Brain-Derived Neurotrophic Factor, Kynurenine Pathway, and Lipid-Profiling Alterations as Potential Animal Welfare Indicators in Dairy Cattle.

Complete animal welfare evaluation in intensive farming is challenging. With this study, we investigate new biomarkers for animal physical and mental health by comparing plasma expression of biochemical indicators in dairy cows reared in three different systems: (A) semi-intensive free-stall, (B) non-intensive tie-stall, and (C) intensive free-stall. Additionally, protein levels of mature brain-derived neurotrophic factor (mBDNF) and its precursor form (proBDNF) and indoleamine 2,3-dioxygenase (IDO1) specific activity were evaluated in brain samples collected from 12 cattle culled between 73 and 138 months of age. Alterations in plasma lipid composition and in the kynurenine pathway of tryptophan metabolism were observed in the tie-stall-reared animals. The total plasma BDNF concentration was higher in tie-stall group compared to the two free-housing groups. Brain analysis of the tie-stall animals revealed a different mBDNF/proBDNF ratio, with a higher level of proBDNF (p < 0.001). Our data are similar to previous studies on animal models of depression, which reported that inhibition of the conversion of proBDNF in its mature form and/or elevated peripheral kynurenine pathway activation may underlie cerebral biochemical changes and induce depressive-like state behavior in animals.

SOMAscan Proteomics Identifies Novel Plasma Proteins in Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex disease characterized by the interplay of genetic and environmental factors for which, despite decades of intense research, diagnosis remains rather delayed, and most therapeutic options fail. Therefore, unravelling other potential pathogenetic mechanisms and searching for reliable markers are high priorities. In the present study, we employ the SOMAscan assay, an aptamer-based proteomic technology, to determine the circulating proteomic profile of ALS patients. The expression levels of ~1300 proteins were assessed in plasma, and 42 proteins with statistically significant differential expression between ALS patients and healthy controls were identified. Among these, four were upregulated proteins, Thymus- and activation-regulated chemokine, metalloproteinase inhibitor 3 and nidogen 1 and 2 were selected and validated by enzyme-linked immunosorbent assays in an overlapping cohort of patients. Following statistical analyses, different expression patterns of these proteins were observed in the familial and sporadic ALS patients. The proteins identified in this study might provide insight into ALS pathogenesis and represent potential candidates to develop novel targeted therapies.

Potential SARS-CoV-2 Susceptibility of Cetaceans Stranded along the Italian Coastline.

Due to marine mammals' demonstrated susceptibility to SARS-CoV-2, based upon the homology level of their angiotensin-converting enzyme 2 (ACE2) viral receptor with the human one, alongside the global SARS-CoV-2 occurrence and fecal contamination of the river and marine ecosystems, SARS-CoV-2 infection may be plausibly expected to occur also in cetaceans, with special emphasis on inshore species like bottlenose dolphins (Tursiops truncatus). Moreover, based on immune and inflammatory responses to SARS-CoV-2 infection in humans, macrophages could also play an important role in antiviral defense mechanisms. In order to provide a more in-depth insight into SARS-CoV-2 susceptibility in marine mammals, we evaluated the presence of SARS-CoV-2 and the expression of ACE2 and the pan-macrophage marker CD68. Aliquots of tissue samples, belonging to cetaceans stranded along the Italian coastline during 2020-2021, were collected for SARS-CoV-2 analysis by real-time PCR (RT-PCRT) (N = 43) and Immunohistochemistry (IHC) (N = 59); thirty-two aliquots of pulmonary tissue sample (N = 17 Tursiops truncatus, N = 15 Stenella coeruleoalba) available at the Mediterranean Marine Mammal Tissue Bank (MMMTB) of the University of Padua (Legnaro, Padua, Italy) were analyzed to investigate ACE2 expression by IHC. In addition, ACE2 and CD68 were also investigated by Double-Labeling Immunofluorescence (IF) Confocal Laser Microscopy. No SARS-CoV-2 positivity was found in samples analyzed for the survey while ACE2 protein was detected in the lower respiratory tract albeit heterogeneously for age, gender/sex, and species, suggesting that ACE2 expression can vary between different lung regions and among individuals. Finally, double IF analysis showed elevated colocalization of ACE2 and CD68 in macrophages only when an evident inflammatory reaction was present, such as in human SARS-CoV-2 infection.

Chronic Wasting Disease Monitoring in Italy 2017-2019: Neuropathological Findings in Cervids.

