Investigation of Prototheca bovis Infection and Its Correlation with Dairy Herd Improvement Data from a Dairy Farm in Central China.

Prototheca bovis (P. bovis), an alga that has attracted considerable attention over the years as a causative microorganism of mastitis in dairy cows, exhibits limited susceptibility to specific aminoglycosides and antifungal agents, and no effective clinical treatment is currently available, thereby posing challenges for both prevention and treatment. To investigate the infection of P. bovis mastitis and its impact on raw milk production, a total of 348 raw milk samples were collected from August to December 2022 from a dairy farm in central China. P. bovis and other bacteria were detected, and the average infection rate of P. bovis in raw milk was 60.34% (210/348). The total number of colonies and the somatic cell count (SCC) of P. bovis positive samples were significantly higher than those of P. bovis negative samples (p < 0.01). The daily milk yield, 305-day milk yield, peak milk yield, and days to peak milk yield of the P. bovis positive samples were significantly lower than those of P. bovis negative samples (p < 0.01). A correlation analysis showed that P. bovis infection was negatively correlated with daily milk yield, 305-day milk yield, peak milk yield, and days to peak milk yield (p < 0.0001), while being positively correlated with the total number of colonies, SCC, milk loss, and protein percentage (p < 0.0001). These findings may help practitioners in comprehending the occurrence of Prototheca mastitis and developing more effective strategies for the prevention of P. bovis infections.

Identification of genes related to sexual differentiation and sterility in embryonic gonads of Mule ducks by transcriptome analysis.

The key genes of avian gonadal development are of great significance for sex determination. Transcriptome sequencing analysis of Mule duck gonad as potential sterile model is expected to screen candidate genes related to avian gonad development. In this study, the embryonic gonadal tissues of Mule ducks, Jinding ducks, and Muscovy ducks were collected and identified. Six sample groups including female Mule duck (A), male Mule duck (B), female Jinding duck (C), male Jinding duck (D), female Muscovy duck (E), and male Muscovy duck (F) were subjected to RNA sequencing analysis. A total of 9,471 differential genes (DEGs) and 691 protein-protein interaction pairs were obtained. Totally, 12 genes (Dmrt1, Amh, Sox9, Tex14, Trim71, Slc26a8, Spam1, Tdrp, Tsga10, Boc, Cxcl14, and Hsd17b3) were identified to be specifically related to duck testicular development, and 11 genes (Hsd17b1, Cyp19a1, Cyp17a1, Hhipl2, Tdrp, Uts2r, Cdon, Axin2, Nxph1, Brinp2, and Brinp3) were specifically related to duck ovarian development. Seven genes (Stra8, Dmc1, Terb1, Tex14, Tsga10, Spam1, and Plcd4) were screened to be specifically involved in the female sterility of Mule ducks; eight genes (Gtsf1, Nalcn, Tat, Slc26a8, Kmo, Plcd4, Aldh4a1, and Hgd) were specifically involved in male sterility; and five genes (Terb1, Stra8, Tex14 Tsga10 and Spam1) were involved in both female and male sterility. This study provides an insight into the differential development between male and female gonads of ducks and the sterility mechanism of Mule ducks through function, pathway, and protein interaction analyses. Our findings provide theoretical basis for the further research on sex determination and differentiation of birds and the sterility of Mule ducks.

Integrated multi-omic data reveal the potential molecular mechanisms of the nutrition and flavor in Liancheng white duck meat.

The Liancheng white (LW) duck is one of the most valued Chinese indigenous poultry breeds. Its meat is rich in nutrients and has distinct flavors, but the molecular mechanisms behind them are unknown. To address this issue, we measured and compared multi-omic data (genome, transcriptome, and metabolome) of breast meat from LW ducks and the Mianyang Shelduck (MS) ducks. We found that the LW duck has distinct breed-specific genetic features, including numerous mutant genes with differential expressions associated with amino acid metabolism and transport activities. The metabolome driven by genetic materials was also seen to differ between the two breeds. For example, several amino acids that are beneficial for human health, such as L-Arginine, L-Ornithine, and L-lysine, were found in considerably higher concentrations in LW muscle than in MS duck muscle (p < 0.05). SLC7A6, a mutant gene, was substantially upregulated in the LW group (p < 0.05), which may lead to excessive L-arginine and L-ornithine accumulation in LW duck meat through transport regulation. Further, guanosine monophosphate (GMP), an umami-tasting molecule, was considerably higher in LW muscle (p < 0.05), while L-Aspartic acid was significantly abundant in MS duck meat (p < 0.05), showing that the LW duck has a different umami formation. Overall, this study contributed to our understanding of the molecular mechanisms driving the enriched nutrients and distinct umami of LW duck meat, which will provide a useful reference for duck breeding.

An insertion and deletion mutant of adenovirus in Muscovy ducks.

Duck adenovirus 3 (DuAdV-3; strain HB) was isolated and sequenced. The genome of the Muscovy-duck-origin virus contains a 54-bp insertion in pVIII, a 3-bp deletion in the overlap region of 100K, 22K, and 33K, a 42-bp deletion at the junction of ORF64 and ORF67, and a 715-bp deletion in right noncoding region of the genome. Notably, HB has a strikingly shorter right inverted terminal repeat (ITR) of 50 bp, whereas all other DuAdV-3 isolates have a 721-bp ITR. These findings demonstrate that HB is an insertion and deletion mutant of DuAdV-3.

The antiviral activity of arctigenin in traditional Chinese medicine on porcine circovirus type 2.

