Proyecto «Uruguay+25»
B. Propuestas: coincidencias y diferencias
Group HFW : HFW and C lambs (Experiment 2)
Eight parasite-na"ive male lambs from each of the HFW and C flocks were infected at 4.5 months-of-age with 50,000 � 0. circumcincta. Four animals per
77 g roup were euthanased on Day 8 p.i. and the remaining eight animals were euthanased on Day 28 p.i. C lambs were #9-1 2 and 1 7-20 and H FW lambs were # 1 3-1 6 and 2 1 -24 (Experiment 2) (ful ly described in Section 2.2. 1 )
Group Ll: Larval Infection Experiment
Twenty-six parasite-na'ive Coopworth lambs, approximately 3 months-of-age, were used. Twenty lambs each received 35,000 0. circumcincta L3 i ntraruminally and were sacrificed 5, 1 0, 1 5, 20 or 30 days after infection. Times of infection were staggered so that one animal was euthanased per day and animals were sacrificed over a period of 7 weeks. Two uninfected control lambs were sacrificed at the start of the experiment and the remaining four controls at the end of the study. Sheep n umbers were: Control #25-30, Day 5 #31 -34, Day 1 0 #35-38, Day 1 5 #39-42, Day 20 #43-46 and Day 30 #47-50. Group AT: Adult Transplant Experiment
Fifteen parasite-na'ive Coopworth lambs, approximately 4 months-of-age, were used. All lambs underwent surgery to implant abomasal cannulae, as described in Section 2.2.2.2. Twelve lambs each received approximately 1 0,000 adult 0. circumcincta through the abomasal cannulae and were sacrificed 6h, 1 2h , 24h and 72h after the transplant. Adult worms were derived from donor sheep, which had been slaughtered 2 1 days after infection with 1 00,000 0. circumcincta l3. Pooled abomasal contents of donor sheep, concentrated by sedimentation, were divided into 1 3 aliquots. Worm numbers were estimated in one aliquot, whilst the remaining 1 2 were given to the recipient animals within 1 - 2h of collection. Three control lambs were sacrificed at the start of the experiment. Sheep numbers were: control #53-55, 6h p . i. #56-58, 1 2h p.i. #59-61 , 24h p.i. #62-64 and 72h p.i. #65-67.
3.2.2 Abomasal
and Serum Gastrin
For G roup H FW, sample collection and assays have been described in Sections 2.2.3 to 2.2.5. Serum gastrin concentrations and abomasal pH are presented in Figures 2. 1 5 to 2.20.
For Group Ll, abomasal fluid for pH measurement and jugular venous blood samples for serum gastrin concentration (Appendix 2.4. 1 ) were collected in a
similar manner to that described in Sections 2.2.4 and 2.2.5. Samples were collected daily for all sheep sacrificed up to Day 1 5 p.i. Sheep sacrificed on Day 20 p.i. were also sampled on Day 1 7 p.i., while animals sacrificed on Day 30 p.i. were sampled every second day from Day 1 5 to Day 27 p.i. All sheep had blood and abomasal fluid taken on the day of necropsy.
For G roup AT, the pH of abomasal fluid col lected at necropsy was used for correlation with histological parameters.
3.2.3
The protocol for necropsy of G roup HFW sheep is described in Section 2.2.2.3. The only difference for the other groups was that, instead of stunning and exanguination, the sheep were euthanased by intravenous injection of a barbiturate (Pentobarb 500®, NZVet, Auckland, NZ) overdose. Fundic tissue from all sheep was fixed in Bouin's solution. For G roup Ll , additional tissue was collected from control sheep and those sacrificed on Days 5 and 1 0 and fixed in 2% glutaraldehyde.
3.2.4 Electron
Tissues fixed in 2% glutaraldehyde were routinely processed and embedded in epoxy resin (EPON81 2®, SPI supplies, West Chester, USA). Sections 1 �-tm thick were also cut and stained with 1 % toluidine blue for light microscopic examination. Sections for transmission electron microscopy were cut 70-90nm thick, mounted on 200 mesh copper grids (SPI supplies, West Chester, USA) and stained with 2% uranyl acetate (Sigma-Aidrich, St. Louis, USA) followed by Reynold's lead citrate (Reynolds, 1 963). Sections were examined using a Phi lips 201 C transmission electron microscope.
