Immune cell kinetics in the ovine abomasal mucosa following hyperimmunization and challenge with Haemonchus contortus.

Abstract

Sheep were sensitized by repeated infection with Haemonchus contortus L3, followed by a 12 week rest period, and an abomasal cannula was surgically implanted in all sheep. Seven of the sensitized sheep were subsequently challenged with 50 000 H. contortus L3 while 4 control sheep were challenged with saline. Biopsy samples were taken using a fibreoptic endoscope on days 0, 1, 2, 3, 5, 7 and 28 after challenge and leukocyte subpopulations quantified by (immuno)histology. Differential blood cell counts were performed on the same days. At the end of the trial, sheep showed significantly reduced worm burdens compared to unsensitized control sheep, confirming their resistance status. Both blood and tissue eosinophils, as well as tissue gammadelta TCR+ cells were rapidly elevated by day 1 post L3 challenge (pc), peaking at day 3 pc. There was a slight increase in tissue CD4 T cells at day 2 pc, peaking at day 3 pc while no significant changes in CD8 T cells were observed. B cells (CD45R+) increased later into challenged tissues with a peak at 5 days pc. All tissue lymphocyte subpopulations as well as tissue and blood eosinophils were reduced by day 7 pc before increasing again at day 28 pc, suggesting separate responses to larval and adult antigens. In contrast, globule leukocytes and mucosal mast cells only showed one peak at day 5 pc and 28 pc, respectively. Unexpectedly, globule leukocytes correlated significantly with tissue eosinophils but not mucosal mast cells. The results are consistent with an early eosinophil-mediated killing of L3, possibly recruited by IL-5 produced by gammadelta T cells. In contrast to post-mortem studies, abomasal cannulation allowed sequential analysis of both early and late time points in the same animal, providing a more complete picture of cellular interactions at both peripheral and local sites, and their correlation with the different stages of parasite development.

Full text

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Original article
Immune cell kinetics in the ovine abomasal mucosa
following hyperimmunization and challenge
with Haemonchus contortus
Nicholas ROBINSON1, David PIEDRAFITA1, Kenneth SNIBSON2,
Paul HARRISON3, Els N. MEEUSEN1*
1Biotechnology Research Laboratories, School of Biomedical Sciences, Monash University,
Clayton, VIC 3800, Australia
2Centre for Animal Biotechnology, School of Veterinary Science, The University of Melbourne, Australia
3Victorian Bioinformatics Consortium, School of Biomedical Sciences, Monash University, Australia
(Received 2 November 2009; accepted 1 February 2010)
Abstract – Sheep were sensitized by repeated infection with Haemonchus contortus L3, followed by a
12 week rest period, and an abomasal cannula was surgically implanted in all sheep. Seven of the sensitized
sheep were subsequently challenged with 50 000 H. contortus L3 while 4 control sheep were challenged
with saline. Biopsy samples were taken using a fibreoptic endoscope on days 0, 1, 2, 3, 5, 7 and 28 after
challenge and leukocyte subpopulations quantified by (immuno)histology. Differential blood cell counts
were performed on the same days. At the end of the trial, sheep showed significantly reduced worm burdens
compared to unsensitized control sheep, confirming their resistance status. Both blood and tissue
eosinophils, as well as tissue cd TCR+cells were rapidly elevated by day 1 post L3 challenge (pc), peaking
at day 3 pc. There was a slight increase in tissue CD4 T cells at day 2 pc, peaking at day 3 pc while no
significant changes in CD8 T cells were observed. B cells (CD45R+) increased later into challenged tissues
with a peak at 5 days pc. All tissue lymphocyte subpopulations as well as tissue and blood eosinophils were
reduced by day 7 pc before increasing again at day 28 pc, suggesting separate responses to larval and adult
antigens. In contrast, globule leukocytes and mucosal mast cells only showed one peak at day 5 pc and
28 pc, respectively. Unexpectedly, globule leukocytes correlated significantly with tissue eosinophils but
not mucosal mast cells. The results are consistent with an early eosinophil-mediated killing of L3, possibly
recruited by IL-5 produced by cd T cells. In contrast to post-mortem studies, abomasal cannulation allowed
sequential analysis of both early and late time points in the same animal, providing a more complete picture
of cellular interactions at both peripheral and local sites, and their correlation with the different stages of
parasite development.
nematode / immunity / sheep / eosinophil
1. INTRODUCTION
There have been several studies characteriz-
ing the cellular changes that occur with primary
andchallengenematodeinfectionsinbothrodent
and ruminant models (reviewed in [1, 9]). Very
few of these studies have investigated the early
period of putative larval rejection and mosthave
concentrated on adult stage interaction with the
host. Several experiments have however shown
that larval rejection can and does occur in both
*Correspondingauthor:els.meeusen@med.monash.
edu.au
Vet. Res. (2010) 41:37
DOI: 10.1051/vetres/2010009
? INRA, EDP Sciences, 2010
www.vetres.org
Article published by EDP Sciences

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Haemonchus contortus and Trichostrongylus
colubriformis [14, 15], and this may be the pre-
dominant mechanism of resistance to gastroin-
testinal nematodesin
Eosinophils, mast cells and globule leukocytes
haveall beenimplicatedas effector cells mediat-
ing resistance to gastrointestinal nematodes,
although their precise role in parasite rejection
has not been elucidated.