Chronic wasting disease (CWD) is a prion disease that affects cervids; it is classified under transmissible spongiform encephalopathies (TSEs). CWD is particularly contagious, making its eradication in endemic areas very difficult and creating serious problems for cervid conservation and breeding. It has recently become an emerging public health risk to be managed by health authorities. Starting in 2017, active CWD surveillance in Italy has intensified with the monitoring of wild and farmed cervids. The present study summarizes findings from a histopathological survey of the brains from wild ruminants collected via CWD monitoring between 2017 and 2019. A total of 113 brains from 62 red deer (Cervus elaphus) and 51 roe deer (Capreolus capreolus) were submitted for analysis at the National Reference Center for Animal Encephalopathies (CEA) to determine major patterns of neuropathological lesions and correlated pathogens. Brain lesions were detected in 20 animals, 10 brain samples were unsuitable for examination, and 83 presented no lesions. Neuropathological examination revealed non-suppurative encephalitis or meningoencephalitis in most cases (15/20). This brain study revealed evidence for the absence of CWD in Italy and provided a reference spectrum of neuropathological lesions for differential diagnosis in cervids.

Neuropathological Characterization of Dolphin Morbillivirus Infection in Cetaceans Stranded in Italy.

Cetacean morbillivirus (CeMV) is responsible for epidemic and endemic fatalities in free-ranging cetaceans. Neuro-inflammation sustained by CeMV is a leading cause of death in stranded cetaceans. A novel dolphin morbillivirus (DMV) strain of Atlantic origin circulating in Italian waters since early 2016 has caused acute/subacute lesions associated with positive immunolabelling of the virus. To date, myelin damage has not been fully documented and investigated in cetaceans. This study describes neuropathological findings in the brain tissue of 31 cetaceans found stranded along the Italian coastline and positive for DMV infection on molecular testing. Cell changes in the areas of myelinopathy were revealed by double indirect immunofluorescence. The most frequent DMV-associated lesions were astro-microgliosis, neuronal necrosis, spongiosis, malacia, and non-suppurative meningoencephalitis. Myelin reduction and areas of demyelination were revealed by means of a specific myelin biomarker. Morbilliviral antigen immunolabelling was mainly observed in neurons and microglial cells, in association with a marked activation of microglia and astrocytes. These findings extend our knowledge of DMV-associated brain lesions and shed light on their pathogenesis.

RT-QuIC detection of pathological prion protein in subclinical goats following experimental oral transmission of L-type BSE.

OBJECTIVE: The spread of bovine spongiform encephalopathy (BSE) agent to small ruminants is still a major issue in the surveillance of transmissible spongiform encephalopathies (TSEs). L-type bovine spongiform encephalopathy (L-BSE) is an atypical form of BSE with an unknown zoonotic potential that is transmissible to cattle and small ruminants. Our current knowledge of bovine atypical prion strains in sheep and goat relies only on experimental transmission studies by intracranial inoculation. To assess oral susceptibility of goats to L-BSE, we orally inoculated five goats with cattle L-BSE brain homogenates and investigated pathogenic prion protein (PrPsc) distribution by an ultrasensitive in vitro conversion assay known as Real-Time Quaking Induced Conversion (RT-QuIC). RESULTS: Despite a prolonged observation period of 80 months, all these animals and the uninfected controls did not develop clinical signs referable to TSEs and tested negative by standard diagnostics. Otherwise, RT-QuIC analysis showed seeding activity in five out of five examined brain samples. PrPsc accumulation was also detected in spinal cord and lymphoreticular system. These results indicate that caprine species are susceptible to L-BSE by oral transmission and that ultrasensitive prion tests deserve consideration to improve the potential of current surveillance systems against otherwise undetectable forms of animal prion infections.

SARS-CoV-2, a Threat to Marine Mammals? A Study from Italian Seawaters.

