Two ovine mitochondrial DNAs harboring a fifth 75/76 bp repeat motif without altered gene expression in Northern Spain.

The Basque Country is home to the Latxa sheep breed, which is divided in several varieties such as Latxa Black Face (LBKF) and Latxa Blonde Face (LBLF). Mitochondrial DNA control region analysis of 174 male sheep (97 LBKF and 77 LBLF) was performed with the objective of characterizing the maternal lineages of these two varieties that are the basis to produce the cheese with Idiazabal quality label. The percentage of unique haplotypes was 77.32% in LBKF and 67.53% in LBLF. Most of the individuals were classified into B haplogroup (98.85%), while A haplogroup was much less frequent. Two Latxa individuals (one LBKF and one LBLF), both belonging to B haplogroup, displayed an additional 75/76 bp tandem repeat motif. Only 33 other sequences with this repeat motif were found among 11 061 sheep sequences included in the GenBank database. Gene expression was analyzed in peripheral blood leukocytes since the additional 75/76 bp repeat motif falls within ETAS1, a domain with a possible function in regulation of replication and transcription. The mRNA expression from four mitochondrial genes (COI, cyt b, ND1, and ND2) was analyzed in the two individuals of this study with a fifth repeat motif and in four without it. Although lower transcription was observed when the additional 75/76 bp repeat motif was present, no statistically significant differences were observed. Therefore, the variation in the number of the 75/76 repeat motif does not seem to modify the gene expression rate in mitochondrial genes.

Paratuberculosis in Latin America: a systematic review.

Latin America is the definition of the American group, where languages of Latin origin are spoken, including countries in South, Central, and North America. Paratuberculosis is a gastrointestinal contagious chronic disease that affects ruminants, whose etiological agent is the bacilli Mycobacterium avium subsp. paratuberculosis (MAP). Paratuberculosis is characterized by intermittent diarrhea, decreased milk production, dehydration, and progressive weight loss and is possibly involved in Crohn's disease, a human intestinal disease. MAP is resistant to environmental factors, pasteurization, and water disinfection, which coupled with the subclinical-clinical nature of the disease, and makes paratuberculosis a relevant socioeconomic and public health issue, justifying the descriptive review of research on the disease carried out in Latin American countries. A survey of articles, published until September 2016, on the Scopus database, PubMed, Agris, and Science Direct, about detection of the agent and the disease in Latin America, without restrictions to the date of the research was performed. The keywords were as follows: "paratuberculosis," "Mycobacterium avium subsp. paratuberculosis," "cattle," "milk," "wildlife," "goat," "ovine," "dairy," and the name of each country in English. Studies found from nine of the 20 Latin America countries, 31 related to Brazil, 17 to Argentina, 14 to Chile, eight to Colombia, six to Mexico, two to Peru, two to Venezuela, and one to Panama and to Bolivia, each. The agent was detected in cattle, goats, sheep, domesticated water buffalo, and wild animals. Microbiological culture, PCR, and ELISA were the frequent techniques. The small number of studies may result in overestimation or underestimation of the real scenario.

SNPs in candidate genes MX dynamin-like GTPase and chemokine (C-C motif) receptor-5 are associated with ovine pulmonary adenocarcinoma progression in Latxa sheep.

Ovine pulmonary adenocarcinoma (OPA) is a contagious lung cancer in sheep caused by Jaagsiekte sheep retrovirus (JSRV). OPA is present in many sheep-rearing countries causing economic and welfare issues, as currently no efficient vaccines or treatments are available. Breed differences suggest a host genetic component may influence the pathogenesis of OPA, but so far few genes have been identified. In this work, a genetic association study was carried out in Latxa dairy sheep which were classified as cases/controls based on the presence/absence of OPA lung tumours. Candidate genes included cytokines and a receptor and innate immunity genes. After SNPs in the candidate genes were identified, the distribution of alleles in cases and controls was compared by means of logistic regression analyses at the allelic, genotypic and haplotypic levels. The association analysis showed that several candidate genes were significantly associated with resistance or susceptibility to OPA; two of the candidates, CCR5 and MX1, remained significantly associated with resistance and susceptibility respectively, even after Bonferroni correction.

