Survival of Mycobacterium avium subsp. paratuberculosis in the intermediate and final digestion products of biogas plants.

AIMS: To evaluate the survival of Mycobacterium avium subsp. paratuberculosis (MAP) during anaerobic digestion (AD), we studied two different biogas plants loaded with manure and slurry from paratuberculosis-infected dairy herds. METHODS AND RESULTS: Both plants were operating under mesophilic conditions, the first with a single digester and the second with a double digester. Mycobacterium avium subsp. paratuberculosis detection was performed by sampling each stage of the process, specifically the prefermenter, fermenter, liquid digestate and solid digestate stages, for 11 months. In both plants, MAP was isolated from the prefermenter stage. Only the final products, the solid and liquid digestates, of the one-stage plant showed viable MAP, while no viable MAP was detected in the digestates of the two-stage plant. CONCLUSIONS: Mycobacterium avium subsp. paratuberculosis showed a significant decrease during subsequent steps of the AD process, particularly in the two-stage plant. We suggest that the second digester maintained the digestate under anaerobic conditions for a longer period of time, thus reducing MAP survival and MAP load under the culture detection limit. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data are unable to exclude the presence of MAP in the final products of the biogas plants, particularly those products from the single digester; therefore, the use of digestates as fertilizers is a real concern related to the possible environmental contamination with MAP.

Review: A review on classical and atypical scrapie in caprine: Prion protein gene polymorphisms and their role in the disease.

Scrapie is a naturally occurring transmissible spongiform encephalopathy in sheep and goat. It has been known for ~250 years and is characterised by the accumulation of an abnormal isoform of a host-encoded prion protein that leads to progressive neurodegeneration and death. Scrapie is recognised in two forms, classical and atypical scrapie. The susceptibility to both types of scrapie is influenced by polymorphisms of the prion protein gene (PRNP). Sheep susceptibility or resistance to classical scrapie is strongly regulated by the polymorphisms at codons 136, 154 and 171 of the PRNP. The genetic role in atypical scrapie in sheep has been defined by polymorphisms at codons 141, 154 and 171, which are associated with different degrees of risk in the occurrence of the ovine disease. Progress has been achieved in the prevention of scrapie in sheep due to efficient genetic breeding programmes based on eradication and control of the disease. In Europe, the success of these programmes has been verified by applying eradication and genetic selection plans. In general terms, the ovine selection plans aim to eliminate and reduce the susceptible allele and to enrich the resistant allele ARR. During outbreaks all susceptible animals are slaughtered, only ARR/ARR resistant rams and sheep and semi-resistant females are preserved. In the occurrence of scrapie positive goats a complete cull of the flock (stamping out) is performed with great economic loss and severe risk of extinction for the endangered breeds. The ability to select scrapie-resistant animals allows to define new breeding strategies aimed to boost genetic progress while reducing costs during scrapie outbreaks. Allelic variants of PRNP can be protective for caprine scrapie, and the knowledge of their distribution in goats has become very important. Over the past few years, the integration of genetic information on goat populations could be used to make selection decisions, commonly referred to as genetic selection. The objective of this review was to summarise the main findings of polymorphisms of the caprine prion protein (PrP) gene and to discuss the possible application of goat breeding schemes integrating genetic selection, with their relative advantages and limitations.

Characterisation of Yersinia pseudotuberculosis isolated from animals with yersiniosis during 1996-2013 indicates the presence of pathogenic and Far Eastern strains in Italy.

Yersinia pseudotuberculosis is a pathogen that infects both animals and humans worldwide. The epidemiology of infection caused by Y. pseudotuberculosis is poorly understood; however, its outbreaks have been traced back to a probable source in wildlife. This study aimed to characterise Y. pseudotuberculosis isolates collected from animals with yersiniosis. This study included 90 isolates of Y. pseudotuberculosis collected from different animals with yersiniosis between 1996 and 2013 in Italy. The isolates were tested for antimicrobial susceptibility and were biotyped. Genes associated with virulence plasmid pYV and those encoding O-antigen, high pathogenicity island (HPI), and superantigenic toxin (YPM) were determined by performing PCR. Pulsed-field gel electrophoresis (PFGE) was performed using NotI and SpeI enzymes, and 3 dendrograms were generated. No antibiotic resistance was found. The presence of pYV was shown in 57 out of 90 isolates. Virulence profiles of majority of the isolates indicated that they belonged to O:1a and O:1b serotypes, biotype 1, and genetic type 2. Isolates belonging to O:2a serotype were detected in sheep and cattle and were found to be associated (for the first time) with septicemia in hares. Y. pseudotuberculosis isolates belonging to O:5a and O:12-O13 serotypes were also detected in hares. To our knowledge, this is the first study to detect Y. pseudotuberculosis isolates belonging to the O:12-O13 serotype from a clinical case in Europe. Results of PFGE indicated that it was a reliable method for investigating the genetic relatedness of Y. pseudotuberculosis isolates. Thus, characterisation of Y. pseudotuberculosis infection in animals should be considered a possible tool for the surveillance of pseudotuberculosis.

