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.

Prion Type-Dependent Deposition of PRNP Allelic Products in Heterozygous Sheep.

UNLABELLED: Susceptibility or resistance to prion infection in humans and animals depends on single prion protein (PrP) amino acid substitutions in the host, but the agent's modulating role has not been well investigated. Compared to disease incubation times in wild-type homozygous ARQ/ARQ (where each triplet represents the amino acids at codons 136, 154, and 171, respectively) sheep, scrapie susceptibility is reduced to near resistance in ARR/ARR animals while it is strongly enhanced in VRQ/VRQ carriers. Heterozygous ARR/VRQ animals exhibit delayed incubation periods. In bovine spongiform encephalopathy (BSE) infection, the polymorphism effect is quite different although the ARR allotype remains the least susceptible. In this study, PrP allotype composition in protease-resistant prion protein (PrP(res)) from brain of heterozygous ARR/VRQ scrapie-infected sheep was compared with that of BSE-infected sheep with a similar genotype. A triplex Western blotting technique was used to estimate the two allotype PrP fractions in PrP(res) material from BSE-infected ARR/VRQ sheep. PrP(res) in BSE contained equimolar amounts of VRQ- and ARR-PrP, which contrasts with the excess (>95%) VRQ-PrP fraction found in PrP in scrapie. This is evidence that transmissible spongiform encephalopathy (TSE) agent properties alone, perhaps structural aspects of prions (such as PrP amino acid sequence variants and PrP conformational state), determine the polymorphic dependence of the PrP(res) accumulation process in prion formation as well as the disease-associated phenotypic expressions in the host. IMPORTANCE: Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative and transmissible diseases caused by prions. Amino acid sequence variants of the prion protein (PrP) determine transmissibility in the hosts, as has been shown for classical scrapie in sheep. Each individual produces a separate PrP molecule from its two PrP gene copies. Heterozygous scrapie-infected sheep that produce two PrP variants associated with opposite scrapie susceptibilities (136V-PrP variant, high; 171R-PrP variant, very low) contain in their prion material over 95% of the 136V PrP variant. However, when these sheep are infected with prions from cattle (bovine spongiform encephalopathy [BSE]), both PrP variants occur in equal ratios. This shows that the infecting prion type determines the accumulating PrP variant ratio in the heterozygous host. While the host's PrP is considered a determining factor, these results emphasize that prion structure plays a role during host infection and that PrP variant involvement in prions of heterozygous carriers is a critical field for understanding prion formation.

Quantitative trait loci for resistance to infection in sheep using a live Salmonella Abortusovis vaccine.

Quantitative trait loci (QTL) mapping for susceptibility to a Salmonella Abortusovis vaccinal strain was performed using an experimental design involving 30 Romane sheep sire families (1216 progenies). Nine QTL corresponding to bacterial load, weight variations and antibody response criteria were mapped on eight chromosomes, including the major histocompatibility complex area on chromosome 20. Surprisingly, none was found to be significant in the SLC11A1 region (formerly NRAMP1) that has been shown to influence Salmonella susceptibility in other species.

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein.

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.

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.

Scavenger, transducer, RNA chaperone? What ligands of the prion protein teach us about its function.

Prion protein, a misfolded isoform of which is the essential component of the agent of prion diseases, still remains an enigmatic protein whose physiological functions are at best hypothetical. To gain a better insight into its putative role, many studies were undertaken to look for molecules that bind prion protein, and have notably identified divalent metal ions, several proteins, and nucleic acids. At first sight, the diversity of prion protein's ligands seems of little help to infer a plausible function. However, the intrinsically disordered property of its N-terminal tail and the potential of the protein to adopt a transmembrane topology, can both be taken into account to predict its different states during its cellular cycle and its possible functions, of which the most promising correspond to a general scavenger, a sensor or adaptor in a signaling cascade, and an RNA chaperone.

Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns.

One of the unsolved problems in prion diseases relates to the physiological function of cellular prion protein (PrP), of which a misfolded isoform is the major component of the transmissible spongiform encephalopathies agent. Knowledge of the PrP-binding molecules may help in elucidating its role and understanding the pathological events underlying prion diseases. Because nucleic acids are known to bind PrP, we attempted to identify the preferred RNA sequences that bind to the ovine recombinant PrP. An in vitro selection approach (SELEX) was applied to a pool of 80-nucleotide(nt)-long RNAs containing a randomised 40-nt central region. The most frequently isolated aptamer, RM312, was also the best ligand (20 nM KD value), according to both surface plasmon resonance and filter binding assays. The fast rates of association and dissociation of RM312 with immobilized PrP, which are reminiscent of biologically relevant interactions, could point to a physiological function of PrP towards cellular nucleic acids. The minimal sequence that we found necessary for binding of RM312 to PrP presents a striking similarity with one previously described PrP aptamer of comparable affinity. In addition, we here identify the two lysine clusters contained in the N-terminal part of PrP as its main nucleic-acid binding sites.