The kinetics of cellular changes with time of
infection and immunity can be informative of
what immune mechanisms are operating at dif-
ferent stages and how these may interact. These
studies have however been limited due to the
large number of animals needed for sequential
killing when working with outbred populations.
Previous studies have shown that abomasal can-
nulation is an effective tool for collecting muco-
sal tissue samples in sheep [12]. Collection of
consecutive samples from the same animal can
reduce individual variation as well as permit
the sequential observation of cell populations
that are recruited early during the histotrophic
stage of the nematode larvae [12, 19, 21]. In
the present study, a surgical technique of insert-
ing an abomasal cannula to take abomasal
mucosal biopsy samples was employed to
observechangesatthesiteofH.contortusinfec-
tion in immune sheep. Tissue samples obtained
via fibreoptic endoscopy allowed both immuno-
histological and histological characterization of
cellular kinetics at early stages of infection with
subsequent enumerationofwormestablishment.
In addition, this procedure allowed direct corre-
lations between cell populations in the same
sheep and revealed significant associations
between critical cell subpopulations.
large animals[1].
2. MATERIALS AND METHODS
2.1. Animals and experimental design
Eighteen non-pregnant merino cross breed ewes
wereusedintheexperiment.Eweswerepasturereared
andacquiredfromacommercialsourceat6monthsof
age. Allsheep were treated initiallywith the manufac-
turersrecommendeddoseofivermectinat8 mL/sheep
(Ivomec 8 g/L, Merial, USA) and housed indoors
under nematode free conditions for 1 month before
commencement of the experiment. Sheep were ran-
domly allocated to 3 groups (Tab. I). Groups 1 and 2
comprised a total of 11 sheep that were sensitized by
oral infection with 5 000 L3 H. contortus L3 larvae
once per week for 12 weeks. The sheep were subse-
quently drenched with ivermectin, and maintained
nematodefreeforafurther12weeks.Group2(7sheep)
was then challenged with50 000 L3 H. contortus and
group 1 (4 sheep) sham challenged withsaline. Group
3comprised7sheepwhichwerehousednematodefree
for 24 weeks without immunization and then chal-
lenged with 50 000 H. contortus L3 larvae (infection
controls). The challenge dose was given just after tak-
ing the Day 0 biopsy sample in groups 1 and 2. Sheep
inallgroupswereeuthanisedat28dayspostchallenge
(pc) by an intravenous injection of lethabarb (Virbac
Pty. Ltd, Australia).
Larvae used for sensitizing and challenging the
sheepwere ensheathed
H. contortus. The experiment was approved by the
University of Melbourne School of Veterinary
Science Animal Ethics Committee (Australia).
McMaster strain L3
2.2. Parasitological measurements
Adult worm counts were performed immediately
following euthanasia of the sheep. The abomasum
was collected and opened along the greater curvature.
The entire abomasal contents were counted for adult
worms in a transparent dish over a lightbox.
2.3. Abomasal cannulation
The abomasal cannula design used was based on
[12] and consisted of a 10 mL syringe, modified by
removing the luer end and placing a circular flange
over the syringe. The flange was molded from silastic
rubber (Dow Corning, Sydney, Australia) and was
approximately 7 cm diameter. This type of rubber
was chosen to limit the possibility of mechanical
abrasion of the mucosal surface of the abomasum.
Feed was withheld for 24 h prior to surgery. After
anaesthesia, a ventral midline incision was made to
locate and exteriorize the abomasum. A 2–3 cm
purse-string suture (Silk 2-0) was placed midway
between the lesser and greater curvature approxi-
mately10 cmfromthepyloricsphincter.Thislocation
waschosentoreducethechanceofcannulafailureand
tominimizethedisturbanceoftheH.contortusL3tis-
sueniche.Astabincisionwasmadeinthecentreofthe
purse-string suture. The cannula and inner flange was
placed inside the abomasum through the stab incision
and the purse-string suture was tightened and tied off.
Afurtherstabincisionwasmadeintheabdominalwall
Vet. Res. (2010) 41:37 N. Robinson et al.
