First detection and phylogenetic analysis of porcine circovirus 3 in female donkeys with reproductive disorders.

BACKGROUND: PCV3 is a pathogen associated with porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, and cardiac and multiorgan inflammation, which was newly identified in 2016 in sows in USA. Recently, PCV3 has also been identified from several non-porcine species like (cattle, dog, wild boar, deer, mice and ticks). However, PCV3 infection in donkey is not well established. Since 2019, 300 blood samples were collected from female donkey, which was characterized by abortion and sterility, in Liaocheng city of China. RESULTS: In the present study, an investigation of PCV3 in donkey blood samples was undertaken employing by real time PCR. Positive rates of PCV3 in donkeys reach to 21.0 %. In addition, one full-length PCV3 genome sequence was obtained, and it had a highest identity with porcine circovirus 3 PCV3/CN/Nanjing2017 strain and is clustered to PCV3a genotype based on ORF2 sequences. CONCLUSIONS: This is the first report of detection of PCV3 from female donkeys presenting reproductive failure in large-scale donkey farms, China. In addition, the PCV3 strain identified in this study shared the closest relationship with those from porcine, suggesting that PCV3 may be transmitted from pigs to donkeys. Totally, PCV3 infection in donkey should be concerned although the association between it and reproductive failure are not better understood.

Identification of a Candidate Gene Regulating Intramuscular Fat Content in Pigs through the Integrative Analysis of Transcriptomics and Proteomics Data.

Pork is a widely consumed source of animal protein worldwide, and the intramuscular fat (IMF) content in pork plays a crucial role in determining its quality. In this study, we sought to identify candidate genes that regulate IMF deposition in pigs. We performed tandem mass tags (TMT)-based quantitative proteomics analysis using Longissimus dorsi (LD) muscle samples obtained from eight pigs with extremely high and low IMF content among a group of 28 Duroc pigs and identified 50 differentially abundant proteins (DAPs). Additionally, we compared the proteomics data with RNA-sequencing data obtained in our previous study and identified TUSC5 as a differentially expressed gene corresponding to the relevant DAP. To investigate the potential role of TUSC5 in adipogenesis, we suppressed TUSC5 expression in mouse 3T3-L1 preadipocytes using short hairpin RNA (shRNA) and observed a significant reduction in the differentiation of 3T3-L1 cells into adipocytes, as indicated by Oil Red O staining and triglyceride content. Moreover, we observed a reduction in the expression of genes associated with adipogenesis (PPARG, CEBPA, FABP4, and FASN) following TUSC5 suppression. Through an integrative analysis of transcriptomics and proteomics data, our study identified TUSC5 as a crucial candidate gene associated with the regulation of IMF content in pigs.

Comparative analysis of newly identified rodent arteriviruses and porcine reproductive and respiratory syndrome virus to characterize their evolutionary relationships.

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses for the global pig industry, but its origins and evolution remain a mystery. In 2018, the genome sequences of seven arteriviruses isolated from rodents were determined, and here we publish new analysis showing that they may be ancestors of PRRSV. The sequence similarity of these viruses to PRRSV was ~60%, with shared genome organization and other characteristics, such as slippery sequences and C-rich motifs in nsp2, and a transactivated protein sequence in nsp1ß. Codon usage basis analysis showed that PRRSV was closer to these rodent arteriviruses than lactate dehydrogenase-elevating virus (LDV) and they were both under pressure of natural selection. Evolutionary analysis revealed that four of the rodent arteriviruses shared the same genus with PRRSV, and were more closely related to PRRSV-2 than PRRSV-1. In addition to this, they all appeared earlier than PRRSV according to evolutionary modeling, and we speculate that they represent an intermediate step in the origin of PRRSV by arterivirus transmission from rodents to swine. Our in-depth analysis furthers our understanding of arteriviruses, and will serve as the basis for subsequent exploration of the evolution of PRRSV and other arteriviruses.

Single-nucleus and bulk RNA sequencing reveal cellular and transcriptional mechanisms underlying lipid dynamics in high marbled pork.

