Identification by a proteomic approach of a plasma protein as a possible biomarker of illicit dexamethasone treatment in veal calves.

Corticosteroids have become the most widespread illegal growth promoters in veal calves and beef cattle. Testing for corticosteroids relies on either direct detection of compounds or their metabolites or indirect detection to identify changes in biological pathways. We used a comparative proteomic approach, based on two-dimensional electrophoresis (2DE), to identify plasma protein markers after short-term dexamethasone administration in veal calves. Twenty-three male Friesian veal calves were treated experimentally with dexamethasone sodium phosphate: 10 received low-dose administration of the drug (0.4 mg day⁻¹ per os) for 20 consecutive days (treatment group); 10 received the drug at therapeutic dosage (2-4 mg kg⁻¹ i.m.) for 3 consecutive days (comparison group). Three animals were not treated (control group). Plasma samples were collected from each animal at six time points (T1-T6; treatment and control group) and at four time points (T1-T4; comparison group) and stored at -80°C until analysis. Plasma proteins were quantified and analysed in triplicate by 2DE. The images were analysed with Bionumerics® software. Comparison of 2DE maps obtained from blood samples at T1 (before treatment) and at T6 (final sampling) showed a significant disappearance (p < 0.001) of two protein spots at T6 in the treatment group. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and immunoblotting identified these isoforms as serum paraoxonase/arylesterase 1 precursor (PON1). Synthesised in the liver and released into the blood, PON1 has an important role in lipid metabolism. The absence of variation of this protein in the comparison group suggests that the marker has good specificity for detecting illicit corticosteroid treatment.

Modeling amyotrophic lateral sclerosis in hSOD1 transgenic swine.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that occurs in two clinically indistinguishable forms: sporadic (SALS) and familial (FALS), the latter linked to several gene mutations, mostly inheritable in a dominant manner. Nearly 20% of FALS forms are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Research on ALS relies on transgenic models and particularly on mice carrying a glycine-to-alanine conversion at the 93rd codon (G93A) of the hSOD1 gene. Although G93A transgenic mice have been widely employed in clinical trials and basic research, doubts have been recently raised from numerous reliable sources about their suitability to faithfully reproduce human disease. Besides, the scientific community has already foreseen swine as an attractive and alternative model to nonhuman primates for modeling human diseases due to closer anatomical, physiological and biochemical features of swine rather than rodents to humans. On this basis, we have produced the first swine ALS model by in vitro transfection of cultured somatic cells combined with somatic cell nuclear transfer (SCNT). To achieve this goal we developed a SOD1(G93A) (superoxide dismutase 1 mutated in Gly93-Ala) vector, capable of promoting a high and stable transgene expression in primary porcine adult male fibroblasts (PAF). After transfection, clonal selection and transgene expression level assessment, selected SOD1(G93A) PAF colonies were used as nuclei donors in SCNT procedures. SOD1(G93A) embryos were transferred in recipient sows, and pregnancies developed to term. A total of 5 piglets survived artificial hand raising and weaning and developed normally, reaching adulthood. Preliminary analysis revealed transgene integration and hSOD1(G93A) expression in swine tissues and 360° phenotypical characterization is ongoing. We believe that our SOD1(G93A) swine would provide an essential bridge between the fundamental work done in rodent models and the reality of treating ALS.

Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk.

Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal reference genes for the identification of genes differentially expressed by qRT-PCR in caprine MSC. Moreover, we provided a set of intron-spanning primer sequences that could be suitable for gene expression experiments using SYBR Green chemistry on other caprine tissues and cells.

Histopathology as a simple and reliable method to detect 17ß-oestradiol illegal treatment in male calves.

