Protecting effect of PrP codons M142 and K222 in goats orally challenged with bovine spongiform encephalopathy prions.

Breeding towards genetic resistance to prion disease is effective in eliminating scrapie. In sheep, classical forms of scrapie have been eradicated almost completely in several countries by breeding programs using a prion protein (PrP) gene (PRNP) amino acid polymorphism. For goats, field and experimental studies have provided evidence for several amino acid polymorphisms that are associated with resistance to scrapie, but only limited data are available concerning the susceptibility of caprine PRNP genotypes to BSE. In this study, goat kids representing five PRNP genotypes based on three polymorphisms (M142, Q211 and K222 and the wild type I142, R211 and Q222) were orally challenged with bovine or goat BSE. Wild type goats were killed with clinical signs between 24-28 months post inoculation (mpi) to both challenges, and goats with genotype R/Q211 succumbed between 29-36 mpi. I/M142 goats developed clinical signs at 44-45 mpi and M/M142 goats remained healthy until euthanasia at 48 mpi. None of the Q/K222 goats showed definite clinical signs. Taken together the highest attack ratios were seen in wild type and R/Q211 goats, and the lowest in I/M142, M/M142 and Q/K222. In all genotype groups, one or more goats remained healthy within the incubation period in both challenges and without detectable PrP deposition in the tissues. Our data show that both the K222 and M142 polymorphisms lengthen the incubation period significantly compared to wild type animals, but only K222 was associated with a significant increase in resistance to BSE infection after oral exposure to both BSE sources.

Genetic control of HgCl2-induced IgE and autoimmunity by a 117-kb interval on rat chromosome 9 through CD4 CD45RChigh T cells.

Gold or mercury salts trigger a dramatic IgE response and a CD4 T-cell-dependent nephropathy in Brown-Norway (BN), but not in Lewis (LEW) rats. We previously identified the 1.1-Mb Iresp3 (immunoglobin response QTL3) locus on chromosome 9 that controls these gold salt-triggered immune disorders. In the present work, we investigated the genetic control of HgCl(2)-induced immunological disorders and assessed the relative contribution of the CD45RC(high) and CD45RC(low) CD4 T-cell subpopulations in this control. By using interval-specific congenic lines, we narrowed down Iresp3 locus to 117-kb and showed that BN rats congenic for the LEW 117-kb were protected from HgCl(2)-triggered IgE response and nephropathy. This 117-kb interval also controls CD45RC expression by CD4 T cells and the ability of CD45RC(high) CD4 T cells to trigger the autoimmune disorders resulting from HgCl(2) administration. This 117-kb region contains four genes, including Vav1, a strong candidate gene according to its cellular function and exclusive expression in hematopoietic cells. Thus, this study highlights the role of the CD45RC(high) CD4 T-cell subpopulation in the opposite susceptibility of BN and LEW rats to HgCl(2)-triggered immune disorders and identifies a 117-kb interval on chromosome 9 that has a key role in their functions.

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 PrP(Sc) in a myocyte subset. In naturally infected sheep, PrP(Sc) is detectable in muscle several months before clinical disease onset. The relative amounts of PrP(Sc) suggest a 5,000-fold lower infectivity for muscle as compared to brain.

Protective effect of the T112 PrP variant in sheep challenged with bovine spongiform encephalopathy.

Sheep with an ARQ/ARQ PRNP genotype at codon positions 136/154/171 are highly susceptible to experimental infection with bovine spongiform encephalopathy (BSE). However, a number of sheep challenged orally or intracerebrally with BSE were clinically asymptomatic and found to survive or were diagnosed as BSE-negative when culled. Sequencing of the full PRNP gene open reading frame of BSE-susceptible and -resistant sheep indicated that, in the majority of Suffolk sheep, resistance was associated with an M112T PRNP variant (TARQ allele). A high proportion (47 of 49; 96%) of BSE-challenged wild-type (MARQ/MARQ) Suffolk sheep were BSE-infected, whereas none of the 20 sheep with at least one TARQ allele succumbed to BSE. Thirteen TARQ-carrying sheep challenged with BSE are still alive and some have survival periods equivalent to, or greater than, reported incubation periods of BSE in ARR/ARR and VRQ/VRQ sheep.

Identification of new quantitative trait Loci (other than the PRNP gene) modulating the scrapie incubation period in sheep.

