Swine Industry Research Articles

Lipoteichoic acids influence cell shape and bacterial division of Streptococcus suis serotype 2, but play a limited role in the pathogenesis of the infection

Streptococcus suis serotype 2 is a major swine pathogen and a zoonotic agent, causing meningitis in both swine and humans, responsible for substantial economic losses to the swine industry worldwide. The pathogenesis of infection and the role of bacterial cell wall components in virulence have not been fully elucidated. Lipoproteins, peptidoglycan, as well as lipoteichoic acids (LTA) have all been proposed to contribute to virulence. In the present study, the role of the LTA in the pathogenesis of the infection was evaluated through the characterisation of a mutant of the S. suis serotype 2 strain P1/7 lacking the LtaS enzyme, which mediates the polymerization of the LTA poly-glycerolphosphate chain. The ltaS mutant was confirmed to completely lack LTA and displayed significant morphological defects. Although the bacterial growth of this mutant was not affected, further results showed that LTA is involved in maintaining S. suis bacterial fitness. However, its role in the pathogenesis of the infection appears limited. Indeed, LTA presence reduces self-agglutination, biofilm formation and even dendritic cell activation, which are important aspects of the pathogenesis of the infection caused by S. suis. In addition, it does not seem to play a critical role in virulence using a systemic mouse model of infection.

Highly pathogenic PRRSV upregulates IL-13 production through nonstructural protein 9–mediated inhibition of N6-methyladenosine demethylase FTO

Porcine reproductive and respiratory syndrome virus (PRRSV), a highly infectious and causes severe losses in the swine industry by regulating the inflammatory response, inducing tissue damage, suppressing the innate immune response, and promoting persistent infection in hosts. Interleukin-13 (IL-13) is a cytokine that plays a critical role in regulating immune responses and inflammation, particularly in immune-related disorders, certain types of cancer, and numerous bacterial and viral infections; however, the underlying mechanisms of IL-13 regulation during PRRSV infection are not well understood. In this study, we demonstrated that PRRSV infection elevates IL-13 levels in porcine alveolar macrophages (PAMs). PRRSV enhances m6A methylated RNA levels while reducing the expression of fat mass and obesity associated protein (FTO, an m6A demethylase), thereby augmenting IL-13 production. PRRSV nonstructural protein 9 (nsp9) was a key factor for this modulation. Furthermore, we found that the residues Asp567, Tyr586, Leu593, and Asp595 were essential for nsp9 to induce IL-13 production via attenuation of FTO expression. These insights delineate PRRSV nsp9's role in FTO-mediated IL-13 release, advancing our understanding of PRRSV's impact on host immune and inflammatory responses.

Humoral and Cellular Immune Responses Induced by Bivalent DNA Vaccines Expressing Fusion Capsid Proteins of Porcine Circovirus Genotypes 2a and 2b.

Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus-associated disease (PCVAD) that profoundly impacts the swine industry worldwide. While most of the commercial PCV vaccines are developed based on PCV genotype 2a (PCV2a), PCV genotype 2b (PCV2b) has become predominant since 2003. In this study, we developed and evaluated DNA-based bivalent vaccines covering both PCV2a and PCV2b. We generated a new immunogen, PCV2b-2a, by combining consensus sequences of the PCV2a and PCV2b capsid proteins (Cap2a and Cap2b) in a form of fusion protein. We also examined whether modifications of the PCV2b-2a fusion protein with a signal sequence (SS) and granulocyte macrophage-colony stimulating factor (GM-CSF) fusing with interleukine-4 (IL-4) (GI) could further improve the vaccine immunogenicity. An immunogenicity study of BALB/cAJcl mice revealed that the DNA vector pVAX1 co-expressing PCV2b-2a and GI (pVAX1.PCV2b-2a-GI) was most potent at inducing both antibody and cellular immune responses against Cap2a and Cap2b. Interestingly, the vaccines skewed the immune response towards Th1 phenotype (IgG2a > IgG1). By performing ELISA and ELISpot with predicted epitope peptides, the three most immunogenic B cell epitopes and five putative T cell epitopes were identified on Cap2a and Cap2b. Importantly, our DNA vaccines elicited broad immune responses recognizing both genotype-specific and PCV2-conserved epitopes. Sera from mice immunized with the DNAs expressing PCV2b-2a and PCV2b-2a-GI significantly inhibited PCV2a cell entry at serum dilution 1:8. All these results suggest a great potential of our PCV2b-2a-based vaccines, which can be further developed for use in other vaccine platforms to achieve both vaccine efficacy and economical production cost.