Pathological findings in young and adult sheep following experimental infection with 2 different doses of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subsp paratuberculosis (Map) is assumed to infect young ruminants; however, little is known concerning the possibility of adult animals becoming infected. An experimental infection was conducted to establish the effect of age and doses of Map on susceptibility to paratuberculosis in sheep. Sixteen of twenty-four 1.5-month-old Churra lambs and 23 of 30 adult ewes (from 2-11 years old) were orally challenged with an ovine field strain of Map. Thirteen ewes and 8 lambs were infected with a high dose (HD) and 10 adult sheep and 8 lambs with a low dose (LD) of Map. The remaining animals were unchallenged controls. Animals were euthanized at 110 to 120 and 210 to 220 days postinfection. Histological, bacteriological, and nested polymerase chain reaction (PCR) studies were conducted in samples of intestine and related lymphoid tissue (Peyer patches, lymph nodes). Animals were classified according to their lesions. The number of granulomas was counted in 3 tissue sections from each sample. Only the HD groups showed lesions associated with paratuberculosis (92.3% of ewes and 100% of lambs). Adults had lesions characterized by few small demarcated focal granulomas restricted to the lymphoid tissue, whereas granulomas were more numerous and larger, appearing in the lamina propria unrelated to lymphoid tissue, in the lambs. Only HD-infected lambs were positive to culture, whereas nested PCR also detected positive HD ewes and some LD animals. These results suggest that adult sheep can become infected by Map, as seen by the development of lesions, but they are focal and restricted to the lymphoid tissue.

Alginate-waterborne polyurethane 3D bioprinted scaffolds for articular cartilage tissue engineering.

Articular cartilage defects comprise a spectrum of diseases associated with degeneration or damage of the connective tissue present in particular joints, presenting progressive osteoarthritis if left untreated. In vitro tissue regeneration is an innovative treatment for articular cartilage injuries that is attracting not only clinical attention, but also great interest in the development of novel biomaterials, since this procedure involves the formation of a neotissue with the help of material support. In this work, functional alginate and waterborne polyurethane (WBPU) scaffolds have been developed for articular cartilage regeneration using 3D bioprinting technology. The particular properties of alginate-WBPU blends, like mechanical strength, elasticity and moistening, mimic the original cartilage tissue characteristics, being ideal for this application. To fabricate the scaffolds, mature chondrocytes were loaded into different alginate-WBPU inks with rheological properties suitable for 3D bioprinting. Bioinks with high alginate content showed better 3D printing performance, as well as structural integrity and cell viability, being most suitable for scaffolds fabrication. After 28 days of in vitro cartilage formation experiments, scaffolds containing 3.2 and 6.4 % alginate resulted in the maintenance of cell number in the range of 104 chondrocytes/scaffold in differentiated phenotypes, capable of synthesizing specialized extracellular matrix (ECM) up to 6 µg of glycosaminoglycans (GAG) and thus, showing a potential application of these scaffolds for in vitro regeneration of articular cartilage tissue.

Presence of Bartonella species in wild carnivores of northern Spain.

The genus Bartonella was detected by PCR in 5.7% (12/212) of wild carnivores from Northern Spain. Based on hybridization and sequence analyses, Bartonella henselae was identified in a wildcat (Felis silvestris), Bartonella rochalimae in a red fox (Vulpes vulpes) and in a wolf (Canis lupus), and Bartonella sp. in badgers (Meles meles).

Immunization of adult dairy cattle with a new heat-killed vaccine is associated with longer productive life prior to cows being sent to slaughter with suspected paratuberculosis.

