This study was aimed to assessing haematological parameters in pigs of different age categories from six farms differing in the presence or absence of selected pathogens. The following categories ...were included: 5 age groups of growers, fatteners and breeding sows. Individual blood samples for determining complete blood count and white blood cell differential count were taken. Group samples of oral fluid and faeces were collected from each farm and tested for detection of Porcine Reproductive and Respiratory Virus (PRRSV), Porcine Circovirus Type 2 (PCV2), and Hepatitis E Virus (HEV) using PCR, RT-PCR, and qRT-PCR protocols. On farms free of PRRSV, PCV2 and HEV, the age of pigs had significant effect on: white blood cell count (WBC), haemoglobin concentration (Hb), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration, platelet count (PLT), percentage of neutrophils, lymphocytes and eosinophils and absolute numbers of neutrophils, lymphocytes, monocytes, basophils and 'large unstained cells' (LUCs). On positive farms all observed blood parameters except the percentage of LUCs were significantly affected by the age. The percentages of lymphocytes, mean corpuscular volume (MCV) and haematocrit were significantly lowered by PRRSV, while WBC, PLT, percentage and absolute numbers of neutrophils, basophils and LUCs were increased. Nine-week-old pigs with PCV2 had significantly lower red blood cell count, PLT, percentage and absolute numbers of monocytes and LUCs, and significantly higher MCV, MCH, percentage of neutrophils and percentage and absolute number of eosinophils than 9-week-old pigs with PCV2 and HEV. Age-related changes in haematological parameters occurred in PCR negative and positive farms.
HIGHLIGHTS
Age-related changes in haematological parameters were present in PCR negative farms and in PCR positive farms in relation to selected pathogens.
Values for several haematological parameters in 9-week-old pigs with a monoinfection differed significantly from those in pigs of the same age with two viral infections.
To interpret results of haematological analyses correctly, it is important to consider the haematological analyser used, the pig age and, health history, and clinical data.
Animal welfare is important; therefore, veterinarians and other animal welfare experts try to use different tools for pig welfare assessment. Several welfare protocols are available for pig welfare ...assessment, and one of the most used is Welfare Quality (WQ) protocol®. Elevated values of cortisol can be indicative of stress and, therefore, poor welfare. Our aim was to assess the correlation between serum cortisol levels from individual samples and oral fluid cortisol levels in group samples with the grades received for pig welfare using the WQ protocol®. Samples were taken at six different commercial pig farms. Animals were divided into age-dependent categories: 5 weeks old (w/o); 7 w/o; 9 w/o; 11 w/o weaners; fatteners; and breeding sows (10 pigs/category). Cortisol was determined in individual sera and group samples of oral fluid (OF), and was compared to values considered to be physiological. Based on WQ protocol® answers, five farms’ welfare level was deemed acceptable, and one was enhanced. Four out of 29 sera and 5 out of 30 OF samples were considered physiological, while in most other samples it was elevated. The correlation between cortisol levels in sera, OF, and WQ protocol® scores was not statistically significant. The cortisol level in OF should be just one of the welfare indicators, i.e., alongside the WQ protocol® filled out by a welfare expert.
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 (
< 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 km
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.
Forkhead box P3
(
) regulatory T cells (Tregs) are a subset of lymphocytes, critical for the maintenance of immune homeostasis. Loss-of-function mutations of the
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
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
in HEK293 and human Jurkat T cell lines through targeting of the core promotor, three known regulatory regions of the
gene (CNS1-3), and two additional regions selected through extensive bioinformatics analysis (Cage1 and Cage2).
Using an activator-domain fusion based dCas9 transcription activator, robust upregulation of
was achieved, and an optimal combination of single guide RNAs was selected, which exerted an additive effect on
gene upregulation. Simultaneous targeting of
and
, a transcription factor known to act in concert with
in initiating a Treg phenotype, resulted in upregulation of
downstream genes
and
. When compared to ectopic expression of
via plasmid electroporation, upregulation of endogenous
via the Cas9-based method resulted in prolonged expression of
in Jurkat cells.
Transfection of both HEK293 and Jurkat cells with dCas9-activators showed that regulatory regions downstream and upstream of
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.
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.
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.
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.
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.
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.
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.
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