17ß-Oestradiol is a steroid hormone banned as a growth promoter in food-producing animals all over Europe because of its carcinogenicity. Despite mandatory monitoring of illegal treatment all over Europe, official analytical methods in use test negative a few days after 17ß-oestradiol administration, requiring new sensitive tools to ensure a high level of protection for consumers. The aim of this work was the evaluation of the accuracy of histopathology and immunohistochemistry for progesterone receptor (PR) as a screening method for the detection of low-dosage illegal treatments with 17ß-oestradiol. Fresian male calves (153) were farmed under controlled conditions, and 89 of them were treated with 17ß-oestradiol (5 mg/animal once a week for 4 weeks). After 15 days of suspension, all animals were slaughtered and sexual accessory glands (prostate and bulbo-urethral glands) were sampled for histological examination and immunohistochemical staining with anti-PR antibody (clone hPRa 2). Microscopically 86 out of 89 bulbo-urethral glands showed mild to severe metaplasia, while mild metaplasia was observed only in 1 control. Eighteen out of 89 samples of prostate did not show metaplastic lesions. Immunopositivity for PR characterised all treated animals, while no signal was detected in controls. These findings show that metaplasia of the sexual accessory glands is a sensitive and specific parameter for illegal 17ß-oestradiol treatment in calves at the slaughterhouse, while the appliance of immunohistochemistry for PR can improve to 100% the accuracy of this highly reliable histological approach.

Molecular characterization of Pseudomonas fluorescens isolates involved in the Italian "blue mozzarella" event.

Between June and September 2010, widespread Italian consumer reports of unusual blue spoilage on fresh dairy products were publicized, resulting in the so-called blue mozzarella event. An inordinately high number of samples from mozzarella and whey cheese products of Italian and German production subsequently tested positive for Pseudomonas fluorescens. The aim of this study was to verify whether a selected P. fluorescens strain was responsible for this apparently unusual event. Molecular characterization of 181 isolated P. fluorescens strains was conducted using a newly optimized pulsed-field gel electrophoresis protocol. Although a high number of pulsotypes was found (132), only four pulsotypes were associated with more than one production plant, and only one German isolate had the same pulsotype as was detected in two Italian plants. This is the only evidence of possible cross-contamination among cheeses from the two countries. The overall results did not support the spread of contamination from German to Italian plants or the presence of one environmental strain that spread in both countries.

Ten years of BSE surveillance in Italy: neuropathological findings in clinically suspected cases.

Between 2001 and 2010, 244 clinically suspected cases of bovine spongiform encephalopathy (BSE) were reported in Italy. This report summarizes the neuropathological findings in cattle displaying clinical signs consistent with a diagnosis of BSE. All animal specimens were submitted for confirmatory testing; samples testing negative underwent neuropathological examination to establish the differential diagnosis. Immunohistochemistry for scrapie prion protein (PrPSc) at the level of frontal cortex was carried out to exclude atypical BSE. Neuropathological changes were detected in 34.9% of cases; no histological lesions were found in 52.3% of subjects; 12.8% of samples were found unsuitable for analysis. BSE was detected in one case, but no cases of atypical BSE were observed. This study identified the diseases most commonly encountered in the differential diagnosis of BSE; furthermore, it demonstrated that the surveillance system is necessary for monitoring neuropathological disease in cattle and for the detection of BSE cases.

Development of an enhanced histopathological approach to detect low-dose dexamethasone illicit treatment in veal calves.

Dexamethasone is one of a number of synthetic corticosteroids illegally used to promote growth in food-producing animals. Since these low-level drug cocktails evade detection by currently available chemical methods, simple biological indicators that can aid in laboratory analysis are needed. In an attempt to devise an accurate biological method that could detect illicit drug treatment in food-producing animals, we characterized microscopic morphologic alterations of the thymus in veal calves administered low-dose dexamethasone versus control animals. For this purpose, 122 male calves were farmed for 6 months in controlled condition: 81 animals were orally administered dexamethasone (0.4 mg day(-1)) for 20 days during the sixth month and the remaining 41 were kept as control. Urine samples were collected systematically during the treatment period, the suspension period and at the slaughterhouse. All animals were slaughtered 10 per day starting from 10 days after the last dexamethasone administration and the thymus was sampled for histological examination. The difference between the two animal groups was evaluated by means of a non-parametric test of hypothesis. No residues were detected in the urines collected since the third day after the last administration, whereas morphometric analysis of the thoracic thymus revealed a significant decrease in the cortex:medulla ratio in the treated animals (p<0.0005). We can conclude that this histological approach offers encouraging prospects as a screening method to overcome current limitations in controlling growth promoter abuse.