The objective of this study was to evaluate the efficacy of a new heat-killed Mycobacterium avium ssp. paratuberculosis (MAP) vaccine for control of premature culling in tuberculosis-free dairy cattle. Feces and gastrointestinal tissues were collected from 50 vaccinated cows and 38 nonvaccinated cows at slaughter and analyzed by bacteriological culture and histopathology. Vaccination was associated with a significant reduction of the frequency of vaccinated animals with MAP in feces and gut tissues compared with the nonvaccinated animals. In addition, the frequency of vaccinated animals with heavy bacterial load in gut tissues was 40% lower than the frequency of the nonvaccinated animals with the same MAP load. The peak age of paratuberculosis-associated culling was from 4.5 to 5 yr old (21%) in the vaccinated animals and from 3 to 4.5 yr old (60%) in the nonvaccinated animals. The vaccinated and nonvaccinated animals with suspected paratuberculosis were culled at an average age of 4.7 and 3.7 yr old, respectively. Therefore, a significant increase in the productive life of the vaccinated animals sent to slaughter with suspected paratuberculosis was observed. In addition, our analysis revealed a positive effect of the vaccine on the carcass weights of the animals with severe histopathological lesions at slaughter compared with the nonvaccinated animals. In summary, our findings suggest a therapeutic effect of the vaccine and a significant attenuation of pre-existing infection in cows naturally infected with paratuberculosis that were adults at the time of vaccination.

Fate of Mycobacterium avium subsp. paratuberculosis after application of contaminated dairy cattle manure to agricultural soils.

Details regarding the fate of Mycobacterium avium subsp. paratuberculosis (basonym, Mycobacterium paratuberculosis) after manure application on grassland are unknown. To evaluate this, intact soil columns were collected in plastic pipes (lysimeters) and placed under controlled conditions to test the effect of a loamy or sandy soil composition and the amount of rainfall on the fate of M. paratuberculosis applied to the soil surface with manure slurry. The experiment was organized as a randomized design with two factors and three replicates. M. paratuberculosis-contaminated manure was spread on the top of the 90-cm soil columns. After weekly simulated rainfall applications, water drainage samples (leachates) were collected from the base of each lysimeter and cultured for M. paratuberculosis using Bactec MGIT ParaTB medium and supplements. Grass was harvested, quantified, and tested from each lysimeter soil surface. The identity of all probable M. paratuberculosis isolates was confirmed by PCR for IS900 and F57 genetic elements. There was a lag time of 2 months after each treatment before M. paratuberculosis was found in leachates. The greatest proportions of M. paratuberculosis-positive leachates were from sandy-soil lysimeters in the manure-treated group receiving the equivalent of 1,000 mm annual rainfall. Under the higher rainfall regimen (2,000 mm/year), M. paratuberculosis was detected more often from lysimeters with loamy soil than sandy soil. Among all lysimeters, M. paratuberculosis was detected more often in grass clippings than in lysimeter leachates. At the end of the trial, lysimeters were disassembled and soil cultured at different depths, and we found that M. paratuberculosis was recovered only from the uppermost levels of the soil columns in the treated group. Factors associated with M. paratuberculosis presence in leachates were soil type and soil pH (P < 0.05). For M. paratuberculosis presence in grass clippings, only manure application showed a significant association (P < 0.05). From these findings we conclude that this pathogen tends to move slowly through soils (faster through sandy soil) and tends to remain on grass and in the upper layers of pasture soil, representing a clear infection hazard for grazing livestock and a potential for the contamination of runoff after heavy rains.

Measuring antibody levels in bulk-tank milk as an epidemiological tool to search for the status of Coxiella burnetii in dairy sheep.

Pooling samples may provide a valuable alternative to individual testing for pathogen surveillance purposes. We studied the reliability of measuring the level of antibodies against Coxiella burnetii in bulk-tank milk (BTM) to estimate the seroprevalence of C. burnetii in dairy sheep in 34 flocks. We then estimated the seroprevalence of C. burnetii in 154 dairy sheep flocks according to the level of antibodies in BTM. We tested for the accuracy of our estimation at the population level by comparing predicted mean C. burnetii flock seroprevalence with that obtained in another survey performed on the same population. Our findings showed that testing BTM by ELISA is a cost-effective and relatively good index of the seroprevalence of C. burnetii in dairy sheep and may be a useful tool for epidemiological surveillance at the population level.

