Pathogenesis of scrapie in ARQ/ARQ sheep after subcutaneous infection: effect of lymphadenectomy and immune cell subset changes in relation to prion protein accumulation.

It is well established that the infectious agent of scrapie can replicate in the lymphoreticular system (LRS). However, the effects of removal of LRS target tissues on the pathogenesis of the infection and the accumulation of disease-associated prion protein (PrP(d)) in LRS tissues on specific immune cell subsets are poorly understood aspects. To address these questions 16 ARQ/ARQ sheep were subcutaneously inoculated in the drainage area of the prefemoral lymph node with brain homogenate derived from Suffolk sheep naturally infected with scrapie. Fourteen sheep were then subjected to either early (14-17 days post-inoculation [dpi]) or late (175-201 dpi) lymphadenectomy and culled at preclinical or clinical stages of infection. Neither late nor even early lymphadenectomy prevented infection or had any effect on the accumulation of PrP(d) in the LRS or CNS suggesting a rapid organic dissemination of the infectious agent after inoculation. Lymph nodes from eight scrapie inoculated sheep selected on the basis of the amount of PrP(d) in their LRS tissues (negative, low or high) were examined for six different immune cell markers. The PrP(d) negative lymph nodes from two sheep with no evidence of scrapie infection showed lower numbers of cluster of determination (CD) 21 positive cells than PrP(d) positive nodes, irrespective of their location (hind leg or head). However, quantitative differences in the expression of this marker were not detected when comparing lymph nodes with low and high levels of PrP(d) accumulation, suggesting that proliferation of CD21 positive cells is related to scrapie infection, but not directly linked to the magnitude of PrP(d) accumulation. An additional observation of the study was that sheep that were methionin-threonine at codon 112 of the prion protein gene showed lower attack rates than methionine homozygotes (67% and 100%, respectively) and also generally lower levels of PrP(d) accumulation in the LRS and brain and increased survival times, suggesting an influence of such polymorphism in the susceptibility to scrapie.

Neuropathological changes correlate temporally but not spatially with selected neuromodulatory responses in natural scrapie.

AIM: Neuropathological changes classically associated with sheep scrapie do not always correlate with clinical disease. We aimed to determine if selected neuromodulatory responses were altered during the course of the infection as it has been described in Creutzfeldt-Jakob disease and experimental bovine spongiform encephalopathy. METHODS: Hemi-brains from healthy sheep and natural scrapie cases at two stages of infection were examined for biochemical alterations related to the expression of type I metabotropic glutamatergic receptors (mGluR(1) ) and type I adenosine receptors I (A(1) R), and of selected downstream intermediate signalling targets. Immunohistochemistry for different scrapie-related neuropathological changes was performed in the contralateral hemi-brains. RESULTS: PrP(d) deposition, spongiform change, astrocytosis and parvalbumin expression were significantly altered in brains from clinically affected sheep compared with preclinical cases and negative controls; the latter also showed significantly higher immunoreactivity for synaptophysin than clinical cases. Between clinically affected and healthy sheep, no differences were found in the protein levels of mGluR(1) , while phospholipase Cß1 expression in terminally ill sheep was increased in some brain areas but decreased in others. Adenyl cyclase 1 and A(1) R levels were significantly lower in various brain areas of affected sheep. No abnormal biochemical expression levels of these markers were found in preclinically infected sheep. CONCLUSIONS: These findings point towards an involvement of mGluR(1) and A(1) R downstream pathways in natural scrapie. While classical prion disease lesions and neuromodulatory responses converge in some affected regions, they do not do so in others suggesting that there are independent regulatory factors for distinct degenerative and neuroprotective responses.

Prevalence, spatial distribution and the effect of control measures on louping-ill virus in the Forest of Bowland, Lancashire.

The complex pathogen-host-vector system of the tick-borne louping-ill virus causes economic losses to sheep and red grouse in upland United Kingdom. This paper examines the spatial distribution, incidence and effect of control measures on louping-ill virus in the Bowland Fells of Lancashire. Seroprevalence in sheep at the beginning of the study varied within the area and was affected significantly by the frequency of acaricide treatment. There was a clear decrease over 5 years in the effective force of infection on farms implementing a vaccination programme, irrespective of acaricide treatment regime, however, only one third of farms apparently eliminated infection. On farms where vaccination did not occur or where vaccination was carried out intermittently, the estimated force of infection was variable or possibly increased. Thus, as befits a complex host-pathogen system, reductions in prevalence were not as dramatic as predicted; we discuss the potential explanations for these observations.

Immunological differences between susceptible and resistant sheep during the preclinical phase of scrapie infection.