Contrasting PRRSV temporal lineage patterns at the individual farm, production system, and regional levels in Ohio and neighboring states from 2017 to 2021

Porcine reproductive and respiratory virus (PRRSV), one of the most significant viruses in the swine industry, has been challenging to control due to its high mutation and recombination rates and complexity. This retrospective study aimed to describe and compare the distribution of PRRSV lineages obtained at the individual farm, production system, and regional levels. PRRSV-2 (type 2) sequences (n = 482) identified between 2017 – 2021 were provided by a regional state laboratory (Ohio Department of Agriculture, Animal Disease Diagnostic Center (ODA-ADDL)) collected from swine farms in Ohio and neighboring states, including Indiana, Michigan, Pennsylvania, and West Virginia. Additional sequences (n = 138) were provided by one collaborating swine production system. The MUSCLE algorithm on Geneious Prime® was used to align the ORF5 region of PRRSV-2 sequences along with PRRSV live attenuated vaccine strains (n = 6) and lineage anchors (n = 169). Sequenced PRRSV-2 were assigned to the most identical lineage anchors/vaccine strains. Among all sequences (n = 620), 29.8% (185/620) were ≥ 98.0% identity with the vaccine strains, where 93.5% (173/185) and 6.5% (12/185) were identical with the L5 Ingelvac PRRS® MLV and L8 Fostera® PRRS vaccine strains, respectively, and excluded from the analysis. At the regional level across five years, the top five most identified lineages included L1A, L5, L1H, L1C, and L8. Among non-vaccine sequences with production system known, L1A sequences were mostly identified (64.3% – 100.0%) in five systems, followed by L1H (0.0% - 28.6%), L1C (0.0% – 10.5%), L5 (0.0% – 14.4%), L8 (0.0% – 1.3%), and L1F (0.0% – 0.5%). Furthermore, among non-vaccine sequences with the premise identification available (n = 262), the majority of sequences from five individual farms were either classified into L1A or L5. L1A and L5 sequences coexisted in three farms, while samples submitted by one farm contained L1A, L1H, and L5 sequences. Additionally, the lineage classification results of non-vaccine sequences were associated with their restriction fragment length polymorphism (RFLP) patterns (Fisher’s exact test, p < 0.05). Overall, our results show that individual farm and production system-level PRRSV-2 lineage patterns do not necessarily correspond to regional-level patterns, highlighting the influence of individual farms and systems in shaping PRRSV occurrence within those levels, and highlighting the crucial goal of within-farm and system monitoring and early detection for accurate knowledge on PRRSV-2 lineage occurrence and emergence.

Diltiazem HCl suppresses porcine reproductive and respiratory syndrome virus infection in susceptible cells and in swine

Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen for swine, resulting in substantial economic losses to the swine industry. However, there has been little success in developing effective vaccines or drugs for PRRSV control. In the present study, we discovered that Diltiazem HCl, an inhibitor of L-type Ca2+ channel, effectively suppresses PRRSV replication in MARC-145, PK-15CD163 and PAM cells in dose-dependent manner. Furthermore, it demonstrates a broad-spectrum activity against both PRRSV-1 and PRRSV-2 strains. Additionally, we explored the underlying mechanisms and found that Diltiazem HCl -induced inhibition of PRRSV associated with regulation of calcium ion homeostasis in susceptible cells. Moreover, we evaluated the antiviral effects of Diltiazem HCl in PRRSV-challenged piglets, assessing rectal temperature, viremia, and gross and microscopic lung lesions. Our results indicate that Diltiazem HCl treatment alleviates PRRSV-induced rectal temperature spikes, pulmonary pathological changes, and serum viral load. In conclusion, our data suggest that Diltiazem HCl could serve as a novel therapeutic drug against PRRSV infection.

