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Bovine spongiform encephalopathy (BSE) can be efficiently transmitted to pigs via intracerebral inoculation. A clear link has been established between the consumption of products of bovine origin contaminated with the BSE agent and the development of variant Creutzfeldt-Jakob disease in humans. Small ruminants can also naturally develop BSE, and sheep-adapted BSE (Sh-BSE) propagates more efficiently than cattle BSE in pigs and in mouse models expressing porcine prion protein. In addition, Sh-BSE shows greater efficiency of transmission to human models than original cow BSE. While infectivity and/or abnormal PrP accumulation have been reported in the central nervous system in BSE-infected pigs, the ability of the agent to replicate in peripheral tissues has not been fully investigated. We previously characterized the presence of prions in a panel of tissues collected at the clinical stage of disease from pigs experimentally infected with Sh-BSE. Western blot revealed low levels of PrPres accumulation in lymphoid tissues, nerves, and skeletal muscles from 4 of the 5 animals analysed. Using protein misfolding cyclic amplification (PMCA), which we found to be 6 log fold more sensitive than direct WB for the detection of pig BSE, we confirmed the presence of the Sh-BSE agent in lymphoid organs, nerves, ileum, and striated muscles from all 5 inoculated pigs. Surprisingly, PrPres positivity was also detected in white blood cells from one pig using this method. The presence of infectivity in lymphoid tissues, striated muscles, and peripheral nerves was confirmed by bioassay in bovine PrP transgenic mice. These results demonstrate the ability of BSE-derived agents to replicate efficiently in various peripheral tissues in pigs. Although no prion transmission has been reported in pigs following oral BSE challenge, our data support the continuation of the Feed Ban measure implemented to prevent entry of the BSE agent into the feed chain. [ABSTRACT FROM AUTHOR]

Background: Anaplasma phagocytophilum is a tick-borne intragranulocytic alpha-proteobacterium. It is the causative agent of tick-borne fever in ruminants, and of human granulocytic anaplasmosis in humans, two diseases which are becoming increasingly recognized in Europe and the USA. However, while several molecular typing tools have been developed over the last years, few of them are appropriate for in-depth exploration of the epidemiological cycle of this bacterium. Therefore we have developed a Multiple-Locus Variable number tandem repeat (VNTR) Analysis typing technique for A. phagocytophilum. Methods: Five VNTRs were selected based on the HZ human-derived strain genome, and were tested on the Webster human-derived strain and on 123 DNA samples: 67 from cattle, 7 from sheep, 15 from roe deer, 4 from red deer, 1 from a reindeer, 2 from horses, 1 from a dog, and 26 from ticks. Results: From these samples, we obtained 84 different profiles, with a diversity index of 0.96 (0.99 for vertebrate samples, i.e. without tick samples). Our technique confirmed that A. phagocytophilum from roe deer or domestic ruminants belong to two different clusters, while A. phagocytophilum from red deer and domestic ruminants locate within the same cluster, questioning the respective roles of roe vs red deer as reservoir hosts for domestic ruminant strains in Europe. As expected, greater diversity was obtained between rather than within cattle herds. Conclusions: Our technique has great potential to provide detailed information on A. phagocytophilum isolates, improving both epidemiological and phylogenic investigations, thereby helping in the development of relevant prevention and control measures. [ABSTRACT FROM AUTHOR]

We report the presence of infectivity in erythrocytes, leukocytes, and plasma of 1 person with variant Creutzfeldt-Jakob disease and in the plasma of 2 in 4 persons whose tests were positive for sporadic Creutzfeldt-Jakob disease. The measured infectivity levels were comparable to those reported in various animals with transmissible spongiform encephalopathies. [ABSTRACT FROM AUTHOR]

Owing to the natural evolution of a disease, several events often arise after a first treatment for the same subject. For example, patients with a primary invasive breast cancer and treated with breast conserving surgery may experience breast cancer recurrences, metastases or death. A certain proportion of subjects in the population who are not expected to experience the events of interest are considered to be ‘cured’ or non-susceptible. To model correlated failure time data incorporating a surviving fraction, we compare several forms of cure rate frailty models. In the first model already proposed non-susceptible patients are those who are not expected to experience the event of interest over a sufficiently long period of time. The other proposed models account for the possibility of cure after each event. We illustrate the cure frailty models with two data sets. First to analyse time-dependent prognostic factors associated with breast cancer recurrences, metastases, new primary malignancy and death. Second to analyse successive rehospitalizations of patients diagnosed with colorectal cancer. Estimates were obtained by maximization of likelihood using SAS proc NLMIXED for a piecewise constant hazards model. As opposed to the simple frailty model, the proposed methods demonstrate great potential in modelling multivariate survival data with long-term survivors (‘cured’ individuals). [ABSTRACT FROM AUTHOR]

