Breed differences in humoral and cellular responses of lambs to experimental infection with the gastrointestinal nematode Teladorsagia circumcincta

Ethical approval

All procedures described in this study were conducted under experimental license from
the Irish Department of Health in accordance with the Cruelty to Animals Act 1876
and the European Communities (Amendments of the Cruelty to Animals Act 1976) Regulations,
2002 and 2005.

Animals

All lambs (32 Texel and 29 Suffolk) were sourced from the flock of purebred Suffolk
and Texel sheep maintained at Athenry Research Centre 6]. Lambs of both the breeds were born indoors from a synchronised mating programme
and then all the lambs were moved to the same pasture for a 6-week period. Lambs were
weaned at about 6 weeks of age and moved indoors where they were maintained on a concentrate-based
diet with free access to water for the remainder of the experiment. Upon housing,
faecal sampling per rectum was attempted on all lambs, but sufficient material was obtained from only 36 individuals
(16 Texel and 20 Suffolk). All lambs were then treated with Ivermectin (Oramec, Merial
Animal Health Limited) according to the manufacturer’s instructions and quarantined
in a slatted pen for a period of 48 h prior to being penned as one group on straw.
Information on age and live weight at housing together with the results from the faecal
samples collected 25] at housing are summarised in Table 1. Five weeks post-housing and anthelmintic treatment, faecal samples were collected
from all animals on 3 consecutive days to determine GIN infection status. Lambs with
a positive FEC for “other” trichostrongyles (FEC_OT), excluding Strongyloides papillosus, (two cases: 1 epg and 2 epg) or for S. papillosus (FEC_SPAP; 7 cases: epg ranged from 1 (5 cases) to 50 (2 cases) on any of these days were treated
with Ivermectin (Noromectin Drench, Norbrook Laboratories Limited) according to the
manufacturer’s instructions and re-sampled to establish the GIN infection status;
all were observed to be GIN negative. Approximately 1 week prior to administration
of the challenge bolus, lambs were weighed and faecal sampled to confirm the GIN free
status.

Table 1. Information (± s.e.) on age, live weight and faecal egg counts for the experimental
animals at housing

Experimental infection

At approximately 20 weeks of age (~14 weeks post housing), the lambs were randomly
assigned within breed, to one of 6 slaughter time points (0, 3, 7, 14, 21 or 35 days
post-infection (pi)), with the restriction that members of a twin pair were not assigned
to the same time point. The slaughter days were chosen based on the expected developmental
stages of T. circumcincta in the sheep abomasum; day 3 and day 7 (L4 stage), day 14 (immature adult), days
21 and 35 (mature adult). Animals assigned to the day 0 time point (6 Texel and 4
Suffolk) did not receive an experimental challenge, while all other lambs (5 per breed
per time point with an exception of 6 Texels for the day 35 time point) received a
single oral dose of 3?×?10⁴ infective T. circumcincta larvae (L3). Blood samples were collected into vacutainers with EDTA, lithium-heparin
or no anticoagulant, respectively, by jugular vene-puncture immediately prior to slaughter.
Plasma was harvested following centrifugation at 2000?g for 5 min at 4 °C and stored at ?20 °C until use. Blood collected for serum antibody
measurement was stored in a refrigerator overnight for clotting. Serum was extracted
following centrifugation at 2000?g for 5 min and stored at ?20 °C until use. Faeces were collected per rectum on day 21 and 35 pi and a 3 g aliquot was used to determine the FEC using the modified
McMaster method 25]. Animals were slaughtered by electrical stunning followed immediately by exsanguination.

Worm count

The abomasum was recovered immediately after sacrifice and the contents recovered;
the abomasum was then dissected along the greater curvature. A saline-digest technique
was used to recover the worms from the abomasal mucosa 26]. Both contents and digest were washed through sieve 1 (75 ?m) and sieve 2 (38 ?m)
followed by preservation in 10% neutral buffered formalin (NBF) 26]. Adult worms were counted from both contents and digests. Extrapolation from two
1% or two 5% aliquots, from sieve 1 and sieve 2 respectively was used for calculating
the worm burden.

