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One challenge for liver-directed gene therapy is sufficient vector delivery to the target tissue while minimizing loss of the applied vector dose to other tissues. Infusion via peripheral veins is the least invasive approach; however, it results in systemic diffusion and substantial vector dilution. Here, we describe a safe and minimally invasive method to deliver adeno-associated virus (AAV) vectors to the liver of small weaned pigs by ultrasound-guided percutaneous trans-hepatic portal vein injection. 4-week-old piglets were infused with ∼2.5×10$^{14}$vector genomes comprising a dual-rAAV2/9 vector system with a split adenine base editor forin vivoinactivation ofPCSK9to reduce LDL-cholesterol levels. Animals had no signs of discomfort and tolerated the procedure well. However, despite 45% editing of the target site with the applied adenine base editor system in cultivated porcine cells, we only found low amounts of AAV vector genomes and neither detectable transgene-expression nor successful editing in the treated pig livers. We hypothesize that rapid proliferation of pig hepatocytes caused AAV vector dilution, leading to a loss of the vectors from the nucleus, and hence insufficient base editor protein expression for achieving detectable editing rates. Nonetheless, ultrasound-guided percutaneous transhepatic injection to the portal vein is well-tolerated in piglets and has potential for human (neonate) application.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory infectious disease responsible for global economic losses in the pig industry. From a monitoring perspective as well as due to the different courses of disease associated with the various serovars, it is essential to distinguish them in different herds or countries. In this study, we developed a novel high resolution melting (HRM) assay based on reference strains for each of the 19 known serovars and additional 15 clinical A. pleuropneumoniae isolates. The novel HRM comprises the species-specific APP-HRM1 and two serovar-specific HRM assays (APP-HRM2 and APP-HRM3). APP-HRM1 allowed polymerase chain reaction (PCR) amplification of apxIV resulting in an A. pleuropneumoniae specific melting curve, while nadV specific primers differentiated biovar 2 from biovar 1 isolates. Using APP-HRM2 and APP-HRM3, 13 A. pleuropneumoniae serovars can be determined by inspecting the assigned melting temperature. In contrast, serovar 3 and 14, serovar 9 and 11, and serovar 5 and 15 have partly overlapping melting temperatures and thus represent a challenge to accurately distinguish them. Consequently, to unambiguously ensure the correct assignment of the serovar, it is recommended to perform the serotyping HRM assay using a positive control for each serovar. This rapid and user-friendly assay showed high sensitivity with 1.25 fg-125 pg of input DNA and a specificity of 100% to identify A. pleuropneumoniae. Characteristic melting patterns of amplicons might allow detecting new serovars. The novel HRM assay has the potential to be implemented in diagnostic laboratories for better surveillance of this pathogen.

Hepatitis E virus (HEV) is an important cause of acute hepatitis in humans worldwide. In industrialised countries, most infections are caused by the zoonotic genotype 3. The main reservoir was found in pigs, with fattening pigs as the main shedders. The aim of this study was to establish a screening tool to detect HEV in pig farms. HEV-positive samples were sequenced using Sanger sequencing. First, different sample materials, including floor swabs, slurry, dust swabs and faeces were tested for HEV. Floor swabs turned out to give the best results and, in the form of sock swabs, were used for the screening of Swiss pig herds. A total of 138 pig farms were tested, with a focus on fattening pigs. Overall, 81 farms (58.8%) were HEV positive. Most sequences belonged to subtype 3h, in which they formed a specific cluster (Swiss cluster). In addition, subtype 3 l and two unassigned sequences were detected. As a conclusion, sock swabs were found to be a helpful tool to screen pig herds for HEV and establish a sequence collection that may enable molecular epidemiology and support outbreak investigation and prevention.

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modifications after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a mass-spectrometry-based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa domesticus), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.

Actinobacillus pleuropneumoniae serotype 19 is a very recently described new serotype with a novel type II capsule synthesis locus. Here, we report the draft genome sequences of two Actinobacillus pleuropneumoniae serotype 19 strains with a serogroup 3/6/8/12/15 O-antigen locus that were isolated in 2018 and 2021 from two different pig farms in Switzerland.

Hepatitis E caused by hepatitis E viruses of the genotype 3 (HEV-3) is a major health concern in industrialized countries and due to its zoonotic character requires a “One Health” approach to unravel routes and sources of transmission. Knowing the viral diversity present in reservoir hosts, i.e., pigs but also wild boars, is an important prerequisite for molecular epidemiology. The aim of this study was to gain primary information on the diversity of HEV-3 subtypes present along the food chain in Switzerland, as well as the diversity within these subtypes. To this end, samples of domestic pigs from slaughterhouses and carcass collection points, as well as from hunted wild boars, were tested for HEV RNA and antibodies. HEV positive meat products were provided by food testing labs. The HEV subtypes were determined using Sanger and next generation sequencing. The genetic analyses confirmed the predominance of a Swiss-specific cluster within subtype HEV-3h in pigs, meat products, and wild boars. This cluster, which may result from local virus evolution due to the isolated Swiss pig industry, supports fast differentiation of domestic and imported infections with HEV.