In order to investigate the relationship between the immune response to scrapie infection and genetic susceptibility to the disease in sheep, immune cell subsets and prion protein (PrP) expression were determined in susceptible and resistant Suffolk sheep in the preclinical phase of infection. At 6 months of age, 12 ARQ/ARQ (susceptible) and nine ARR/ARR (resistant) scrapie-free Suffolk lambs were challenged subcutaneously with scrapie inoculum. Prefemoral lymphadenectomies were carried out at 14 and 180 days post-inoculation (p.i.) and serial bleeds were collected at monthly intervals for up to 1 year p.i. An indirect double-labelling procedure was carried out on peripheral blood mononuclear cells (PBMCs) and lymph node cell preparations and analysed using flow cytometry. Prior to scrapie challenge, significantly more PrP(+) cells were detected in PBMCs from the susceptible sheep. Furthermore, following challenge, significantly more CD8(+) and gammadelta(+) T cells were detected in the PBMCs of the resistant sheep. However, at both 14 and 180 days p.i, CD21(+) cell expression was significantly higher in the lymph node preparations of the susceptible sheep. In contrast, more CD4(+) cells were detected in the lymph nodes of the resistant sheep at both time points. It was concluded that significant differences in immune cell subsets and PrP expression occur between ARQ/ARQ and ARR/ARR Suffolk sheep in the preclinical phase of infection.

Genome re-arrangements associated with loss of pathogenicity of the gamma-herpesvirus alcelaphine herpesvirus-1.

The alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in ruminants. Previous work had shown that serial passage of AlHV-1 in culture resulted in genome alterations that are associated with a loss in pathogenicity. Here we have analysed the re-arrangements that occur in more detail. None of the observed re-arrangements was entirely consistent. However, they did all involve translocation of a similar region of DNA from around the centre of the genome to areas either next to or in between terminal repeat elements at either end of the genome. There was also a concomitant loss of the wild-type locus. These re-arrangements appeared to be associated with the loss of virulence and the appearance of cell-free virus.

Outbreak of malignant catarrhal fever in Welsh Black cattle in Carmarthenshire.

An outbreak of malignant catarrhal fever (MCF) resulted in the deaths of 12 cattle in a herd of 77 animals during seven weeks in 1999; in addition, one cow developed a milder disease which was confirmed as MCF by PCR for ovine herpesvirus 2 DNA and an immunofluorescent antibody test for antibodies to the virus, but recovered. Further PCR and serological testing revealed the infection in three other animals, none of which developed clinical disease. Hypocuprosis and the possibility of a genetic predisposition were identified as factors which may have contributed to the outbreak.

Detection of cellular cytokine mRNA expression during orf virus infection in sheep: differential interferon-gamma mRNA expression by cells in primary versus reinfection skin lesions.

In sheep infected with the parapoxvirus orf virus, primary infection orf skin lesions developed and resolved within 8 weeks. Reinfection lesions were smaller and resolved within 3 weeks. The host response in the skin was characterized by an accumulation of neutrophils, dendritic cells, CD4+ T cells, CD8+ T cells, B cells and T19+ gammadelta T cells. The magnitude of this accumulation paralleled orf virus replication in the skin. In situ hybridization was used to detect cells expressing interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-4 (IL-4) mRNAs in orf skin. Cells expressing IL-4 mRNA were not detected at any time after infection. Cells expressing IFN-gamma mRNA were detected after reinfection but not after primary infection. Cells expressing TNF-alpha mRNA included epidermal cells, vascular endothelium and uncharacterized cells that increased more rapidly in the skin after reinfection compared to primary infection. The results are consistent with a prominent role for IFN-gamma in the host immune response controlling the severity of the disease.

A recombinant Semliki Forest virus particle vaccine encoding the prME and NS1 proteins of louping ill virus is effective in a sheep challenge model.

This study has examined the efficacy following intramuscular administration of a recombinant Semliki Forest virus (rSFV) vaccine, encoding the prME and NS1 proteins of louping ill virus (LIV), in sheep. Administration of rSFV-LIV vaccine resulted in transient detection at the injection site and draining lymph node only and no dissemination to distal sites. In addition, the recombinant vaccine offered complete protection against subcutaneous challenge with LIV, and partial protection following intranasal administration of LIV. Protected animals had no pathological changes normally associated with LIV infection, and had developed high antibody titres. In contrast, the two animals not protected exhibited classical clinical signs and neuropathological lesions of LIV infection. These findings indicate that rSFV-based vaccines have the potential to be developed as effective prototype vaccines for LIV.

Role of small mammals in the persistence of Louping-ill virus: field survey and tick co-feeding studies.

