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In the equestrian world, two different types of management can be distinguished: traditional management and natural boarding. The aim of this research was to compare hormonal, hematological and immunological parameters of 47 horses kept in these two different managements. Blood and horsehair of the horses were sampled to determine DHEA (dehydroepiandrosterone) and cortisol concentration through RIA. Moreover, blood count was conducted, and flow cytometry was employed to phenotype lymphocyte subpopulations. Results showed that, in horsehair, DHEA concentration was significantly higher in natural horses, whereas cortisol concentration and cortisol/DEHA ratio significantly lower. These hormonal parameters are used to assess the stress condition and the welfare of animals. The most favorable endocrine framework found in horses kept in natural boarding suggests that this management conveys most with ethological and physiological needs of the species. The research underlines the need of a modification of horses' husbandry systems. For the first time, this study validates the assay of DHEA in horsehair. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is an issue of global concern that includes human, animal, and environmental health. To tackle this phenomenon, a One Health approach is required through the involvement of all these interconnected elements. The environment poses challenges for investigation, but wildlife animals, not directly exposed to antibiotic treatments and interacting with their habitats, can serve as indicators of AMR contamination. Specifically, wild birds could play a significant role in dissemination of AMR, as they can acquire AMR bacteria from wildlife reservoirs and disperse them through environments. This study aims to assess the prevalence of AMR in commensal bacteria isolated from wild birds and their role as bioindicators of environmental AMR. A total of 73 birds belonging to various species were sampled in the Emilia-Romagna region with buccal, cloacal and feather samplings. The samples were cultured on selective media, colonies were identified using MALDI-TOF technology and antimicrobial susceptibility to different drugs was assessed using the Kirby-Bauer method. The birds' data were statistically evaluated in relation to AMR percentages. In total, 117 bacterial strains were isolated, belonging to 23 genera and 46 different bacterial species. The highest non-susceptibility percentages were observed for tetracycline (12.2 %) and enrofloxacin (8.6 %) considering all bacterial isolates, as well as for oxacillin (46.8 %), clindamycin (29.3 %) and rifampicin (20.8 %), among Gram-positive isolates. In the statistical analysis, a higher AMR percentage was correlated with Gram-positive isolates from birds belonging to rural/urban habitat (p = 0.01). Among Gram-positives, a higher oxacillin non-susceptibility percentage was found to be associated with isolates from birds sampled in province of Bologna (p = 0.007), a higher enrofloxacin non-susceptibility percentage revealed an association with rural/urban habitat (p = 0.02), while a higher non-susceptibility percentage towards rifampicin resulted associated with isolates from migratory birds (p = 0.031). In conclusion, this preliminary study suggests a potential role of wild birds as bioindicators for monitoring AMR contamination in the environment., Competing Interests: Declaration of competing interest We hereby declare no financial/personal interest or belief that could affect the objectivity of this research., (Copyright © 2024. Published by Elsevier Ltd.)

Colistin is a polymyxin antimicrobic mainly used to treat infection caused by multi-drug resistant Gram-negative bacteria. Mechanisms of colistin resistance are linked to the mobile colistin resistance (mcr) genes, which are transferable within mobile plasmids. Currently, there is limited research on the environmental dissemination of these genes. The behavioural and morphological characteristics of Apis mellifera L. make honey bees effective environmental bioindicators for assessing the prevalence of antimicrobial-resistant bacteria. This study aims to evaluate the colistin phenotypic and genotypic resistance in environmental Gram-negative bacteria isolated from foraging honey bees, across a network of 33 colonies distributed across the Emilia-Romagna region in Italy. Phenotypic resistances were determined through a microdilution assay using the minimum inhibitory concentration (MIC) with dilutions ranging from 0.5 μg/ml to 256 μg/ml. Strains with MIC values gather than 2 μg/ml were classified as resistant. Also, the identification of the nine mcr genes was carried out using two separate multiplex PCR assays. The study found that 68.5% of isolates were resistant and the genus with the higher resistance rates observed in Enterobacter spp. (84.5%). At least one mcr gene was found in 137 strains (53.3%). The most detected gene was mcr5 (35.3%), which was the most frequently detected gene in the seven provinces, while the least observed was mcr4 (4.8%), detected only in two provinces. These results suggested the feasibility of detecting specific colistin resistance genes in environmentally spread bacteria and understanding their distribution at the environmental level, despite their