Agenti di encefalopatie spongiformi trasmissibili e zoonosi: tipizzazione molecolare

Transmissible spongiform encephalopathies (TSE) are neurodegenerative diseases caused by the conversion of the host-encoded cellular protein (PrPC) to a disease-associated isoform (PrPSc). The agent responsible for prion diseases may exist as different strains with specific biological and biochemical properties. According to the protein-only hypothesis, prion strain diversity is enciphered in PrPSc conformation. Molecular strain typing methods are based on the electrophoretic mobility of protease resistant core of PrPSc, on the susceptibility to protease digestion, on the glycosylation profile of PrPres and on the conformational stability of PrPSc. In this study a new conformational stability assay was developed based on the differential solubility of PrPC and PrPSc: CSSA (conformational stability and solubility assay). The conformational stability assay was performed by measuring PrPSc solubility in homogenates treated with increasing concentrations of GdnHCl, in the absence of proteinase K. Indeed, dose-response curves allowed estimation of the concentration of GdnHCl able to solubilise 50% of PrPSc. The results showed that this method is valuable for the biochemical typing of strains in bank voles and it is also a promising tool for molecular analysis of natural prion isolates. CSSA also revealed strain-specific PrPSc conformational stabilities of ovine natural isolates so that this feature, combined with the N-terminal PrPSc cleavage, allowed differentiation of classical scrapie, including CH1641-like, from natural goat BSE and experimental sheep BSE. In view of the implications concerning strain similarity between animal and human TSEs, the physico-chemical properties of the Nor98 with two human prion diseases (VPSPr and GSS) were compared in order to investigate the extent of the similarity between animal and human prion strains. The results showed an unexpected heterogeneity of the molecular features among human and sheep TSEs associated with internal PrPres fragments with the possible exception of Nor98 and a case of GSS P102L. These similarities and differences need further investigation by N- and C-terminal sequencing and biological characterization.

Identification of CNV and QTL for productive and functional traits in dairy cattle using dense SNP chips

The thesis identify CNV structural variants as possible markers for genomic selection and identify QTL regions for Fatty Acid Content in the Italian Brown Swiss population. Additionally it maps the QTL for mastitis resistance in the Valdostana Red Pied cattle.

Breast abscess due to Actinomyces europaeus

Actinomyces europaeus was first described in 1997 as a new species causing predominantly skin and soft-tissue infections. Mastitis due to A. europaeus is an unusual condition. This article reports a case of primary breast abscess caused by A. europaeus in a postmenopausal woman.

Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

BACKGROUND: During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection. METHODS: Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture. RESULTS: Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele 'Q' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele 'q'. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways 'dendritic cell maturation' and 'acute phase response signaling', whereas cell culture affected biological processes involved in 'cellular development'. CONCLUSIONS: The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.

Distinct proteinase K-resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak

Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrP(res)) in a highly scrapie-affected goat flock in Greece. The PrP(res) profile by Western immunoblotting in most animals was that of classical scrapie in sheep. However, in a series of clinically healthy goats we identified a unique C- and N-terminally truncated PrP(res) fragment, which is akin but not identical to that observed for atypical scrapie. These findings reveal novel aspects of the nature and diversity of the molecular PrP(res) phenotypes in goats and suggest that these animals display a previously unrecognized prion protein disorder.

Molecular characterization and phylogenetic analysis of small ruminant lentiviruses isolated from Canadian sheep and goats

Background: Small Ruminant Lentiviruses (SRLV) are widespread in Canadian sheep and goats and represent an important health issue in these animals. There is however no data about the genetic diversity of Caprine Arthritis Encephalitis Virus (CAEV) or Maedi Visna Virus (MVV) in this country. Findings: We performed a molecular and phylogenetic analysis of sheep and goat lentiviruses from a small geographic area in Canada using long sequences from the gag region of 30 infected sheep and 36 infected goats originating from 14 different flocks. Pairwise DNA distance and phylogenetic analyses revealed that all SRLV sequences obtained from sheep clustered tightly with prototypical Maedi visna sequences from America. Similarly, all SRLV strains obtained from goats clustered tightly with prototypical US CAEV-Cork strain. Conclusions: The data reported in this study suggests that Canadian and US SRLV strains share common origins. In addition, the molecular data failed to bring to light any evidence of past cross species transmission between sheep and goats, which is consistent with the type of farming practiced in this part of the country where single species flocks predominate and where opportunities of cross species transmissions are proportionately low.

