L. González

Lack of a specific immune response against a recombinant capsid protein of Jaagsiekte sheep retrovirus in sheep and goats naturally affected by enzootic nasal tumour or sheep pulmonary adenomatosis

Enzootic nasal tumour (ENT) and sheep pulmonary adenomatosis (SPA) are two contagious adenocarcinomas of the respiratory tract of sheep and goats. Both diseases are associated with related, but distinct, type-D-retroviruses (ENTV and JSRV respectively). No evidence of circulating antibodies has been described in animals affected by either ENT or SPA using antigens from natural sources. We evaluated the usefulness of a recombinant JSRV capsid protein (JSRV-CA) as antigen to study the antibody responses of animals naturally affected by ENT or SPA, using immunoblotting. Positive reactions were detected in the sera of both affected and unaffected sheep and goats. The reactivity was abolished completely by absorption with the GST fusion partner but not by JSRV-CA, suggesting that it was not specific. The results support prior observations indicating that sheep and goats infected by JSRV and ENTV do not develop specific humoral responses to these retroviruses.

Pathology of Ovine Pulmonary Adenocarcinoma

Clinical, gross pathology, histopathology and electron microscopy of the ovine pulmonary adenocarcinoma (OPA, jaagsiekte) either natural or experimentally induced in sheep, goat and moufflon are described. OPA is caused by an oncogenic betaretrovirus,jaagsiekte sheep retrovirus (JSRV). Most natural cases of OPA appear in animals 1-4 years old. There is no evidence of sex or breed susceptibility. Sheep affected by OPA show an afebrile respiratory illness associated with loss of weight. A very characteristic clinical sign is moist rales caused by the accumulation of fluid in the respiratory airways which is discharged from the nostrils when the head is lowered. Gross lesions are confined to the lungs but occasionally thoracic or extrathoracic structures are also affected. Two pathologic forms of OPA are currently recognized, classical and atypical. In classical forms the neoplastic lesions occurs particularly in the cranioventral parts of all lung lobes. They are diffuse or nodular, light grey or light purple in colour. On the cut surface the tumour is moist, and frothy fluid may pour from the airways on slight pressure. Atypical forms tend to be more nodular in both early and advanced tumours. They are pearly white in colour, very hard in consistency, very well demarcated from the surrounding parenchyma and their surface is dry. Histology of the lung sections reveals the presence of several foci of epithelial cell neoplastic proliferation in both alveolar or bronchiolar regions. The tumours, derived from type II pneumocytes and Clara cells, proliferate into mostly papillary but also acinar or occasionally solid growths. The tumour generally shows a benign histological pattern but intra- and extrathoracic metastases have been detected in some cases. Several considerations suggest that the tumour should be classified as an adenocarcinoma of the lung. The histology of atypical OPA is similar to that of the classical disease, with an increase in the stromal reaction accompanying the epithelial proliferations. Pathological features of OPA induced experimentally in sheep, or of OPA in goats and moufflon are similar to those described in sheep. Detailed electron microscopy of tumour material confirms that type II pneumocytes and Clara bronchiolar epithelial cells are the origin of the neoplasia. Also included in this chapter is a description of the morphology of the viral particles associated with OPA.

Postmortem diagnosis of preclinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa

Samples of tissue from the central nervous system (CNS), the lymphoreticular system (LRS) and the rectal mucosa of a large number of scrapie-exposed sheep, with and without signs of clinical disease, were examined immunohistochemically for evidence of disease-associated prion protein (PrPd). The rectal mucosa has received almost no attention so far in scrapie diagnosis, despite its abundant rectoanal mucosa-associated lymphoid tissue, and its accessibility. The scrapie-confirmed cases included 244 with clinical disease, of which 237 (97·1 per cent) were positive in the rectal mucosa, and 121 apparently healthy sheep, of which 104 (86 per cent) were positive in the rectal mucosa. PrPd was detected in 86·4 to 91·5 per cent of the other LRS tissues of the healthy sheep examined and in 77·7 per cent of their CNS tissues. The stage of infection, therefore, affected the probability of a positive result in the rectal mucosa, whereas the breed, PrP genotype, age and sex had little or no independent effect. Accumulations of PrPd were observed in the rectal mucosa and other LRS tissues of VRQ/ARR sheep with preclinical and clinical scrapie, albeit with a lower frequency and magnitude than in sheep of other PrP genotypes. Western immunoblotting analyses of samples of rectal mucosa gave the characteristic PrP glycoprofile, with a sensitivity similar to that of immunohistochemistry.