Effect of two candidate genes on the Salmonella carrier state in fowl.

Selection for increased resistance to Salmonella carrier-state (defined as the persistency of the bacteria 4 wk after inoculation) could reduce the risk for the consumer of food toxi-infections. The effects of two genomic regions on chromosomes 7 and 17 harboring two genes, NRAMP1 (SLC11A1) and TLR4, known to be involved in the level of chicken infection 3 d after inoculation by Salmonella were thus tested on a total of 331 hens orally inoculated at the peak of lay with 10(9) bacteria. The animals and their parents were genotyped for a total of 10 microsatellite markers mapped on chromosomes 7 and 17. Using maximum likelihood analysis and interval mapping, it was found that the SLC11A1 region was significantly involved in the control of the probability of spleen contamination 4 wk after inoculation. Single nucleotide polymorphisms (SNP) within the SLC11A1 and TLR4 gene were tested on those animals as well as on a second batch of 279 hens whose resistance was assessed in the same conditions. As the former was significantly associated with the risk of spleen contamination and the number of contaminated organs, SLC11A1 appears to be involved in the control of resistance to Salmonella carrier state. The involvement of the TLR4 gene was also highly suspected as a significant association between SNP within the gene, and the number of contaminated organs was detected.

Establishment and characterisation of ovine blood monocyte-derived cell lines.

Studies of the important functions in host defense assured by macrophages, both as functional elements and as potential targets for intracellular pathogens, are often inhibited by the lack of a source of large numbers of uniform, well-characterised cells. To address this lack for ovine studies, we have established cell lines from spontaneously-proliferating adherent mononuclear cells from sheep blood. Eight such lines which have been continuously cultured for over 400 passages have phagocytic activities and cytochemical characteristics indicating that they retain the nature of mononuclear phagocytes. They display typical functional membrane proteins such as CD14, Fc receptors and MHC class II. Such cells can facilitate in vitro studies of pathogen-monocyte interactions and can furnish copious amounts of cells for transfer experiments.

Cytokine gene expression in lymph node and spleen of sheep in response to Salmonella infection by two serotypes displaying different host specificity.

In order to investigate the determinism of the host specificity and to better understand the host resistance mechanisms, infections of sheep were performed with either S. abortusovis, serotype specific for ovine species, or with S. dublin, serotype adapted to cattle and accidentally transmissible to human. Following a subcutaneous challenge, S. dublin disseminated more rapidly towards lymphoid tissues than S. abortusovis. However, S. abortusovis tended to persist in spleen more efficiently than S. dublin. Using a quantitative RT-PCR method, the expression level of ovine cytokines genes was measured in the draining lymph node and in the spleen, in the course of infection. Inflammatory cytokine response was characterised by an early and strong increase of IL-1beta and TNFalpha mRNA in both lymphoid organs following S. dublin infection, while S. abortusovis challenge only induced IL-1beta mRNA increase in the spleen at day 3 post-inoculation. Likewise, S. dublin infection provoked a marked increase of IL-12 mRNA and a slight up-regulation of IFNgamma gene transcription in the local lymphoid site, in contrast to S. abortusovis infection. Elsewhere, both serotypes induced a strong and early IL-10 mRNA production and had no effect on IL-4 gene expression. Finally, taken together, these data suggest that the intensity of inflammatory and anti-infectious cytokine responses, but not the type 2 cytokine response, is serotype-dependent. They also suggest that the host-specific serotype, by limiting the host cytokine-mediated defence, could favour its persistence within lymphoid organs.

Effect of the mouse Nramp1 genotype on the expression of IFN-gamma gene in early response to Salmonella infection.

