeks (Balic & Meeusen, unpublished
data). Further studies using sheep of similar age and back-
ground are needed to examine the requirements for the
induction of these two immune response profiles in detail.
In summary, the present study has shown that different
responses to challenge infection can be generated in sensit-
ized sheep. In particular, when challenge larvae reach their
tissue niche, pronounced changes in lymphocyte populations
and activation of CD4 T cells occur in the draining lymph
nodes accompanied by recruitment of CD4 and T cells
and B cells in the infected tissues. In addition, eosinophil
numbers increase progressively in the tissue after challenge
and localize to sites of parasite invasion in the tissues and
mucus layer. In contrast, in some sensitized sheep, challenge
larvae are rejected before they reach their tissue niche, and
very little lymphocyte activity is observed in the lymph
nodes and tissues with no recruitment of eosinophils. These
results suggest that the different rejection kinetics (delayed
or rapid rejection) previously described for gastrointestinal
nematode infections may be associated with different
immune mechanisms generated in immunized sheep. The
detailed immunological characterization of two immune
response profiles provided in the present study, combined
with quantitative parasitological data, should allow this
hypothesis to be tested in further experiments.
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