Evaluation of Biosecurity Measures in Pig Holdings in Slovenia as a Risk Assessment for the Introduction of African Swine Fever Virus.

African Swine Fever (ASF) is persistently spreading and hindering pork production in Europe. Slovenia is one of the last countries in Central Europe without a confirmed ASF case in domestic pigs or in wild boar. The aim of this study was to assess the current biosecurity implementation on different types of pig farms. Internal and external biosecurity status was determined in 17 commercial (CF), 15 non-commercial (NC), and 15 outdoor (O) farms. Data were collected using the Biocheck.UGent questionnaire and assessed in combination with the latest information on the wild boar population in Slovenia. Biosecurity was compared between farm types based on the assessment of 12 subcategories. Statistically significant differences (p < 0.05) were found in six subcategories: (i) purchase of pigs and semen, (ii) visitors and farmworkers, (iii) vermin and bird control, (iv) finishing unit, (v) measures between compartments and use of equipment, and (vi) cleaning and disinfection. The highest total biosecurity score (0-100%) was determined on CF with 64.59 ± 16.47%, followed by NC with 55.73 ± 10.67%, and O with 48.47 ± 8.20%. The density of the wild boar population was estimated from the number of wild boars per km2 per year, with 3 or more hunted wild boars per unit representing the highest density. Geolocation of farms on the wild boar population map showed that two O farms are at high risk and seven farms (1 O, 5 NC, and 1 CF) are at medium risk for disease transmission from wild to domestic pigs. Biosecurity measures must be tightened in some subcategories, especially in areas with a high density of wild boar.

Discrepancies between farmers' perceptions and actual animal welfare conditions on commercial pig farms.

Animal welfare is a multiparameteral concept that encompasses the physical and mental health of animals and includes various aspects such as physical wellbeing, absence of hunger and thirst, and ability to express motivated behavior, to which farmers usually attach different importance. The objectives of this study were to evaluate animal welfare on Slovenian commercial pig farms, to determine whether farmers' perceived importance of animal welfare differ from actual animal welfare on farms and to determine, if farmer's age, gender, their level of education and participation in vocational training have an influence. For that purpose, we created an Animal Welfare Protocol/Questionnaire for Pig Farms (AWQ/P-P) that assessed several parameters of animal welfare: (1) general status, (2) animal behavior, (3) health status, (4) living conditions, and (5) environmental conditions. Each parameter included at least five observation points and was scored on a 5-point scale. The same observation points were used to measure farmers' perceived importance of animal welfare and for observational assessment. Consequently, we were able to compare both statistically. Farmers from 14 (N = 14) large Slovenian pig farms participated in the study. Results show that farmers rate all parameters of animal welfare very highly. For them, animal health status is the most important, and environmental conditions are the least important factors for animal welfare. Observational inspections yielded significantly lower scores for animal welfare conditions than those obtained from farmer ratings. The highest correlations between farmers' perceptions and observational inspections were found for the parameters of animal behavior and environmental conditions. The results of this study also suggest that vocational training is a significant variable in increasing levels of pig welfare. Age, gender, and education level are not significant variables, however, farms led by older male farmers with lower level of education but involved in vocational training from different sources had slightly better welfare on the farm. This should be further investigated before making conclusions, due to our small sample size. The significance of the study is to identify deficiencies in pig welfare as perceived by farmers and consequently improve pig welfare.

A molecular survey and phylogenetic analysis of porcine circovirus type 3 using oral fluid, faeces and serum.

BACKGROUND: Porcine circovirus type 3 is the most recently discovered porcine circovirus, and an emerging pathogen. In this study the status of its presence on some Slovenian farms is reported. The effectiveness of the vaccine against porcine circovirus type 2 was assessed against porcine circovirus type 3. Group samples of oral fluid, faeces and individual serum samples were taken from six different pig categories and tested for presence of viral DNA, using both real time and conventional PCR. Positive samples were subjected to direct Sanger sequencing. Nucleotide sequences were analyzed and compared to GenBank PCV3 sequences. RESULTS: Positive samples were sent for genome sequencing, which confirmed the presence of virus in all different pig categories on five farms. A high to moderate correlation of strong statistical significance was found between individual serum samples, oral fluid and faeces. Slovenian PCV3 was found to be distributed in a way similar to that of other countries. Slovenian PCV3 nt sequences are highly related, sharing more than 99.5% nt identity. On one farm a commercially available vaccine against porcine circovirus type 2 was used on 3-week-old pigs. It did not affect the presence of porcine circovirus type 3 in oral fluid or sera of any of the seven age groups of pigs, each with two control groups. CONCLUSIONS: The results constitute the first discovery of the virus in Slovenia. Genome sequencing has revealed a high degree of similarity between Slovenian and GenBank isolates.