3.2.5.1 Tissue
Gastric mucosa was fixed overnight in Bouin's solution and stored in 70% ethanol (vlv) until further processing. Samples were routinely processed for paraffin wax embedding. They were dehydrated in 70% ethanol, cleared in
79 chloroform and two changes of xylene and infiltrated with paraffin wax at 59°C, according to the standard schedule on a TP1 050 automatic tissue processor (Leica Jung, Wetzlar, Germany). The samples were embedded on a Tissue Tek® embedding console (Miles Scientific, Chicago, USA) and then sectioned on a rotary microtome at 5J.!m thickness. The sections were floated in a waterbath at 40°C, attached to microscope slides and oven-dried at 60°C. Sections were dewaxed and hydrated using a standard method.
3.2.5.2 Standard Stains
Paraffin-embedded sections 5Jlm thick were stained with haematoxylin and eosin (H&E) from all tissues. Additional sections from Group Ll sheep (control and Days 5 and 1 0 p.i.) and Group AT were stained using Luna's method for eosinophils (Luna, 1 993}. For G roup Ll sheep, resin-embedded semithin sections of 111m thickness were stained with 1 % toluidine blue.
3.2.5.3
Dewaxed and hydrated 5Jlm thick sections were pre-treated with 0.05% saponin (Sigma-Aidrich, St. Louis, USA), washed with phosphate buffered saline ( PBS) and exposed to 0.5% bovine serum albumin (BSA) (Fraktion V, Boehringer Mannheim GmBH, Germany). Sections from all tissues were stained for TGF-a. Sections form G roup HFW sheep only were stained for eos inophils.
Immunohistochemical staining for TG F-a was done using a mouse monoclonal antibody (lgG2a) (Calbiochem, San Diego, CA, U SA) in a 1 /500 dilution in P BS containing 0.5% BSA. I mmunohistochemical staining for sheep eosinophils was performed using a mouse monoclonal (lgG1 ) antibody (the kind gift of Dr Wayne Hein, AgResearch Ltd, Wallaceville, NZ) in a 1 /500 dilution in phosphate-buffered saline (PBS) containing 0.01 % BSA. The second antibody in each case was an ovine anti-mouse antibody (whole lg) (Amersham Life Science, Little Chalfont, U K) in a 1 /200 dilution in PBS contain ing 0.5% BSA. The streptavidin-biotin method was used to amplify binding of antibodies using 1 /200 dilution of a streptavidin-biotinylated horseradish peroxidase complex (Amersham Life Science, Little Chalfont, U K) in PBS contain ing 0.5% BSA.
Antibody binding was visualised using a 0.4% (w/v) d iaminobenzidine solution containing heavy metal intensification and 0. 1 2% (v/v) hydrogen peroxidase. All sections were counterstained with Mayer's haematoxylin. Some of the TGF-a stained sections from the larval infection experiment were also counterstained with H&E to assess the number of parietal cells (if any) that did not bind the antibody.
3.2.5.4 and TGF-a Parietal Cell Counts
Nucleated parietal cells and eosinophils were counted in a 258pm wide column of mucosa (a location), from the muscularis mucosae to the luminal surface at 40x magnification using a 1 cm2 eyepiece graticule. Nucleated parietal cells were counted within the full m ucosal depth from base to surface in five locations within each section. When counting parietal cells, nodular areas associated with larval development within glands were avoided. Tissue thickness was also recorded.
G roup H FW, two sections from each of two folds and for G roups Ll and AT, five sections, one from each of five gastric folds, were examined per animal. 3.2.5.5 TGF-a Parietal Cel l Counts
From the groups of animals killed on each day in G roup Ll , TGF-a stained sections counterstained with H&E from two or three animals were used to establish the number of TGF-a negative parietal cells. Parietal cells that were clearly labelled only for H&E were counted and added to the TGF-a (only) counts and expressed as the total parietal cell cou nt. TGF-a negative parietal cells were then expressed as a percentage of the total count. For Group AT, tissues from one or two animals per group were used to establish the n umber of TGF-a negative parietal cells.
3.2.6 Statistical
Data are presented mean ± SEM (J1 ± S EM).
For G roup H FW, data were examined for normality using the Shapiro-Wilk test in the S PSS version 1 1 .0 . If necessary, data were log1o transformed. In all
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