Vaccine strain of virus
Icelandic Maedi-Visna strain K796 was selected for preparation of vaccine, because long-term animal experiments carried out 1960–1975 had demonstrated that this strain was highly immunogenic and caused both Visna and Maedi in the experimental sheep [6
]. Strain K796 was originally isolated from the brain of a symptomatic sheep in the 5th
brain-to-brain laboratory passage of natural Visna. In preparation for this vaccine trial in Cyprus, WB tests on a few sera from older sheep in the flock that had been selected to house the experiment tested positive against strain K796, indicating that related strains circulated in the flock.
Virus was grown in sheep choroid plexus tissue culture from 4 month-old Icelandic lambs. Cells were grown in tissue-culture flasks on a flat surface, (80cm2), in medium 199 with Earl’s salts (199E, Gibco, UK) and 20% Icelandic lamb serum. When the cell cultures were confluent the medium was changed to 199E with 2% lamb serum and the tissue cultures infected. Cytopathic lesions were at their maximum, 8–9 days after infection and then the tissue culture fluid was harvested, cell debris removed by centrifugation at 3332G for 40 minutes and the supernatant inactivated in formalin (Merk, Germany) 1:4000 at 37°C for 4–5 days. At the end of the inactivation period the fluid was divided into 200 ml aliquots, which were centrifuged at 22524G for 4 hrs. All supernatant fluid was then discarded and the pellet in each bottle resuspended in 2 ml. medium 199E without serum, thus concentrating the virus preparation 100x. In order to test for failure of inactivation, 2 ml. of concentrate from one pellet were inoculated into 2 bottles of tissue culture and incubated 5–6 weeks with frequent microscopic examinations for cytopathic lesions. When this control was finished, sterile aluminium hydroxide (Fisher Scientific Company, USA. E&A Tested Purity Reagent) powder, 5 mg/ml, was added to the vaccine. After mild shaking twice a day for one week the vaccine was ready for use.
The vaccinees were one twin lamb in each of 40 female twin pairs born to mothers of the Chios breed of sheep into an infected flock of about 700 adult sheep on one well maintained sheep farm in Cyprus. The other twin served as an unvaccinated control throughout the experiment. Mothers of these twins were selected according to results of AGID screening tests on all pregnant ewes in the flock a month before birth of these lambs: 30 mothers seronegative and, for comparison, 10 mothers seropositive in the AGID test. Blood samples were collected from mothers and both twins at birth of each twin pair when one twin in each pair was vaccinated. Blood samples from both twins in each pair were also collected 3 weeks after the first, second and third vaccination, then again 6 weeks after the 3rd vaccination and thereafter at 5–7 months intervals during the next 2 years. Serum was harvested from all blood samples and frozen at – 20°C. The original plan for this experiment was to vaccinate one twin lamb in 30 female twin pairs born into a naturally infected sheep flock by seronegative mothers and, for comparison, one twin in 10 pairs born by seropositive mothers. When sera were recollected from the mothers at birth of their lambs, 13 of the 30 mothers negative in AGID tests 4–5 weeks earlier were positive in WB tests against the vaccine strain K796. All the 10 mothers previously positive in the AGID tests were positive in the WB tests. In accordance with different immunological experience of the mothers the vaccinees were divided into 3 groups:
Group 1) 17 twin pairs born by mothers seronegative both in AGID and WB tests.
Group 2) 13 twin pairs born by mothers negative in AGID, but positive in WB tests.
Group 3) 10 twin pairs born by mothers positive both in AGID and WB tests.
The flock was kept and fed on the sheep farm under natural farming conditions throughout the experiment. According to farming traditions, newborn lambs are kept with their mothers for their first 35 days of life, then taken away from the mothers and moved to the main flock. This applied to the experimental lambs as well as all other lambs born into this flock.
This experiment was approved of the Chief Veterinary Officer in Cyprus, who also was the Director of the Department of Veterinary Services at the Ministry of Agriculture, Natural Resources and Environment. The Ministry supported this work by a grant for maintenance of the experimental sheep, as two branches of the National Veterinary Services, the Central Virus Laboratory in Nicosia and the National Veterinary Services in Limassol, were major participants in this work.
Vaccine, 1 ml per dose, was given at the birth of the vaccinee, a second injection 3 weeks later, then a third injection when the lambs were 3 months old. There were no later boosters. Vaccine was injected intramuscularly with no side effects.
Diagnostic laboratory work
The AGID screening tests, then used for diagnostic work at the Virus Laboratory of the National Veterinary Services in Cyprus were performed on over 600 pregnant ewes on the farm during the last month of their pregnancy in order to select mothers for the vaccinees. Commercially available test plates (Weybridge Laboratories, England) were used in this work and the tests were performed by senior technicians according to instructions for use of the plates. Methods for Western Blots (WB) and Complement Fixation (CF) tests done in Iceland have been previously described and compared [7
]. The CF test was chosen for laboratory diagnosis of infection in vaccinees and their unvaccinated siblings because it is a reliable diagnostic test [3
]. It is comparable with the WB test in time of antibody response after infection [8
]. Good CF antigens, that is high titer untreated tissue culture fluid, could be prepared from local Maedi-Visna strains isolated from other sheep in the flock. Their primary isolation in choroid plexus cells from Icelandic lambs was time consuming and their growth low and slow as compared with K796. Serial 2 fold dilutions of sera were prepared and mixed with antigen and 4 units of complement (Guinea Pig Complement, Harlan Sera Lab, UK). This mixture was kept overnight at + 4°C, the hemolytic system added next morning and reading done 3–4 hours later. Four-fold or larger difference in titer is significant in a CF test [9