Pork is the most consumed meat in the world, and its quality is associated with human health. Intramuscular fat (IMF) deposition (also called marbling) is a key factor positively correlated with various quality traits and lipo-nutritional values of meat. However, the cell dynamics and transcriptional programs underlying lipid deposition in highly marbled meat are still unclear. Here, we used Laiwu pigs with high (HLW) or low (LLW) IMF contents to explore the cellular and transcriptional mechanisms underlying lipid deposition in highly-marbled pork by single-nucleus RNA sequencing (snRNA-seq) and bulk RNA sequencing. The HLW group had higher IMF contents but less drip loss than the LLW group. Lipidomics results revelled the changes of overall lipid classes composition (e.g., glycerolipids including triglycerides, diglycerides, and monoglycerides; sphingolipids including ceramides and monohexose ceramide significantly increased) between HLW and LLW groups. SnRNA-seq revealed nine distinct cell clusters, and the HLW group had a higher percentage of adipocytes (1.40% vs. 0.17%) than the LLW group. We identified 3 subpopulations of adipocytes, including PDE4D+/PDE7B+ (in HLW and LLW), DGAT2+/SCD+ (mostly in HLW) and FABP5+/SIAH1+ cells (mostly in HLW). Moreover, we showed that fibro/adipogenic progenitors could differentiate into IMF cells and contribute to 43.35% of adipocytes in mice. In addition, RNA-seq revealed different genes involved in lipid metabolism and fatty acid elongation. Our study provides new insights into the cellular and molecular signatures of marbling formation; such knowledge may facilitate the development of new strategies to increase IMF deposition and the lipo-nutritional quality of high marbled pork.

Tannic acid attenuates intestinal oxidative damage by improving antioxidant capacity and intestinal barrier in weaned piglets and IPEC-J2 cells.

Tannic acid (TA) has received widespread attention for its beneficial biological function with antioxidant capacity. This study investigated the protective role of TA on the intestinal antioxidant capacity and intestinal barrier in weaned piglets and porcine intestinal epithelial cells (IPEC-J2). A total of 18 weaned piglets were randomly allocated into two groups (n = 9) and fed with a basal diet (control, CON) and a basal diet containing 1,000 mg/kg TA for two weeks. The in vivo results showed that treatment with TA increased both glutathione peroxidase (GSH-PX) activity and the protein expression of ZO-1 in the jejunum of weaned piglets, and reduced the level of malondialdehyde (MDA) in the serum and the mRNA and protein expression of Keap1 in the jejunum of weaned piglets. Furthermore, in vitro results indicated that TA treatment effectively alleviated tert-butyl hydroperoxide (TBH)-induced oxidative stress in IPEC-J2 cells, improved the antioxidant capacity by elevating the cell redox state and activating the Nrf2 pathway, and improved the intestinal barrier by upregulating the mRNA and protein expression of intestinal tight junction proteins and increasing the transepithelial electrical resistance (TEER) value. In conclusion, these results confirmed that TA relieves oxidative injury and improves intestinal barrier function and intestinal antioxidant capacity by activating the Nrf2 signaling pathway. These findings suggest that TA has the potential application in alleviating oxidative stress in the intestine of weaned piglets.

CLA improves the lipo-nutritional quality of pork and regulates the gut microbiota in Heigai pigs.

Conjugated linoleic acid (CLA) is a potential nutritional strategy to regulate meat quality in pigs and produce high-quality pork. However, the effects of CLA on nutritional quality, lipid dynamics, microbiota, and their metabolites in the gut of pigs remain unclear. Our study explored the effects of CLA on lipo-nutritional quality based on a Heigai pig model and investigated the regulatory mechanism using an integrated analysis of multiple omics. A total of 58 Heigai finishing pigs (body weight: 85.58 ± 10.39 kg) were randomly divided into 2 treatments and fed diets containing 1% soyabean oil and 1% CLA for 40 days. 1% CLA significantly decreased the backfat thickness (P < 0.05) and increased the intramuscular fat (IMF) content (P < 0.05). The expression of lipid metabolism-related genes was significantly changed (P < 0.05) and lipidome analysis showed the alternations of lipid dynamics in the longissimus dorsi muscle (LDM). In addition, based on the microbiome and metabolomic analyses, the relative abundances of Parabacteroides, Bacteroides, and Lachnospiraceae_UCG-010 increased and CLA changed the metabolome profiles and the short-chain fatty acid (SCFA) composition in the gut, which were significantly increased (P < 0.05). Additionally, Pearson's correlation analysis indicated that differential microbial genera and SCFAs induced by CLA had tight correlations with the backfat thickness, IMF content and lipids in the LDM. CLA enhances the lipid accumulation and metabolism in muscle and these changes are associated with the production and functions of the differential bacteria and SCFAs in the gut of pigs.