The early interferon-gamma (IFN-gamma) response after Salmonella infection was compared in resistant and susceptible congenic mice strains differing by the allele of Nramp1. IFN-gamma gene expression in vivo was studied by quantitative reverse transcription-PCR performed from the spleen of mice challenged by Salmonella either in multiplication or in resting phase. The spleen colonisation by Salmonella in vivo was investigated and was shown to be identical in a genetically defined host, whatever the initial phase of the bacteria. However, the bacterial load of the spleen was significantly reduced in Nramp1(r) (resistant) mice compared to Nramp1(s) (susceptible) mice. The background level of IFN-gamma mRNA was higher in the spleen of resistant mice than in the susceptible mice, before infection. Interestingly, the early upregulation of IFN-gamma gene transcription, which was observed after infection with Salmonella, was reproducibly delayed in susceptible mice compared to resistant mice. Finally, the kinetics of the host IFN-gamma response seems to be Nramp1 dependent. Resistant mice present the advantage of being more prompt to express this anti-infectious cytokine gene than susceptible mice in response to Salmonella infection.

Quantification of ovine cytokine gene expression by a competitive RT-PCR method.

A quantitative competitive RT-PCR method was developed in order to measure IL-1beta, IL-4, IL-12, IFNgamma, TNFalpha and G(3)PDH mRNA from samples of ovine tissue such as lymph node or spleen. The main advantage of the method relies on the use, for each target sequence, of an internal competitor construct similar to the relevant target, but 4-bp different in size. This competitive strategy is validated by the equivalence of the amplification process, observed separately between competitor DNA and target DNA species. Furthermore, the copy number of each cytokine cDNA is normalized to a fixed copy number of G(3)PDH cDNA. The cDNA level of this constitutive gene was effectively shown to remain constant whatever the tissue studied and independently of the experimental conditions used. The accurate and reproducible data obtained permit the application of this quantitative RT-PCR method to measure the sheep cytokine response to Salmonella infection. Early induction of IFNgamma mRNA was observed in the draining lymph node 1 day after infection. At the same time, a strong increase of IL-1beta mRNA was observed in local and systemic lymphoid organs, suggesting the initiation of the inflammatory response. Finally, the overall results demonstrate the efficiency of the method and its suitability for further studies of the immune response in the ovine species.

Statistical power of QTL mapping methods applied to bacteria counts.

Most QTL mapping methods assume that phenotypes follow a normal distribution, but many phenotypes of interest are not normally distributed, e.g. bacteria counts (or colony-forming units, CFU). Such data are extremely skewed to the right and can present a high amount of zero values, which are ties from a statistical point of view. Our objective is therefore to assess the efficiency of four QTL mapping methods applied to bacteria counts: (1) least-squares (LS) analysis, (2) maximum-likelihood (ML) analysis, (3) non-parametric (NP) mapping and (4) nested ANOVA (AN). A transformation based on quantiles is used to mimic observed distributions of bacteria counts. Single positions (1 marker, 1 QTL) as well as chromosome scans (11 markers, 1 QTL) are simulated. When compared with the analysis of a normally distributed phenotype, the analysis of raw bacteria counts leads to a strong decrease in power for parametric methods, but no decrease is observed for NP. However, when a mathematical transformation (MT) is applied to bacteria counts prior to analysis, parametric methods have the same power as NP. Furthermore, parametric methods, when coupled with MT, outperform NP when bacteria counts have a very high proportion of zeros (70.8%). Our results show that the loss of power is mainly explained by the asymmetry of the phenotypic distribution, for parametric methods, and by the existence of ties, for the non-parametric method. Therefore, mapping of QTL for bacterial diseases, as well as for other diseases assessed by a counting process, should focus on the occurrence of ties in phenotypes before choosing the appropriate QTL mapping method.

Cytogenetical anchoring of sheep linkage map and syntenic groups using a sheep BAC library.

In order to simultaneously integrate linkage and syntenic groups to the ovine chromosomal map, a sheep bacterial artificial chromosome (BAC) library was screened with previously assigned microsatellites using a sheep-hamster hybrid panel and genetic linkage. Thirty-three BACs were obtained, fluorescently labelled and hybridised on sheep-goat hybrid metaphases (2n = 57). This study allowed us, (i), to anchor all linkage groups on sheep chromosomes, (ii), to give information on the probable position of the centromere on the linkage map for the centromeric chromosomes, (iii), to contradict the previous orientation of the ovine X linkage group by the mapping of BMS1008 on OARXq38. Concerning our somatic cell hybrid panel, this study resulted in the assignment of all the previously unassigned groups to ovine chromosomes and a complete characterisation of the hybrid panel. In addition, since hybridisations were performed on a sheep-goat hybrid, new marker/anchoring points were added to the caprine cytogenetic map.

Regional characterization of a hamster-sheep somatic cell hybrid panel.