Lipidomics reveals the significance and mechanism of the cellular ceramide metabolism for rotavirus replication.

As one of the most important causative agents of severe gastroenteritis in children, piglets, and other young animals, species A rotaviruses have adversely impacted both human health and the global swine industry. Vaccines against rotaviruses (RVs) are insufficiently effective, and no specific treatment is available. To understand the relationships between porcine RV (PoRV) infection and enterocytes in terms of the cellular lipid metabolism, we performed an untargeted liquid chromatography mass spectrometry (LC-MS) lipidomics analysis of PoRV-infected IPEC-J2 cells. Herein, a total of 451 lipids (263 upregulated lipids and 188 downregulated lipids), spanning sphingolipid, glycerolipid, and glycerophospholipids, were significantly altered compared with the mock-infected group. Interestingly, almost all the ceramides among these lipids were upregulated during PoRV infection. LC-MS analysis was used to validated the lipidomics data and demonstrated that PoRV replication increased the levels of long-chain ceramides (C16-ceramide, C18-ceramide, and C24-ceramide) in cells. Furthermore, we found that these long-chain ceramides markedly inhibited PoRV infection and that their antiviral actions were exerted in the replication stage of PoRV infection. Moreover, downregulation of endogenous ceramides with the ceramide metabolic inhibitors enhanced PoRV propagation. Increasing the levels of ceramides by the addition of C6-ceramide strikingly suppressed the replication of diverse RV strains. We further found that the treatment with an apoptotic inhibitor could reverse the antiviral activity of ceramide against PoRV replication, demonstrating that ceramide restricted RV infection by inducing apoptosis. Altogether, this study revealed that ceramides played an antiviral role against RV infection, providing potential approaches for the development of antiviral therapies.IMPORTANCERotaviruses (RVs) are among the most important zoonosis viruses, which mainly infected enterocytes of the intestinal epithelium causing diarrhea in children and the young of many mammalian and avian species. Lipids play an essential role in viral infection. A comprehensive understanding of the interaction between RV and lipid metabolism in the enterocytes will be helpful to control RV infection. Here, we mapped changes in enterocyte lipids following porcine RV (PoRV) infection using an untargeted lipidomics approach. We found that PoRV infection altered the metabolism of various lipid species, especially ceramides (derivatives of the sphingosine). We further demonstrated that PoRV infection increased the accumulation of ceramides and that ceramides exerted antiviral effects on RV replication by inducing apoptosis. Our findings fill a gap in understanding the alterations of lipid metabolism in RV-infected enterocytes and highlight the antiviral effects of ceramides on RV infection, suggesting potential approaches to control RV infection.

Strategies for Transboundary Swine Disease Management in Asian Islands: Foot and Mouth Disease, Classical Swine Fever, and African Swine Fever in Taiwan, Japan, and the Philippines.

Swine transboundary diseases pose significant challenges in East and Southeast Asia, affecting Taiwan, Japan, and the Philippines. This review delves into strategies employed by these islands over the past two decades to prevent or manage foot and mouth disease (FMD), classical swine fever (CSF), and African swine fever (ASF) in domestic pigs and wild boars. Despite socio-economic differences, these islands share geographical and climatic commonalities, influencing their thriving swine industries. Focusing on FMD eradication, this study unveils Taiwan's success through mass vaccination, Japan's post-eradication surveillance, and the Philippines' zoning strategy. Insights into CSF in Japan emphasize the importance of wild boar control, whereas the ASF section highlights the multifaceted approach implemented through the Philippine National ASF Prevention and Control Program. This review underscores lessons learned from gained experiences, contributing to a comprehensive understanding of swine disease management in the region.