Transforming UTE-mDixon MR Abdomen-Pelvis Images Into CT by Jointly Leveraging Prior Knowledge and Partial Supervision.

Computed tomography (CT) provides information for diagnosis, PET attenuation correction (AC), and radiation treatment planning (RTP). Disadvantages of CT include poor soft tissue contrast and exposure to ionizing radiation. While MRI can overcome these disadvantages, it lacks the photon absorption information needed for PET AC and RTP. Thus, an intelligent transformation from MR to CT, i.e., the MR-based synthetic CT generation, is of great interest as it would support PET/MR AC and MR-only RTP. Using an MR pulse sequence that combines ultra-short echo time (UTE) and modified Dixon (mDixon), we propose a novel method for synthetic CT generation jointly leveraging prior knowledge as well as partial supervision (SCT-PK-PS for short) on large-field-of-view images that span abdomen and pelvis. Two key machine learning techniques, i.e., the knowledge-leveraged transfer fuzzy c-means (KL-TFCM) and the Laplacian support vector machine (LapSVM), are used in SCT-PK-PS. The significance of our effort is threefold: 1) Using the prior knowledge-referenced KL-TFCM clustering, SCT-PK-PS is able to group the feature data of MR images into five initial clusters of fat, soft tissue, air, bone, and bone marrow. Via these initial partitions, clusters needing to be refined are observed and for each of them a few additionally labeled examples are given as the partial supervision for the subsequent semi-supervised classification using LapSVM; 2) Partial supervision is usually insufficient for conventional algorithms to learn the insightful classifier. Instead, exploiting not only the given supervision but also the manifold structure embedded primarily in numerous unlabeled data, LapSVM is capable of training multiple desired tissue-recognizers; 3) Benefiting from the joint use of KL-TFCM and LapSVM, and assisted by the edge detector filter based feature extraction, the proposed SCT-PK-PS method features good recognition accuracy of tissue types, which ultimately facilitates the good transformation from MR images to CT images of the abdomen-pelvis. Applying the method on twenty subjects' feature data of UTE-mDixon MR images, the average score of the mean absolute prediction deviation (MAPD) of all subjects is 140.72 ± 30.60 HU which is statistically significantly better than the 241.36 ± 21.79 HU obtained using the all-water method, the 262.77 ± 42.22 HU obtained using the four-cluster-partitioning (FCP, i.e., external-air, internal-air, fat, and soft tissue) method, and the 197.05 ± 76.53 HU obtained via the conventional SVM method. These results demonstrate the effectiveness of our method for the intelligent transformation from MR to CT on the body section of abdomen-pelvis.

Liquefaction of porcine hoof shell to prepare peptone substitute by instant catapult steam explosion.

Instant catapult steam explosion (ICSE) was proposed as a method to liquefy porcine hoof shell (PHS) and prepare a peptone substitute for fermentation culture, achieving environmentally friendly animal by-product recycling. The liquefaction of PHS was conducted at various pressures (0.5-2.3 MPa) for 5-30 min. As evidenced by the scanning electron microscopy analysis, ICSE caused randomly cracks changing the morphological structure of the solid fraction, and ultimately led to protein migration from the solid to liquid phase. Moreover, the chromatographic analysis revealed that the main constituents of the liquid fraction were short peptides (<2 kDa, 84.72%) and amino acids (1.68 mg/mL) at the pressure of 2.3 MPa for 30 min. Subsequently, liquid fractions were prepared as a PHS peptone substitute for fermentation culture. Results suggested the PHS peptone substitute as the main nitrogen source in media was more suitable for the growth of fungus. Therefore, ICSE provides a possibility of large-scale environmentally sustainable management of animal by-products through liquefaction.

Ultrasensitive and eco-friendly immunoassays based monoclonal antibody for detection of deoxynivalenol in cereal and feed samples.