restricted clinical use. In a One-Health approach, this capability enables valuable environmental monitoring, considering the significant role of colistin in the context of public health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The role of small animal veterinary hospitals in the onset and dissemination of antimicrobial-resistant organisms (AMROs) is still not clear, and the implementation of an internal surveillance systems is a cost-effective tool to better understand their impact. The aim of this study was to describe a pilot program of active surveillance in a Spanish Veterinary Teaching Hospital, developed to estimate the detection frequency of AMROs in the commensal flora of patients and in the environment. Surveillance was focused on Methicillin-resistant Staphylococci (MRS), third generation cephalosporins resistant gram-negative bacteria (3GCR-GNB), and carbapenems-resistant gram-negative bacteria (CR-GNB). Oral and perirectal swabs were collected in the same dogs and cats hospitalized > 48 h, at their admission and before their discharge. Out of 50 patients sampled, 24% (12/50) were carriers at admission of at least one of the three investigated AMROs. Twenty-eight percent of patients (14/50) acquired at least one AMRO during the hospital stay. MRS detection frequency at admission was 12% (6/50), while acquisition was 6% (3/50). 3GCR-GNB detection frequency was 14% at admission (7/50) and acquisition 22% (11/50), while CR-GNB detection frequency was 2% at admission (1/50) and acquisition 2% (1/50). Environmental surveillance (98 samples) showed a total detection frequency of 22.4% for MRS (22/98), 2% for 3GCR-GNB and CR-GNB (2/98). Clinical staff' shoe soles showed high detection frequency for MRS (50%). 3GCR Escherichia coli was the most isolated species in patients (n = 17). The results show how active surveillance can be used as a tool to assess the impact of AMROs in veterinary hospitals to subsequently build up tailored control plans based on specific issues., Competing Interests: Declaration of Competing Interest All the authors disclose any financial and personal relationships with other people or organizations that could inappropriately influence (bias) the work. They all declare that there's no financial/personal interest or belief that could affect their objectivity., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The rise of antimicrobial resistance (AMR) is one of the most relevant problems for human and animal health. According to One Health Approach, it is important to regulate the use of antimicrobials and monitor the spread of AMR in the environment as well. Apis mellifera (L. 1758) colonies were used as bioindicators thanks to their physical and behavioural characteristics. During their foraging flights, bees can intercept small particles, including atmospheric particulate matter, etc., and also microorganisms. To date, the antimicrobial surveillance network is limited to the sanitary level but lacks into environmental context. This study aimed to evaluate the use of A. mellifera colonies distributed throughout the Emilia-Romagna region (Italy) as indicators of environmental antimicrobial-resistant bacteria. This was performed by creating a statistical predictive model that establishes correlations between environmental characteristics and the likelihood of isolating specific bacterial genera and antimicrobial-resistant strains. A total of 608 strains were isolated and tested for susceptibility to 19 different antimicrobials. Aztreonam-resistant strains were significantly related to environments with sanitary structures, agricultural areas and wetlands, while urban areas present a higher probability of trimethoprim/sulfamethoxazole-resistant strains isolation. Concerning genera, environments with sanitary structures and wetlands are significantly related to the genera Proteus spp., while the Escherichia spp. strains can be probably isolated in industrial environments. The obtained models showed maximum values of Models Accuracy and robustness (R 2 ) of 55 % and 24 %, respectively. The results indicate the efficacy of utilizing A. mellifera colonies as valuable bioindicators for estimating the prevalence of AMR in environmentally disseminated bacteria. This survey can be considered a good basis for the development of further studies focused on monitoring both sanitary and animal pathology, creating a specific network in the environments of interest., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Laura Bortolotti reports financial support was provided by Italian Ministry of Agriculture, Food Sovereignty and Forestry (MASAF). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Paratuberculosis is considered one of the most economically devastating infectious diseases of domestic livestock, and the most effective control strategy is a combination of 'test-and-cull' and on-farm biosecurity measures. In Italy, a Voluntary National Control Plan (VNCP) and guidelines have been introduced to reduce the impact of the disease, and farmers can voluntarily enroll in the control plan. The main aims of this study were: i) the description of the trend over a 4-year period on total, within-herd (WH) and between herd (BH) apparent seroprevalences observed in 64 dairy herds members of a mutual company located in Italy after the introduction of a proposed "Customized Control Plan" (CCP); ii) the evaluation of its effectiveness in terms of percentage of participating farms that decided to join the VNCP. Analyses on serum samples were performed with Enzyme-Linked Immuno Sorbent Assay (ELISA) method and revealed a general decrease in both total, WH and BH apparent seroprevalence. Total average apparent seroprevalence decreased from 2.39% in 2017 to 1% in 2020. Negative herds raised from 51.9% in 2017 to 71.1% in 2020, while farms with WH apparent seroprevalence > 5% decreased from 17.3% in 2017 to 4.4% in 2020. BH apparent seroprevalence decreased from 51.2% in 2017 to 29.2% in 2020. Among the 52 out of 64 herds that accepted to continue the proposed CCP after the first year, 41 (78.8%) joined in 2020 the VNCP, that assessed the health ranking of the herds. The results provide evidence that a control plan based on a farm-specific strategy and a subsidized testing process can effectively reduce the impact of paratuberculosis in dairy herds, especially in convincing farmers to continue in paratuberculosis control by joining the VNCP, including them in a National context and increasing their awareness of the disease., Competing Interests: Declaration of Competing Interest All the authors disclose any financial and personal relationships with other people or organizations that could inappropriately influence (bias) the work. They all declare that there's no financial/personal interest or belief that could affect their objectivity., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Multiple Sclerosis is a chronic neurological disease characterized by demyelination and axonal loss. This pathology, still largely of unknown etiology, carries within it a complex series of etiopathogenetic components of which it is difficult to trace the origin. An inflammatory state is likely to be the basis of the pathology. Crucial elements of the inflammatory process are the interactions between platelets and mast cells as well as the bacterial component of the intestinal microbiota. In addition, the involvement of mast cells in autoimmune demyelinating diseases has been shown. The present work tries to hang up on that Ariadne's thread which, in the molecular complexity of the interactions between mast cells, platelets, microbiota and inflammation, characterizes Multiple Sclerosis and attempts to bring the pathology back to the causal determinism of psychopathological phenomenology. Therefore, we consider the possibility that the original error of Multiple Sclerosis can be investigated in the genetic origin of the depressive pathology.

Alkaline electrolyzed water (REW) is known for its cleaning action. The aim of this work was to assess REW effectiveness in reducing microbial load on surfaces intended for contact with food. Stainlesssteel surfaces were experimentally contaminated, bacterial inactivation was tested before and after treatment with REW. Treatment with REW was operated spraying it on the contaminated plates until drying. Tests were conducted for Salmonella spp., Listeria spp., Staphylococcus aureus and Escherichia coli . The treatment revealed different degrees of sanitizing activity of REW on different bacterial species, with higher efficacy on E . coli and Salmonella spp. than S. aureus , Listeria spp.. Statistical analysis revealed a significant microbial load reduction (p<0.01) after treatment with REW, suggesting that it has a good disinfectant activity which, along with its easy and safe use, makes it a good alternative to many other more widely used disinfectants., Competing Interests: Conflict of interest: The authors declare no conflict of interest., (©Copyright: the Author(s).)

Staphylococcus aureus infection led to a case of goat abortion, and four veterinarians contracted S. aureus infection from the goat during and after the abortion. Three veterinarians assisted a doe during the dystocic delivery of a dead foetus. Seventy-two hours after the dystocia, which ended with the goat's death, the veterinarians who assisted during the kidding and the veterinarian who performed the necropsy showed the presence of multiple, isolated, painful pustules 1-5 mm in diameter located along their forearms and knees. S. aureus was isolated from the pustules of the veterinarians, the placenta and uterus of the goat, the organs (brain, thymus gland, abomasum, liver and spleen) of the foetus, the scrotum and eye swabs of the buck, and mammary pustules of another goat from the same herd. Histological analysis revealed purulent metritis and inflammation of the placental cotyledons. Additional investigations eliminated the chances of other infections. S. aureus isolates recovered from the veterinarians, goats, foetus and buck were sensitive to the tested anti-microbials and did not encode staphylococcal enterotoxin genes (sea, ser, sep, see, seg and sei). The isolates were closely related, as indicated by the results of Fourier-transform infrared spectroscopy and comparative whole-genome sequencing analysis. The results of this study clearly support the hypothesis that an episode of professional zoonosis was caused by S. aureus infection during the abortion and also highlight the need for bacterial subtyping in epidemiological surveys., (© 2021 Crown copyright. Zoonoses and Public Health published by Wiley-VCH GmbH.)