Nanoparticle vaccines based on the receptor binding domain of porcine deltacoronavirus elicit robust protective immune responses in mice.

Porcine deltacoronavirus (PDCoV), a novel swine enteropathogenic coronavirus, challenges the global swine industry. Currently, there are no approaches preventing swine from PDCoV infection. A new PDCoV strain named JS2211 was isolated. Next, the dimer receptor binding domain of PDCoV spike protein (RBD-dimer) was expressed using the prokaryotic expression system, and a novel nanoparticle containing RBD-dimer and ferritin (SC-Fe) was constructed using the SpyTag/SpyCatcher system. Finally, the immunoprotection of RBD-Fe nanoparticles was evaluated in mice. The novel PDCoV strain was located in the clade of the late Chinese isolate strains and close to the United States strains. The RBD-Fe nanoparticles were successfully established. Immune responses of the homologous prime-boost regime showed that RBD-Fe nanoparticles efficiently elicited specific humoral and cellular immune responses in mice. Notably, high level PDCoV RBD-specific IgG and neutralizing antibody (NA) could be detected, and the histopathological results showed that PDCoV infection was dramatically reduced in mice immunized with RBD-Fe nanoparticles. This study effectively developed a candidate nanoparticle with receptor binding domain of PDCoV spike protein that offers protection against PDCoV infection in mice.

Pseudorabies Virus HLJ Strain Suppresses Peripheral Blood T and B Lymphocytes in Mice

Background: Pseudorabies virus (PRV), responsible for pseudorabies, is one of the most important pathogen in swine industry. PRV infection suppresses interferon-β (IFN-β) and tumor necrosis factor-α (TNF-α) expression, which consequently results in an immune escape in the host. Methods: In this study, distribution of peripheral blood T and B cell sub-populations was analyzed by flow cytometry after infecting mice with 1×105 TCID50 PRV. Result: We found that the levels of CD3+ T, CD4+ T and CD8+ T lymphocyte in infection group were significantly decreased at 24 hpi (p less than 0.01) and remained at a low level until 72 hpi compared to the control group. Interestingly, the levels of CD19+ T lymphocyte were significantly lower after 24 hpi (p less than 0.01) and lasted until 48 hpi, then increased sharply and then dropped at 96 hpi. Collectively, these results indicated that PRV infection could play a negative role in adaptive immunity in mice by decreasing the levels of B and T lymphocytes.

Unveiling Shared Immune Responses in Porcine Alveolar Macrophages during ASFV and PRRSV Infection Using Single-Cell RNA-seq.

African swine fever virus (ASFV) and porcine reproductive and respiratory syndrome virus (PRRSV) infections lead to severe respiratory diseases in pigs, resulting in significant economic losses for the global swine industry. While numerous studies have focused on specific gene functions or pathway activities during infection, an investigation of shared immune responses in porcine alveolar macrophages (PAMs) after ASFV and PRRSV infections was lacking. In this study, we conducted a comparison using two single-cell transcriptomic datasets generated from PAMs under ASFV and PRRSV infection. Pattern recognition receptors (PRRs) RIG-I (DDX58), MDA5 (IFIH1), and LGP2 (DHX58) were identified as particularly recognizing ASFV and PRRSV, triggering cellular defense responses, including the upregulation of four cytokine families (CCL, CXCL, IL, and TNF) and the induction of pyroptosis. Through weighted gene co-expression network analysis and protein-protein interaction analysis, we identified thirteen gene and protein interactions shared by both scRNA-seq analyses, suggesting the ability to inhibit both ASFV and PRRSV viral replication. We discovered six proteins (PARP12, PARP14, HERC5, DDX60, RSAD2, and MNDA) in PAMs as inhibitors of ASFV and PRRSV replication. Collectively, our findings showed detailed characterizations of the immune responses in PAMs during ASFV and PRRSV infections, which may facilitate the treatments of these viral diseases.