Ultrasensitive immunoassays, including an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a lateral-flow immunochromatographic assay (ICA), were developed based on a monoclonal antibody for the analysis of deoxynivalenol in food and feed samples. With 0.01 M PBS, 20% ethanol-PBS, and 60% ethanol-PBS extraction, which are environmentally safe, the 50% inhibitory concentration (IC50) and limit of detection (LOD) values were 1.83-4.68 µg/kg and 0.241-0.664 µg/kg, respectively, with recovery rates of 87.7%-137% and coefficient variation values of 3.99-9.88% (intra-assay) and 4.17-9.81% (inter-assay) for the ic-ELISA relative to the results obtained by liquid chromatography-tandem mass spectrometry (LC-MS). For the ICA strip, the visual LODs were 10-150 µg/kg, the cut-off values were 50-750 µg/kg, and the calculated LODs were 1.97-46.8 µg/kg, with different sample extraction solutions, and the recovery rates were 66.7%-127%. These methods are sensitive, simple and safe, providing an auxiliary analytical tool for screening the massive samples in markets.

Development of Indirect Competitive Enzyme-Linked Immunosorbent and Immunochromatographic Strip Assays for Tiamulin Detection in Chicken.

Tiamulin (TML) is a diterpenoid antibiotic used in animals. In this study, a gold nanoparticle immunochromatographic strip assay and an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) were developed to detect the residue of TML in chicken. TML aminobutyric was synthesized and conjugated to keyhole limpet hemocyanin by mixed anhydride method as immunogen, whereas TML was connected to ovalbumin with 1,1'-carbonyldiimidazole as coating antigen. Under optimized conditions, the ultrasensitive monoclonal antibody-based ic-ELISA exhibited a half-maximal inhibitory concentration (IC50) value of 0.36 ng/mL with a working range of 0.14-0.9 ng/mL for TML. A rapid and sensitive immunochromatographic strip assay was developed with a TML cutoff value of 2.5 ng/mL. On the basis of these results, both developed methods are useful for TML detection in chicken.

Emergence of pathogenic and multiple-antibiotic-resistant Macrococcus caseolyticus in commercial broiler chickens.

Macrococcus caseolyticus is generally considered to be a non-pathogenic bacterium that does not cause human or animal diseases. However, recently, a strain of M. caseolyticus (SDLY strain) that causes high mortality rates was isolated from commercial broiler chickens in China. The main pathological changes caused by SDLY included caseous exudation in cranial cavities, inflammatory infiltration, haemorrhages and multifocal necrosis in various organs. The whole genome of the SDLY strain was sequenced and was compared with that of the non-pathogenic JCSC5402 strain of M. caseolyticus. The results showed that the SDLY strain harboured a large quantity of mutations, antibiotic resistance genes and numerous insertions and deletions of virulence genes. In particular, among the inserted genes, there is a cluster of eight connected genes associated with the synthesis of capsular polysaccharide. This cluster encodes a transferase and capsular polysaccharide synthase, promotes the formation of capsules and causes changes in pathogenicity. Electron microscopy revealed a distinct capsule surrounding the SDLY strain. The pathogenicity test showed that the SDLY strain could cause significant clinical symptoms and pathological changes in both SPF chickens and mice. In addition, these clinical symptoms and pathological changes were the same as those observed in field cases. Furthermore, the anti-microbial susceptibility test demonstrated that the SDLY strain exhibits multiple-antibiotic resistance. The emergence of pathogenic M. caseolyticus indicates that more attention should be paid to the effects of this micro-organism on both poultry and public health.

A novel multi-variant epitope ensemble vaccine against avian leukosis virus subgroup J.