Background: Respiratory diseases are the second most common cause of illnesses in horses, their etiology can be viral, bacterial, immune-mediated, or mechanical (Racklyeft and Love DN, Aust Vet J 78:549-59, 2000; Austin et al., J Am Vet Med Assoc 207:325-328, 1995; Arroyo et al., J Vet Intern Med 31:894-900, 2017). Klebsiella variicola is a Gram-negative bacterium that was initially identified as an endophyte in soil and plants such as bananas, rice, sugar cane and maize but recent studies have identified this microorganism as an emerging pathogen in humans (Rodríguez-Medina et al., Emerg Microbes Infect 8:973-988, 2019; Fontana et al., J Clin Microbiol 57:e00825-18, 2019; Rosenblueth et al., Syst Appl Microbiol 27:27-35, 2004). This paper describes, for the first time to our knowledge, the isolation of K. variicola from pleural effusion in a male adult horse., Case Presentation: 17-years Italian Saddle Horse with respiratory distress and fever was admitted to the Veterinary Teaching Hospital of the Department of Veterinary Medical Sciences, University of Bologna. At home, the patient had undergone antibiotic therapy without clinical improvement. Vital signs on admission revealed an increased respiratory rate, tachycardia, pyrexia and weight loss. The animal was submitted for collateral examination including thoracic radiology and ultrasound and thoracoscopy that showed bilateral pleural effusion associated with multifocal pulmonary atelectasis. During the thoracoscopic examination, that confirmed the presence of a seropurulent pleural effusion, a sample of pleural fluid was collected and Gram-negative bacteria were isolated and subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that allowed the identification of K. variicola. The isolate was sensitive to amikacin, cefazolin, enrofloxacin, marbofloxacin, tetracycline, and trimethoprim-sulfamethoxazole;the horse was treated with Oxytetracycline and amikacin. Despite a general health improvement of the subject, the pleural effusion did not resolve after treatment., Conclusions: This paper describes, for the first time, the isolation of K. variicola in a horse with respiratory disease. The misidentification between K. variicola and K. pneumoniae has caused unawareness about significant aspects of this bacterial species. In fact, even though in animals the role of this bacterium is not clear, in humans it has been recognized as an emerging pathogen. The use of new methods for bacterial identification will probably lead to the isolation of a greater number of strains which will have to be studied to acquire knowledge that will be useful to clarify the clinical importance and relevance of K. variicola also in animals.

Although cells of mushroom-producing fungi typically contain paired haploid nuclei (n + n), most Armillaria gallica vegetative cells are uninucleate. As vegetative nuclei are produced by fusions of paired haploid nuclei, they are thought to be diploid (2n). Here we report finding haploid vegetative nuclei in A. gallica at multiple sites in southeastern Massachusetts, USA. Sequencing multiple clones of a single-copy gene isolated from single hyphal filaments revealed nuclear heterogeneity both among and within hyphae. Cytoplasmic bridges connected hyphae in field-collected and cultured samples, and we propose nuclear migration through bridges maintains this nuclear heterogeneity. Growth studies demonstrate among- and within-hypha phenotypic variation for growth in response to gallic acid, a plant-produced antifungal compound. The existence of both genetic and phenotypic variation within vegetative hyphae suggests that fungal individuals have the potential to evolve within a single generation in response to environmental variation over time and space.

