Les marqueurs de l'inflammation chez les bovins : nature, physiopathologie et intérêt diagnostique

La réaction inflammatoire non spécifique est un mécanisme de défense de l'organisme face aux micro-organismes et aux destructions tissulaires. Elle se traduit par des modifications locales et générales visant essentiellement à éliminer l'agent agresseur et restaurer l'homéostasie. Les variations de certains paramètres biochimiques et hématocytologiques induites par la réaction inflammatoire peuvent être utilisées à des fins diagnostiques. Pour être interprétables, ces paramètres doivent dépendre uniquement du processus inflammatoire et présenter une variation significative par rapport aux valeurs de base. Leur intégration dans une démarche clinique raisonnée nécessite, de plus, le développement de techniques d'analyses rapides et précises. Dans cette étude bibliographique, l'auteur passe en revue les principaux marqueurs de l'inflammation répondant à ces critères dans l'espèce bovine. L'hématologie et les différentes protéines de la réaction inflammatoire sont étudiées comme marqueurs systémiques de l'inflammation. Les modifications du LCR, du lait et du liquide de ponction péritonéal sont aussi abordées en tant que marqueurs locaux. Les variations physiologiques et pathologiques, les techniques d'analyse et les implications diagnostiques en médecine bovine sont détaillées. Une attention particulière est portée aux protéines de la phase aiguë, dont l'importance est déjà largement établie chez l'homme. Malgré une connaissance partielle de leur physiopathologie chez les bovins, l'utilisation de ces marqueurs se révèle prometteuse en termes de diagnostic, de suivi et de traitement de certaines affections inflammatoires

Advanced survival models for risk-factor analysis in scrapie

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

Highly Efficient Prion Transmission by Blood Transfusion

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

Experimental transmission to a calf of an isolate of Spanish classical scrapie

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 (PrPSc) 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 PrPSc was detected in the CNS, the corresponding phenotype differed from that of BSE. Immunohistochemistry and PMCA also revealed the presence of PrPSc 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 PrPSc in tissues other than the CNS after experimental transmission of scrapie to cattle

Prions in Milk from Ewes Incubating Natural Scrapie

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 PrPSc 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 µg 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

Atypical/Nor98 Scrapie Infectivity in Sheep Peripheral Tissues

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrPSc negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed

Modèles de mélange en analyse de survie en présence de données groupées : application à la tremblante du mouton

Although they are of major interest in the understanding of the classical scrapie dynamic in infected flocks, the factors influencing the contamination by the infectious agent and incubation period remain poorly know. The absence of antemortem diagnostic tools and the confounding effects of long incubation periods and flock management practices yield a partial knowledge of the infectious status of animals. Moreover, we must take into account that only an unknown fraction of animals become infected, even in heavily affected flocks.To deal with issues, we use a class of particular survival models, which take account for the presence of long term survivors. We propose a penalized likelihood approach, allowing for the estimation of a smooth risk function. We also develop some parametric models with shared frailties to deal with the presence of grouped data. These different models are evaluated through simulations studies.These statistical approaches are then applied to the analysis of real data collected during the following-up of infected flocks in Pyrénées Atlantiques, France. The key influence of the PRP genotype on the contamination risk and incubation periods is confirmed. Our results also suggest that, at the individual level, the infection mainly takes place around birth. Finally, the strong heterogeneity in the contamination risk and incubation periods observed between flocks could be partially explained by their PRP genetic structure and the number of incubating animals.Les facteurs de risque individuels et environnementaux associés à la contamination par l'agent de la tremblante classique du mouton et à la durée d'incubation des animaux infectés demeurent mal connus. En l'absence de diagnostic antemortem précoce, la longue durée d'incubation de la maladie et les durées de vie limitées des animaux conduisent à une connaissance partielle du statut sanitaire des animaux. De plus, l'analyse doit tenir compte du fait qu'une partie seulement des animaux se contamine. Nous utilisons des modèles d'analyse des données de survie prenant en compte l'existence d'une fraction non à risque. Nous proposons une approche par vraisemblance pénalisée, qui allie les avantages des modèles paramétriques et semi paramétriques existants. Nous nous intéressons ensuite aux modèles paramétriques de survie avec fraction non à risque et effets aléatoires afin de tenir compte du regroupement des animaux dans les élevages. Ces différentes approches sont évaluées à l'aide d'études de simulations.L'application des ces modèles aux données issues du suivi longitudinal d'élevages infectés des Pyrénées Atlantiques (France) confirme le rôle déterminant du génotype au gène PRP sur le risque de contamination et les durées d'incubation. Nos résultats suggèrent de plus que la contamination par l'agent infectieux a principalement lieu en période néonatale. Enfin la forte hétérogénéité des risques de contamination et des durées d'incubation mise en évidence entre troupeaux pourrait être partiellement expliquée par la prise en compte de la structure génétique des élevages et du nombre d'animaux infectés présents

A SAS macro for parametric and semiparametric mixture cure models.

International audienceCure 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. The aim of this paper is to propose a SAS macro to estimate parametric and semiparametric mixture cure models with covariates. The cure fraction can be modelled by various binary regression models. Parametric and semiparametric models can be used to model the survival of uncured individuals. The maximization of the likelihood function is performed using SAS PROC NLMIXED for parametric models and through an EM algorithm for the Cox's proportional hazards mixture cure model. Indications and limitations of the proposed macro are discussed and an example in the field of cancer clinical trials is shown