Table 1.
Inoculation routes and Clone 13 titers used in this study.
Fig 1.
Schematic representation of the DSS study.
Lambs (n = 14) were inoculated with Clone 13 via SC and ID route. A group of the same size functioned as sentinels. On days 2, 4, 6, 8, 10, 12 and 14, two lambs from each group were euthanized. The following samples were collected for detection of Clone 13: saliva, nasal mucus, lacrimal fluid, urine and faeces. During necropsy, gonades, kidney, adrenal gland, spleen, Ln. ileocaecalis, small and large intestines, bladder, pancreas, liver, lung, thymus, heart muscle, injection sites, M. tripceps brachii, Ln prescapularis left and right, salivary gland and brain samples were collected.
Fig 2.
Schematic representation of the RTV study.
Two lambs were inoculated via SC and ID routes and blood samples were collected on D0, 1, 2, and 3. On day 7, lambs were euthanized and prescapular lymph nodes were collected. Samples containing the highest amount of Clone 13 RNA were inoculated via the same routes into two naïve lambs (Passage 1). No virus RNA was detected in similar samples collected from these lambs. The passage was repeated with another group of 10 lambs (Repeat passage 1). In none of these lambs, Clone 13 RNA was detected.
Fig 3.
Schematic representation and major findings of the safety study with pregnant ewes.
Ewes were inoculated with Clone 13 via SC and ID routes at 50 days or 120 days after mating. Ewes were monitored for clinical signs until delivery of the lambs, which were subsequently investigated for clinical signs, malformations, precolostral antibodies and presence of virus in blood and organs upon necropsy at the end of the study. Ewes seropositive by ELISA are indicated with a blue Ig symbol. The small symbol indicates a doubtful result. 1Ewe was euthanized after signs of imminent abortion; 2Ewe was euthanized because lambing had not occurred at the anticipated time point; 3Lamb was born alive, but showed signs of central nervous system dysfunction such as ataxic movements and disorientation and was euthanized to minimise suffering, 4Lamb corresponding to Fig 4A, 4B and 4D, 5Lamb corresponding to Fig 4E. Coloured dots refer to the following findings in lambs: viral RNA in blood or organ (red dots), malformations (blue dots) and precolostral antibodies (green dots).
Table 2.
Detection of Clone 13 by RT-qPCR in samples taken from lambs during the dissemination, shedding and spreading (DSS) study1.
Fig 4.
In utero infection of fetuses is associated with malformations of the skeletal- and central nervous system.
(A) Arthrogryposis and scoliosis and (B) malformations of the central nervous system i.e. hydranencephaly and hypoplasia of cerebrum, and cerebellum in a stillborn lamb from an ewe vaccinated with the Clone 13 approximately 50 days after mating. (C) Myelin staining of the spinal cord from a fetus with normal morphology or (D and E) myelin staining of a spinal cord displaying hypoplasia of the grey matter (micromyelia) at (D) 20x magnification (E) 400x magnification. Myelin staining was performed with a rabbit anti-myelin antibody and a horseradish peroxidase-labelled anti-rabbit conjugate. The identities of the lambs are indicated by the footnotes in Fig 3.