The aim of the study was to investigate the combined effect of the manufacturing process followed by HPP treatment on the inactivation of Salmonella spp. in artificially contaminated coppa samples, in order to verify the ability of the combined processes to achieve the objective of a 5-log reduction of Salmonella spp. needed for exportation to the U.S. Fresh anatomical cuts intended for coppa production were supplied by four different delicatessen factories located in Northern Italy. Raw meat underwent experimental contamination with Salmonella spp. using a mixture of 3 strains. Surface contamination of the fresh anatomical cuts was carried out by immersion into inoculum containing Salmonella spp . The conditions of the HPP treatment were: pressure 593 MPa, time 290 seconds, water treatment temperature 14°C. Surface and deep samples were performed post contamination (T0), end of the cold phase (T1), end of process (Tend), and after HPP treatment (postHPP) and Salmonella spp. Enumerated. The results of this study show a significant reduction of Salmonella spp. all through the production process (P<0.01) for all companies, followed by an additional reduction of bacterial counts due to HPP treatment (P<0.01), both in superficial and deep contaminations (P<0.01). The superficial overall reduction resulted of 1.58 to 5.04 log CFU/g during the production process. HPP treatment resulted in a significant (P<0.01) superficial and deep decrease in Salmonella spp. enumeration varying from 0.61 to 4.01 log and from 1.49 to 4.13 log. According to the data presented in this study, only the combined approach of coppa manufacturing process followed by HPP treatment always led to a 5-log reduction of Salmonella spp. required by USDA/FSIS guidelines., Competing Interests: Conflict of interest: The authors declare no conflict of interest., (©Copyright: the Author(s).)

Microglia, a resident CNS macrophage, are dynamic cells, constantly extending and retracting their processes as they contact and functionally regulate neurons and other glial cells. There is far less known about microglia-vascular interactions, particularly under healthy steady-state conditions. Here, we use the male and female mouse cerebral cortex to show that a higher percentage of microglia associate with the vasculature during the first week of postnatal development compared with older ages and that the timing of these associations is dependent on the fractalkine receptor (CX3CR1). Similar developmental microglia-vascular associations were detected in the human brain. Using live imaging in mice, we found that juxtavascular microglia migrated when microglia are actively colonizing the cortex and became stationary by adulthood to occupy the same vascular space for nearly 2 months. Further, juxtavascular microglia at all ages associate with vascular areas void of astrocyte endfeet, and the developmental shift in microglial migratory behavior along vessels corresponded to when astrocyte endfeet more fully ensheath vessels. Together, our data provide a comprehensive assessment of microglia-vascular interactions. They support a mechanism by which microglia use the vasculature to migrate within the developing brain parenchyma. This migration becomes restricted on the arrival of astrocyte endfeet such that juxtavascular microglia become highly stationary and stable in the mature cortex. SIGNIFICANCE STATEMENT We report the first extensive analysis of juxtavascular microglia in the healthy, developing, and adult brain. Live imaging revealed that juxtavascular microglia within the cortex are highly motile and migrate along vessels as they are colonizing cortical regions. Using confocal, expansion, super-resolution, and electron microscopy, we determined that microglia associate with the vasculature at all ages in areas lacking full astrocyte endfoot coverage and motility of juxtavascular microglia ceases as astrocyte endfeet more fully ensheath the vasculature. Our data lay the fundamental groundwork to investigate microglia-astrocyte cross talk and juxtavascular microglial function in the healthy and diseased brain. They further provide a potential mechanism by which vascular interactions facilitate microglial colonization of the brain to later regulate neural circuit development., (Copyright © 2020 the authors.)

In this study the effect of the application of High Pressure Treatment (HPP) combined with four different manufacturing processes on the inactivation of Listeria innocua, used as a surrogate for L. monocytogenes, in artificially contaminated coppa samples was evaluated in order to verify the most suitable strategy to meet the Listeria inactivation requirements needed for the exportation of dry-cured meat in the U.S. Fresh anatomical cuts intended for coppa production were supplied by four different delicatessen factories located in Northern Italy. Raw meat underwent experimental contamination with Listeria innocua using a mixture of 5 strains. Surface contamination of the fresh anatomical cuts was carried out by immersion into inoculum containing Listeria spp . The conditions of the HPP treatment were: pressure 593 MPa, time 290 seconds, water treatment temperature 14°C. Listeria innocua was enumerated on surface and deep samples post contamination, resting, ripening and HPP treatment. The results of this study show how the reduction of the microbial load on coppa during the production process did not vary among three companies (P>0.05) ranging from 3.73 to 4.30 log CFU/g, while it was significantly different (P<0.01) for the fourth company (0.92 log CFU/g). HPP treatment resulted in a significant (P<0.01) deep decrease of L. innocua count with values ranging between 1.63-3.54 log CFU/g with no significant differences between companies. Regarding superficial contamination, HPP treatment resulted significant (P<0.01) only in Coppa produced by two companies. The results highlight that there were processes less effective to inhibit the pathogen; in particular for company D an increase of L. innocua count was shown during processing and HPP alone cannot be able to in reaching the Listeria inactivation requirements needed for exportation of dry-cured meat in the U.S. According to the data reported in this paper, HPP treatment increases the ability of the manufacturing process of coppa in reducing Listeria count with the objective of a lethality treatment., Competing Interests: Conflict of interest., (©Copyright: the Author(s).)