The hypervariable antigenicity and immunosuppressive features of avian leukosis virus subgroup J (ALV-J) has led to great challenges to develop effective vaccines. Epitope vaccine will be a perspective trend. Previously, we identified a variant antigenic neutralizing epitope in hypervariable region 1 (hr1) of ALV-J, N-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-C. BLAST analysis showed that the mutation of A, E, T and H in this epitope cover 79% of all ALV-J strains. Base on this data, we designed a multi-variant epitope ensemble vaccine comprising the four mutation variants linked with glycine and serine. The recombinant multi-variant epitope gene was expressed in Escherichia coli BL21. The expressed protein of the variant multi-variant epitope gene can react with positive sera and monoclonal antibodies of ALV-J, while cannot react with ALV-J negative sera. The multi-variant epitope vaccine that conjugated Freund's adjuvant complete/incomplete showed high immunogenicity that reached the titer of 1:64,000 at 42 days post immunization and maintained the immune period for at least 126 days in SPF chickens. Further, we demonstrated that the antibody induced by the variant multi-variant ensemble epitope vaccine recognized and neutralized different ALV-J strains (NX0101, TA1, WS1, BZ1224 and BZ4). Protection experiment that was evaluated by clinical symptom, viral shedding, weight gain, gross and histopathology showed 100% chickens that inoculated the multi-epitope vaccine were well protected against ALV-J challenge. The result shows a promising multi-variant epitope ensemble vaccine against hypervariable viruses in animals.

Identification of a variant antigenic neutralizing epitope in hypervariable region 1 of avian leukosis virus subgroup J.

Avian leukosis virus subgroup J (ALV-J) is a hypervariable oncogenic retrovirus that causes great economic loss in poultry. Antigenic variations in the variable regions make the development of an effective vaccine a challenging task. In the present study, we identified a variant antigenic neutralizing epitope using reverse vaccinology methods. First, we predicted the B-cell epitopes in gp85 gene of ALV-J strains by DNAman and bioinformatics. Fourteen candidate epitopes were selected and linked in tandem with glycines or serines as a multi-epitope gene. The expressed protein of multi-epitope gene can induce high-titer antibody that can recognize nature ALV-J and neutralize the infectivity of ALV-J strains. Next, we identified a high effective epitope using eight overlapping fragments of gp85 gene reacting with mAb 2D5 and anti-multi-epitope sera. The identified epitope contained one of the predicted epitopes and localized in hyervariable region 1 (hr1), indicating a variant epitope. To better understand if the variants of the epitope have a good antigenicity, we synthesized four variants to react with mAb 2D5 and anti-ALV-J sera. The result showed that all variants could react with the two kinds of antibodies though they showed different antigenicity, while could not react with ALV-J negative sera. Thus, the variant antigenic neutralizing epitope was determined as 137-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-158. The result shows a potential use of this variant epitopes as a novel multi-epitope vaccine against ALV-J in poultry.

Transcriptome sequencing and comparative analysis reveal long-term flowing mechanisms in Hevea brasiliensis latex.

BACKGROUND: The rubber tree, Hevea brasiliensis, is a major commercial source of natural rubber. Increasing the rubber yield of rubber trees is a very serious problem since the demands for high quality rubber materials are great. Establishment of a tapping system is based on an estimate of tapping intensity from the rubber tree. Latex flowing time is one of the most critical factors that determine the rubber yield. Long-term flow is a type of phenomenon of the rubber tree latex with longer flowing time than normal latex flow, and is always caused by intensive tapping. Thus, transcriptome and expression profiling data for long-term flowing latex (LFL) are needed as an important resource to identify genes and to better understand the biological mechanisms of latex flow in rubber trees. RESULTS: The transcripts were sequenced using the Illumina sequencing platform. After cleaning, quality checks and sequencing, 98,697 transcripts and 38,584 unigenes were assembled with the mean size of 1437.31bp and 923.86bp, respectively. In BLAST searches of our database against public databases, 65.17% (25,147) of the unigenes were annotated with gene descriptions, conserved protein domains, or gene ontology terms. Functional categorization further revealed 853 individual unigenes related to long-term flow. According to KEGG classification, the clusters for "cysteine and methionine metabolism", "energy", "oxidative phosphorylation", "terpenoid backbone biosynthesis", "plant hormone signal transduction" and "copper, potassium transporter" were significantly enriched metabolic pathways. CONCLUSIONS: We conducted high-resolution transcriptome profiling related to LFL in H. brasiliensis. The research facilitates further studies on gene discovery and on the molecular mechanisms related to the estimation of tapping intensity and prolonging latex flowing time. We concluded that it was necessary to improve energy supplies for intensive tapping and the copper ion content of rubber tree latex could be considered as a standard to estimate tapping intensity.