Small ruminant post-mortem testing programs were initially designed for monitoring the prevalence of prion disease. They are now considered as a potential alternative to genetic selection for eradicating/controlling classical scrapie at population level. If such policy should be implemented, its success would be crucially dependent on the efficiency of the surveillance system used to identify infected flocks. In this study, we first determined the performance of post-mortem classical scrapie detection in eight naturally affected goat herds (total n = 1961 animals) according to the age at culling. These results provided us with necessary parameters to estimate, through a Monte Carlo simulation model, the performance of scrapie detection in a commercial population. According to this model, whatever the number of tests performed, post mortem surveillance will have limited success in identifying infected herds. These data support the contention that scrapie eradication programs relying solely on post mortem testing in goats will probably fail. Considering the epidemiological and pathological similarities of scrapie in sheep and goats, the efficiency of scrapie surveillance in both species is likely to be similar. [ABSTRACT FROM AUTHOR]

It is now clearly established that the transfusion of blood from variant CJD (v-CJD) infected individuals can transmit the disease. Since the number of asymptomatic infected donors remains unresolved, inter-individual v-CJD transmission through blood and blood derived products is a major public health concern. Current risk assessments for transmission of v-CJD by blood and blood derived products by transfusion rely on infectious titers measured in rodent models of Transmissible Spongiform Encephalopathies (TSE) using intra-cerebral (IC) inoculation of blood components. To address the biological relevance of this approach, we compared the efficiency of TSE transmission by blood and blood components when administrated either through transfusion in sheep or by intra-cerebral inoculation (IC) in transgenic mice (tg338) over-expressing ovine PrP. Transfusion of 200 µL of blood from asymptomatic infected donor sheep transmitted prion disease with 100% efficiency thereby displaying greater virulence than the transfusion of 200 mL of normal blood spiked with brain homogenate material containing 10³ID50 as measured by intracerebral inoculation of tg338 mice (ID50 IC in tg338). This was consistent with a whole blood titer greater than 103.6 ID50 IC in tg338 per mL. However, when the same blood samples were assayed by IC inoculation into tg338 the infectious titers were less than 32 ID per mL. Whereas the transfusion of crude plasma to sheep transmitted the disease with limited efficacy, White Blood Cells (WBC) displayed a similar ability to whole blood to infect recipients. Strikingly, fixation of WBC with paraformaldehyde did not affect the infectivity titer as measured in tg338 but dramatically impaired disease transmission by transfusion in sheep. These results demonstrate that TSE transmission by blood transfusion can be highly efficient and that this efficiency is more dependent on the viability of transfused cells than the level of infectivity measured by IC inoculation. [ABSTRACT FROM AUTHOR]

Fetal/neonatal alloimmune thrombocytopenia is a frequent disease in humans where alloantibodies against platelet Ags lead to platelet destruction and hemorrhage. Although a role in the disease for Abs against MHC has been suspected, this has not been formally demonstrated. Since 2007, a hemorrhagic syndrome due to thrombocytopenia and designated as bovine neonatal pancytopenia (BNP) has been recognized in calves in several European countries. An inactivated antiviral vaccine is strongly suspected to be involved in this syndrome because of its highly frequent use in the dams of affected calves. In this study, we show that BNP is an alloimmune disease, as we reproduced the signs by transferring serum Abs from vaccinated BNP dams into healthy neonatal calves. Ab specificity was strongly associated with the presence of allogeneic MHC class I Abs in the dams. MHC class I staining was also observed on Madin-Darby bovine kidney cells, a cell line related to the one used to produce the vaccine Ag. Our report emphatically demonstrates that alloimmunization against MHC class I is associated with a substantial risk of developing cytopenia-associated syndromes in neonates when a cell line of the same species is used to produce an inactivated vaccine injected into the mother. [ABSTRACT FROM AUTHOR]