Co-existence of classical scrapie and Nor98 in a sheep from an Italian outbreak.

Nor98 is an atypical scrapie strain characterized by a molecular pattern and brain distribution of the pathological prion protein (PrP(Sc)) different from classical scrapie. In Italy, 69 atypical cases have been identified so far and all were characterized as Nor98 strain. In this paper we report an unusual case in a sheep which showed immunohistochemical and molecular features of PrP(Sc) different from the other atypical cases. The sheep was from an outbreak where the index and the other four cases were affected by classical scrapie. Histopathological, immunohistochemical and Western blot analyses on the brain of the unusual case revealed the simultaneous presence of pathological features characteristic of Nor98 and classical scrapie. Interestingly, the prevalent disease phenotype in the brainstem was classical scrapie-like, while in the cerebral cortex and cerebellum the Nor98 phenotype was dominant. The sub-mandibular lymph node was positive and showed a PrP(Sc) molecular pattern referable to classical scrapie. The PrP genotype was AL(141)RQ/AF(141)RQ. Taken together, the occurrence of classical scrapie in the outbreak, the PrP genotype, the involvement of different cellular targets in the brain and the pathological and molecular PrP(Sc) features observed suggest that this unusual case may result from the co-existence of Nor98 and classical scrapie.

Neuropathology of italian cats in feline spongiform encephalopathy surveillance.

Feline spongiform encephalopathy (FSE) is a transmissible spongiform encephalopathy associated with the consumption of feedstuffs contaminated with tissue from bovine spongiform encephalopathy-affected cattle and characterized by the accumulation in the central nervous system of an abnormal isoform of the prion protein (PrP(sc)). Clinically, it presents as a progressive fatal neurologic syndrome that is not easily distinguished from other feline neurologic conditions. Most cases of FSE have been reported in England, where it was first detected in 1990, but a few cases have been reported from other European countries. To identify possible cases of FSE in Italy, the Italian Ministry of Health funded a 2-year surveillance project during which the brains from 110 domestic cats with neurologic signs were evaluated histologically for spongiform encephalopathy and immunohistochemically to detect PrP(sc). Although no cases of FSE were found, the study proved useful in monitoring the Italian cat population for other neurologic diseases: neoplasia (21.8%), toxic-metabolic encephalopathy (18.2%), granulomatous encephalitis (15.5%), suppurative encephalitis (4.6%), trauma (3.6%), circulatory disorders (3.6%), degeneration (2.7%), nonsuppurative encephalitis (2.7%), and neuromuscular diseases (1.8%). No histologic lesions were found in 20% of the brains, and samples from 5.5% of the cats were rejected as unsuitable.

Genetic variability of the PRNP gene in goat breeds from Northern and Southern Italy.

AIMS: To determine the variability of the prion protein gene (PRNP) in goats from Northern and Southern Italy. METHODS AND RESULTS: Genomic DNA isolated from goat blood was polymerase chain reaction (PCR)-amplified for the coding region of the PRNP gene and then sequenced. In total, 13 polymorphic sites were identified: G37V, T110P, G127S, M137I, I142M, I142T, H143R, R154H, P168Q, T194P, R211Q, Q222K and S240P (substitutions I142T and T194P are novel) giving rise to 14 haplotypes. Clear frequency differences between Northern and Southern breeds were found and confirmed by genetic distance analysis. CONCLUSIONS: Differences in allele distribution were found between Northern and Southern goats, in particular regarding the M142 and K222 alleles, possibly associated to scrapie resistance; philogeographical analysis supported the idea that Northern and Southern breeds may be considered as separate clusters. SIGNIFICANCE AND IMPACT OF THE STUDY: In Italy only limited studies have been carried out on caprine PRNP genotype distribution; this study is important to fill this lack of information. Moreover the finding of significant differences among allele distributions in Northern and Southern goats, especially if involved in modulating resistance/susceptibility, need to be carefully considered for the feasibility of selection plans for resistance to scrapie.