Zoonotically transmitted coronaviruses were responsible for Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), causing the dramatic Coronavirus Disease-2019 (CoViD-19) pandemic, which affected public health, the economy, and society on a global scale. The impact of the SARS-CoV-2 pandemic permeated into our environment and wildlife as well; in particular, concern has been raised about the viral occurrence and persistence in aquatic and marine ecosystems. The discharge of untreated wastewaters carrying infectious SARS-CoV-2 into natural water systems that are home to sea mammals may have dramatic consequences on vulnerable species. The efficient transmission of coronaviruses raises questions regarding the contributions of virus-receptor interactions. The main receptor of SARS-CoV-2 is Angiotensin Converting Enzyme-2 (ACE-2), serving as a functional receptor for the viral spike (S) protein. This study aimed, through the comparative analysis of the ACE-2 receptor with the human one, at assessing susceptibility to SARS-CoV-2 for different species of marine mammals living in Italian waters. We also determined, by means of immunohistochemistry, ACE-2 receptor localization in the lung tissue from different cetacean species, in order to provide a preliminary characterization of ACE-2 expression in the marine mammal respiratory tracts. Furthermore, to evaluate if and how Italian wastewater management and coastal exposition to extreme weather events may led to susceptible marine mammal populations being exposed to SARS-CoV-2, geomapping data were carried out and overlapped. The results showed the potential SARS-CoV-2 exposure for marine mammals inhabiting Italian coastal waters, putting them at risk when swimming and feeding in specific risk areas. Thus, we highlighted the potential hazard of the reverse zoonotic transmission of SARS-CoV-2 infection, along with its impact on marine mammals regularly inhabiting the Mediterranean Sea, while also stressing the need for appropriate action in order to prevent further damage to specific vulnerable populations.

Evidence for Unknown Sarcocystis-Like Infection in Stranded Striped Dolphins (Stenella coeruleoalba) from the Ligurian Sea, Italy.

Two striped dolphins (SD1, SD2), stranded along the Ligurian coast of Italy, were diagnosed with a nonsuppurative meningoencephalitis associated with previously undescribed protozoan tissue cysts. As tissue cysts were morphologically different from those of Toxoplasma gondii, additional histopathological, immunohistochemical, ultrastructural, and biomolecular investigations were performed, aiming to fully characterize the organism. Histopathology revealed the presence of large Sarcocystis-like tissue cysts, associated with limited inflammatory lesions in all CNS areas studied. IHC was inconclusive, as positive staining with polyclonal antisera did not preclude cross-reaction with other Sarcocystidae coccidia. Applied to each animal, 11 different PCR protocols precluded a neural infection by Sarcocystis neurona, Sarcocystis falcatula, Hammondia hammondi, and Neospora caninum. T. gondii coinfection was confirmed only in dolphin SD2. Sarcocystis sp. sequences, showing the highest homology to species infecting the Bovidae family, were amplified from SD1 myocardium and SD2 skeletal muscle. The present study represents the first report of Sarcocystis-like tissue cysts in the brain of stranded cetaceans along with the first description of Sarcocystis sp. infection in muscle tissue of dolphins from the Mediterranean basin.

Ticks infesting humans and associated pathogens: a cross-sectional study in a 3-year period (2017-2019) in northwest Italy.

BACKGROUND: Tick-borne diseases are common throughout Europe. Ticks transmit pathogens to the host while feeding and together with mosquitoes, they are major vectors of infectious agents worldwide. In recent years, there has been a marked increase in the incidence of tick-bite events and tick-borne disease in northwest Italy, but information on the prevalence of tick-borne pathogens in ticks removed from humans remains scarce. To fill this gap, we report here the prevalence of tick bites and tick-borne pathogens documented for humans in Piedmont, northwest Italy, in the 3-year period 2017-2019. METHODS: Ticks attached to humans during 2017-2019 were collected from residents of urban and rural area by physicians and veterinarians working with local veterinary agencies. All ticks (n = 1290) were morphologically identified to the species level. A subset of ticks removed from children (age 0-18 years) and the elderly (> 70 years), both age groups considered to be at-risk populations, was screened by biomolecular analysis to detect pathogens (e.g. Rickettsia spp., Borrelia spp., Anaplasma spp.). Pathogen identity was confirmed by Sanger sequencing. RESULTS: Ticks were taxonomically assigned to ten species of six genera (Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus). Most belonged to the genus Ixodes: 1009 ticks (78.22%) were classified as Ixodes ricinus. A subset of 500 ticks collected from the two at-risk populations were subjected to PCR assay to determine the presence of Rickettsia spp., Borrelia spp., and Anaplasma spp. The overall prevalence of infection was 22.8% (n = 114; 95% confidence interval [CI]: 19.19-26.73%), meaning that at least one pathogen was detected: Rickettsia spp. (prevalence 15%, n = 76; 95% CI 12.17-18.65%); Borrelia spp. (prevalence 6.4%, n = 32; 95% CI 4.42-8.92%); and Anaplasma spp. (prevalence 1.2%, n = 6; 95% CI 0.44-2.6%). CONCLUSIONS: Our data underline the importance of surveillance in the epidemiology of tick-borne diseases and the implementation of strategies to control tick infestation and associated pathogens.