Neuroinvasion in sheep transmissible spongiform encephalopathies: the role of the haematogenous route

Background: It is generally believed that after oral exposure to transmissible spongiform encephalopathy (TSE) agents, neuroinvasion occurs via the enteric nervous system (ENS) and the autonomic nervous system. As a result, the dorsal motor nucleus of the vagus nerve is the initial point of disease‐associated prion protein (PrPd) accumulation in the brain. Hypothesis and aim: If direct ENS invasion following oral infection results in an early and specific brain targeting for PrPd accumulation, such topographical distribution could be different when other routes of infection were used, highlighting distinct routes for neuroinvasion. Methods: An immunohistochemical study has been conducted on the brain of 67 preclinically infected sheep exposed to natural scrapie or to experimental TSE infection by various routes. Results: Initial PrPd accumulation consistently occurred in the dorsal motor nucleus of the vagus nerve followed by the hypothalamus, regardless of the breed of sheep, PrP genotype, TSE source and, notably, route of infection; these factors did not appear to affect the topographical progression of PrPd deposition in the brain either. Moreover, the early and consistent appearance of PrPd aggregates in the circumventricular organs, where the blood–brain barrier is absent, suggests that these organs can provide a portal for entry of prions when infectivity is present in blood. Conclusions: The haematogenous route, therefore, can represent a parallel or alternative pathway of neuroinvasion to ascending infection via the ENS/autonomic nervous system.

Evaluation of a PCR technique for the detection of Maedi-Visna proviral DNA in blood, milk and tissue samples of naturally infected sheep

Abstract A novel, simple polymerase chain reaction (PCR) protocol based on the amplification of a 291 base pair DNA fragment in the long terminal repeat (LTR) region of the Maedi-Visna (MV) provirus has been evaluated on samples collected from 115 sheep at the time of slaughter. The sheep came from Maedi-Visna virus (MVV) non-infected ( n =18) or MVV infected ( n =97) flocks, and the samples examined included peripheral blood leukocytes (PBLs; 115 sheep), milk cells (MCs; 64 sheep) and several tissue samples (TSs; 91 sheep). The LTR-PCR results were compared with the results of an indirect enzyme-linked immunosorbent assay (ELISA) and an agar-gel immunodiffusion test (AGIDT) performed on sera from the same animals obtained not only at the time of slaughter, but also in previous samplings. The LTR-PCR showed 100% specificity and an overall sensitivity of 98% in comparison with the two serological methods combined. Its sensitivity was lower when single types of samples were considered (84% in PBLs, 67% in MCs and 88% in TSs). It is concluded that this LTR-PCR can be used to complement serological tests and to perform studies of the pathogenesis of this lentiviral infection.

Diagnosis of preclinical scrapie in live sheep by the immunohistochemical examination of rectal biopsies.

In most sheep infected with a transmissible spongiform encephalopathy (tse) the disease-associated prion protein (PrPd) accumulates in tissues of the lymphoreticular system, suggesting that it might be detected in biopsy specimens. A procedure has been developed to obtain biopsy specimens of rectal mucosa in which PrPd has been detected by immunohistochemistry in preclinically infected sheep of all susceptible PrP genotypes. It is probable that PrPd increases with the age of sheep or period of incubation. PrPd was detectable approximately halfway through the incubation period, with sheep of some PrP genotypes showing positive results earlier than others. For a preclinical diagnosis, the risk of a false negative result was approximately 9 per cent for samples containing 10 follicles, a figure that was reached in 87 per cent of the biopsies. The rectal biopsies had the same sensitivity and time of onset of PrPd accumulation as biopsies of the palatine tonsil, but provided larger numbers of follicles. The procedure is simple and quick, does not require dedicated specific instruments, sedation or general anaesthesia, and can be performed repeatedly on the same sheep without detrimental effects to either the animal or the number of follicles obtained.

Natural transmission of BSE between sheep within an experimental flock.