Although susceptibility to scrapie is largely controlled by the PRNP gene, we have searched for additional genomic regions that affect scrapie incubation time in sheep, using two half-sib families with a susceptible PRNP genotype and naturally infected by scrapie. Quantitative trait loci were detected on OAR6 and OAR18.

Discrimination of sheep susceptible and resistant to transmissible spongiform encephalopathies by an haplotype specific monoclonal antibody.

In the present report, the selective detection of sheep PrP haplotypes by monoclonal antibody 2A11 is described. It is showed that the substitution of glutamine by arginine but not by histidine at ovine PrP position 171 abolishes completely the recognition of either PrP(c) or PrP(d) by mAb 2A11, in such a way that the application of this antibody allows the unambiguous discrimination of R(171) homozygotes. On the basis of the high resistance to classical scrapie and bovine spongiform encephalophaty (BSE) infection associated to the R(171) PrP haplotype, animals bearing the ARR allele are currently selected within the scrapie national plan initiated in Great Britain. A 2A11-based immuno enzymatic test have been developed and evaluated using a panel of plasma and sera from sheep of different PrP genotypes and breeds. The test allows the efficient discrimination of R(171) homozygotes, R(171) heterozygotes and non-R(171) carriers, therefore offering a rapid, cheap and easy to use alternative method to select sheep for their resistance to scrapie.

Major hypercorticism is an endocrine feature of ewes with naturally occurring scrapie.

The aim of this study was to identify the origin of scrapie-induced hypercortisolism. Cortisol and ACTH kinetics and production rate were measured in 14 ewes (6 healthy and 8 scrapie-affected). It was shown that cortisol plasma clearance remained unmodified but that cortisol production rate and plasma concentrations of free cortisol were increased by a factor of 5, whereas the total cortisol plasma concentrations were only doubled. The apparent discrepancy between adrenal secretion rate and the corresponding total cortisol plasma levels was attributable to the scrapie-induced lower corticosteroid-binding globulin (CBG) binding capacity, which altered the ratio of free-to-bound cortisol. The secretion rate of ACTH from diseased ewes was increased by a factor of 1.5, in comparison with healthy ewes, and 4 of the 8 scrapie-affected ewes exhibited a decreased response to a low dexamethasone suppression test. The administration of tetracosactide induced a 2-fold increase in the cortisol production in diseased ewes, compared with that of healthy ewes, but the pituitary sensitivity to ovine CRF was not modified by the prion disease. In conclusion, natural scrapie displays a syndrome of hypercorticism associated with increased ACTH secretion, hyperresponsiveness of the adrenals, and lower CBG binding capacity, which leads to overexposure to CBG-free cortisol.

Corticoid concentrations are increased in the plasma and urine of ewes with naturally occurring scrapie.

The 24-h pattern of corticoid plasma concentrations was determined in scrapie-affected ewes during the clinical phase of the disease. Twenty one ewes (8 healthy and 13 scrapie-affected ewes) were subjected to 24-h blood sampling sessions. Urine samples were simultaneously obtained during the clinical stage of the disease and in healthy ewes. The scrapie diagnosis was performed by histopathology. Plasma and urinary corticoids were assayed using HPLC methods. Mean plasma and urinary levels of corticoid (cortisol, 20beta-dihydrocortisol cortisone) of scrapie-affected ewes were greater than those observed in healthy ewes. 20Beta-dihydrocortisol appeared to be the main cortisol metabolite in ewes. The intra-individual variations of 20beta-dihydrocortisol plasma concentrations were lower than the corresponding plasma cortisol concentrations due to the dampening effect of the metabolic process on the short term variations of cortisol secretion. This dampening mechanism was amplified in urine, the urinary concentrations integrating cortisol production over the period preceding sampling. For these reasons, 20beta-dihydrocortisol could present a potential interest for a non invasive diagnostic test of transmissible spongiform encephalopathies. The pathophysiological consequences of an excessive exposure to cortisol on development of the neurogenerative process are discussed.

Unexpected high testis-specific transcriptional activity of the cyclin T1 promoter in transgenic mice.

The ubiquitously expressed cyclin T1 gene encodes for a protein involved in human immunodeficiency virus type 1 (HIV-1) transcription activation. The goat gene was recently shown to share an expression pattern similar to that of its endogenous counterpart when incorporated into mice using a BAC insert. To assess if its promoter could target ubiquitous expression of the bovine Prnp in transgenic mice, two constructs carrying either 1 or 30 kb of cyclin T1 5'-flanking sequences were built and microinjected. Both constructs resulted in the unexpected high male germ cell-specific expression of the prion protein. These data re-question the suspected location of the cyclin T1 gene regulatory elements.