Inhibition of PEDV viral entry upon blocking N-glycan elaboration

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the global swine industry, demanding a thorough understanding of its cellular invasion mechanism for effective interventions. This study meticulously investigates the impact of O- and N-linked glycans on PEDV proteins and host cell interaction, shedding light on their influence on the virus's invasion process.Utilizing CRISPR-Cas9 technology to inhibit cell surface O- and N-linked glycan synthesis demonstrated no discernible impact on virus infection. However, progeny PEDV strains lacking these glycans exhibited a minor effect of O-linked glycans on virus infection. Conversely, a notable 40% reduction in infectivity was observed when the virus surface lacked N-linked glycans, emphasizing their pivotal role in facilitating virus recognition and binding to host cells.Additionally, inhibition studies utilizing kifunensine, a natural glycosidase I inhibitor, reaffirmed the significant role of N-linked glycans in virus infection. Inhibiting N-linked glycan synthesis with kifunensine substantially decreased virus entry into cells and potentially influenced spike protein expression.Assessment of the stability and recovery potential of N-linked glycan-deficient strains underscored the critical importance of N-glycans at various stages of the virus lifecycle. In vivo experiments infecting piglets with N-glycan-deficient strains exhibited milder clinical symptoms, reduced virus excretion, and less severe pathological lesions compared to conventional strains.These findings offer promising translational applications, proposing N-glycosylation inhibitors as potential therapeutic interventions against PEDV. The utilization of these inhibitors might mitigate virus invasion and disease transmission, providing avenues for effective antiviral strategies and vaccine development. Nonetheless, further research is warranted to elucidate the precise mechanisms of N-linked glycans in PEDV infection for comprehensive clinical applications.

Identification of Three Novel Linear B-Cell Epitopes in Non-Structural Protein 3 of Porcine Epidemic Diarrhea Virus Using Monoclonal Antibodies.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic swine coronavirus that causes diarrhea and high mortality in piglets, resulting in significant economic losses within the global swine industry. Nonstructural protein 3 (Nsp3) is the largest in coronavirus, playing critical roles in viral replication, such as the processing of polyproteins and the formation of replication-transcription complexes (RTCs). In this study, three monoclonal antibodies (mAbs), 7G4, 5A3, and 2D7, targeting PEDV Nsp3 were successfully generated, and three distinct linear B-cell epitopes were identified within these mAbs by using Western blotting analysis with 24 truncations of Nsp3. The epitope against 7G4 was located on amino acids 31-TISQDLLDVE-40, the epitope against 5A3 was found on amino acids 141-LGIVDDPAMG-150, and the epitope against 2D7 was situated on amino acids 282-FYDAAMAIDG-291. Intriguingly, the epitope 31-TISQDLLDVE-40 recognized by the mAb 7G4 appears to be a critical B-cell linear epitope due to its high antigenic index and exposed location on the surface of Nsp3 protein. In addition, bioinformatics analysis unveiled that these three epitopes were highly conserved in most genotypes of PEDV. These findings present the first characterization of three novel linear B-cell epitopes in the Nsp3 protein of PEDV and provide potential tools of mAbs for identifying host proteins that may facilitate viral infection.

Research on the Measurement and Influencing Factors of Carbon Emissions in the Swine Industry from the Perspective of the Industry Chain