Atypical Yersinia pseudotuberculosis serotype O:3 isolated from hunted wild boars in Italy.

Atypical Yersinia pseudotuberculosis serotype O:3 was isolated from rectal contents of two wild boars hunted in Italy within a regional wildlife management program. No outbreak of yersiniosis was reported in this area in the same period and no lesions were found by the veterinarian at post-mortem inspection. Nevertheless, after histological examination, granulomatous lesions were detected in submandibular lymph nodes of one of the two wild boars. Microbiological and bio molecular characterization of the isolates revealed a melibiose-negative, biotype 2, wbyK+O:3 genotype, carrying inv, yop (yopH and yopB), virF, and R-HPI. Strains showing the same profile, matching to the criteria of genetic group 5, have been recently reported in fatal cases of yersiniosis in cynomolgus macaques and in farmed deer and atypical O:3 serotype has been suggested as a pathogenic subtype of O:3. This is the third report of an atypical O:3 Y. pseudotuberculosis strain, the first outside the American continent and the first one not associated to fatal yersiniosis. Wild boars could be a possible reservoir of this emerging pathogen.

The genetic resistance to gastro-intestinal strongylids in Appenninica sheep: preliminary results.

Heritability parameters of resistance to gastro-intestinal strongylids, measured as FEC (Faecal Egg Count), were evaluated in the Appenninica sheep breed. FEC heritability coefficient was 0.11 +/- 0.061 while FEC repeatability coefficients were 0.58 +/- 0.085 and 0.76 +/- 0.223 in adult females and lambs respectively. Subjects were classified, based on FEC, into three different levels of resistance to strongylids. Ewes belonging to the 'resistant class' should be conveniently exploited in mating schemes, in order to provide a method, alternative to drug administration, for a long-term parasite control; this would result particularly helpful under those production systems, such as organic farming, where the use of drugs is not allowed or limited.

Statistical analysis of individual assignment tests among four cattle breeds using fifteen STR loci.

Assignment tests based on multilocus genotypes are becoming increasingly important to certify quality and origin of livestock products and assure food safety and authenticity. The purpose of this study was to determine the potential of microsatellites (STR) for determining the breed origin of beef products among cattle breeds present in the market. We typed 19 STR in 269 animals from 4 cattle breeds. Based on Wright's F-statistics, 4 loci were discarded, and the remaining 15 loci (FIT = 0.101, FST = 0.089, and FIS = 0.013) were used to compute the likelihood that each multilocus genotype of the total sample was drawn from its true breed instead of another breed. To avoid occurrence of zero likelihood when one or more alleles were missing from a tested breed, sample allele frequencies were estimated assuming uniform prior distributions. Log-likelihood ratio [log(LR)] distributions of the individual assignments were determined for all possible breed contrasts, and their means and SD were used to infer the true-positive and false-positive rates at several values of the log(LR). The posterior probability that the animals of a presumed breed were actually drawn from that breed instead of any another breed was then calculated. Given an observed value of log(LR) > 0 and assuming equal priors, these probabilities were > 99.5% in 10 of 12 possible breed contrasts. For the 2 most closely related breeds (FST = 0.041), this probability was 96.3%, and the probability of excluding the origin of an animal from an alleged breed when it was actually derived from another breed was similar.

Common themes in the pathogenesis of acute myeloid leukemia.

The pathogenesis of acute myeloid leukemia is associated with the appearance of oncogenic fusion proteins generated as a consequence of specific chromosome translocations. Of the two components of each fusion protein, one is generally a transcription factor, whereas the other partner is more variable in function, but often involved in the control of cell survival and apoptosis. As a consequence, AML-associated fusion proteins function as aberrant transcriptional regulators that interfere with the process of myeloid differentiation, determine a stage-specific arrest of maturation and enhance cell survival in a cell-type specific manner. The abnormal regulation of transcriptional networks occurs through common mechanisms that include recruitment of aberrant co-repressor complexes, alterations in chromatin remodeling, and disruption of specific subnuclear compartments. The identification and analysis of common and specific target genes regulated by AML fusion proteins will be of fundamental importance for the full understanding of acute myeloid leukemogenesis and for the implementation of disease-specific drug design.