Management effects on colostrogenesis in small ruminants: a review

Colostrum feeding in small ruminants is crucial during the first hours after birth due to the lack of Ig transfer during pregnancy via the placenta. In addition the immature immune system of the neonate is slow to produce its own Ig during the first weeks of life. Colostrogenesis, i.e. the transfer of Ig from blood into mammary secretions, starts several weeks prepartum. In goat plasma, immunoglobulin G (IgG) concentration decreases by around 38% from the third month of gestation until partum, which coincides with the dry period. Thus, management during the dry period is crucial for the course of colostrogenesis. The colostrum synthesis is determined by the nutrition during the prepartum period, but the transfer of Ig is obviously independent of nutritional influences. The administration of conjugated linoleic acid during the dry period to dairy goats causes a less pronounced decrease of blood plasma IgG concentration (6%) but it did not change colostral IgG levels. In cattle, IgG1 is transported from blood into colostrum by an IgG1 specific receptor located on the surface of alveolar epithelial cells during colostrogenesis, and this is most likely similar in small ruminants. Via inactivation of this receptor, the Ig transfer is downregulated by increasing prolactin (PRL) during lactogenesis. It was recently observed in goats treated with PGF2 alpha, in order to induce parturition, lower colostrum IgG concentrations occurred concomitantly with an earlier increase of plasma PRL as compared to untreated animals. The effect of litter size and number of lactations on colostral IgG concentration in small ruminants has not been made fully clear until now most likely due to the different breeds used in the published studies.

Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland

Different pathogens, such as Escherichia coli and Staphylococcus aureus, can be responsible for different outcomes of mastitis; that is, acute and severe or chronic and subclinical. These differences in the disease could be related to different mammary responses to the pathogens. The objective of this study was to determine if intramammary challenge with the endotoxins lipopolysaccharide (LPS), from E. coli, and lipoteichoic acid (LTA), from Staph. aureus, induce different immune responses in vivo in milk cells and mammary tissue. To provide a reference level for comparing the challenge and to show the different stimulation of the mammary immune system on a quantitatively similar level, dosages of LPS and LTA were chosen that induced an increase of somatic cells in milk to similar maxima. One udder quarter in each of 21 lactating dairy cows was challenged with 0.2 mug of LPS or 20 mug of LTA. From these quarters and from respective control quarters, milk cells or tissue biopsies were obtained at 0, 6, and 12h relative to the challenge to measure mRNA expression of tumor necrosis factor-alpha (TNFalpha), IL-1beta, IL-8, lactoferrin, and RANTES (regulated upon activation, normal T-cell expressed and secreted). Furthermore, if no biopsies were performed, hourly milk samples were taken for measurement of somatic cell count, lactate dehydrogenase (LDH), and TNFalpha. Somatic cell count increased in all treatments to similar maxima with LPS and LTA treatments. Concentrations of TNFalpha in milk increased with LPS but not with LTA. The activity of LDH in milk increased in both treatments and was more pronounced with LPS than with LTA. The mRNA expression of TNFalpha, IL-1beta, IL-8, and RANTES showed increases in milk cells, and LPS was a stronger inducer than LTA. Lactoferrin mRNA expression decreased in milk cells with LPS and LTA treatments. The measured factors did not change in either treatment in mammary tissue. Challenge of udder quarters with dosages of LPS and LTA that induce similar increases in SCC stimulate the appearance of different immune factor patterns. This dissimilar response to LPS and LTA may partly explain the different course and intensity of mastitis after infection with E. coli and Staph. aureus, respectively.

Direct detection of Mycoplasma bovis in milk and tissue samples by real-time PCR

Bovine mastitis caused by Mycoplasma bovis is of great economic importance to the beef and dairy industry. Here we describe a new specific real-time PCR assay targeting the uvrC gene that was developed to directly detect M. bovis from milk and tissue samples without laborious DNA purification.