Louping-ill (LI) is a tick-borne viral disease of red grouse, Lagopus lagopus scoticus Lath. (Tetraonidae: Galliformes), and sheep, Ovis aries L. (Bovidae: Artiodactyla), that causes economic loss to upland farms and sporting estates. Unvaccinated sheep, grouse and mountain hares, Lepus timidus L. (Leporidae: Lagomorpha), are known to transmit LI virus, whereas red deer, Cenrus elaphus L. (Cervidae: Artiodactyla), and rabbits, Oryctolagus cuniculus L. (Leporidae: Lagomorpha), do not. However, the role of small mammals is unknown. Here, we determine the role of small mammals, in particular field voles, Microtus agrestis L. (Muridae: Rodentia), in the persistence of LI virus on upland farms and sporting estates in Scotland, using field sampling and non-viraemic transmission trials. Small mammals were not abundant on the upland sites studied, few ticks were found per animal and none of the caught animals tested seropositive to LI virus. Laboratory trials provided no evidence that small mammals (field voles, bank voles, Clethrionomys glareolus L. (Muridae: Rodentia), and wood mice, Apodemus sylvaticus L. (Muridae: Rodentia), can transmit LI virus between cofeeding ticks and, in the field, LI virus was prevalent only in areas with known LI virus competent hosts (grouse, mountain hares or unvaccinated sheep) and absent elsewhere. In contrast to the case of tick-borne encephalitis (TBE) virus in Europe, it is concluded that small mammals seem to be relatively unimportant in LI virus persistence.

Sequence and functional analysis of a homolog of interleukin-10 encoded by the parapoxvirus orf virus.

Orf virus is a large DNA virus and is the type species of the Parapoxvirus genus of the family Poxviridae. Orf virus infects the epithelium of sheep and goats and is transmissible to humans. Recently we discovered a gene in orf virus that encodes a polypeptide with remarkable homology to mammalian interleukin (IL-10) and viral encoded IL-10s of herpes viruses. The predicted polypeptide sequence shows high levels of amino acid identity to IL-10 of sheep (80%), cattle (75%), humans (67%) and mice (64%), as well as IL-10-like proteins of Epstein-Barr virus (63%) and equine herpes virus (67%). The C-terminal region, comprising two-thirds of the orf virus protein, is identical to ovine IL-10 which suggests that this gene has been captured from its host sheep during the evolution of orf virus. In contrast the N-terminal region shows little homology with cellular IL10s and in this respect resembles other viral IL-10s. IL-10 is a pleiotrophic cytokine that can exert either immunostimulatory or immunosuppressive effects on many cell types. IL-10 is a potent anti-inflammatory cytokine with inhibitory effects on non-specific immunity in particular macrophage function and Thl effector function. Our studies so far, indicate, that the functional activities of orf virus IL-10 are the same as ovine IL-10. Orf virus IL-10 stimulates mouse thymocyte proliferation and inhibits cytokine synthesis in lipopolysaccharide-activated ovine macrophages, peripheral blood monocytes and keratinocytes. Infection of sheep with an IL-10 deletion mutant of orf virus has shown that interferon-gamma levels are higher in tissue infected with the mutant virus than the parent virus. The functional activities of IL-10 and our data on orf virus IL-10 suggest a role in immune evasion.

The role of lambs in louping-ill virus amplification.

In some areas of Scotland, the prevalence of louping-ill virus has not decreased despite the vaccination of replacement ewes for over 30 years. The role of unvaccinated lambs in viral persistence was examined through a combination of an empirical study of infection rates of lambs and mathematical modelling. Serological sampling revealed that most lambs were protected by colostral immunity at turnout in May/June but were fully susceptible by the end of September. Between 8 and 83% of lambs were infected over the first season, with seroconversion rates greater in late rather than early summer. The proportion of lambs that could have amplified the louping-ill virus was low, however, because high initial titres of colostral antibody on farms with a high force of infection gave protection for several months. A simple mathematical model suggested that the relationship between the force of infection and the percentage of lambs that became viraemic was not linear and that the maximum percentage of viraemic lambs occurred at moderately high infection rates. Examination of the conditions required for louping-ill persistence suggested that the virus could theoretically persist in a sheep flock with over 370 lambs, if the grazing season was longer than 130 days. In practice, however, lamb viraemia is not a general explanation for louping-ill virus persistence as these conditions are not met in most management systems and because the widespread use of acaracides in most tick-affected hill farming systems reduces the number of ticks feeding successfully.

Parapoxviruses are strongly inhibited in vitro by cidofovir.

Three parapoxviruses which cause orf or related diseases in humans and animals and the orthopoxvirus, vaccinia virus, were tested for their in vitro sensitivity to cidofovir. The 50% inhibitory concentration for the three parapoxviruses was between 0.21 and 0.27 microg/ml and for vaccinia was 1.32 microg/ml. The selectivity index varied from 198 to 264 for the parapoxviruses and was 42 for vaccinia virus. Virus yield assays confirmed the ability of cidofovir to reduce ortho- and parapoxvirus replication. The efficacy of cidofovir against parapoxviruses justifies its evaluation as a candidate drug for the treatment of parapoxvirus infections in humans and animals.