Cure models have been developed to analyze failure time data with a cured fraction. For such data, standard survival models are usually not appropriate because they do not account for the possibility of cure. Mixture cure models assume that the studied population is a mixture of susceptible individuals, who may experience the event of interest, and non-susceptible individuals that will never experience it. Important issues in mixture cure models are estimation of the baseline survival function for susceptibles and estimation of the variance of the regression parameters. The aim of this paper is to propose a penalized likelihood approach, which allows for flexible modeling of the hazard function for susceptible individuals using M-splines. This approach also permits direct computation of the variance of parameters using the inverse of the Hessian matrix. Properties and limitations of the proposed method are discussed and an illustration from a cancer study is presented. Copyright © 2008 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The aim of our study was to evaluate the flock sensitivity and specificity of fecal qPCR and serum ELISA using pooled samples for screening paratuberculosis in French sheep. Using individual feces with low or high qPCR Ct values from ewes sampled in 14 infected flocks, a total of 555 pools of size 5, 10 and 20 were created by diluting individual materials in negative feces and analysed using a commercial IS900 qPCR kit. The relative performances of pooled serum ELISA analysis were evaluated based on the analysis of 181 different pools of size 5 and 10, composed of individual serum samples of various individual S/P values. Results showed that for pools of size 5, 10 or 20, individual fecal samples with low Ct values were invariably detected. Conversely fecal samples with high Ct values were associated with a lower detection rate in both pools of size 5 (87.0% to 90.0%), 10 (63.0% to 70.7%) and 20 (46.7% to 60.0%). After lowering the decision threshold to 25% and 15% for serum pools of size 5 and 10 respectively, the pooled serum ELISA relative sensitivity ranged between 62.2% and 100.0% depending on the composition of the pools. Finally, a simulation study was carried out to evaluate the performances of 16 screening strategies at flock level, with varying pool size (5 to 20) and number (5 to 60). The use of pooled serum ELISA led to very false positive detection rate ranging between 37.6% and 91.8% in paratuberculosis free flocks and prevents its further use in that context. For infection prevalence ≤ 5%, the flock sensitivity based on pooled fecal qPCR ranged between 39.0% (5 pools of size 10) and 99.9% (300 sampled individuals, with pools of size 5,10 or20), and was always above 93% when the infection prevalence was greater or equal to 15%. We conclude that pooled-fecal qPCR but not pooled-serum ELISA could be a useful tool to detect sheep flocks infected with paratuberculosis. [ABSTRACT FROM AUTHOR]

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M142, N/D146, S/D146, R/Q211, and Q/K222) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I142R154R211Q222) goats and homozygous and/or heterozygous I/M142, R/H154, R/Q211, and Q/K222 goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M142 PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H154, Q211, and K222 PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H154 and Q211 allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K222 and a small proportion of homozygous K222 goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K222 goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie. [ABSTRACT FROM AUTHOR]

Mice overexpressing the prion protein (PrP) sequence from various host species are widely used for measuring infectious titers in prion disease. However, the impact that the transgene expression level might have on the susceptibility to infection raises some concerns about the final biological relevance of these models. Here we report that endpoint titration of a sheep scrapie isolate in sheep and in mice overexpressing the ovine PrP results in similar estimates of the infectious titer. [ABSTRACT FROM AUTHOR]

Previous experiments carried out in a sheep scrapie model demonstrated that the transfusion of 200 μl of prion-infected whole blood has an apparent 100% efficacy for disease transmission. These experiments also indicated that, despite the apparent low infectious titer, the intravenous administration of white blood cells (WBC) resulted in efficient disease transmission. In the study presented here, using the same transmissible spongiform encephalopathy (TSE) animal model, our aim was to determine the minimal number of white blood cells and the specific abilities of mononucleated cell populations to transmit scrapie by the transfusion route. Our results confirmed that the transfusion of 100 μl, but not 10 μl, of fresh whole blood collected in asymptomatic scrapie-infected donor sheep can transmit the disease. The data also show that the intravenous administration of 105 WBCs is sufficient to cause scrapie in recipient sheep. Cell-sorted CD45R+ (predominantly B lymphocytes), CD4+/CD8+ (T lymphocytes), and CD14+ (monocytes/macrophages) blood cell subpopulations all were shown to contain prion infectivity by bioassays in ovine PrP transgenic mice. However, while the intravenous administration of 106 CD45+ or CD4+/8+ living cells was able to transmit the disease, similar numbers of CD14+ cells failed to infect the recipients. These data support the contention that mononucleated blood cell populations display different abilities to transmit TSE by the transfusion route. They also represent an important input for the risk assessment of blood-borne prion disease transmission and for refining the target performance of leukoreduction processes that currently are applied to mitigate the transmission risk in transfusion medicine. [ABSTRACT FROM AUTHOR]