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approximately 10–15 cm from the laparotomy inci-
sionontherightparamedianareatoenablethecannula
to be passed freely through while maintaining the
abomasum in an anatomically correct position. An
external flange was placed over the external syringe
barrel which was then secured in place by the use of
an elastrator ring (NASCO Export, Fort Atkinson,
WI, USA) and metal bolt that was placed transversely
throughthesyringetobothkeeptheflangeinplaceand
the stopper (a shortened 10 mL syringe plunger) from
externally dislodging. There were no problems with
cannula failure during the experiment. The external
parts of the cannula did not affect the sheep’s ability
to ambulate or assume a sternally recumbent position.
Post anaesthetic monitoring was carried out with
sheep housed individually for 24 h and then returned
to group housing. Procaine penicillin and benzathine
penicillin was administered during the surgical proce-
dure at the manufacturers recommended dose (1 mL/
20 kg bodyweight, Duplocillin, Intervet Ltd, Upper
Hutt, New Zealand).
2.4. Biopsy procedure
Biopsy specimens were taken from both group 1
(sensitized, saline challenged) and group 2 (sensi-
tized, L3 challenged) sheep using a flexible fibreoptic
endoscope (Olympus, Sydney, Australia). The biopsy
forceps used through the endoscope biopsy port pro-
duced a tissue specimen measuring 2 · 2 · 3 mm.
Sheep were restrained in a custom made sling
which exposed the ventral region of the sheep’s abdo-
men including the cannula. As in previous studies
[12], we found no need for sedation as the sheep
appeared relaxed and did not show any signs of pain
or discomfort during the procedure. While sheep are
restrained in the harness, the cannula stopper was
removed and the abomasal contents collected. The
endoscope was introduced into the abomasum and
the fundic region located which was the furthest dis-
tance from the cannula. Due to the design of the
biopsy forceps, it was found that the most productive
biopsy specimens were taken from the tip of the large
abomasal folds of the fundic region. The fibreoptic
endoscope allowed sufficient visibility (in color) to
differentiate structures within a 3–4 cm distance from
the tip of the scope, and characteristic abomasal folds
were readily visible. Four biopsy specimens were
taken for each time point. At subsequent time points
there was never any evidence of tissue injury from
previous biopsy collection, as observed macroscopi-
cally by eye or examined histologically for signs of
inflammation or regenerative changes. The flexible
endoscope also allowed different regions of the
abomasum to be sampled and, considering the size
of the biopsy relative to the surface area of the
abomasums, the probability of resampling the same
site is minute.
2.5. Peripheral blood
Peripheral venous blood was collected by jugular
venipuncture and placed into 5 mmol of ethylenedi-
aminetetraacetic acid (EDTA). Total WBC count was
performed by adding 10 lL of EDTA treated blood
with 190 lL Turk’s solution (1% glacial acetic acid
and 0.01% gentian violet in distilled H2O). Enumera-
tion of cells was performed using a haemocytometer
and results expressed as cells per litre of blood.
Differential white cell counts were performed on
peripheral blood (PB) smears stained with Diff Quik
(Dade Behring, Inc., IL, USA). For differential cell
counts, the differential percentage were multiplied
by the total WBC count and results expressed as cells
per litre of blood.
2.6. Histological procedures
For paraffin sections, tissue samples were placed
in 10% neutral buffered formalin and processed at
the University of Melbourne Veterinary Histology
Laboratory. Eosinophils and globule leukocytes were
differentiated morphologically on haematoxylin and
eosin stained sections.
For immunostaining, tissue biopsies were embed-
ded in Optimal Cutting Temperature (OCT) medium
Table I. Experimental protocol.
Group number (n) Weeks of
sensitization*
Challenge*
(Day 0)
Weeks between last
sensitization and challenge
Sampling time points
(days post challenge)
Group 1 (4)
Group 2 (7)
Group 3 (7)
12
12
0
?
+
+
12
12
?
0, 1, 2, 3, 5, 7, 28
0, 1, 2, 3, 5, 7, 28
28
*Sheep were sensitized by weekly infection with 5 000 H. contortus L3 and challenged with 50 000 L3.
Cellular kinetics after H. contortus infection
Vet. Res. (2010) 41:37
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(TissueTek, Miles Inc., USA), frozen on liquid nitro-
gen and stored at ?70 ?C until used. Five microns of
frozen tissue sections were fixed in 200 mL 100%
ethanol with 2 mL H2O2for 10 min at 4 ?C and
stained for cell surface markers using ovine-specific
monoclonal antibodies (mAb) as described previ-
ously [2, 3]. Briefly, mAbs were anti-sheep IgE,
CD4, CD8, CD45, CD45R, cd TCR (86D) and
WC1 (T19), a cd-TCR subset.