Porcine circovirus type 2 (PCV2) infections and resulting diseases are a worldwide threat to pig production. PCV2 bears a uniqueness that allows for us to understand more about chronic infections and the immune system in general. The virus can be phylogenetically subdivided into PCV2a to PCV2h genotypes. Although vaccination against PCV2 has been seen to prevent the manifestation of PCV disease, PCV2 still lingers as subclinical infections in all developmental stages of pigs. The “slow and low” tactic gives PCV2 a particular advantage in a host’s immune surveillance. Since the inception of the PCV2 associated panzootic, research scientists have been trying to understand the pathogenicity of PCV2. Different research groups found that one genotype group member was more pathogenic than others. We found, in our weaner infection model with in vivo transfection of different recombinant PCV2 genotype group members that these viruses alter T cell maturation in the thymus, including host’s central tolerance. Here, we extend these original observations by showing that PCV2 infected cells were also found in proximity within the female and male reproductive organs of stillborn pig fetuses. These PCV2 pools were sufficient in infecting three and half-day-old embryos in sows. Furthermore, the dominant PCV2 group member was more pathogenic in our weaner infection model. PCV2 pre-immunocompetence infection makes PCV2 recognized by central immune tolerance as belonging to the host. This also explains why pathogenicity is not a genetically intrinsic characteristic of PCV2; however, the dominance of any one PCV2 genotype group member leads to a more efficient deletion of the T cells against that specific genotype group member in the thymus.

Introduction: While treatment frequency as an indicator of antimicrobial consumption is often assessed using defined doses, it can also be calculated directly as an Animal Treatment Index (ATI). In this study, the correlation of calculating antimicrobial usage on Swiss pig farms using either national Defined Daily Doses (DDDch) or an ATI (number of treatments per animal per year) and the agreement between the different methods for the identification of high usage farms were investigated. Material and Methods: The antimicrobial consumption of 893 Swiss pig herds was calculated separately for suckling piglets, weaned piglets, fattening pigs, lactating and gestating sows using the indicators nDDDch (number of DDDch) per animal per year and ATI. Correlations between the indicators were investigated by calculating Spearman's Rho coefficients. The 5, 10, and 25% highest usage farms were determined by applying both methods and the interrater reliability was described using Cohen's Kappa coefficients and visualized by Bland-Altman plots. Results: The Spearman's Rho coefficients showed strong correlations (r > 0.5) between nDDDch/animal/year and ATI. The lowest coefficient was shown for the correlation of both indicators in gestating sows (r = 0.657) and the highest in weaned piglets (r = 0.910). Kappa coefficients identifying high usage farms were the highest in weaned piglets (k = 0.71, 0.85, and 0.91, respectively for 5, 10, and 25% most frequent users) and the lowest in gestating sows (k = 0.54, 0.58, and 0.55 for 5, 10, and 25% most frequent users). Conclusions: In general, the investigated indicators showed strong correlations and a broad agreement in terms of the calculated levels of antimicrobial usage and the identification of high usage farms. Nevertheless, a certain proportion of the farms were defined differently depending on the indicator used. These differences varied by age category and were larger in all age categories except weaned piglets when a higher perc

Background: In 2015, in Switzerland the Suissano Health Programme was implemented in pig production to improve transparency for antimicrobial usage (AMU) and to reduce the usage of fluoroquinolones (FQ), macrolides and cephalosporins, representing highest priority critically important antimicrobials. Methods: In the presented cohort study, the impact of the Suissano programme on the AMU of 291 pig farms between 2016 and 2017 was investigated. AMU was calculated in total numbers of defined course doses (nDCDch) for all farms in the programme. For each single farm the nDCDch/animal/year was determined for four different age categories (suckling piglets, weaned piglets, fattening pigs, sows) as well as each antimicrobial substance separately. Trends between 2016 and 2017 were investigated for all farms as well as the 25 per cent with the highest usage of antimicrobials (high users) separately. Results: Total AMU measured in nDCDch declined by 23 per cent between 2016 and 2017, but statistically significant differences could not be observed when comparing the data sets of the individual farms. A significantly reduced usage of FQ could be demonstrated in suckling piglets (P=0.003), weaned piglets (P=0.006) and sows (P=0.008) in 2017 compared with 2016. For high users, a significant reduction of total AMU could be shown in suckling piglets (P=0.02), weaned piglets (P=0.0004) and fattening pigs (P=0.01). Conclusion: This study demonstrated a significant reduction in the usage of FQs in suckling piglets, weaned piglets and sows as well as total AMU in suckling piglets, weaned piglets and fattening pigs on high-usage farms.