Early and Pre-Clinical Detection of Prion Seeding Activity in Cerebrospinal Fluid of Goats using Real-Time Quaking-Induced Conversion Assay.

Since 2005, two cases of natural bovine spongiform encephalopathies (BSE) have been reported in goats. Furthermore, experimental transmissions of classical (C-BSE) and atypical (L-BSE) forms of BSE in goats were also reported. To minimize further spreading of prion diseases in small ruminants the development of a highly sensitive and specific test for ante-mortem detection of infected animals would be of great value. Recent studies reported high diagnostic value of a second generation of cerebrospinal fluid (CSF) Real-Time Quaking-Induced Conversion (RT-QuIC) assay across a wide spectrum of human prions. Here, we applied this improved QuIC (IQ-CSF) for highly efficient detection of TSEs prion protein in goat cerebrospinal fluid. IQ-CSF sensitivity and specificity were evaluated on CSF samples collected at disease endpoint from goats naturally and experimentally infected with scrapie or bovine isolates of C-BSE and L-BSE, respectively. Next, CSF samples collected from L-BSE infected goats during pre-symptomatic stage were also analysed. PrPL-BSE associated seeding activity was detected at early time points after experimental inoculation, with an average time of 439 days before clinical symptoms appeared. Taken together these data are indicative of the great potential of this in vitro prion amplification assay as ante-mortem TSE test for live and asymptomatic small ruminants.

Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS.

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices.

Analysis of Gluten Content in Gluten-Free Pizza from Certified Take-Away Pizza Restaurants.

Currently, a strict gluten-free diet is the only treatment for celiac disease. In Italy, food service establishments and restaurants can be certified for providing gluten-free foods, including pizza restaurants that make both gluten-free pizza and traditional wheat-based pizza. With this study we analyzed the gluten content in samples of gluten-free pizza prepared and purchased at certified restaurants in the Turin metropolitan area. All samples, from 28 pizzas and 28 cooked dough bases, produced results below the test limit of detection, except for one sample of cooked dough, that tested positive for gluten but still below the warning level for celiac consumers (<20 ppm). Gluten-free pizza, as advertised in the restaurants surveyed, can be considered a safe option for gluten-free consumption. Attention to and compliance with good manufacturing practices, a requisite for obtaining gluten-free certification for restaurants, were noted to have a positive effect on the final product.

Outbreak of febrile gastroenteritis caused by Listeria monocytogenes 1/2a in sliced cold beef ham, Italy, May 2016.

In May 2016, two separate clusters of febrile gastroenteritis caused by Listeria monocytogenes were detected by the local health authority in Piedmont, in northern Italy. We carried out epidemiological, microbiological and traceback investigations to identify the source. The people affected were students and staff members from two different schools in two different villages located in the Province of Turin; five of them were hospitalised. The epidemiological investigation identified a cooked beef ham served at the school canteens as the source of the food-borne outbreak. L. monocytogenes was isolated from the food, the stools of the hospitalised pupils and the environment of the factory producing the cooked beef ham. All isolates except one were serotype 1/2a, shared an indistinguishable PFGE pattern and were 100% identical by whole genome sequencing (WGS). By combining a classical epidemiological approach with both molecular subtyping and WGS techniques, we were able to identify and confirm a Listeria gastroenteritis outbreak associated with consumption of sliced cold beef ham.

Thymus atrophy is an efficient marker of illicit treatment with dexamethasone in veal calves: Results from a triennial experimental study.

Glucocorticoids, used in a wide range of pathologies thank to their therapeutical properties, are also illegally used as growth-promoters in animal breeding even if the European Union regulates their use to protect consumers' health from the adverse effects of residues in food. The first aim of the study was to establish the applicability of two histological parameters - atrophy and cortex-medulla ratio - to detect glucocorticoids misuse in calves. The second aim was to concurrently test the potentiality of both parameters to discriminate between treated and untreated animals. One hundred and seventy-two male Friesian veal calves were raised for six months and divided into two groups: Group A (106 calves) was given dexamethasone per os for twenty days (0.4mg/day), Group B (66 calves) used as control. Thymic samples were microscopically examined. Fat infiltration was evaluated and a degree of atrophy, ranging from 1 to 3 (mild, moderate, severe) was attributed; thymic cortex-medulla ratio was calculated too. Fisher's exact test and a Wilcoxon-Mann-Whitney test were performed to investigate the differences in thymic atrophy and cortex-medulla ratio between the groups. Results demonstrate that the thymic atrophy grading was significantly increased in group A (p=0,006), whereas the cortex-medulla ratio was decreased (p<0,004) when compared to group B; moreover, the parallel testing with fixed degree of atrophy and cortex-medulla ratio cut-off thresholds optimize the sensitivity (90%) in the detection of glucocorticoids anabolic treatments. These data suggest that microscopic thymus analysis represent a valid tool for the screening and monitoring of glucocorticoid illicit treatments.