Abomasal mast cell and eosinophil counts

A section (approximately 2 cm²) of abomasal tissue was taken from the midline fold after the recovery of the abomasal
contents. The tissue sample was fixed in 10% NBF and processed in a Sakura Tissue-Tek®
VIP processor (Clinical Distributor, Dublin, Ireland). The tissue was sectioned using
a Leitz® 1512 Microtome (GMI Inc, mounted on microscopic slides. These sections were
stained using GIEMSA (Merck, UK) and examined using a QImaging colour camera (Nikon,
Japan) under a 40X objective. The images were visualised using the program Image-Pro.
Mast cells and eosinophils were counted in a field area of 0.023 mm². A total of 50 such fields were counted per slide and the mean figure was expressed
as total number per 0.023 mm².

Mucosal antibody recovery

Immediately after slaughter, the surface layer together with the mucus epithelial
layer of a portion of abomasal fold tissue (~3 cm²), excised from the midline of the dissected abomasum, was removed by scraping with
a microscope slide, placed in cryovials and snap frozen in liquid nitrogen followed
by storage at ?80 °C until use. The samples were prepared for antibody recovery according
to the method described previously 27]. Briefly, thawed samples in 3 volumes of phosphate buffered saline (PBS) containing
5 ?g/mL of protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA) were homogenised
using a Retsch® tissue lyser (Qiagen, Crawley, UK). After centrifugation of the homogenate
at 12 000?g for 30 min the supernatant was removed and protein concentration determined using
the BCA protein assay reagent kit (Pierce, IL, USA). The remaining supernatant was
stored at ?20 °C.

IgA ELISA

Antigen from T. circumcincta L3 was freshly prepared as previously described 26]. ELISA assays to measure IgA in serum or mucosa were performed as described previously
23]. The wells of 96-well polystyrene ELISA plates (BD FalconTM) were coated with 100 ?L
of L3 antigen (5 ?g/mL) in carbonate buffer at pH 9.6 and stored at 4 °C overnight.
The plates were washed 4 times using PBS-T (PBS?+?1% Tween). An aliquot of 100 ?L
of either serum sample (diluted 1:50) or mucosal sample (adjusted to 500 ?g/mL) was
placed in each well. After plates were washed in PBS 4 times using PBS-T, 100 ?L of
monoclonal mouse anti-ovine IgA (AbD Serotec, UK), diluted 1:50, was added to each
well. Plates were then washed 4 times using PBS-T and 100 ?L of a secondary goat anti-mouse
horse-radish peroxidase (HRP) conjugate (Dako Diagnostics, Dublin, Ireland) was added
to each well and incubated at 37 °C for 30 min. After 4 washes with PBS-T, 100 ?L
of chromogen, tetramethylbenzidine (TMB) (Dako Diagnostics, Dublin, Ireland) was added
to each well and incubated for 15 min at room temperature. The reaction was stopped
using 100 ?L of 10% 1 M HCl. The AsysTM UVM-240 microplate reader (VWR International,
Dublin, Ireland) was used to read the optical density at 450 nm of each plate. Each
plate included 3 wells with PBS (TBSA) instead of plasma as a blank, a single serum
sample yielding low levels of nematode specific antibodies as a negative control and
a single serum sample yielding high levels of nematode specific antibodies as a positive
control.

Pepsinogen and haematology

Plasma pepsinogen concentration was measured as described previously 28]. Whole blood was used for haematology analysis using an ADVIA2120 haematology system
(Bayer Healthcare, Leverkusen, Germany) as per manufacturer’s instructions. Neutrophils,
eosinophils, monocytes, lymphocytes, basophils, red blood cells (RBC), haemoglobin
concentration (Hgb), platelets and mean corpuscular volume (MCV) were measured.

Statistical analysis

All data were analysed using Proc MIXED (SAS 2003) to fit a model that had effects
for breed, time and their interaction. Observations on eosinophils in abomasal sections
and on FEC were log transformed prior to analysis to normalise the residuals. Data
on differential white cell counts were subjected to the arcsine transformation prior
to analysis. Because of the large variation in mean values among subclasses (day or
day-by-breed) for many of the variables the heterogeneity of residual variances was
tested and accommodated in the mixed model when significant. The results for the F
tests of breed and day effects and their interaction are reported but the main interest
is the evidence for breed differences in the pattern of change following experimental
infection. Thus, the differences among slaughter days were evaluated on a within-breed
basis using Dunnett’s test with the lambs slaughtered on day 0 as the control group.
Orthogonal polynomials were used to partition the variation between days into single
degree of freedom components to describe the pattern of change with time.