Naturally occurring scrapie is associated with a lower CBG binding capacity in ewes.

Naturally scrapie-affected ewes present a syndrome of hypercortisolism as evaluated by measuring total plasma cortisol concentrations. The objective of this study was to investigate the plasma protein binding of cortisol and to evaluate the concentration of the biologically active free fraction of cortisol in scrapie-affected ewes. Corticosteroid binding globulin (CBG) binding parameters were evaluated by equilibrium dialysis in 13 naturally scrapie-affected ewes and nine healthy ewes, during two periods of the clinical evolution of the disease. The hypercortisolism of the scrapie-affected ewes was confirmed by a significant increase of the plasma 20 beta-dihydrocortisol and cortisone concentrations, while total cortisol concentrations, obtained from an isolated sample, did not differ between scrapie-affected and control ewes. The scrapie diagnosis was confirmed by histopathology. The CBG maximal capacity (B(max)) was two times lower in scrapie-affected ewes than in healthy ewes (37+/-32 nM and 73+/-28 nM respectively). The dissociation constant K(d) (8.8+/-3.7 nM and 9.8+/-3.0 nM respectively) and the non-specific constant value of binding to albumin (1.13+/-0.18 and 1.14+/-0.23 respectively) did not differ significantly between diseased and control ewes. The significant increased concentrations of CBG-free cortisol (i.e. both albumin-bound and free cortisol fractions) in scrapie-affected ewes indicates that total plasma cortisol concentration is not an appropriate index of pituitary-adrenocortical hyperactivity. In conclusion, ewes with naturally occurring scrapie display a syndrome of hypercortisolism associated with a lower CBG binding capacity which leads to an overexposure of glucocorticoid-sensitive targets to CBG-free cortisol. The physiopathological consequences of this overexposure on the development of the neurodegenerative process in prion disease are discussed.

Discriminant value of blood and urinary corticoids for the diagnosis of scrapie in live sheep.

The mean (sd) concentration of plasma 20beta-dihydrocortisol in 126 scrapie-affected sheep was 5-5 (7.0) ng/ml compared with 1.1 (0.7) ng/ml in 52 healthy sheep. The mean (sd) concentration of creatinine in the urine of 93 scrapie-affected sheep was 2.43 (1.56) microg/ml compared with 0.94 (0.86) pg/ml in 49 healthy sheep and 1.10 (0-95) pg/ml in 25 sheep with other diseases. These discriminant analyses carried out on healthy and scrapie-affected sheep showed that plasma 20beta-dihydrocortisol and urinary creatinine were the best predictors of the disease, and classified correctly 98 per cent of healthy sheep and 82 per cent of scrapie-affected sheep.

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.

Which PrP haplotypes in a French sheep population are the most susceptible to atypical scrapie?

A French sheep case control study has been organised to estimate the effects of the PrP haplotypes on resistance to atypical scrapie. The ALHQ and AFRQ haplotypes are significantly more susceptible than the others.

Dynamics and genetics of PrPSc placental accumulation in sheep.

Placentae from scrapie-affected ewes are an important source of contamination. This study confirmed that scrapie-incubating ewes bearing susceptible genotypes could produce both abnormal prion protein (PrPSc)-positive and -negative placentae, depending only on the PRP genotype of the fetus. The results also provided evidence indicating that scrapie-incubating ARR/VRQ ewes may be unable to accumulate prions in the placenta, whatever the genotype of their progeny. Multinucleated trophoblast cells appeared to play a key role in placental PrPSc accumulation. PrPSc accumulation began in syncytiotrophoblasts before disseminating to uninucleated trophoblasts. As these result from trophoblast/uterine epithelial cell fusion, syncytiotrophoblast cells expressed maternal and fetal PrPC, whilst uninucleated trophoblast cells only expressed fetal PrPC. In ARR/VRQ scrapie-infected ewes, expression of the ARR allele by syncytiotrophoblasts appeared to prevent initiation of PrPSc placental deposition. The absence of prions in affected ARR/VRQ sheep placentae reinforces strongly the interest in ARR selection for scrapie control.

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.

Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein.

