Caroline Lacroux

PrPSc accumulation in myocytes from sheep incubating natural scrapie.

Because variant Creutzfeldt–Jakob disease (vCJD) in humans probably results from consumption of products contaminated with tissue from animals with bovine spongiform encephalopathy, whether infectious prion protein is present in ruminant muscles is a crucial question. Here we show that experimentally and naturally scrapie-affected sheep accumulate the prion protein PrPSc in a myocyte subset. In naturally infected sheep, PrPSc is detectable in muscle several months before clinical disease onset. The relative amounts of PrPSc suggest a 5,000-fold lower infectivity for muscle as compared to brain.

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.

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.

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.

Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French Alpine and Saanen breeds and their association with classical scrapie

In sheep, susceptibility to scrapie is mainly influenced by polymorphisms of the PrP gene. In goats, there are to date few data related to scrapie susceptibility association with PrP gene polymorphisms. In this study, we first investigated PrP gene polymorphisms of the French Alpine and Saanen breeds. Based on PrP gene open reading frame sequencing of artificial insemination bucks (n=404), six encoding mutations were identified at codons 127, 142, 154, 211, 222 and 240. However, only seven haplotypes could be detected: four (GIH(154)RQS, GIRQ(211)QS, GIRRK(222)S and GIRRQP(240)) derived from the wild-type allele (G(127)I(142)R(154)R(211)Q(222)S(240)) by a single-codon mutation, and two (S(127)IRRQP(240) and GM(142)RRQP(240)) by a double-codon mutation. A case-control study was then implemented in a highly affected Alpine and Saanen breed herd (90 cases/164 controls). Mutations at codon 142 (I/M), 154 (R/H), 211 (R/Q) and 222 (Q/K) were found to induce a significant degree of protection towards natural scrapie infection. Compared with the baseline homozygote wild-type genotype I(142)R(154)R(211)Q(222)/IRRQ goats, the odds of scrapie cases in IRQ(211)Q/IRRQ and IRRK(222)/IRRQ heterozygous animals were significantly lower [odds ratio (OR)=0.133, P<0.0001; and OR=0.048, P<0.0001, respectively]. The heterozygote M(142)RRQ/IRRQ genotype was only protective (OR=0.243, P=0.0186) in goats also PP(240) homozygous at codon 240. However, mutated allele frequencies in French Alpine and Saanen breeds were low (0.5-18.5 %), which prevent us from assessing the influence of all the possible genotypes in natural exposure conditions.

Immune response to Haemonchus contortus infection in susceptible (INRA 401) and resistant (Barbados Black Belly) breeds of lambs.

The immune responses to Haemonchus contortus were compared in studies in resistant Barbados Black Belly (BBB) and susceptible INRA 401 (INRA) breeds of lambs. The cytokine patterns indicated a Th2‐biased response in both breeds. A more persistent and elevated Th2 cytokine mRNA transcription and blood eosinophilia were noted in the BBB lambs. However, at days 4 and 30 post‐infection, abomasal recruitment of eosinophils and mast cells were similar between the two breeds. Following primary infections, the BBB demonstrated a substantially lower faecal egg count compared to the INRA lambs. Furthermore, worm counts at 4 and 30 days post‐infection, and adult female worm size and in utero egg counts 30 days after the first infection were significantly lower in the BBB than in the INRA breed. In the INRA breed, re‐infection caused a significant reduction in most parasitological parameters compared with those observed after the primary infection. A similar response was not observed in the BBB sheep. In conclusion, while the major driving force in the response to H. contortus infection is a Th2‐biased immunity in which the BBB showed its maximal performance during the primary infection, the INRA breed performed better after re‐infection compared to its response to first exposure.

Preclinical Detection of Variant CJD and BSE Prions in Blood

The emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy (BSE) agent. More recently, secondary vCJD cases were identified in patients transfused with blood products prepared from apparently healthy donors who later went on to develop the disease. As there is no validated assay for detection of vCJD/BSE infected individuals the prevalence of the disease in the population remains uncertain. In that context, the risk of vCJD blood borne transmission is considered as a serious concern by health authorities. In this study, appropriate conditions and substrates for highly efficient and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding Cyclic Amplification (PMCA) were first identified. This showed that whatever the origin (species) of the vCJD/BSE agent, the ovine Q171 PrP substrates provided the best amplification performances. These results indicate that the homology of PrP amino-acid sequence between the seed and the substrate is not the crucial determinant of the vCJD agent propagation in vitro. The ability of this method to detect endogenous vCJD/BSE agent in the blood was then defined. In both sheep and primate models of the disease, the assay enabled the identification of infected individuals in the early preclinical stage of the incubation period. Finally, sample panels that included buffy coat from vCJD affected patients and healthy controls were tested blind. The assay identified three out of the four tested vCJD affected patients and no false positive was observed in 141 healthy controls. The negative results observed in one of the tested vCJD cases concurs with results reported by others using a different vCJD agent blood detection assay and raises the question of the potential absence of prionemia in certain patients.

Prionemia and Leukocyte-Platelet-Associated Infectivity in Sheep Transmissible Spongiform Encephalopathy Models

ABSTRACT 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 106.5-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.

Endocarditis in Cattle Caused by Bartonella bovis

This study aimed to determine the role of Bartonella as an endocarditis agent in cattle. Bartonella bovis was identified by PCR, gene sequences analysis, and specific internal transcribed spacer amplicon product size in 2 bovine endocarditis cases with high antibody titers, which demonstrates that B. bovis is a pathogen for cattle.

PrP-associated resistance to scrapie in five highly infected goat herds.

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.