SIR, – The recognition of bovine spongiform encephalopathy (BSE) in a French goat ([Eloit and others 2005][1]) has heightened the debate in Europe as to whether BSE has been maintained in small ruminants following historical exposure via feed. Key to the debate and associated risk assessments,

Oral transmission of bse to vrq/vrq sheep in an experimental flock

SIR, — We report here the first confirmed case of oral transmission of bovine spongi-form encephalopathy (bse) to sheep homozygous for valine at codon 136 of the prion protein (PrP) gene. Although intracerebral transmission has been previously reported ([Martin and others 2005][1]), our finding is

Variability in disease phenotypes within a single PRNP genotype suggests the existence of multiple natural sheep scrapie strains within Europe

Variability of pathological phenotypes within classical sheep scrapie cases has been reported for some time, but in many instances it has been attributed to differences in the PRNP genotype of the host. To address this issue we have examined by immunohistochemistry (IHC) and Western blotting (WB) for the disease-associated form of the prion protein (PrP(d)), the brains of 23 sheep from five European countries, all of which were of the same ARQ/ARQ genotype. As a result of IHC examinations, sheep were distributed into five groups with different phenotypes and the groups were the same regardless of the scoring method used, long or short PrP(d) profiling. The groups made did not respond to the geographical origin of the cases and did not correlate with the vacuolar lesion profiles, which showed a high individual variability. Discriminatory IHC and WB methods coincided to detect a CH1641-like case but otherwise correlated poorly in the classification of disease phenotypes. No other polymorphisms of the PRNP gene were found that could account for the pathological differences, except perhaps for a sheep from Spain with a mutation at codon 103 and a unique pathological phenotype. Preliminary evidence indicates that those different IHC phenotypes correlate with distinct biological properties on bioassay, suggesting that they are indicative of strain diversity. We therefore conclude that natural scrapie strains exist and that they can be revealed by detailed pathological examinations, which can be harmonized between laboratories to produce comparable results.

Influence of Polymorphisms in the Prion Protein Gene on the Pathogenesis and Neuropathological Phenotype of Sheep Scrapie after Oral Infection

The prion protein gene (Prnp) plays a crucial role in the susceptibility of sheep to scrapie in terms of attack rate and/or incubation period. However, the influence of Prnp on the pathogenesis of the disease, specifically the involvement of tissues of the lymphoreticular system (LRS), pathways of neuroinvasion and neuropathological phenotypes, remains controversial. This study reports the onset and progression of disease-associated prion protein (PrP(d)) accumulation in the LRS and nervous tissues of sheep of six different Prnp genotypes infected by oral administration of the same mixed scrapie brain homogenate. Sheep homozygous for glutamine (Q) at codon 171 of PrP, with either valine (V) or alanine (A) at codon 136 (i.e. VRQ/VRQ, VRQ/ARQ and ARQ/ARQ), showed early and consistent PrP(d) accumulation in LRS tissues of the pharynx and gut. In contrast, LRS involvement was minimal, inconsistent and occurred late in the incubation period in sheep heterozygous for arginine (R) at codon 171 (i.e. VRQ/ARR and ARQ/ARR). Despite this difference, all five groups were susceptible to infection and developed clinical disease, albeit with significantly different incubation periods (shortest in VRQ/VRQ and longest in ARQ/ARR sheep). The remaining group of ARR/ARR homozygous sheep did not show evidence of infection at the end of the experiment or at previous predetermined time points. As for LRS tissues, the sites of initial PrP(d) accumulation in the brain were determined immunohistochemically. These were the same in all susceptible sheep (except for ARR/ARR sheep), regardless of their Prnp genotype which, together with an early and consistent accumulation of PrP(d) in circumventricular organs and a late or inconsistent involvement of the enteric and autonomic nervous system and of the spinal cord, suggests neuroinvasion occurring via the blood. The neuropathological phenotype (PrP(d) profile in the central nervous system) of clinically affected sheep was similar in the three V136 carrier groups, but showed some differences in the two A136 homozygous groups, suggesting a codon 136-driven selection of different strains from the mixture contained in the inoculum. ARQ/ARR sheep showed an irregular distribution of brain PrP(d), contrasting with the more widespread distribution of the other four groups. The results indicate that (1) ARQ/ARR sheep are more susceptible to oral scrapie infection than would be predicted from incidence figures in natural disease, (2) amplification and accumulation of PrP(d) in LRS tissues is host genotype dependent, but does not necessarily have a marked effect on the outcome of the infection and (3) the neuropathological phenotype of scrapie is related to the host genotype, but possibly in combination with the infecting source.