From the perspective of the industry chain, this paper uses the life cycle assessment (LCA) method to divide the swine industry into six production stages: feed crop cultivation, feed crop transportation and processing, intestinal fermentation, manure management, energy consumption in pig farming, and slaughtering and processing. Using the LCA method, the carbon emissions from the swine industry are measured from 2001 to 2020 for the whole country and 31 provincial regions. Based on the measurement results, this paper analyzes the dynamic evolution of carbon emissions from the national swine industry during the study period. Meanwhile, the spatial divergence in carbon emissions from the swine industry and the share of carbon emissions from each production stage were further analyzed by combining different provincial regions and production stages. Afterward, this paper uses the Logarithmic Mean Divisia Index (LMDI) model to decompose the influencing factors of carbon emissions at the national and provincial levels, and in each production stage. It is found that (1) The dynamic evolution of China’s swine industry carbon emissions from 2001 to 2020 roughly follows a trend of “slow growth—sharp decline—fluctuating rise—fluctuating decline.” The fluctuations are influenced by multiple factors, including the industry structure, agricultural policy, and farming scale. The primary driver for the increase in carbon emissions from the swine industry is the growth in demand for pork consumption, leading to the rise in swine supply. (2) In terms of spatial divergence at the provincial level, the regional differences in carbon emissions from the swine industry are significant, the total carbon emissions and unit carbon emissions of Jiangsu, Anhui, and Henan are higher than the national average. (3) In the production stages of the swine industry, feed crop cultivation and manure management are the primary sources of carbon emissions, associated with factors such as substantial feed consumption, crop production patterns, and backward manure management practices. (4) Regarding influencing factors, production efficiency, industry structure, and urbanization level have inhibiting effects on carbon emissions in the swine industry. Economic development and population scale have promoting effects. Production efficiency is the most significant inhibiting factor, and economic development is the most significant promoting factor. Finally, suggestions are made to curb carbon emissions in China’s swine industry, including strengthening environmental control, formulating long-term plans for carbon emission reduction, delineating key areas and demonstration bases for carbon emission reduction, enhancing expertise in fertilizer application and manure treatment, and improving agricultural machinery and equipment.

Development and characterization of monoclonal antibodies against p37 protein of African swine fever virus

African Swine Fever Virus (ASFV) is a highly contagious pathogen posing a serious threat to the global swine industry. Despite this, there is currently no effective vaccine against this virus. Within ASFV's core shell structure, p37, a product of polyprotein pp220, shares sequence similarity with SUMO-1 proteases. Localization studies show p37 in various nuclear regions during early infection, shifting to the cytoplasm later on. Research indicates active export of p37 from the nucleus, mediated by CRM1-dependent and -independent pathways. Hydrophobic amino acids in p37 are crucial for these pathways, highlighting their importance throughout the ASFV replication cycle. Additionally, p37 serves as the first nucleocytoplasmic shuttle protein encoded by ASFV, participating in the intranuclear material transport process during ASFV infection of host cells. In this study, we successfully screened five murine monoclonal antibodies targeting p37. Through the truncated expression method, we identified four dominant antigenic epitopes of p37 for the first time. Furthermore, utilizing alanine scanning technology, we determined the key amino acid residues for each epitope. This research not only provides essential information for a deeper understanding of the protein's function but also establishes a significant theoretical foundation for the design and development of ASFV vaccines.

The role of vehicle movement in swine disease dissemination: Novel method accounting for pathogen stability and vehicle cleaning effectiveness uncertainties

SummarySeveral propagation routes drive animal disease dissemination, and among these routes, contaminated vehicles traveling between farms have been associated with indirect disease transmission. In this study, we used near-real-time vehicle movement data and vehicle cleaning efficacy to reconstruct the between-farm dissemination of the African swine fever virus (ASFV). We collected one year of Global Positioning System data of 823 vehicles transporting feed, pigs, and people to 6363 swine production farms in two regions in the U.S. Without cleaning, vehicles connected up to 2157 farms in region one and 437 farms in region two. Individually, in region one vehicles transporting feed connected 2151 farms, pigs to farms 2089 farms, pigs to market 1507 farms, undefined vehicles 1760 farm, and personnel three farms. The simulation results indicated that the contact networks were reduced the most for crew transport vehicles with a 66% reduction, followed by vehicles carrying pigs to market and farms, with reductions of 43% and 26%, respectively, when 100% cleaning efficacy was achieved. The results of this study showed that even when vehicle cleaning and disinfection are 100% effective, vehicles are still connected to numerous farms. This emphasizes the importance of better understanding transmission risks posed by vehicles to the swine industry and regulatory agencies.

