Assessing the susceptibility of transgenic mice overexpressing deer prion protein to bovine spongiform encephalopathy.

Several transgenic mouse models have been developed which facilitate the transmission of chronic wasting disease (CWD) of cervids and allow prion strain discrimination. The present study was designed to assess the susceptibility of the prototypic mouse line, Tg(CerPrP)1536(+/-), to bovine spongiform encephalopathy (BSE) prions, which have the ability to overcome species barriers. Tg(CerPrP)1536(+/-) mice challenged with red deer-adapted BSE resulted in 90% to 100% attack rates, and BSE from cattle failed to transmit, indicating agent adaptation in the deer.

The interpretation of disease phenotypes to identify TSE strains in mice: characterisation of BSE using PrPSc distribution patterns in the brain.

In individual animals affected by transmissible spongiform encephalopathies, different disease phenotypes can be identified which are attributed to different strains of the agent. In the absence of reliable technology to fully characterise the agent, classification of disease phenotype has been used as a strain typing tool which can be applied in any host. This approach uses standardised data on biological parameters, established for a single host, to allow comparison of different prion sources. Traditionally prion strain characterisation in wild type mice is based on incubation periods and lesion profiles after the stabilisation of the agent into the new host which requires serial passages. Such analysis can take many years, due to prolonged incubation periods. The current study demonstrates that the PrPSc patterns produced by one serial passage in wild type mice of bovine or ovine BSE were consistent, stable and showed minimal and predictable differences from mouse-stabilised reference strains. This biological property makes PrPSc deposition pattern mapping a powerful tool in the identification and definition of TSE strains on primary isolation, making the process of characterisation faster and cheaper than a serial passage protocol. It can be applied to individual mice and therefore it is better suited to identify strain diversity within single inocula in case of co-infections or identify strains in cases where insufficient mice succumb to disease for robust lesion profiles to be constructed. The detailed description presented in this study provides a reference document for identifying BSE in wild type mice.

A retrospective immunohistochemical study reveals atypical scrapie has existed in the United Kingdom since at least 1987.

Atypical scrapie is a relatively recent discovery, and it was unknown whether it was a new phenomenon or whether it had existed undetected in the United Kingdom national flock. Before 1998, the routine statutory diagnosis of transmissible spongiform encephalopathy (TSE) in sheep relied on the presence of TSE vacuolation in the brainstem. This method would not have been effective for the detection of atypical scrapie. Currently, immunohistochemistry (IHC) and Western blot are commonly used for the differential diagnosis of classical and atypical scrapie. The IHC pattern of PrPd deposition in atypical scrapie is very different from that in classical scrapie using the same antibody. It is thus possible that because of a lack of suitable diagnostic techniques and awareness of this form of the disease, historic cases of atypical scrapie remain undiagnosed. Immunohistochemistry was performed on selected formalin-fixed, paraffin-embedded (FFPE) blocks of ovine brain from the Veterinary Laboratories Agency archives that were submitted for various reasons, including suspect neurological disorders, between 1980 and 1989. It was found that PrPd deposits in a single case were consistent with atypical scrapie. A method was developed to obtain a PrP genotype from FFPE tissues and was applied to material from this single case, which was shown to be AHQ/AHQ. This animal was a scrapie suspect from 1987, but diagnosis was not confirmed by the available techniques at that time.

Parenteral adenoviral boost enhances BCG induced protection, but not long term survival in a murine model of bovine TB.

Boosting BCG using heterologous prime-boost represents a promising strategy for improved tuberculosis (TB) vaccines, and adenovirus (Ad) delivery is established as an efficacious boosting vehicle. Although studies demonstrate that intranasal administration of Ad boost to BCG offers optimal protection, this is not currently possible in cattle. Using Ad vaccine expressing the mycobacterial antigen TB10.4 (BCG/Ad-TB10.4), we demonstrate, parenteral boost of BCG immunised mice to induce specific CD8(+) IFN-γ producing T cells via synergistic priming of new epitopes. This induces significant improvement in pulmonary protection against Mycobacterium bovis over that provided by BCG when assessed in a standard 4week challenge model. However, in a stringent, year-long survival study, BCG/Ad-TB10.4 did not improve outcome over BCG, which we suggest may be due to the lack of additional memory cells (IL-2(+)) induced by boosting. These data indicate BCG-prime/parenteral-Ad-TB10.4-boost to be a promising candidate, but also highlight the need for further understanding of the mechanisms of T cell priming and associated memory using Ad delivery systems. That we were able to generate significant improvement in pulmonary protection above BCG with parenteral, rather than mucosal administration of boost vaccine is critical; suggesting that the generation of effective mucosal immunity is possible, without the risks and challenges of mucosal administration, but that further work to specifically enhance sustained protective immunity is required.

Boosting BCG with inert spores improves immunogenicity and induces specific IL-17 responses in a murine model of bovine tuberculosis.

Tuberculosis (TB) remains a global pandemic, in both animals and man, and novel vaccines are urgently required. Heterologous prime-boost of BCG represents a promising strategy for improved TB vaccines, with respiratory delivery the most efficacious to date. Such an approach may be an ideal vaccination strategy against bovine TB (bTB), but respiratory vaccination presents a technical challenge in cattle. Inert bacterial spores represent an attractive vaccine vehicle. Therefore we evaluated whether parenterally administered spores are efficacious when used as a BCG boost in a murine model of immunity against Mycobacterium bovis. Here we report the use of heat-killed, TB10.4 adsorbed, Bacillus subtilis spores delivered via subcutaneous injection to boost immunity primed by BCG. We demonstrate that this approach improves the immunogenicity of BCG. Interestingly, this associated with substantial boosting of IL-17 responses; considered to be important in protective immunity against TB. These data demonstrate that parenteral delivery of spores represents a promising vaccine vehicle for boosting BCG, and identifies potential for optimisation for use as a vaccine for bovine TB.