Epidemiological study of the decline of BSE in Italy.

In this paper, data derived from the national database of the Italian surveillance system for bovine spongiform encephalopathy (BSE) are used to describe the Italian epidemic of bse. Two data flows were established to collect the results of active and passive surveillance, and 25 regional laboratories were involved. The National Reference Centre (CEA) was in charge of the data analysis. Crude and age-standardised estimates of the prevalence and incidence of bse were obtained to describe the distribution of the disease in terms of the main risk factors (age, breed and herd size), year of birth, time of diagnosis and geographical location. The increased risk was calculated in terms of the incidence rate ratio. During the five years since January 2001, 128 cases of bse were identified in domestic cows and four were identified in imported cattle. All but one of the cases were detected through active surveillance. The risk of the disease was highest in dairy stock and in large herds. The northern regions of Italy had an incidence of bse 2.6 times higher than the southern regions. There was a clear decline in the age-standardised prevalence, from one positive case per 10,000 tests in 2001 to one per 100,000 tests in 2005.

Immunohistochemistry for PrPSc in natural scrapie reveals patterns which are associated with the PrP genotype.

Immunohistochemistry for PrPSc is used widely in scrapie diagnosis. In natural scrapie cases the use of immunohistochemistry (IHC) has revealed the existence of up to 12 different morphological types of immunostained deposits. The significance of this pattern variability in relation to genotype has not been studied extensively in natural disease. In this study we recorded in detail PrPSc patterns at the obex level of the medulla oblongata from 163 animals derived from 55 flocks which presented through passive surveillance in the UK and Italy. A strong association was seen between PrPSc patterns and PrP genotype, particularly in relation to codon 136. In a blind assessment of this association we were able to predict, with over 80% accuracy, the genotype of 151 scrapie cases which were presented through passive surveillance from 13 farms. The genotype of these cases was ARQ/ARQ or VRQ/VRQ. The association of PrPsc patterns with genotype was generally stronger in those farms where all the affected animals belonged to a single genotype compared with farms where both genotypes were identified, with the exception of one farm in which the genotype of all affected sheep was ARQ/ARQ and the PrPSc patterns were of the VRQ/VRQ type. Our observations support the hypothesis that the observed association between specific IHC patterns and genotypes may in fact be strain driven but in natural disease individual scrapie strains may demonstrate a genotypic tropism.

Evaluation of an enzyme immunoassay for the detection of central nervous system tissue contamination at the slaughterhouse.

To protect public health from bovine spongiform encephalopathy, European Commission Regulation EC 1139/2003 on monitoring programs and specified risk material requires that as of 1 October 2003, each member state has in place a sampling plan with an appropriate laboratory test to detect central nervous system (CNS) tissue in bovine head meat harvested at slaughterhouses or cutting plants. With this study, we wanted to evaluate the accuracy and reliability of an enzyme immunoassay, the RIDASCREEN Risk Material 10/5, in targeting a CNS-specific marker, the glial fibrillary acidic protein. A receiver operating characteristics curve was plotted to identify the best cutoff of CNS concentration. Reliability was calculated by Cohen's kappa on data from two diagnostic sessions. Test performance showed high sensitivity and specificity (97.9 and 97.4%, respectively) for a cutoff value between positive and negative at a CNS concentration of 0.049%; reliability of test precision was also very good. When these criteria are applied, the RIDASCREEN Risk Material 10/5 test appears to be a reliable tool for monitoring CNS tissue contamination in meat. This diagnostic procedure should therefore be recommended for national application in monitoring programs.

BSE immunohistochemical patterns in the brainstem: a comparison between UK and Italian cases.