To stain for mucosal mast cells, frozen sections
were stained with 1% toluidine blue in a solution
of 50% PBS and 50% methanol for approximately
30 s.
2.7. Morphometric analysis
To maximise the number of cells counted per
biopsy in the abomasal mucosa, the entire biopsy
was counted using a counting graticule (Leica Micro-
systems, Wetzlar, Germany) of area 0.2 mm2. This
technique was used for both frozen and paraffin sec-
tions. Each biopsy was recorded as the mean number
of cells per graticule field (± SEM).
2.8. Statistical analysis
Group comparisons were conducted by compar-
ing group 1 with group 2 at each time point utilizing
unpaired student’s t-test. The linear dependence
between 2 variables within the experimental group
2 was calculated using the Pearson product-moment
correlation coefficient (r). p < 0.05 was considered
significant.
3. RESULTS
3.1. Changes in peripheral blood WBC
after challenge
Analysis of PB samples taken on days ?24
and day 0 before challenge show that both
group 2 (L3 challenged) and group 1 (sham
challenged) did not significantly differ for any
parameter measured (eosinophil, neutrophils
and lymphocytes) (Fig. 1). On day 1 pc, group
2 had significantly elevated PB eosinophils.
The upward trend continued on day 3 and
5 pc with significant difference between eosin-
ophils in group 2 compared to group 1. At the
remaining time points, days 7 and 28 pc, eosin-
ophils remained elevated above the control
group but not significantly. There were no sig-
nificant differences observed in the other PB
leukocytes at any time point.
3.2. Lymphocyte kinetics into abomasal tissues
after challenge
No difference was observed in total leuko-
cyte (CD45+cells) numbers between groups 1
and 2 before challenge (Fig. 2). Following chal-
lenge, group 2 showed an upward trend which
reached significance at day 3 before reaching a
plateau for the remaining time points.
CD4+cells were only slightly and not signif-
icantly differentbetween
2 at days 0, 1 and 2 pc (Fig. 2). CD4+cells
were significantly above controls on days 3, 7
and 28 pc, with a peak at day 3 and a second
increase from day 7 to day 28 pc. CD8+cells
were not significantly different at any time
point.
cd TCR+cells in group 2 increased from day
1 pc and peaked at day 3 pc before decreasing
slightly at days 5 and 7 pc (Fig. 2). Like the
CD4 T cells, cd TCR cells increased again by
day 28 pc. The WC1 cd TCR (T19+) cell sub-
set showed a similar trend with two peaks, one
occurring at day 3 pc decreasing to the lowest
point at day 7 pc before rising again at day
28 pc.
CD45R+cells which comprise mostly B
cells in the abomasum [7] were similar in both
groups 1 and 2 over days 0, 1 and 2 pc (Fig. 2).
At day 3 pc, B cells were significantly higher in
group 2 than group 1 and reached a peak at day
5 pc. CD45R+numbers appreciable declined by
day 7, followed by a slight increase in numbers
to day 28.
groups1and
3.3. Kinetics of mucosal effector cells
after challenge
Tissue eosinophils, mast cells and globule
leukocytenumberswerelowinallsheepfollow-
ing the rest period before the challenge (Fig. 3).
Group 1 maintained low levels of these cells
throughout the post saline challenge. Group 2
showed a significant increase in eosinophils by
day 2 pc, reaching a peak at day 3 pc before
declining, but remaining above control levels,
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on days 5 and 7 pc. On day 28 pc eosinophil
counts intheL3challenge groupwere againsig-
nificantly increased above the control group.
Globule leukocytes started to increase on
day 2 pc reaching a peak on day 5 pc
(Fig. 3). Even though there was a steady decline
after day 5 pc, globule leukocyte numbers on
both days 7 and 28 pc were significantly higher
than controls.
Mucosal mast cells showed no change from
day 0 to day 4 pc, and only started to increase
on day 5 pc reaching significance on day 7 and
continuing their increase to day 28 pc (Fig. 3).
IgE+cells in the L3 challenge group started an
0.0
0.2
0.4
0.6
0
1
2
3
4
5
Neutrophils
Days
-24
Cells x 109/L blood
Cells x 109/L blood
Cells x 109/L blood
0
2
4
6
8
10
Lymphocytes
Eosinophils
*
*
*
01235728
-24012357 28
-24012357 28
Figure 1. Peripheral blood eosinophils, neutrophils and lymphocytes (mean ± SEM) in saline challenged
(d) and L3 challenged (.) sensitized sheep, at various days post challenge. * Indicates significant
difference (p < 0.05).
Cellular kinetics after H. contortus infection
Vet. Res. (2010) 41:37
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