Paraffin-embedded tissue blot as a sensitive method for discrimination between classical scrapie and experimental bovine spongiform encephalopathy in sheep.

The paraffin-embedded tissue (PET) blot was modified for use as a tool to differentiate between classical scrapie and experimental bovine spongiform encephalopathy (BSE) in sheep. Medulla (obex) from 21 cases of classical scrapie and 6 cases of experimental ovine BSE were used to develop the method such that it can be used as a tool to differentiate between BSE and scrapie in the same way that differential immunohistochemistry (IHC) has been used previously. The differential PET blot successfully differentiated between all of the scrapie and ovine BSE cases. Differentiation was permitted more easily with PET blot than by differential IHC, with accurate observations possible at the macroscopic level. At the microscopic level, sensitivity was such that discrimination by the differential PET blot could be made with more confidence than with differential IHC in cases where the immunohistochemical differences were subtle. The differential PET blot makes use of harsh epitope demasking conditions, and, because of the differences in the way prion protein is processed in different prion diseases, it can serve as a new, highly sensitive method to discriminate between classical scrapie and experimental BSE in sheep.

Studies of the transmissibility of the agent of bovine spongiform encephalopathy to the domestic chicken.

BACKGROUND: Transmission of the prion disease bovine spongiform encephalopathy (BSE) occurred accidentally to cattle and several other mammalian species via feed supplemented with meat and bone meal contaminated with infected bovine tissue. Prior to United Kingdom controls in 1996 on the feeding of mammalian meat and bone meal to farmed animals, the domestic chicken was potentially exposed to feed contaminated with the causal agent of BSE. Although confirmed prion diseases are unrecorded in avian species a study was undertaken to transmit BSE to the domestic chicken by parenteral and oral inoculations. Transmissibility was assessed by clinical monitoring, histopathological examinations, detection of a putative disease form of an avian prion protein (PrP) in recipient tissues and by mouse bioassay of tissues. Occurrence of a progressive neurological syndrome in the primary transmission study was investigated by sub-passage experiments. RESULTS: No clinical, pathological or bioassay evidence of transmission of BSE to the chicken was obtained in the primary or sub-passage experiments. Survival data showed no significant differences between control and treatment groups. Neurological signs observed, not previously described in the domestic chicken, were not associated with significant pathology. The diagnostic techniques applied failed to detect a disease associated form of PrP. CONCLUSION: Important from a risk assessment perspective, the present study has established that the domestic chicken does not develop a prion disease after large parenteral exposures to the BSE agent or after oral exposures equivalent to previous exposures via commercial diets. Future investigations into the potential susceptibility of avian species to mammalian prion diseases require species-specific immunochemical techniques and more refined experimental models.

Cytokine-mediated inhibition of apoptosis in non-transformed T cells and neutrophils can be dissociated from protein kinase B activation.

In the absence of survival-inducing cytokines activated T cells and neutrophils enter apoptosis spontaneously. Phosphatidylinositol 3-kinase (PI3 K) activation and signaling through PKB/AKT have been widely linked to the inhibition of apoptosis by cytokines. Here we have investigated the role of PKB in the inhibition of spontaneous apoptosis of activated human CD4+ T cells and neutrophils. We used a range of cytokines known to induce survival and/or activation of PKB. We found activation of PKB in T cells treated with IL-2 and insulin, and neutrophils cultured with N-formyl-Met-Leu-Phe (fMLP), insulin or granulocyte-macrophage colony-stimulating factor. Insulin did not inhibit apoptosis in neutrophils or T cells and fMLP did not delay neutrophil apoptosis. Intriguingly, IFN-beta induced PI3 K-dependent survival in both cell types, but did not activate PKB. IL-2 mediated rescue of T cells from apoptosis but no induction of proliferation occurred in thepresence of LY294002, an inhibitor of PI3 K, which also blocked subsequent PKB activation. The main role of PI3 K in IL-2-mediated signaling may therefore be in the regulation of proliferation. These findings suggest that activation of PKB and inhibition of apoptosis can be dissociated in cytokine-mediated rescue of non-transformed CD4+ T cells and neutrophils.

Reactive oxygen species limit neutrophil life span by activating death receptor signaling.

Neutrophils are abundant, short-lived leukocytes, and their death by apoptosis is central to hemostasis and the resolution of inflammation, yet the trigger for their entry into apoptosis is unknown. We show here that death receptor signaling, including CD95 death-inducing signaling complex (DISC) formation and caspase 8 activation, occurred early in neutrophil apoptosis. However, death receptor ligation was not required for apoptosis, suggesting a novel mechanism for caspase 8 activation. We detected ceramide generation and clustering of CD95 in lipid rafts early in neutrophil apoptosis, and neutrophil apoptosis and ceramide generation were both significantly inhibited in acid sphingomyelinase knockout (ASM(-/-)) mice compared to wild-type littermates. Further studies revealed that ceramide generation, CD95 clustering, and neutrophil apoptosis were dependent on reactive oxygen species (ROSs) and were preceded by a fall in reduced glutathione levels. We propose that accumulation of ROSs, as a consequence of altered redox status, initiates ligand-independent death receptor signaling via activation of ASM and clustering of preformed DISC components in lipid rafts and is therefore a primary factor limiting neutrophil life span.