Differences in questing tick species distribution between Atlantic and continental climate regions in Spain.

Climate and vegetation in Spain vary from north to south, affecting tick distribution and consequently the presence of tick-borne diseases. The aim of this study was to investigate throughout a 2-yr study the distribution of the different exophilic questing tick species present in 18 areas: eight located in central and 10 in northern Spain. The same methodology was used in both areas, sampling vegetation on a monthly basis by blanket dragging for 20- to 30-min intervals. A total of 12 species belonging to the genera Ixodes, Haemaphysalis, Rhipicephalus, Dermacentor, and Hyalomma was identified. Differences in species distribution and prevalence were dramatically different. The most frequent and abundant species in northern Spain were Ixodes ricinus (67% of adult ticks) and Haemaphysalis punctata (8%), whereas Hyalomma lusitanicum (86%) and Dermacentor marginatus (12%) were the most abundant in central Spain. There were important differences in the monthly seasonal patterns for the different tick species. These results highlight important differences in tick distribution in neighboring areas and underline the need for ongoing surveillance programs to monitor tick population dynamics and the prevalence of tick-borne pathogens.

Genetic variation of toll-like receptor genes and infection by Mycobacterium avium ssp. paratuberculosis in Holstein-Friesian cattle.

Toll-like receptors (TLR) are membrane proteins that play a key role in innate immunity, by recognizing pathogens and subsequently activating appropriate responses. Mutations in TLR genes are associated with susceptibility to inflammatory and infectious diseases in humans. In cattle, 3 members of the TLR family, TLR1, TLR2, and TLR4, are associated with Mycobacterium avium ssp. paratuberculosis infection, although the extent of this association for the TLR1 and TLR4 receptors has not yet been determined. Moreover, the causal variant in the TLR2 gene has not yet been unequivocally established. In this study, 24 single nucleotide polymorphisms (SNP) in the bovine TLR1, TLR2, and TLR4 genes were selected from the literature, databases, and in silico searches, for a population-based genetic association study of a Spanish Holstein-Friesian sample. Whereas previous results regarding the TLR1 gene were not corroborated, a risk haplotype was detected in TLR2; however, its low frequency indicates that this detected association should be interpreted with caution. In the case of the TLR4 gene, 3 tightly linked SNP were found to be associated with susceptibility to M. avium ssp. paratuberculosis infection. Moreover, one of these SNP, the SNP c.-226G>C, which is localized in the 5'UTR region of the TLR4 gene, has been reported to be able to alter TLR4 expression, raising the possibility that this mutation may contribute to the response of the individual to infection.

Paratuberculosis vaccination specific and non-specific effects on cattle lifespan.

Records of cattle vaccination against paratuberculosis (PTB) have been analyzed to determine whether or not non-specific effect (NSE) on overall mortality similar to that observed in BCG vaccinated humans occurs in animals. The results of a previously reported slaughterhouse study on PTB prevalence were used as a reference on the age incidence of advanced patent (clinical) epidemio-pathogenic forms. In the proper vaccine study, cows in 30 cattle farms in the Basque Country, Spain were followed-up for between 1 and 13 years. Vaccinated groups were composed by 1008 (592 right-censored) animals younger than 3 months treated as calves and by 3761 (3160 right-censored) vaccinated at any older age. Controls were 339 (157 right-censored) and 4592 (2213 right-censored) age matched animals, respectively. Individual last year presence in the annual testing was considered age at culling or death. A survival analysis was carried out according age at vaccination of vaccinated versus non-vaccinated animals. PTB age incidence in the slaughterhouse study was subtracted from the difference between vaccinated and non-vaccinated animals at the same age in order to estimate PTB-specific and non-specific effects. The maximum difference was observed at the 2-3 years interval with a 33.9% mortality reduction in the calf vaccinated group. This corresponded also with the maximum NSE that was 24.5% for a PTB incidence of 9.5%. Overall, vaccination afforded to calves a 26.5% yearly mortality protection, split between 11.1% PTB-specific and 15.4% NSE. These results support a NSE on total mortality associated with PTB vaccination that appeared to persist for up to 6-7 years. This confirms for the first time in an animal field study the innate immune system memory predicted by the recently proposed trained immunity theory. Contrasting the literature, no deleterious effects of killed vaccines on females were observed. Mortality reduction would offset vaccination costs and could improve livestock systems efficiency and potentially reduce antibiotic use. Clinical trial registered with Spanish Agency for Drugs and Sanitary products (AEMPS) as 11/012/ECV.