CRISPRa-mediated FOXP3 gene upregulation in mammalian cells.

BACKGROUND: Forkhead box P3+ (FOXP3 +) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis. Loss-of-function mutations of the FOXP3 gene in animal models and humans results in loss of differentiation potential into Treg cells and are responsible for several immune-mediated inflammatory diseases. Strategies of increasing FOXP3 expression represent a potential approach to increase the pool of Tregs within the lymphocyte population and may be employed in therapies of diverse autoimmune conditions. In the present study, a dCas9 CRISPR-based method was systematically employed to achieve upregulation and sustained high expression of endogenous FOXP3 in HEK293 and human Jurkat T cell lines through targeting of the core promotor, three known regulatory regions of the FOXP3 gene (CNS1-3), and two additional regions selected through extensive bioinformatics analysis (Cage1 and Cage2). RESULTS: Using an activator-domain fusion based dCas9 transcription activator, robust upregulation of FOXP3 was achieved, and an optimal combination of single guide RNAs was selected, which exerted an additive effect on FOXP3 gene upregulation. Simultaneous targeting of FOXP3 and EOS, a transcription factor known to act in concert with FOXP3 in initiating a Treg phenotype, resulted in upregulation of FOXP3 downstream genes CD25 and TNFR2. When compared to ectopic expression of FOXP3 via plasmid electroporation, upregulation of endogenous FOXP3 via the Cas9-based method resulted in prolonged expression of FOXP3 in Jurkat cells. CONCLUSIONS: Transfection of both HEK293 and Jurkat cells with dCas9-activators showed that regulatory regions downstream and upstream of FOXP3 promoter can be very potent transcription inducers in comparison to targeting the core promoter. While introduction of genes by conventional methods of gene therapy may involve a risk of insertional mutagenesis due to viral integration into the genome, transient up- or down-regulation of transcription by a CRISPR-dCas9 approach may resolve this safety concern. dCas9-based systems provide great promise in DNA footprint-free phenotype perturbations (perturbation without the risk of DNA damage) to drive development of transcription modulation-based therapies.

Prochloraz and coumaphos induce different gene expression patterns in three developmental stages of the Carniolan honey bee (Apis mellifera carnica Pollmann).

The Carniolan honey bee, Apis mellifera carnica, is a Slovenian autochthonous subspecies of honey bee. In recent years, the country has recorded an annual loss of bee colonies through mortality of up to 35%. One possible reason for such high mortality could be the exposure of honey bees to xenobiotic residues that have been found in honey bee and beehive products. Acaricides are applied by beekeepers to control varroosis, while the most abundant common agricultural chemicals found in honey bee and beehive products are fungicides, which may enter the system when applied to nearby flowering crops and fruit plants. Acaricides and fungicides are not intrinsically highly toxic to bees but their action in combination might lead to higher honey bee sensitivity or mortality. In the present study we investigated the molecular immune response of honey bee workers at different developmental stages (prepupa, white-eyed pupa, adult) exposed to the acaricide coumaphos and the fungicide prochloraz individually and in combination. Expression of 17 immune-related genes was examined by quantitative RT-PCR. In treated prepupae downregulation of most immune-related genes was observed in all treatments, while in adults upregulation of most of the genes was recorded. Our study shows for the first time that negative impacts of prochloraz and a combination of coumaphos and prochloraz differ among the different developmental stages of honey bees. The main effect of the xenobiotic combination was found to be upregulation of the antimicrobial peptide genes abaecin and defensin-1 in adult honey bees. Changes in immune-related gene expression could result in depressed immunity of honey bees and their increased susceptibility to various pathogens.

Phenotypic characterization of Mycoplasma synoviae induced changes in the metabolic and sensitivity profile of in vitro infected chicken chondrocytes.

In infectious synovitis caused by Mycoplasma synoviae chicken chondrocytes (CCH) may come into direct contact with these bacteria that are also capable of invading CCH in vitro. In this study, phenotype microarrays were used to evaluate the influence of Mycoplasma synoviae on the global metabolic activity of CCH. Therefore, CCH were cultured in the presence of 504 individual compounds, spotted in wells of 11 phenotype microarrays for eukaryotic cells, and exposed to Mycoplasma synoviae membranes or viable Mycoplasma synoviae. Metabolic activity and sensitivity of normal cells versus infected cells were evaluated. Metabolic profiles of CCH treated with viable Mycoplasma synoviae or its membranes were significantly different from those of CCH alone. CCH treated with Mycoplasma synoviae membranes were able to use 48 carbon/nitrogen sources not used by CCH alone. Treatment also influenced ion uptake in CCH and intensified the sensitivity to 13 hormones, 5 immune mediators, and 29 cytotoxic chemicals. CCH were even more sensitive to hormones/immune mediators when exposed to viable Mycoplasma synoviae. Our results indicate that exposure to Mycoplasma synoviae or its membranes induces a wide range of metabolic and sensitivity modifications in CCH that can contribute to pathological processes in the development of infectious synovitis.