Phenotypical Variability in Bovine Spongiform Encephalopathy: Epidemiology, Pathogenesis, and Diagnosis of Classical and Atypical Forms.

After thirty years, bovine spongiform encephalopathy (BSE) still represents the biggest crisis in the field of food safety. Initially detected in the United Kingdom in 1986, BSE spread to many other countries all over the world, involving approximately 200,000 cattle. The origin of BSE is uncertain, but epidemiological studies suggest that the source was cattle feed prepared from prion-infected animal tissues. The implementation of the drastic measures, including the ban of meat and bone meal from livestock feed and the removal of specified risk material from the food chain, has eventually resulted in a significant decline of the epidemic. For many years, it was believed that the disease was caused by transmission of a single prion strain. However, since 2004 two types of BSE, with distinct phenotypical characteristics, have been detected in Italy and France. These atypical types are characterized by distinct Western Blot profiles of abnormal protease-resistant prion protein, named high-type (H-BSE) and low-type (L-BSE). At present, there is no comprehensive information about the origin of the atypical BSEs (sporadic vs. acquired), and data about the pathogenesis of both atypical forms are very limited as compared to the classical type (C-BSE). This chapter will provide a well-organized overview of what is known about classical and atypical BSE. It will review information on the main epidemiological features, pathogenesis, and the criteria for routine diagnosis based on rapid tests, histological, immunohistochemical, and Western blot analysis. Furthermore, a brief overview about the most recently in vitro techniques will be also provided.

Whole Blood Gene Expression Profiling in Preclinical and Clinical Cattle Infected with Atypical Bovine Spongiform Encephalopathy.

Prion diseases, such as bovine spongiform encephalopathies (BSE), are transmissible neurodegenerative disorders affecting humans and a wide variety of mammals. Variant Creutzfeldt-Jakob disease (vCJD), a prion disease in humans, has been linked to exposure to BSE prions. This classical BSE (cBSE) is now rapidly disappearing as a result of appropriate measures to control animal feeding. Besides cBSE, two atypical forms (named H- and L-type BSE) have recently been described in Europe, Japan, and North America. Here we describe the first wide-spectrum microarray analysis in whole blood of atypical BSE-infected cattle. Transcriptome changes in infected animals were analyzed prior to and after the onset of clinical signs. The microarray analysis revealed gene expression changes in blood prior to the appearance of the clinical signs and during the progression of the disease. A set of 32 differentially expressed genes was found to be in common between clinical and preclinical stages and showed a very similar expression pattern in the two phases. A 22-gene signature showed an oscillating pattern of expression, being differentially expressed in the preclinical stage and then going back to control levels in the symptomatic phase. One gene, SEL1L3, was downregulated during the progression of the disease. Most of the studies performed up to date utilized various tissues, which are not suitable for a rapid analysis of infected animals and patients. Our findings suggest the intriguing possibility to take advantage of whole blood RNA transcriptional profiling for the preclinical identification of prion infection. Further, this study highlighted several pathways, such as immune response and metabolism that may play an important role in peripheral prion pathogenesis. Finally, the gene expression changes identified in the present study may be further investigated as a fingerprint for monitoring the progression of disease and for developing targeted therapeutic interventions.

Confirmation of the progesterone receptor as an efficient marker of treatment with 17ß-estradiol in veal calves.

