3. MATERIALS AND METHODS
3.2. Parasitological Techniques
3.2.1. Infection of Donor Sheep
The nematodes H. contortus and O. circumcincta had been donated to the Institute of Veterinary, Animal and Biomedical Sciences at Massey University by AgResearch, Palmerston North in 1990, where they had been selected from sheep carrying field infections. In the laboratory where this research was carried out, each species had been separately passaged through sheep for several years. L3 of these parasites were stored in distilled water at 4°C for O.
circumcincta and at 10°C for H. contortus, respectively. Larval motility as a measure for their viability was assessed at room temperature under a microscope. Only batches containing at least 98% motile larvae showing strong movements were used to infect the donor sheep, which were given either 6,000 H. contortus or 10,000 O. circumcincta L3 intraruminally by tube.
Donor sheep, 6-12 months old, were used to collect eggs for the maintenance of larval cultures of the parasite strains or to raise adult worms. The sheep had either been raised parasite-free indoors or were brought indoors 2 weeks before infection. They were fed lucerne chaff ad libitum and had water freely available.
When brought indoors, the sheep were drenched with a double dose of Leviben® (15 mg/kg levamisole + 8 mg/kg ricobendazole; Young’s Animal Health Ltd., New Zealand) and a single dose of Ivomec® (0.4 mg/kg ivermectin; Merck and Co., USA). Faecal samples were taken 5 days later and faecal floats in saturated saline were carried out to confirm that the sheep were free of nematodes.
Intramuscular injections of dexamethasone (Dex 5®, 0.15 mg/kg; Virbac, New Zealand) were given twice weekly for the duration of the infection, beginning one week before infection with H. contortus or O. circumcincta.
3. Materials and Methods
3.2.2. Recovery of Adult Parasites
To obtain adult nematodes for further experiments, the infected sheep were killed between 19 and 26 days p.i., the day with the highest amount of adult parasites determined from daily faecal egg counts performed according to the McMaster method modified by STAFFORD et al. (1994) from day 19 p.i.
onwards. Donor animals were killed by stunning with a captive bolt, followed by exsanguination. Their abdomen was opened immediately and the abomasum ligated and removed with its entire contents.
Adult worms were collected from the abomasum using the technique of SIMPSON et al. (1999), which is a modification of the method of VAN WYK and GERBER (1978). The isolated abomasum was opened along its greater curvature and the contents collected. The luminal surface was washed with saline (0.9% NaCl solution, at 37°C), warmed to 37°C to remove adherent worms. The washings were combined with the abomasal contents and solid material was allowed to settle in a measuring cylinder in a 37°C waterbath. After settling, some of the surface fluid was carefully poured off to reduce the volume.
A 3% agar (Bacto Agar, DIFCO Laboratories, USA) solution in water at 40°C was mixed with the worm solution in a ratio of two parts abomasal contents to one part agar. This mixture was poured immediately into shallow plastic trays, setting rapidly in blocks about 1 cm deep, which were covered with pre-warmed saline and placed in a 37°C room. Soon after covering, the worms started emerging from the agar block into the saline where they proceeded to form clumps. Most worms had migrated from the agar by 30 min. The clumps of worms were picked up carefully with fine forceps and placed in PBS. They were washed three times in sterile PBS, followed by two washes in the incubation medium. Incubation to produce E/S products began immediately (Section 3.3).
3. Materials and Methods
3.2.3. Larval Culture
To maintain pure cultures of L3, male donor sheep were infected with either H.
contortus or O. circumcincta larvae (Section 3.2.1). Faeces were collected into bags held in place by a harness. After the presence of nematode eggs in the faeces had been detected by faecal floats carried out from 19 days p.i. onwards, faecal egg counts were carried out using the McMaster method modified by STAFFORD et al. (1994). When there were more than 100 eggs per gram, faeces were collected daily, put into trays and mixed with water and about half their volume of Vermiculite® (Cartann Horticultural Supplies, New Zealand). The covered trays were incubated for at least 14 days in a room with a constant temperature of 24°C, keeping them moist by watering them regularly. L3 were then harvested by putting the faeces in sieves lined with tissue paper and immersing them overnight in bowls of deionised water. In this time, the larvae emerged from the faeces and sedimented in the bowl. The water was poured through a 22 µm mesh filter and the retained larvae poured into tissue culture flasks containing deionised water at a density of about 1,000 larvae/ml. They were stored in the dark, O. circumcincta at a temperature of 4°C and H.
contortus at 10°C, until used in experiments or for the infection of sheep.
3.2.4. Exsheathing and Counting L3
Larvae to be used for the generation of E/S products were exsheathed prior to incubation by mixing them with 0.2% sodium hypochlorite solution and monitoring their exsheathing under a microscope. The exsheathed larvae were removed from the solution by centrifugation at 200 x g for 2 min and washed four times, twice in deionised water, then twice in the incubation medium in which they were finally resuspended. They were counted under the microscope by mixing 100 µl of the suspension with a drop of iodine on a microscope slide and calculating the mean of ten replicate estimations to obtain larvae/ml suspension.
3. Materials and Methods