The nucleocapsid protein facilitates p53 ubiquitination-dependent proteasomal degradation via recruiting host ubiquitin ligase COP1 in PEDV infection

Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of the coronavirus family and caused severe economic losses to the global swine industry. Previous studies have established that p53 is a host restriction factor for PEDV infection, and p53 degradation occurs in PEDV-infected cells. However, the underlying molecular mechanisms through which PEDV viral proteins regulate p53 degradation remain unclear. In this study, we found that PEDV infection or expression of the nucleocapsid protein downregulates p53 through a post-translational mechanism: increasing the ubiquitination of p53 and preventing its nuclear translocation. We also show that the PEDV N protein functions by recruiting the E3 ubiquitin ligase COP1 and suppressing COP1 self-ubiquitination and protein degradation, thereby augmenting COP1-mediated degradation of p53. Additionally, COP1 knockdown compromises N-mediated p53 degradation. Functional mapping using truncation analysis showed that the N-terminal domains of N protein was responsible for interacting with COP1 and critical for COP1 stability and p53 degradation. The results presented here suggest the COP1-dependent mechanism for PEDV N protein to abolish p53 activity. This study significantly increases our understanding of PEDV in antagonizing the host antiviral factor p53 and will help initiate novel antiviral strategies against PEDV.

108 Discriminating highly resilient spermatozoa during long-term chilled storage

Abstract Artificial insemination (AI) is the most important assisted reproductive technique devised to enhance the genetic potential of animals by enabling widespread utilization of elite males. In the swine industry, AI with extended semen is highly effective, with several extenders developed for long-term storage. Regardless, the gradual decline of sperm quality during storage remains a major concern for rapid genetic dissemination. We hypothesized that existing sire-to-sire variations could reflect on sperm survival during prolonged storage. Therefore, this study examined boar sperm motility and morphology profiles to determine potential disparities during long-term storage within a commercial extender. Freshly collected Duroc boar semen samples (n = 58 boars) were extended at a commercial boar stud (Prestage Farms, MS), and sample doses (n = 29) were appropriately transported to the laboratory for experiments. Semen samples were aliquoted in tubes (filled to the top) and immediately stored in an incubator set at 17°C for up to 10 d post-collection. Daily, samples were assessed for total motility (TM), progressive motility (PM), velocities [average path velocity (VAP), curvilinear velocity (VCL), and straight-line velocity (VSL)], and normal morphology (NM) using the computer-assisted sperm analyzer (CASA, CEROS II). The deviations of TM means were used for survival discrimination between so-called “good” and “bad” semen. Data were log-transformed and analyzed (repeated ANOVA and Bonferroni adjustment) to determine the effect of time during storage. Results are expressed as mean ± SEM and P &amp;lt; 0.05 indicates significant differences. As expected, all data demonstrated a consistent and significant decline over time (P &amp;lt; 0.05), from day-0 to day-10 (TM: 78.2 ± 1.0% to 42.8 ± 4.3%; PM: 46.1 ± 1.8% to 16.2 ±2.3%, and NM: 85.1 ± 1.0% to 76.1 ± 1.9%). Notably, velocity parameters (VAP, VSL, and VCL) remained relatively stable during the storage period, while the distal droplets contributed the most to morphological defects (54.1±1.5% vs. 28.3±1.5%, 18.3±0.5%, and 2.4±0.2% for the proximal droplets, bent and coiled tailed, respectively; P&amp;lt; 0.05). Survival discrimination indicated the highest deviations from the overall averages on day-7.Consequently, good (n = 8) and bad (n = 7) semen samples had significantly different (P &amp;lt; 0.05) in TM (80.2 ± 1.4% and 21.7 ± 4.6%, respectively), PM (39.7 ± 2.7% and 4.7 ± 1.7%, respectively), and NM (87.9 ± 1.9 and 66.4 ± 2.6%, respectively).Similarly, the velocity parameters were significantly different (P &amp;lt; 0.05) between both sample groups, including VAP (74.9 ± 2.6 µm/s vs. 40.4 ± 8.7 µm/s), VCL (135.2 ± 6.0µm/s vs. 81.9 ± 17.3 µm/s), and VSL (59.0 ± 1.8 µm/s vs. 31.3 ± 6.8 µm/s). These findings indicate that specific subsets of boar semen may be well-suited for survival during long-term chilled preservation. The development of tools for early discrimination of semen doses, immediately following collection, could significantly enhance their management and facilitate appropriate shipments to remote areas or during adverse weather conditions. Ongoing studies are dedicated to unraveling the cellular mechanisms underlying the resilience of such spermatozoa. - Research funded by USDA-ARS, grant#58-6066-3-038.