Retrovirus-mediated transfer of the herpes simplex virus thymidine kinase and enhanced green fluorescence protein genes in primary T lymphocytes.

The EGFP-tk retroviral vector, encoding enhanced green fluorescent protein (EGFP) and the herpes simplex virus thymidine kinase (HSV-tk) packaged in a Phoenix amphotropic cell line, was used to transduce healthy donor T lymphocytes. Infection yielded a mean of 41.8 +/- 9.3% SD (range 31.1-48.4%) EGFP-positive cells and a mean of 92 +/- 2% SD (range 90-94%) after cell sorting. EGFP expression remained stable for 30 d after infection. The entire gene transfer procedure had no significant effect on lymphocyte subsets and slightly reduced clonogenicity. Ganciclovir (gcv) treatment (1 microg/ml x 10 d) killed all EGFP-positive cells in the transduced and transduced/sorted populations, but had no effect on untransduced controls. Our results show that primary T lymphocytes can be transduced using an EGFP-tk vector that yields a homogeneous infected population without affecting lymphocyte subsets, function and clonogenicity.

PML regulates p53 acetylation and premature senescence induced by oncogenic Ras.

The tumour suppressor p53 induces cellular senescence in response to oncogenic signals. p53 activity is modulated by protein stability and post-translational modification, including phosphorylation and acetylation. The mechanism of p53 activation by oncogenes remains largely unknown. Here we report that the tumour suppressor PML regulates the p53 response to oncogenic signals. We found that oncogenic Ras upregulates PML expression, and overexpression of PML induces senescence in a p53-dependent manner. p53 is acetylated at lysine 382 upon Ras expression, an event that is essential for its biological function. Ras induces re-localization of p53 and the CBP acetyltransferase within the PML nuclear bodies and induces the formation of a trimeric p53-PML-CBP complex. Lastly, Ras-induced p53 acetylation, p53-CBP complex stabilization and senescence are lost in PML-/- fibroblasts. Our data establish a link between PML and p53 and indicate that integrity of the PML bodies is required for p53 acetylation and senescence upon oncogene expression.

A monoclonal antibody (MUM1p) detects expression of the MUM1/IRF4 protein in a subset of germinal center B cells, plasma cells, and activated T cells.

A new monoclonal antibody (MUM1p) was used to study the cell/tissue expression of human MUM1/IRF4 protein, the product of the homologous gene involved in the myeloma-associated t(6;14) (p25;q32). MUM1 was expressed in the nuclei and cytoplasm of plasma cells and a small percentage of germinal center (GC) B cells mainly located in the "light zone." Its morphologic spectrum ranged from that of centrocyte to that of a plasmablast/plasma cell, and it displayed a phenotype (MUM1(+)/Bcl-6(-)/Ki67(-)) different from that of most GC B cells (MUM1(-)/Bcl-6(+)/Ki67(+)) and mantle B cells (MUM1(-)/Bcl-6(-)/Ki67(-)). Polymerase chain reaction (PCR) analysis of single MUM1(+ )cells isolated from GCs showed that they contained rearranged Ig heavy chain genes with a varying number of V(H) somatic mutations. These findings suggest that these cells may represent surviving centrocytes and their progeny committed to exit GC and to differentiate into plasma cells. MUM1 was strongly expressed in lymphoplasmacytoid lymphoma, multiple myeloma, and approximately 75% of diffuse large B-cell lymphomas (DLCL-B). Unlike normal GC B cells, in which the expression of MUM1 and Bcl-6 were mutually exclusive, tumor cells in approximately 50% of MUM1(+) DLCL-B coexpressed MUM1 and Bcl-6, suggesting that expression of these proteins may be deregulated. In keeping with their proposed origin from GC B cells, Hodgkin and Reed-Sternberg cells of Hodgkin's disease consistently expressed MUM1. MUM1 was detected in normal and neoplastic activated T cells, and its expression usually paralleled that of CD30. These results suggest that MUM1 is involved in the late stages of B-cell differentiation and in T-cell activation and is deregulated in DLCL-B. (Blood. 2000;95:2084-2092)