Design of drug-loaded 3D printing biomaterial inks and tailor-made pharmaceutical forms for controlled release.

Curcumin and chloramphenicol are drugs with different solubility properties in physiological conditions due to their hydrophobic and hydrophilic structure, respectively. In this work, sodium alginate-cellulose nanofibers (SA-CNF) based inks loaded with curcumin and/or chloramphenicol have been developed for syringe extrusion 3D printing technology. Printability and shape fidelity of the drug-loaded inks were analyzed through rheological characterization. Suitable drug-loaded inks were 3D printed showing shape fidelity, and samples were either freeze-dried or crosslinked with Ca2+ and air-dried to achieve functional pharmaceutical forms with different morphological characteristics. In vitro drug delivery tests were carried out from the resulted forms and it was observed that the release performed faster in freeze-dried than in Ca2+ crosslinked/air-dried ones for all cases, resulting in two different methods for controlling drug delivery over time. The differences in aqueous solubility of the drugs, the different CNF content of the inks and the surface area of the samples also played an important role during drug delivery, involving strategies to control the release over an extended duration.

3D printed alginate-cellulose nanofibers based patches for local curcumin administration.

Alginate and nanocellulose are potential biomaterials to be employed as bioinks for three-dimensional (3D) printing. Alginate-cellulose nanofibers (A-CNF) formulations with CNF amounts up to 5 wt% were developed and rheologically characterized to evaluate their printability. Results showed that formulations with less than 3 wt% CNF did not present suitable characteristics to ensure shape fidelity after printing. Selected A-CNF bioinks were 3D printed and freeze-dried to obtain porous scaffolds. Morphological and mechanical analysis were performed, showing that CNF contributed to the reinforcement of the scaffolds and modulated their porosity. The applicability for drug delivery was evaluated by the addition of curcumin to printable A-CNF formulations. The curcumin loaded bioinks were successfully 3D printed in patches and the in vitro release tests showed that alginate and CNF played an important role in curcumin stabilization, whereas the CNF content and the disintegration of the scaffold were essential in the release kinetics.

Genetic association between bovine NOD2 polymorphisms and infection by Mycobacterium avium subsp. paratuberculosis in Holstein-Friesian cattle.

Nucleotide-Binding Oligomerization Domain 2 (NOD2) has been reported to be a candidate gene for Mycobacterium avium subsp. paratuberculosis (MAP) infection in a Bos taurus × Bos indicus mixed breed based on a genetic association with the c.2197T>C single nucleotide polymorphism (SNP). Nevertheless, this SNP has also been reported to be monomorphic in the B. taurus species. In the present work, 18 SNPs spanning the bovine NOD2 gene have been analysed in a genetic association study of two independent populations of Holstein-Friesian cattle. We found that the C allele of SNP c.*1908C>T, located in the 3'-UTR region of the gene, is significantly more frequent in infected animals than in healthy ones, which supports the idea that the bovine NOD2 gene plays a role in susceptibility to MAP infection. However, in silico analyses of the NOD2 nucleotide sequence did not yield definitive data about a possible direct effect of SNP c.*1908C>T on susceptibility to infection and led us to consider its linkage disequilibrium with the causative variant. A more exhaustive genetic association study including all putative, functional SNPs from this gene and subsequent functional analyses needs to be conducted to achieve a more complete understanding of how different variants of NOD2 may affect susceptibility to MAP infection in cattle.