Tissue expression of porcine transient receptor potential mucolipin protein channels and their differential responses to porcine reproductive and respiratory syndrome virus infection in vitro

Abstract Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) infection results in a serious disease, posing a huge economic threat to the global swine industry. The transient receptor potential mucolipin proteins (TRPMLs) have been shown to be strongly associated with virus infection and other physiological processes in humans, but their tissue distribution and responses to PRRSV in pigs remain unknown. Material and Methods Quantitative reverse-transcription PCR analysis was undertaken to determine the optimal primer for TRPML expression detection and for quantifying that expression individually in different pig tissue samples. Meat Animal Research Center 145 (MARC-145) monkey kidney cells and the TRPML-specific activator mucolipin synthetic agonist 1 (ML-SA1) were used to reveal the relationship between TRPML and PRRSV-2 infection. Results The best primers for each TRPML gene used in a fluorescence-based quantitative method were identified and TRPML1 was found to be highly expressed in the kidneys and liver of pigs, while TRPML2 and TRPML3 were observed to be primarily expressed in the kidney and spleen tissues. The expression of TRPML2 was upregulated with the rise in PRRSV-2 infection titre but not the expression of TRPML1 or TRPML3, and ML-SA1 inhibited PRRSV-2 in a dose-dependent manner. Conclusion Our research revealed the gene expression of TRPMLs in pigs and identified that TRPML channels may act as key host factors against PRRSV infection, which could lead to new targets for the prevention and treatment of pig infectious diseases.

Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with GP5 in PRRSV-Infected PAMs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus causing a large economic impact on the swine industry. The structural protein GP5 of PRRSV plays a pivotal role in its pathogenicity and immune evasion. Virus-host interactions play a crucial part in viral replication and immune escape. Therefore, understanding the interactions between GP5 and host proteins are significant for porcine reproductive and respiratory syndrome (PRRS) control. However, the interaction network between GP5 and host proteins in primary porcine alveolar macrophages (PAMs) has not been reported. In this study, 709 GP5-interacting host proteins were identified in primary PAMs by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these proteins were involved in multiple cellular processes, such as translation, protein transport, and protein stabilization. Subsequently, immunoprecipitation and immunofluorescence assay confirmed that GP5 could interact with antigen processing and presentation pathways related proteins. Finally, we found that GP5 may be a key protein that inhibits the antigen processing and presentation pathway during PRRSV infection. The novel host proteins identified in this study will be the candidates for studying the biological functions of GP5, which will provide new insights into PRRS prevention and vaccine development.

Genetic analysis of African swine fever virus in Bali based on p72 and CD2v Genes

African Swine Fever (ASF) has caused negative effects in the swine industry in various countries of the world, including Indonesia. Bali is one of the most severely affected provinces in Indonesia. Phylogenetic study to determine the genotype and serogroup of the ASF virus found in Bali has never been done before. In this study, virus was obtained from an infected spleen sample, designated as Bali/2023, and two gene fragments, namely p72 (B646L) and CD2v (EP402R), were then amplified by PCR and sequenced by Sanger method. The DNA sequences of p72 and CD2v virus was then compared with sequences from other countries for genetic analysis. The results showed that Bali/2023 virus is genetically very close to viruses found in other Asian countries, mainly viruses isolated in China and Vietnam in 2019. These genetic analyses results placed Bali/2023 virus in genotype II and serogroup 8.