The continuous monitoring of bovine spongiform encephalopathy (BSE) cases is an integral component of European research and surveillance programmes, to ensure that any changes in the presentation of transmissible spongiform encephalopathies (TSE) in cattle can be detected and defined. Monitoring is generally limited to the brainstem at the level of the obex, for reasons of practicality, safety and cost. Demonstration of disease-specific prion protein (PrP(d)) by immunohistochemistry is currently the most widely used confirmatory tool for both active and passive surveillance. This study assessed PrP(d) immunostaining in the brainstems (obex) of cattle with BSE in the UK and Italy. Immunoreactivity 'profiles' were created for each case based on the nature of the immunostaining, its relative intensity and precise neuroanatomical location. This study compares the obex immunostaining patterns of Italian cases (only active surveillance) and two UK groups (both active and passive surveillance). The neuroanatomical distribution and relative intensity of PrP(d) was highly reproducible in all cases. The overall staining intensity varied widely but was generally stronger in the active than in the passive surveillance populations. The conclusion to be drawn from this comparative study is that the pattern of immunopathology in these routine screening samples for BSE diagnosis and surveillance is the same in the UK and Italy, whether or not the animal was displaying typical, or indeed any, clinical signs at the time of sampling. This indicates that the current confirmatory diagnostic strategy remains appropriate for active surveillance applications.

A case of scrapie in a sheep carrying the lysine-171 allele of the prion protein gene.

Susceptibility to scrapie in sheep depends on the host PrP genotype. No data about the linkage of the rare ARK allele to differential scrapie susceptibility are currently available. Several tissues isolated from sheep from an Italian scrapie outbreak and carrying the ARK allele were examined for the presence of the pathological prion protein. A weak positivity was detected only by Western blot in the brainstem of one ARK/ARH sheep. This result shows that the ARK allele does not confer full resistance against scrapie and that the allele needs to be studied further before it can be considered for breeding purposes.

Identification of prion protein gene polymorphisms in goats from Italian scrapie outbreaks.

Susceptibility to scrapie in sheep is influenced by polymorphisms of the prion protein (PrP) gene, whereas no strong association between genetics and scrapie has yet been determined in goats due to the limited number of studies on these animals. In this case-control study on 177 goats from six Italian scrapie outbreaks, the association between PrP alleles and the occurrence of scrapie was studied. Three silent mutations and 11 PrP polymorphisms were identified, of which two polymorphisms (L133Q and M137I) and one silent mutation (T202T) have not been reported previously. Twelve alleles were determined by cloning. Statistical analysis suggested a possible protective role against scrapie for the glutamine to lysine mutation at codon 222.

Low frequency of the scrapie resistance-associated allele and presence of lysine-171 allele of the prion protein gene in Italian Biellese ovine breed.

Frequencies of polymorphisms at codons 136, 154 and 171 of the prion protein (PrP) gene were studied in 1207 pure-bred and cross-bred Italian Biellese rams, a small ovine breed of about 65 000 head in Italy. Aside from the five most common alleles (VRQ, ARQ, ARR, AHQ and ARH), the rare ARK allele was also found, with the highest frequency reported so far in an ovine breed (2.5 %). ARK/--- genotypes had a total frequency of 4.9 %. The resistance-associated ARR allele was seen at a low frequency (8.3 %). Only 1.4 % of animals examined had a resistant ARR/ARR PrP genotype. Semi-resistant (ARR/ARQ, ARR/ARH and ARR/AHQ) PrP genotypes had a total frequency of 12.6 % and PrP genotypes that are associated with high scrapie susceptibility (e.g. VRQ/VRQ and ARQ/ARQ) had a total frequency of 81.1 %. Statistical analysis comparing PrP allele frequencies between pure-bred and cross-bred animals showed that the ARR allele occurred at a significantly lower frequency in pure-bred rams. Furthermore, comparison of PrP allele frequencies between pure-bred rams over 18 months of age and those below 18 months of age showed a significant decrease in the ARR allele in breeding rams over 18 months of age. Based on these results, breeding for scrapie resistance in the Biellese breed will have to take into account the low frequency of the ARR allele, which also seems to be subject to negative selection by farmers. Further investigation is required to understand whether the ARK allele is also associated with resistance to transmissible spongiform encephalopathies.