Border disease virus seroprevalence correlates to antibodies in bulk-tank milk and reproductive performance of dairy sheep flocks.

There is a great need to establish effective tools to control border disease virus (BDV) in European dairy sheep flocks. Hence, our main aim was to investigate the accuracy of analyzing anti-BDV antibodies in bulk-tank milk (BTM) in detecting the real BDV seroprevalence in dairy sheep flocks. Furthermore, the relevance of BDV to reproductive performance of dairy sheep flocks prompted us to search for the association between BDV seroprevalence and reproductive parameters. For these purposes, 34 flocks were selected based on different percentages of antibody inhibition (AIP) values in BTM as estimated by ELISA. Serum samples from 10 replacement lambs older than 6 mo, 10 ewes 1 to 2 yr old, and 10 ewes > 2 yr old were collected and analyzed for the presence of anti-BDV antibodies by ELISA. A negative relationship between BDV AIP in BTM and within-flock seroprevalence was observed. Flocks with a high AIP (> 80%) had an average of 2.5% seropositive animals; flocks with a moderate AIP (46-79%) had 11.4% seropositive animals; and finally, flocks with an AIP < or = 45% showed a high flock seroprevalence (57.2%). Ten out of 34 flocks showed a high BDV seroprevalence in lambs, suggesting the presence of persistently infected animals in the flock. The observed AIP values in BTM from these likely BDV-infected flocks were indicative of a high seroprevalence. The analysis of reproductive-parameters data collected from these flocks showed no differences in fertility or prolificacy in relation to BDV circulation rates. Nonetheless, lamb mortality was significantly greater in flocks with low-moderate seroprevalence (10-30%), probably as a result of a first-time contact with BDV of previously naïve ewes. These findings suggest that testing of BTM samples may be useful in inferring the BDV seroprevalence in a flock.

SP110 as a novel susceptibility gene for Mycobacterium avium subspecies paratuberculosis infection in cattle.

The intracellular pathogen resistance 1 (Ipr1) gene has been reported to play a role in mediating innate immunity in a mouse model of Mycobacterium tuberculosis infection, and polymorphisms of its human ortholog, SP110 nuclear body protein, have been suggested to be associated with tuberculosis. Thus, the bovine SP110 gene was considered to be a promising candidate for a genetic association study of bovine paratuberculosis, or Johne's disease, a chronic granulomatous enteritis caused by Mycobacterium avium ssp. paratuberculosis (MAP). Initially, single nucleotide polymorphisms (SNP) within the bovine SP110 gene were identified, and subsequently a population-based genetic association study was carried out. Seventeen new SNP along the SP110 gene were identified in Holstein-Friesian cattle, and 6 more were compiled from public databases. A total of 14 SNP were included in the association study of 2 independent populations. The SNP c.587A>G was found to be significantly associated with MAP infection, with the major allele A appearing to confer greater disease susceptibility in one of the analyzed populations. In addition, 2 haplotypes containing this SNP were also found to be associated with infection in the same population. The SNP c.587A>G is a nonsynonymous mutation that causes an amino acid change in codon 196 from asparagine to serine. In silico analyses point to SNP c.587A>G as a putative causal variant for susceptibility to MAP infection. The elucidation of the precise mechanism by which this SNP can exert its effect in the protein and, as a result, in the risk of infection, requires future functional analyses. Likewise, the absence of genetic association in one of the analyzed populations renders it necessary to carry out this study in other independent populations, with the aim of substantiating the repeatability of the present results. Nevertheless, the present results deepen our understanding of the genetic basis of susceptibility and resistance mechanisms related to MAP infection in cattle and, in turn, constitute a step forward toward the implementation of marker-assisted selection in breeding programs aimed at controlling paratuberculosis.