Caprine PRNP polymorphisms at codons 171, 211, 222 and 240 in a Greek herd and their association with classical scrapie

The association between PRNP variation and scrapie incidence was investigated in a highly affected Greek goat herd. Four mutations were identified at codons 171Q/R, 211R/Q, 222Q/K and 240P/S. Lysine at codon 222 was found to be associated with the protection from natural scrapie (P=0.0111). Glutamine at codon 211 was observed in eight animals, all of them being scrapie-negative, indicating a possible protective role of this polymorphism although statistical analysis failed to support it (P=0.1074). A positive association (P=0.0457) between scrapie-affected goats and the wild-type Q(171)R(211)Q(222)S(240) allele is presented for the first time. In addition, a novel R(171)RQS allele, which is identical to the A(136)R(154)R(171) allele that has been associated with resistance to classical scrapie in sheep, was observed in low frequency. Resistant alleles that include K(222) and Q(211) are absent or rare in sheep and can provide the basis for the development of a feasible breeding programme for scrapie eradication in goats.

The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study

Notochordal cells and nucleus pulposus cells are co-existing in the intervertebral disc at various ratios among different mammalians. This fact rises the question about the interactions and the evolutionary relevance of this phenomenon. It has been described that these relatively large notochordal cells are mainly dominant in early lifetime of all vertebrates and then differences occur with ageing. Human, cattle, sheep, and goat lose the cells with age, whereas rodents and lagomorphs maintain these throughout their lifetime.

Development of an in vivo experimental model for percutaneous vertebroplasty in sheep

Several studies have described 'open' approach techniques for cementation of sheep and goat vertebrae; however, no percutaneous technique has been developed so far for use in non-primates. The aim of this study was to develop an animal model for percutaneous vertebroplasty under clinical conditions.

Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of beta- and alpha(S1)-casein during incubation of milk samples at peak somatic cell counts, with that of beta-casein being faster than that of alpha(S1)-casein. Two-dimensional gel electrophoretograms of milk 6h after infusion with the toxin confirmed hydrolysis of beta- and alpha(S1)-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C(18) columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from alpha(s1)- and beta-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.

Cross-sectional study of Streptococcus species in quarter milk samples of dairy cows in the canton of Bern, Switzerland

A total of 2538 quarter milk samples from 638 lactating dairy cows from 47 farms in the canton of Bern, Switzerland, were investigated for streptococci. A novel, simple and inexpensive laboratory method was used for the differentiation of Streptococcus species, and a risk factor analysis was carried out. The prevalence in the quarter milk samples was 0.2 per cent for Streptococcus agalactiae, 1.3 per cent for Streptococcus uberis, 1.3 per cent for Streptococcus dysgalactiae, 0.1 per cent for Enterococcus species and 2.9 per cent for minor Streptococcus species (designated Streptococcus-Lactococcus-Enterococcus [SLE] group). Based on the somatic cell count (SCC), S uberis and S dysgalactiae were classified as 'major' pathogens and the bacteria in the SLE group as 'minor' pathogens. For S uberis, S dysgalactiae and bacteria in the SLE group, the most significant risk factor was an intramammary infection (IMI) of a neighbouring quarter by the same pathogen. Other significant risk factors for S uberis infection were a positive California Mastitis Test (CMT) result and a SCC of more than 100,000 cells/ml. Significant risk factors for IMI with S dysgalactiae were a positive CMT result, teat injury and palpable abnormalities in the udder. Infection with bacteria in the SLE group was significantly associated with a SCC of more than 100,000 cells/ml, a lactation number of more than 2, the right rear quarter (as the location of infection) and a positive CMT result.

The MHC-haplotype influences primary, but not memory, immune responses to an immunodominant peptide containing T- and B-cell epitopes of the caprine arthritis encephalitis virus Gag protein

In this report, we describe a short peptide, containing a T helper- and a B-cell epitope, located in the Gag protein of the caprine arthritis encephalitis virus (CAEV). This T-cell epitope is capable of inducing a robust T-cell proliferative response in vaccinated goats with different genetic backgrounds and to provide help for a strong antibody response to the B-cell epitope, indicating that it may function as a universal antigen-carrier for goat vaccines. The primary immune response of goats homozygous for MHC class I and II genes showed an MHC-dependent partitioning in rapid-high and slow-low responses, whereas the memory immune response was strong in both groups, demonstrating that a vaccine based on this immunodominant T helper epitope is capable to overcome genetic differences.