Arctigenin (ACT) is a phenylpropanoid dibenzylbutyrolactone lignan extracted from the traditional herb Arctium lappa L. (Compositae) with anti-viral and anti-inflammatory effects. Here, we investigated the antiviral activity of ACT found in traditional Chinese medicine on porcine circovirus type 2 (PCV2) in vitro and in vivo. Results showed that dosing of 15.6-62.5µg/mL ACT could significantly inhibit the PCV2 proliferation in PK-15 cells (P<0.01). Dosing of 62.5µg/mL ACT 0, 4 or 8h after challenge inoculation significantly inhibited the proliferation of 1MOI and 10MOI in PK-15 cells (P<0.01), and the inhibitory effect of ACT dosing 4h or 8h post-inoculation was greater than 0h after dosing (P<0.01). In vivo test with mice challenge against PCV2 infection demonstrated that intraperitoneal injection of 200µg/kg ACT significantly inhibited PCV2 proliferation in the lungs, spleens and inguinal lymph nodes, with an effect similar to ribavirin, demonstrating the effectiveness of ACT as an antiviral agent against PCV2 in vitro and in vivo. This compound, therefore, may have the potential to serve as a drug for protection of pigs against the infection of PCV2.

Fluorescent labeling for clonal selection of Marc 145 cells secreting high levels of recombinant protein PBD-1.

Despite the powerful impact gene expression markers like the green fluorescent protein (GFP) or enhanced GFP (EGFP) exert on linking the expression of recombinant protein for selection of high producers in recent years, there is still a strong incentive to develop more economical and efficient methods for isolating mammalian cell clones secreting high levels of recombinant proteins. Here we present a new method based on the co-expression of EGFP that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the related protein are linked by an internal ribosome entry site and thus are transcribed into the same mRNA in an independent translation process. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein in each clone. By expressing recombinant porcine ß-defensin 1 in Marc 145 cells, we demonstrate the robustness and performance of this technique. The method can be served as an alternative to identify high-producer clones with various cell sorting methods.

Functional analysis of pig myostatin gene promoter with some adipogenesis- and myogenesis-related factors.

Myostatin (MSTN) is primarily expressed in muscle and plays an important role in muscle and fat development in pigs. However, there is little information about the regulation of pig MSTN. In order to elucidate whether pig MSTN could be regulated by muscle- and fat-related factors, the porcine MSTN promoter was amplified and cloned into pGL3-basic vector, and transfected into cells to analyze the transcriptional activity of promoter with muscle- and fat-related factors through dual-luciferase reporter assays. 5'-deletion expression showed that there was a negative-regulatory region located between nucleotides -1519 and -1236 bp, and there were some positive-regulatory regions located between -1236 and -568 bp. The longest fragment (1.7 kb) was cotransfected with muscle-related transcription factor myogenic differentiation 1 (MyoD), resulting in promoter transcriptional activity upregulation. The fragment was treated by the adipogenic agents (DIM) including dexamethasone, insulin, and isobutyl-1-methylxanthine (IBMX). We found that MSTN promoter transcriptional activity can be regulated by IBMX, but not by DIM. CCAAT/enhancer binding protein (C/EBP) α and C/EBPß, two proteins which are induced by DIM during adipogenesis were cotransfected with the 1.7-kb fragment, respectively, resulting in promoter transcriptional activity downregulation. Treating the fragment with rosiglitazone which induce the expression of peroxisome proliferator-activated receptor γ (PPARγ), resulting in promoter transcriptional activity upregulation. Cotransfection experiments confirmed this result. Taken together, we showed that porcine MSTN could be upregulated by IBMX, MyoD, and PPARγ but downregulated by C/EBPα and C/EBPß.

Survival and engraftment of mouse embryonic stem cells in the mammary gland.

Embryonic stem (ES) cells have been investigated in many animal models of severe injury and degenerative disease. However, few studies have examined the ability of ES cells to improve functional outcome following mammary gland injury. This study investigates the feasibility of implanting mouse ES cells labeled with enhanced green fluorescence protein in the developing mammary glands in order to acquire lineage-committed cells in mammary (mammary gland epithelial cell or luminal cell). Cells implanted in high numbers (5 × 10(6) cells per mammary gland) survived in the majority of the mice and nearly 38.4% of the surviving cells were CK18(+) at 15th week following the transplantation. Our results may provide a technique instrument on advanced therapy of breast diseases and the mammary regeneration after breast ablated partly.

Recombinant PBD-1 (porcine beta-defensin 1) expressed in the milk by transplanting transgenic mES-like-derived cells into mouse mammary gland.

ES (embryonic stem)-derived cells have been investigated in many animal models of severe injury and degenerative disease. However, few studies have examined the ability of ES-derived cells to improve functional outcome following partially damaged breast and also the modification of mammary tissue to produce costly proteins. This study investigates the feasibility of implanting mES-dK (mouse ES-derived keratinocytes-like) cells stably transfected with a mammary gland special expression vector for the PBD-1 (porcine beta-defensin 1) in developing mammary glands. Our aim was to assess the ability of cell grafting to improve functional outcome following partial damage of the breast, also on the breast modification mammary tissue in mice for the production of PBD-1 protein secreted in the milk. Our results showed that the ratios of the surviving cells labelled with the myoepithelial or luminal cell markers, EMA (epithelial membrane antigen) and CALLA, were 41.7 +/- 15.2% and 28.4 +/- 9.6%, respectively, which revealed that transplanted mES-dK cells survived, integrated in vivo and differentiated into myoepithelial or luminal cells. In addition, Western blot analysis showed that 37.5% (3 out of 8) female transplanted mice had PBD-1 expression in their milk and reached 0.4998, 0.5229 and 0.5195 microg/ml, respectively.