Transmissible spongiform encephalopathies, or prion diseases, are fatal degenerative disorders of the central nervous system that affect humans and animals. Prions are nonconventional infectious agents whose replication depends on the host prion protein (PrP). Transmission of prions to cultured cells has proved to be a particularly difficult task, and with a few exceptions, their experimental propagation relies on inoculation to laboratory animals. Here, we report on the development of a permanent cell line supporting propagation of natural sheep scrapie. This model was obtained by stable expression of a tetracycline-regulatable ovine PrP gene in a rabbit epithelial cell line. After exposure to scrapie agent, cultures were repeatedly found to accumulate high levels of abnormal PrP (PrPres). Cell extracts induced a scrapie-like disease in transgenic mice overexpressing ovine PrP. These cultures remained healthy and stably infected upon subpassaging. Such data show that (i) cultivated cells from a nonneuronal origin can efficiently replicate prions; and (ii) species barrier can be crossed ex vivo through the expression of a relevant PrP gene. This approach led to the ex vivo propagation of a natural transmissible spongiform encephalopathy agent (i.e., without previous experimental adaptation to rodents) and might be applied to human or bovine prions.

Persistent infection of B lymphocytes by bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in young cattle. Here we demonstrate BRSV persistence at low levels in tracheobronchial and mediastinal lymph nodes up to 71 days after the experimental infection of calves. Positive results were obtained on viral genomic RNA and messenger RNA coding for the nucleoprotein, glycoprotein (G), and fusion protein (F). G and F proteins were also detected in the pulmonary lymph nodes by immunohistochemistry. Double-staining experiments revealed that viral antigen was present in B-lymphocytes. Coculture experiments with the lymph node cells showed that the virus was still able to infect permissive target cells, even though no cytopathic effect was recorded. In vitro studies indicate that BRSV was still able to replicate in bovine B-lymphocyte cell lines 6 months after infection. These results may also be relevant to the understanding not only of the epidemiology and the peculiarities of the immune response of BRSV infections but also of human respiratory syncytial virus infections.

Melatonin and prolactin secretion profile in naturally occurring scrapie in ewe.

The 24 hr pattern of melatonin secretion was determined in scrapie-affected ewes during the clinical course of the disease. The melatonin response to a night interruption by a 1 hr period of illumination was also measured. Fourteen ewes (seven control and seven scrapie-affected ewes) were subjected to artificial short days (9L:15D). Four 24 hr blood sampling sessions separated by about 10 days were performed. Ewes were sacrificed when clinical signs had progressed to irreversible recumbency and the scrapie diagnosis was confirmed by histopathology. Plasma melatonin was assayed in all samples and prolactin was analysed in samples obtained during the second sampling session using RIA methods. The instantaneous amplitude of elevation of plasma melatonin concentrations was calculated for each ewe and each sampling session and the within-ewe repeatability of this parameter was evaluated. The within-ewe repeatability of instantaneous amplitude of melatonin secretion was apparently greater in control than in scrapie-affected ewes (72% vs. 39%). The light stimulus induced an abrupt decrease of night melatonin concentrations in all ewes. Prolactin secretion was not affected by the disease. It was concluded that the 24 hr pattern of melatonin secretion was maintained in scrapie-affected ewes. The retino-hypothalamic tract transducing light information remained functional in diseased ewes despite some evidence of histopathological changes of the pineal gland. The instability of melatonin secretion during the clinical course of scrapie could reflect a disturbance of pineal function. However, whether this effect exists or not, it could not be used to discriminate scrapie-affected ewes from control ones.

Genetic susceptibility and transmission factors in scrapie: detailed analysis of an epidemic in a closed flock of Romanov.

Information from a scrapie epidemic in a closed INRA Romanov flock is presented. Performances, pedigree, histopathological diagnoses and PrP genotypes were recorded from the beginning of the outbreak (in 1993). Between 1st of April, 1993 and 1st of May, 1997, 1015 animals were exposed to scrapie, and 304 died from this disease. A major influence of the polymorphisms at codons 136, 154 and 171 is shown, A136H154Q171 allele carriers proving to be nearly as resistant as A136R154R171 carriers. A possible relationship between gastrointestinal parasitism and scrapie is discussed. There is evidence of maternal transmission, with a risk ratio for artificially fed lambs of 67 percent of the risk of lambs fed by their mother. Our results strongly suggest that resistant animals were not healthy carriers or at least were less infectious when comparing risk for lambs born to healthy dams either of resistant (risk = 0.431) or of susceptible (risk = 1.000) genotype.