Bovine spongiform encephalopathy (BSE) can be efficiently transmitted to pigs via intracerebral inoculation. A clear link has been established between the consumption of products of bovine origin contaminated with the BSE agent and the development of variant Creutzfeldt-Jakob disease in humans. Small ruminants can also naturally develop BSE, and sheep-adapted BSE (Sh-BSE) propagates more efficiently than cattle BSE in pigs and in mouse models expressing porcine prion protein. In addition, Sh-BSE shows greater efficiency of transmission to human models than original cow BSE. While infectivity and/or abnormal PrP accumulation have been reported in the central nervous system in BSE-infected pigs, the ability of the agent to replicate in peripheral tissues has not been fully investigated. We previously characterized the presence of prions in a panel of tissues collected at the clinical stage of disease from pigs experimentally infected with Sh-BSE. Western blot revealed low levels of PrPres accumulation in lymphoid tissues, nerves, and skeletal muscles from 4 of the 5 animals analysed. Using protein misfolding cyclic amplification (PMCA), which we found to be 6 log fold more sensitive than direct WB for the detection of pig BSE, we confirmed the presence of the Sh-BSE agent in lymphoid organs, nerves, ileum, and striated muscles from all 5 inoculated pigs. Surprisingly, PrPres positivity was also detected in white blood cells from one pig using this method. The presence of infectivity in lymphoid tissues, striated muscles, and peripheral nerves was confirmed by bioassay in bovine PrP transgenic mice. These results demonstrate the ability of BSE-derived agents to replicate efficiently in various peripheral tissues in pigs. Although no prion transmission has been reported in pigs following oral BSE challenge, our data support the continuation of the Feed Ban measure implemented to prevent entry of the BSE agent into the feed chain., Competing Interests: The authors have declared that no competing interests exist.

Multiple theories exist regarding the origin of bovine spongiform encephalopathy (BSE). An early and prominent theory proposed that BSE was the result of the adaptation of sheep scrapie to cattle. The reports to date indicate that the distribution of the pathological prion protein (PrP Sc ) in experimental bovine scrapie is largely restricted to the central nervous system (CNS). Here, we describe pathological findings in a calf intracerebrally inoculated with a Spanish classical scrapie isolate. While clinical disease was observed 30 months after inoculation and PrP Sc was detected in the CNS, the corresponding phenotype differed from that of BSE. Immunohistochemistry and PMCA also revealed the presence of PrP Sc in the peripheral nerves, lymphoid tissues, skeletal muscle and gastrointestinal tract, suggesting centrifugal spread of the scrapie agent from the brain. To the best of our knowledge, this is the first report describing the detection of PrP Sc in tissues other than the CNS after experimental transmission of scrapie to cattle.

The ARR allele is considered to provide a very strong resistance against classical scrapie infection in sheep. In this study, we report the occurrence of clinical transmissible spongiform encephalopathy in ARR/ARR sheep, following their inoculation by the intracerebral route with a classical scrapie isolate. On first passage, the disease displayed an incomplete attack rate transmission, with incubation periods exceeding 6 years. On second passage, the obtained prion did not display better abilities to propagate than the original isolate. These transmission results contrasted with the 100 % attack rate and the short incubation periods observed in ARQ/ARQ sheep challenged with the same isolate. These data confirm that ARR/ARR sheep cannot be considered to be fully resistant to classical scrapie. However, they also support the contention that classical scrapie has a very limited capacity to transmit and adapt to ARR/ARR sheep.