Effect of Mycoplasma synoviae and lentogenic Newcastle disease virus coinfection on cytokine and chemokine gene expression in chicken embryos.

Mycoplasma synoviae and Newcastle disease virus (NDV) are 2 avian pathogens that cause modulation in expression of a variety of cytokine and chemokine genes in chickens. However, there is limited data about gene modulation after coinfection with these 2 pathogens and even less data about gene modulation after infection of chicken embryos. In this study, the effect of M. synoviae type strain WVU 1853 and lentogenic LaSota vaccine strain of NDV infection on cytokine and chemokine gene expression in chicken embryos was analyzed in the liver, spleen, bursa of Fabricius, and thymus by using quantitative real-time PCR. Three types of infection were performed; infection with M. synoviae on d 10, infection with NDV on d 17; and consecutive infection with both pathogens, where M. synoviae was inoculated on d 10 and NDV on d 17. Thus, simulation of consecutive infection that may occur after NDV infection of the M. synoviae-infected host was performed. Mycoplasma synoviae infection of embryos resulted in intensive upregulation of cytokine and chemokine genes, including interferon (IFN)-γ, IL-1ß, IL-6, IL-12p40, IL-16, IL-18, MIP-1ß (CCL4), inducible nitric oxide synthase (iNOS), XCL1, and lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF), with different expression profiles in the 4 organs. Inoculation of lentogenic NDV significantly upregulated IFN-γ, IL-6, and IL-16 genes in spleen and IFN-γ, IL-1ß, IL-2, IL-16, IL-21, XCL1, and MIP-1ß (CCL4) genes in the thymus, but to a lesser extent than M. synoviae. However, no genes were upregulated by NDV in the liver and bursa of Fabricius. Overall effect of NDV inoculation, regarding the number of modulated cytokine and chemokine genes and the extent of expression, was lower than M. synoviae. When NDV was introduced after on-going M. synoviae infection, most M. synoviae-induced cytokine and chemokine genes were significantly downregulated. This study provides the first evidence in chicken embryos that consecutive infection with NDV could suppress expression of cytokine and chemokine genes being significantly upregulated by the previous M. synoviae infection.

Diacylated lipopeptide from Mycoplasma synoviae mediates TLR15 induced innate immune responses.

Avian-specific toll like receptor 15 (TLR15) is functionally equivalent to a group of TLR2 family proteins that the mammalian innate immune system utilizes to recognize a broad spectrum of microbe-associated molecular patterns, including bacterial lipoproteins. In this study we examined the role of chicken TLR2 family members in the innate immune response to the avian pathogenic bacterium, Mycoplasma synoviae. We found that Mycoplasma synoviae, and specifically the N-terminal diacylated lipopeptide (MDLP) representing the amino-terminal portion of its mature haemagglutinin protein, significantly induces the expression of TLR15, but not TLR1 and TLR2 in chicken macrophages and chondrocytes. TLR15 activation is specific and depends on diacylation of the lipopeptide. Activation of TLR15 after stimulation with Mycoplasma synoviae and MDLP triggers an increase in the expression of transcription factor nuclear factor kappa B and nitric oxide production. Moreover, transfection of avian macrophage cells with small interfering RNA reduces the expression of TLR15 after stimulation with MDLP. This leads to decreased activation of the innate immune response, as measured by nitric oxide production. Additionally, pretreatment of cells with neutralizing anti-TLR15 antibody results in a notable attenuation of MDLP-driven release of nitric oxide. This positive correlation may constitute a mechanism for stimulating the innate immune response against avian mycoplasmas in chicken cells via TLR15.

Mycoplasma synoviae induces upregulation of apoptotic genes, secretion of nitric oxide and appearance of an apoptotic phenotype in infected chicken chondrocytes.