Mammalian gastrointestinal tract is colonized by a large number of microorganisms, known as gut microbiota, that play a key role in the physiological and pathological states. In particular, the gastrointestinal tract of dogs and cats harbors a complex and highly biodiverse microbial ecosystem. Recent studies see it involved in a wide range of life processes, including energy needs, metabolism, immunological activity, and neuro-behavioral development. This review focuses on the role of the microbiota on the health of pets and will discuss changes that occur in the disease.

Microglia rapidly respond to changes in neural activity and inflammation to regulate synaptic connectivity. The extracellular signals, particularly neuron-derived molecules, that drive these microglial functions at synapses remain a key open question. Here we show that whisker lesioning, known to dampen cortical activity, induces microglia-mediated synapse elimination. This synapse elimination is dependent on signaling by CX3CR1, the receptor for microglial fractalkine (also known as CXCL1), but not complement receptor 3. Furthermore, mice deficient in CX3CL1 have profound defects in synapse elimination. Single-cell RNA sequencing revealed that Cx3cl1 is derived from cortical neurons, and ADAM10, a metalloprotease that cleaves CX3CL1 into a secreted form, is upregulated specifically in layer IV neurons and in microglia following whisker lesioning. Finally, inhibition of ADAM10 phenocopies Cx3cr1 -/- and Cx3cl1 -/- synapse elimination defects. Together, these results identify neuron-to-microglia signaling necessary for cortical synaptic remodeling and reveal that context-dependent immune mechanisms are utilized to remodel synapses in the mammalian brain.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a genetic expansion of the CAG repeat region in the huntingtin (HTT) gene. Studies in HD mouse models have shown that artificial miRNAs can reduce mutant HTT, but evidence for their effectiveness and safety in larger animals is lacking. HD transgenic sheep express the full-length human HTT with 73 CAG repeats. AAV9 was used to deliver unilaterally to HD sheep striatum an artificial miRNA targeting exon 48 of the human HTT mRNA under control of two alternative promoters: U6 or CβA. The treatment reduced human mutant (m) HTT mRNA and protein 50-80% in the striatum at 1 and 6 months post injection. Silencing was detectable in both the caudate and putamen. Levels of endogenous sheep HTT protein were not affected. There was no significant loss of neurons labeled by DARPP32 or NeuN at 6 months after treatment, and Iba1-positive microglia were detected at control levels. It is concluded that safe and effective silencing of human mHTT protein can be achieved and sustained in a large-animal brain by direct delivery of an AAV carrying an artificial miRNA.