The PrP gene polymorphisms at codons 142 (I/M), 154 (R/H), 211 (R/Q), 222 (Q/K) and 240 (S/P) and their association with susceptibility to classical scrapie infection were investigated in five French goat herds displaying a high disease prevalence (>10%). On the basis of PrP(Sc) detection in the central nervous system and in various lymphoid tissues, 301 of 1343 goats were found to be scrapie infected. The statistical analyses indicated that while P(240) mutation had no direct impact on scrapie infection risk, the H(154), Q(211) and K(222) mutations were associated with high resistance to scrapie. The M(142) mutated allele was associated with a limited protection level against the disease. These results further reinforce the view that, like in sheep, the control and eradication of classical scrapie through the selection of certain PrP alleles could be envisaged in commercial goat population.

The dynamics of the circulation and distribution of transmissible spongiform encephalopathy (TSE) agents in the blood of infected individuals remain largely unknown. This clearly limits the understanding of the role of blood in TSE pathogenesis and the development of a reliable TSE blood detection assay. Using two distinct sheep scrapie models and blood transfusion, this work demonstrates the occurrence of a very early and persistent prionemia. This ability to transmit disease by blood transfusion was correlated with the presence of infectivity in white blood cells (WBC) and peripheral blood mononucleated cells (PBMC) as detected by bioassay in mice overexpressing the ovine prion protein PrP (tg338 mice) and with the identification of abnormal PrP in WBC after using protein misfolding cyclic amplification (PMCA). Platelets and a large variety of leukocyte subpopulations also were shown to be infectious. The use of endpoint titration in tg338 mice indicated that the infectivity in WBC (per ml of blood) was 10(6.5)-fold lower than that in 1 g of posterior brainstem sample. In both WBC and brainstem, infectivity displayed similar resistance to PK digestion. The data strongly support the concept that WBC are an accurate target for reliable TSE detection by PMCA. The presence of infectivity in short-life-span blood cellular elements raises the question of the origin of prionemia.

Oral inoculation is currently considered as the best approach to mimic natural TSE contamination in ruminants. In this study, we compared the timing of abnormal prion protein (PrP(Sc)) dissemination and accumulation in the organism of susceptible sheep either orally inoculated or naturally infected with classical scrapie. Both animal groups shared a similar PrP(Sc) dissemination scheme and accumulation dynamics in lymphoid tissues. However, orally challenged animals displayed an earlier neuro-invasion and a dramatically shorter incubation period than naturally exposed sheep. No differences were observed between the groups with regards to the neuro-invasion route. These results unambiguously indicate that oral inoculation can have an impact on both the earliness of neuro-invasion and the incubation period. They also support the statement that oral inoculation is a relevant model for investigating transmissible spongiform encephalopathy pathogenesis. Nevertheless, data obtained under such experimental conditions should be used with some caution.

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrP(Sc) accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 microg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species.

Because of the confounding effects of long incubation duration and flock management, accurate epidemiological studies of scrapie outbreaks are difficult to carry out. In this study, 641 Manech red-faced sheep from six scrapie-affected field flocks in Pyrénées Atlantiques, France, were monitored for clinical scrapie over a 6-9 year period. Over this period, 170 scrapie clinical cases were recorded and half of the culled animals were submitted for post-mortem transmissible spongiform encephalopathy diagnosis to assess their infectious status. Collected data were analysed using a 'mixture cure model' approach, which allowed for the discriminating effect of PrP genotype and flock origin on incidence and incubation period. Simulations were performed to evaluate the applicability of such a statistical model to the collected data. As expected, ARR heterozygote sheep were less at risk of becoming infected than ARQ/ARQ individuals and had a greater age at clinical onset. Conversely, when compared with ARQ/ARQ, the VRQ haplotype was associated with an increased infection risk, but not a shorter incubation period. Considering the flock effect, we observed that a high incidence rate was not associated with shorter incubation periods and that the incubation period could be significantly different in flocks harbouring similar infection risks. These results strongly support the conclusion that other parameters, such as the nature of the agent or flock management, could interfere with epidemiological dynamics of the infection in scrapie-affected flocks.

Oral contamination with bovine spongiform encephalopathy (BSE) agent in susceptible PRNP genotype sheep results in widespread distribution of prion in the host. Because ARR homozygous sheep are considered to be resistant to transmissible spongiform encephalopathies, they have been selected to eradicate scrapie from sheep flocks and to protect the human food chain from small ruminant BSE risk. However, results presented here show that several months after an oral challenge with BSE agent, healthy ARR/ARR sheep can accumulate significant amounts of PrP(Sc) in the spleen.