Clinical, pathological, and molecular features of classical and L-type atypical-BSE in goats

<div><p>Monitoring of small ruminants for transmissible spongiform encephalopathies (TSEs) has recently become more relevant after two natural scrapie suspected cases of goats were found to be positive for classical BSE (C-BSE). C-BSE probably established itself in this species unrecognized, undermining disease control measures. This opens the possibility that TSEs in goats may remain an animal source for human prion diseases. Currently, there are no data regarding the natural presence of the atypical BSE in caprines. Here we report that C-BSE and L-type atypical BSE (L-BSE) isolates from bovine species are intracerebrally transmissible to goats, with a 100% attack rate and a significantly shorter incubation period and survival time after C-BSE than after L-BSE experimental infection, suggesting a lower species barrier for classical agentin goat. All animals showed nearly the same clinical features of disease characterized by skin lesions, including broken hair and alopecia, and abnormal mental status. Histology and immunohistochemistry showed several differences between C-BSE and L-BSE infection, allowing discrimination between the two different strains. The lymphoreticular involvement we observed in the C-BSE positive goats argues in favour of a peripheral distribution of PrPSc similar to classical scrapie. Western blot and other currently approved screening tests detected both strains in the goats and were able to classify negative control animals. These data demonstrate that active surveillance of small ruminants, as applied to fallen stock and/or healthy slaughter populations in European countries, is able to correctly identify and classify classical and L-BSE and ultimately protect public health.</p></div

Non-quantitative diagram of neuroanatomical distribution of PrP immunolabelling in L-BSE group.

<p>This distribution refers to one animal (69540) representative of all animals in the group. A: Telencephalon; B: Diencephalon; C: Mesencephalon; D: Pons; E: Cerebellum; F: Brainstem.</p

Antibodies to PrP used for IHC and WB analysis, indicating the corresponding epitopes.

<p>Antibodies to PrP used for IHC and WB analysis, indicating the corresponding epitopes.</p

Mean values of vacuolar lesion profiles.

<p>Square = BSE i.c. goats; rhombus = L-BSE i.c. goats (Brain areas, Medulla (at obex): 1 Dorsal nucleus of the vagus nerve, 2 Nucleus of the hypoglossal nerve, 3 Reticular formation, 4 Midline Raphe, 5 Accessory cuneate nucleus, 6 Olivary nuclei; Rostral medulla: 7 Vestibular nuclear complex, 8 Cochlear nucleus, 9 Nucleus of the spinal tract of the trigeminal nerve, 10 Midline raphe; Cerebellar vermis: 11 Nodulus±granular layer, 12 Nodulus±molecular layer; Midbrain: 13 Central grey matter, 14 Red nucleus, 15 Substantia nigra, 16 Lateral geniculate nucleus; Thalamus: 17 Dorsomedial thalamic nucleus, 18 Ventral thalamic nuclei, 19 Area hypothalamica; Frontal: 20 Caudate nucleus, 21 Nucleus accumbens, 22 Frontal cortex).X-axis: brain areas; Y-axis: mean vacuolation score with error bars (standard deviation).</p

Haematoxylin and eosin.

<p>A: Frontal cortex of C-BSE (10X); B: Frontal cortex of L-BSE (10X); C: Dorsal nucleus of vagus nerve, brainstem of C-BSE (10X); D: Dorsal nucleus of vagus nerve, brainstem of L-BSE (10X). The single image shows one animal (81556 for C-BSE and 69540 for L-BSE) representative of all animals in the group. Scale bar: 100 μm.</p

PrP immunohistochemistry of muscle samples from the Holstein-Friesian cow affected by natural BASE (#126752/09).

<p>PrP immunohistochemistry of muscle samples from the Holstein-Friesian cow affected by natural BASE (#126752/09).</p

Transmission of BASE to Tgbov XV mice following inoculation of muscles from experimentally and naturally affected cattle.

<p>(A) Lesion profiles of mice infected with brain tissue from natural BASE (blue line) and BSE (green line), <i>longissimus dorsi</i> muscle from experimental BASE (red line) and <i>intercostalis</i> (bordeaux line) and <i>gluteus</i> (yellow line) muscles from natural BASE. Vacuolation was scored on a scale of 0–5 in the following brain areas: 1, dorsal medulla; 2, cerebellar cortex; 3, superior colliculus; 4, hypothalamus; 5, thalamus; 6, hippocampus; 7, septum; 8, retrosplenial and adjacent motor cortex; and 9, cingulated and adjacent motor cortex. Data are mean ± s.e.m. (B) Western blot analysis of proteinase K-digested brain samples of mice infected with brain homogenates from cattle with natural BASE (#12966/07) and BSE, <i>longissimus dorsi</i> muscle from cattle with experimental BASE (#995) and two different muscles from natural BASE (#12699/07). (C–M) Neuropathological changes of mice infected with brain (C–E) and muscle from cattle with experimental BASE (F–H), and muscle from cattle with natural BASE (I–M). Micrographs were obtained from corresponding areas of the thalamic region stained with haematoxylin-eosin (C,F,I) or labeled with the anti-PrP antibody 6H4. The severity of vacuolation and the type of PrP deposition, characterized by diffuse immunostaining of the neuropil with focal enhancement, is similar in all the samples analyzed. Scale bar = 100 µm.</p

Kaplan–Meier survival curves for survival times.

<p>Kaplan–Meier survival curves for survival times.</p

PrP immunohistochemistry of muscle samples from Alpine brown cow experimentally infected with BASE (#995).

<p>PrP immunohistochemistry of muscle samples from Alpine brown cow experimentally infected with BASE (#995).</p

PrP deposition in the muscles of natural BASE cattle.

<p>(A–C) PrP deposits in <i>peroneus</i> muscle from a cattle with natural BASE (#126752/09). The PrP-imunoreactive material was found in isolated muscle fibers with a scattered distribution (A,C) and was localized inside the cytoplasm in the form of small amorphous aggregates or granular deposits (B). Scale bar = 100 µm for figure A and C;  = 20 µm for figure B.</p