Under current European Union legislation the use of anabolic steroids in food-producing livestock is banned because of their long-term adverse effects on human health. We examined the expression profile of the immunohistochemical marker progesterone receptor in veal calves' sex accessory glands following experimental administration of anabolic compounds. The aim was to confirm the accuracy of the immunohistochemical approach in the detection of the over-expression of the progesterone receptor induced by the administration of sexual steroids at low levels (17ß-estradiol and nandrolone alone or in combination). A total of 217 male veal calves were randomly divided into four groups: group A (104 calves) treated with 17ß-estradiol (5 mg/head; 4 weekly injections); group B (20 calves) treated with nandrolone (50 mg/head; 4 weekly injections); group C (20 calves) treated with the association of the two steroids (5 mg estradiol + 50 mg nandrolone; 4 weekly injections); and group K (73 calves) kept as a control. All the sexual accessory glands were collected at the slaughterhouse (15 days after the last administration) and subjected to immunohistochemical staining with anti-progesterone receptor antibody. All the calves treated with 17ß-estradiol alone or in association with nandrolone (groups A and C) showed strong positivity, while nandrolone-treated calves and controls (groups B and K) gave negative results to the immunohistochemical investigation. The statistical analysis showed that the progesterone receptor is a significant predictor of 17ß-estradiol treatment alone or in association with nandrolone (p < 0.001): the immunohistochemical study resulted in 100% sensitivity (CI = 95%: 97.1-100%) and specificity (CI = 95%: 95.1-100%) for prostate and 99% sensitivity (CI = 95%: 95.6-100%) and 100% specificity (CI = 95%: 95.1-100%) for bulbo-urethral glands. The data confirm that this innovative biological approach offers a reliable tool to enhance the efficacy of the histological test to detect illegal treatments with estrogens alone or in association with androgens.

Egg and Milk Proteins as Hidden Allergens in Food: 5-Year (2010 to 2014) Results of Food Allergen Monitoring in Piedmont, Italy.

Cow's milk and egg allergies are two of the most common food allergies. Manufacturers of food products containing milk or eggs or their derivatives as an ingredient are required by European Union regulations to list their presence on the ingredient label. Under European Union legislation, member states are mandated to carry out food safety monitoring programs to verify compliance with food labeling requirements. Through the Regional Integrated Plan for Food Safety, the Piedmont (Italy) regional authority carries out an annual program to determine the presence of undeclared allergens in foods. In the 5-year period from 2010 to 2014, a total of 1,566 food samples were analyzed for the presence of hidden egg and milk proteins. The average positive percentage was 2.8% (3.6% egg and 2% milk proteins). Comparison between the allergen concentration and the published eliciting dose (ED) for egg proteins (0.03 mg) and for total milk proteins (0.1 mg) indicated a high risk of allergen exposure for sensitized consumers. The calculated exposure was up to 135× (for milk) the ED01 reported in the literature. Food manufacturers will need to improve their allergen control programs to reduce allergen exposure and risk.

Detection of cellular prion protein in exosomes derived from ovine plasma.

Prion protein (PrP) is present at extremely low levels in the blood of animals and its detection is complicated by the poor sensitivity of current standard methodologies. Interesting results have been obtained with recent advanced technologies that are able to detect minute amounts of the pathological PrP (PrPSc), but their efficiency is reduced by various factors present in blood. In this study, we were able to extract cellular PrP (PrPC) from plasma-derived exosomes by a simple, fast method without the use of differential ultracentrifugation and to visualize it by Western blotting, reducing the presence of most plasma proteins. This result confirms that blood is capable of releasing PrP in association with exosomes and could be useful to better study its role in the pathogenesis of transmissible spongiform encephalopathies.

Detection and discrimination of classical and atypical L-type bovine spongiform encephalopathy by real-time quaking-induced conversion.

Statutory surveillance of bovine spongiform encephalopathy (BSE) indicates that cattle are susceptible to both classical BSE (C-BSE) and atypical forms of BSE. Atypical forms of BSE appear to be sporadic and thus may never be eradicated. A major challenge for prion surveillance is the lack of sufficiently practical and sensitive tests for routine BSE detection and strain discrimination. The real-time quaking-induced conversion (RT-QuIC) test, which is based on prion-seeded fibrillization of recombinant prion protein (rPrPSen), is known to be highly specific and sensitive for the detection of multiple human and animal prion diseases but not BSE. Here, we tested brain tissue from cattle affected by C-BSE and atypical L-type bovine spongiform encephalopathy (L-type BSE or L-BSE) with the RT-QuIC assay and found that both BSE forms can be detected and distinguished using particular rPrPSen substrates. Specifically, L-BSE was detected using multiple rPrPSen substrates, while C-BSE was much more selective. This substrate-based approach suggests a diagnostic strategy for specific, sensitive, and rapid detection and discrimination of at least some BSE forms.