Background: Transgenic sheep are currently the only large animal model of Huntington's disease expressing full-length mutant human huntingtin. These transgenic sheep provide an opportunity to test adeno associated virus (AAV) therapies directly targeting the huntingtin gene. A recent study demonstrated that self-complementary (sc) AAV with artificial miRNA against human huntingtin reduced mutant human huntingtin in caudate and putamen after a single injection near the internal capsule., Objective: To identify an AAV serotype among AAVrh8, AAV9 and AAVrh10 with the highest neuronal uptake and distribution, with no obvious cell loss in the neostriatum of the sheep., Methods: We tested AAVrh8, AAV9 and AAVrh10 by stereotactic direct unilateral injection into the neostriatum of sheep, near the internal capsule. Four weeks after administration, we examined the viral spread and neuronal uptake of each serotype of AAV containing GFP. We compared single stranded (ss) and scAAVs. Further, we measured the distribution of AAVrh8 and AAV9 to a variety of tissues outside the brain., Results: Sc AAV9 had the best combination of neuronal uptake and distribution throughout the neostriatum. scAAVrh10 demonstrated good spread, but was not taken up by neurons. scAAVrh8 demonstrated good spread, but had less neuronal uptake than AAV9. Six hours after convection-enhanced administration to the neostriatum, both AAVrh8 and AAV9 viral genomes were detected in blood, saliva, urine, feces and wool. By four weeks, viral genomes were detected in wool only. Administration of AAVrh8, AAV9 and AAVrh10 was not associated with loss of neostriatal, medium spiny neuron number as measured by DARPP32 immunohistochemistry., Conclusions: Altogether, we found scAAV9 had the best neuronal uptake and spread, showed no loss of neurons at one-month post-injection, and was not measurable in body fluids one month after injection. This information will guide future clinical experiments requiring brain injection of AAV for therapeutics for gene or miRNA deliveries in sheep transgenic for the human huntingtin gene.

Background: Membranoproliferative glomerulonephritis (MPGN) is an uncommon cause of chronic nephropathy recently reclassified into immunoglobulin-associated MPGN (Ig-MPGN) and C3 glomerulopathy (C3G). In this study we aimed: (1) to evaluate the complement genetic and biochemical profile in patients with Ig-MPGN/C3G; (2) to investigate whether genetic variants and different patterns of complement activation (i.e., fluid versus solid phase) correlate with disease manifestations and outcomes., Methods: In 140 patients with idiopathic Ig-MPGN or C3G we performed complement biochemical and genetic screening and correlated genetic, biochemical and histology data with clinical features., Results: Mutations in genes encoding alternative pathway complement proteins were found in both Ig-MPGN and C3G, and mutations in the two components of the C3 convertase are the most prevalent. We also report a mutation in THBD encoding thrombomodulin in a C3G patient. The presence of mutations alone does not significantly increase the risk of Ig-MPGN or C3G, but it does so when combined with common susceptibility variants (CD46 c.-366A in Ig-MPGN; CFH V62 and THBD A473 in C3G). Finally, patients without complement gene mutations or C3NeFs--autoantibodies that stabilize the alternative pathway C3 convertase--have a higher risk of progressing to end-stage renal disease than patients with identified mutations and/or C3NeFs, suggesting the existence of different pathogenetic mechanisms that lead to renal disease., Conclusions: We provide new insights into the pathogenesis of Ig-MPGN/C3G that underscore the complex nature of these diseases and suggest that the current C3G classification may miss many cases associated with abnormalities of the complement alternative pathway., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: The immune system In Huntington's disease (HD) is activated and may overreact to some therapies. RNA interference using siRNA lowers mutant huntingtin (mHTT) protein but could increase immune responses., Objective: To examine the innate immune response following siRNA infusion into the striatum of wild-type (WT) and HD transgenic (YAC128) mice., Methods: siRNAs (2'-O-methyl phosphorothioated) were infused unilaterally into striatum of four month-old WT and YAC128 mice for 28 days. Microglia number and morphology (resting (normal), activated, dystrophic), cytokine levels, and DARPP32-positive neurons were measured in striatum immediately or 14 days post-infusion. Controls included contralateral untreated striatum, and PBS and sham treated striata., Results: The striata of untreated YAC128 mice had significantly fewer resting microglia and more dystrophic microglia than WT mice, but no difference from WT in the proportion of activated microglia or total number of microglia. siRNA infusion increased the total number of microglia in YAC128 mice compared to PBS treated and untreated striata and increased the proportion of activated microglia in WT and YAC128 mice compared to untreated striata and sham treated groups. Cytokine levels were low and siRNA infusion resulted in only modest changes in those levels. siRNA infusion did not change the number of DARPP32-positive neurons., Conclusion: Findings suggest that siRNA infusion may be a safe method for lowering mHTT levels in the striatum in young animals, since treatment does not produce a robust cytokine response or cause neurotoxicity. The potential long-term effects of a sustained increase in total and activated microglia after siRNA infusion in HD mice need to be explored.