The regional characterization of a previously obtained hamster-sheep hybrid panel is reported. Using data available from ruminant maps (sheep, cattle, and goat), we have selected a set of 300 markers and have analyzed them by PCR in this hybrid panel. Results obtained for 204 markers show the presence of all sheep chromosomes (including gonosomes) in entire or fragmented form. Analysis of syntenies has given 130 types of answer defining segments of variable sizes. This study has led to the regional characterization of this panel and provides comparative data on a set of bovine and caprine markers. With the level of characterization now achieved for this hybrid panel, the regional assignment of new genes or markers to sheep chromosomes can be rapidly obtained. Finally, this panel will help to collect new data for comparative mapping of domestic animals and to highlight the conservation of syntenic groups between closely related species, that is, sheep, cattle, and goat.

Host cytokine response and resistance to Salmonella infection.

Knowledge of the host response, of the resistance process, and of the mediators committed against Salmonella infection is essential to progress towards better means of prophylaxis and eradication. In this context, the present contribution attempts to interconnect, with the pivotal role of the macrophage, the early resistance process under the control of the Nramp1 gene and the cytokine response for resolving infection. IL-12 produced by macrophages is an inducer of IFN-gamma production, which in turn activates the macrophage antibacterial activity and synergizes its effects with TNF-alpha. All three of these cytokines are powerful actors in the first line of anti-Salmonella defence. It can be pointed out that susceptible and resistant individuals do not seem to see the cytokine environment the same way, the former being unresponsive to IL-1 or GM-CSF treatment and deficient in IFN-gamma production. These discrepancies may rely on cell signalling events that could be defective in macrophages of the susceptible phenotype.

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.

Variability in the resistance of four chicken lines to experimental intravenous infection with Salmonella enteritidis phage type 4.

The capacity of four chicken lines (Y11, L2, B13, PA12) to control Salmonella enteritidis (SE) phage type 4 (PT4) systemic colonization was investigated. Thirteen-week-old chickens were intravenously inoculated with 10(6) SE colony-forming units, and the levels of SE colonization were determined at various time intervals after inoculation in liver, spleen, genital organs, and ceca. The course of SE infection showed a rapid contamination of liver, spleen, and genital organs, whereas the ceca were infected later. A significant (P < 0.001) effect of the chicken line on levels of SE was detected on day 3 postinoculation (PI) in liver and ceca, on day 10 PI in ceca, and on day 15 PI in spleen. Because an early control of systemic Salmonella infection by the Ity/Nramp1 gene has been demonstrated in mice, we aimed to study the early resistance of chickens to SE. As a consequence, we then focused our study on the between- and within-line variabilities of SE levels on day 3 PI. According to the SE levels in liver on day 3 PI, the chicken lines could be classified as susceptible (Y11 and L2) or resistant (PA12 and B13). This early variability was explored in resistant B13 and susceptible L2 lines. Differences between these two lines were confirmed in liver but not in ceca. A large within-line variability was observed in all organs of these two lines. The genetic origin of this variability will have to be determined as a prerequisite to an eventual selection.

Mouse susceptibility to infection by the Salmonella abortusovis vaccine strain Rv6 is controlled by the Ity/Nramp 1 gene and influences the antibody but not the complement responses.

Early growth of Salmonella typhimurium in spleen and liver of mice is controlled by the mouse chromosome 1 locus Ity/Nramp 1. Genetic control of resistance to the attenuated vaccine strain Rv6 of Salmonella abortusovis was studied in mice infected by the intravenous route. Comparison of kinetics of bacterial colonization of spleen and liver in two congenic BALB/c-susceptible (Itys) and -resistant (Ityr) mouse lines showed that BALB/c mice (Itys) were significantly more susceptible to infection than C.D2 mice (Ityr) suggesting that infection by this vaccine strain is controlled by a gene which is close or identical to Ity/Nramp 1. Congenic mice also differed in their anti-Salmonella antibody response, measured by ELISA: susceptible mice had a significantly higher antibody level than resistant mice, whatever the immunoglobulin isotype (IgM, IgG1, IgG2a, IgG3, IgA, and total immunoglobulins). The two congenic BALB/c mouse lines had equal serum C3c levels in response to infection. However, we observed a highly significant difference according to the sex of mice, suggesting a role of sex hormones in the regulation of the level of some complement factors. These results, obtained with congenic mice, strongly suggest that the Ity/Nramp 1 locus controls susceptibility to infection by the S. abortusovis vaccine strain Rv6 and influences the antibody response.