The role of chondrocytes in the development of infectious arthritis is not well understood. Several examples of mycoplasma-induced arthritis in animals indicate that chondrocytes come into direct contact with bacteria. The objective of this study was to analyze the interaction of an arthrogenic Mycoplasma synoviae strain WVU 1853 with chicken chondrocytes. We found that M. synoviae significantly reduces chondrocyte respiration. This was accompanied by alterations in chondrocyte morphology, namely cell shrinkage and cytoplasm condensation, as well as nuclear condensation and formation of plasma membrane invaginations containing nuclear material, which appeared to cleave off the cell surface. In concordance with these apoptosis-like events in chondrocytes, transcription was increased in several pro-apoptotic genes. Twenty-four hours after infection, strong upregulation was assayed in NOS2, Mapk11, CASP8 and Casp3 genes. Twenty-four and 72 h incubation of chondrocytes with M. synoviae induced upregulation of AIFM1, NFκB1, htrA3 and BCL2. Casp3 and NOS2 remained upregulated, but upregulation ceased for Mapk11 and CASP8 genes. Increased production of nitric oxide was also confirmed in cell supernates. The data suggests that chicken chondrocytes infected with M. synoviae die by apoptosis involving production of nitric oxide, caspase 3 activation and mitochondrial inactivation. The results of this study show for the first time that mycoplasmas could cause chondrocyte apoptosis. This could contribute to tissue destruction and influence the development of arthritic conditions. Hence, the study gives new insights into the role of mycoplasma infection on chondrocyte biology and development of infectious arthritis in chickens and potentially in humans.

Mechanisms of an autoimmunity syndrome in mice caused by a dominant mutation in Aire.

Homozygous loss-of-function mutations in AIRE cause autoimmune polyglandular syndrome type 1 (APS 1), which manifests in a classic triad of hypoparathyroidism, adrenal insufficiency, and candidiasis. Interestingly, a kindred with a specific G228W AIRE variant presented with an autosomal dominant autoimmune phenotype distinct from APS 1. We utilized a novel G228W-knockin mouse model to show that this variant acted in a dominant-negative manner to cause a unique autoimmunity syndrome. In addition, the expression of a large number of Aire-regulated thymic antigens was partially inhibited in these animals, demonstrating the importance of quantitative changes in thymic antigen expression in determining organ-specific autoimmunity. Furthermore, the dominant-negative effect of the G228W variant was exerted through recruitment of WT Aire away from active sites of transcription in the nucleus of medullary thymic epithelial cells in vivo. Together, these results may demonstrate a mechanism by which autoimmune predisposition to phenotypes distinct from APS 1 can be mediated in a dominant-negative fashion by Aire.

AIRE recruits P-TEFb for transcriptional elongation of target genes in medullary thymic epithelial cells.

AIRE is a transcriptional activator that directs the ectopic expression of many tissue-specific genes in medullary thymic epithelial cells, which plays an important role in the negative selection of autoreactive T cells. However, its mechanism of action remains poorly understood. In this study, we found that AIRE regulates the step of elongation rather than initiation of RNA polymerase II. For these effects, AIRE bound and recruited P-TEFb to target promoters in medullary thymic epithelial cells. In these cells, AIRE activated the ectopic transcription of insulin and salivary protein 1 genes. Indeed, by chromatin immunoprecipitation, we found that RNA polymerase II was already engaged on these promoters but was unable to elongate in the absence of AIRE. Moreover, the genetic inactivation of cyclin T1 from P-TEFb abolished the transcription of AIRE-responsive genes and led to lymphocytic infiltration of lacrimal and salivary glands in the CycT1-/- mouse. Our findings reveal critical steps by which AIRE regulates the transcription of genes that control central tolerance in the thymus.

Oligomerization of HEXIM1 via 7SK snRNA and coiled-coil region directs the inhibition of P-TEFb.

Transcriptional elongation of most eukaryotic genes by RNA polymerase II requires the kinase activity of the positive transcription elongation factor b (P-TEFb). The catalytically active P-TEFb complex becomes inactive when sequestered into the large complex by the cooperative actions of 7SK snRNA and HEXIM1. In this study, we report that HEXIM1 forms oligomers in cells. This oligomerization is mediated by its predicted coiled-coil region in the C-terminal domain and 7SK snRNA that binds a basic region within the central part of HEXIM1. Alanine-mutagenesis of evolutionary conserved leucines in the coiled-coil region and the digestion of 7SK snRNA by RNase A treatment prevent this oligomerization. Importantly, mutations of the N-terminal part of the coiled-coil region abrogate the ability of HEXIM1 to bind and inhibit P-TEFb. Finally, the formation of HEXIM1 oligomers via the C-terminal part of the coiled-coil or basic regions is critical for the inhibition of transcription. Our results suggest that two independent regions in HEXIM1 form oligomers to incorporate P-TEFb into the large complex and determine the inhibition of transcriptional elongation.