Identification of single nucleotide polymorphisms in the bovine solute carrier family 11 member 1 (SLC11A1) gene and their association with infection by Mycobacterium avium subspecies paratuberculosis.

Johne's disease is a chronic enteritis caused by Mycobacterium avium ssp. paratuberculosis (MAP) that causes substantial financial losses for the cattle industry. Susceptibility to MAP infection is reported to be determined in part by genetic factors, so marker-assisted selection could help to obtain bovine populations that are increasingly resistant to MAP infection. Solute carrier family 11 member 1 (SLC11A1) was adjudged to be a potential candidate gene because of its role in innate immunity, its involvement in susceptibility to numerous intracellular infections, and its previous association with bovine MAP infection. The objectives of this study were to carry out an exhaustive process of discovery and compilation of polymorphisms in SLC11A1 gene, and to perform a population-based genetic association study to test its implication in susceptibility to MAP infection in cattle. In all, 57 single nucleotide polymorphisms (SNP) were detected, 25 of which are newly described in Bos taurus. Twenty-four SNP and two 3'-untranslated region polymorphisms, previously analyzed, were selected for a subsequent association study in 558 European Holstein-Friesian animals. The SNP c.1067C>G and c.1157-91A>T and a haplotype formed by these 2 SNP yielded significant association with susceptibility to MAP infection. The c.1067C>G is a nonsynonymous SNP that causes an amino acid change in codon 356 from proline to alanine (P356A) that could alter SLC11A1 protein function. This association study supports the involvement of SLC11A1 gene in susceptibility to MAP infection in cattle. Our results suggest that SNP c.1067C>G may be a potential causal variant, although functional studies are needed to assure this point.

Somatic mosaicism in a case of apparently sporadic Creutzfeldt-Jakob disease carrying a de novo D178N mutation in the PRNP gene.

Transmissible spongiform encephalopathies (TSEs) are a group of rare fatal neurodegenerative disorders. Creutzfeldt-Jakob disease (CJD) represents the most common form of TSE and can be classified into sporadic, genetic, iatrogenic and variant forms. Genetic cases are related to prion protein gene mutations but they only account for 10-20% of cases. Here we report an apparently sporadic CJD case with negative family history carrying a mutation at codon 178 of prion protein gene. This mutation is a de novo mutation as the parents of the case do not show it. Furthermore the presence of three different alleles (wild type 129M-178D and 129V-178D and mutated 129V-178N), confirmed by different methods, indicates that this de novo mutation is a post-zygotic mutation that produces somatic mosaicism. The proportion of mutated cells in peripheral blood cells and in brain tissue was similar and was estimated at approximately 97%, suggesting that the mutation occurred at an early stage of embryogenesis. Neuropathological examination disclosed spongiform change mainly involving the caudate and putamen, and the cerebral cortex, together with proteinase K-resistant PrP globular deposits in the cerebrum and cerebellum. PrP typing was characterized by a lower band of 21 kDa. This is the first case of mosaicism described in prion diseases and illustrates a potential etiology for apparently sporadic neurodegenerative diseases. In light of this case, genetic counseling for inherited and sporadic forms of transmissible encephalopathies should take into account this possibility for genetic screening procedures.

Improvements in the detection of small ruminant lentivirus infection in the blood of sheep by PCR.

The polymerase chain reaction on blood samples has been considered a complement to serological methods for the detection of small ruminant lentiviruses (SRLV) infections in sheep and goats. This is a report on the results of a study to evaluate the use of the same blood sample for the detection of infected animals by ELISA and PCR. A comparison between the results obtained by applying PCRs targeting LTR and gag sequences on blood clot, serum and peripheral blood leucocytes was made. In addition to simplifying sampling and laboratory work, the use of blood clot samples with the gag-PCR improved remarkably the detection of infected animals. Finally, this study has shown the existence of a cell-free viremia in the serum of SRLV-infected sheep.