Characterization of RKZ isolate of ovine herpesvirus 1.

Cytopathic effect (CPE) characterized mainly by foci of rounded cells was observed in cultures of primary plexus choroideus cells from healthy lamb following cryopreservation. It was possible to transmit the infectious agent to other primary cells of ovine origin by co-cultivation with infected cells. By indirect immunofluorescence microscopy it was found that high percentage of sheep (65-80% in 3 different herds from Slovakia) are infected with this infectious agent. Electron microscopy of cells with CPE revealed the presence of herpesvirus particles. Viral DNA was isolated from infected cells using pulse-field gel electrophoresis and further used as probe in Southern blot analysis. The probe reacted specifically only with DNA from cells infected with Ovine herpesvirus 1 (OvHV-1) but not with DNA of other ruminant herpesviruses. Some of the HindIII restriction fragments of DNA of the obtained OvHV-1 isolate denominated RKZ were cloned. Part of the H9 clone was sequenced identifying a gene that encoded a polypeptide homologous to conserved herpesvirus VP23 structural protein. From comparison of the sequence of this clone with VP23 sequences of other herpesviruses it was deduced that OvHV-1 might be classified within the Rhadinovirus genus of the Gammaherpesvirinae subfamily. The sequencing of the H9 clone of DNA of RKZ isolate enabled establishment of sensitive and highly specific polymerase chain reaction (PCR) assay for detection of OvHV-1.

Effects of pulsed or continuous infusion of cortisol on immune function in sheep.

It was postulated that frequent pulses of cortisol such as might be induced by a repeated or chronic stressor, could induce immune suppression and that the effect would be greater than in animals subjected to less frequent increases. Four groups of nine adult Scottish Blackface ewes were infused for 14 d with saline or hydrocortisone hemisuccinate (cortisol) delivered continuously or in pulses. Plasma concentrations of cortisol were significantly elevated (to between approximately 100 and 1000 nmol/liter; P < 0.001) for about 30 or 75 min after infusion of pulses of hydrocortisone hemisuccinate at intervals of 1 hr (P1) or 6 hr (P6), respectively. In animals continuously infused (CI), they were consistently elevated (P < 0.001), compared with concentrations in control animals infused with saline only (S), to approximately 1000 nmol/liter or more. Antibody production in response to ovalbumin injection was not affected by any of the infusion regimes. At Days 10, 24, and 31 after injection of ovalbumin and initiation of the infusion, rates of multiplication of unstimulated lymphocytes, in vitro, were greater (P < 0.05) in P6 animals than in saline-infused, control animals and this resulted in a reduction in the stimulated lymphocyte response. As a consequence of the increased basal lymphocyte activity, after Day 0, the corrected, stimulated lymphocyte response of P6 animals was consistently below that of controls (P < 0.05 at Day 24). Both mean basal and stimulated lymphocyte activities in CI and P1 animals were similar to those of controls. The gamma interferon (IFN-gamma) response was generally small and not affected by treatment. It is concluded that large, relatively infrequent increases in circulating cortisol concentrations can modify the cell mediated immune response such that the response to a specific antigen challenge is compromised but smaller, more frequent pulses had no effect. Elevated cortisol concentrations per se did not have a significant inhibitory effect on the immune system.

Phenotype, growth regulation and cytokine transcription in Ovine Herpesvirus-2 (OHV-2)-infected bovine T-cell lines.

The causal agent of sheep-associated malignant catarrhal fever (MCF), Ovine Herpesvirus-2 (OHV-2), can be propagated in IL-2-dependent lymphoblastoid cell lines derived from diseased cattle and deer providing a useful model for the investigation of the pathogenesis of MCF. In this study, five interleukin-2 (IL-2)-dependent cell lines were established from affected cattle to examine their growth regulation and cytokine transcription. All cell lines expressed CD2, CD5 and CD25. Three of the cell lines were CD4+ and one CD8+, whereas one cell was of mixed CD4 and CD8 phenotye. The growth of these cell lines was reduced when cultured with antibody against CD25, the IL-2 receptor alpha subunit. All cell lines showed a lack of response to Con A and their cell growth was inhibited by Cyclosporin A which is known to inhibit cytokine promoters. It was decided therefore, to examine the cell lines for the presence of mRNA of different cytokines. The results showed that the cell lines transcribed message for IFNgamma, TNFalpha, IL-4 and IL-10 whereas no mRNA for IL-2 or IL-1beta was detected. In conclusion, the OHV-2-immortalised cell lines resemble anergic T-cells which may be activated giving rise to the characteristic lesions of MCF.