Rapid detection of pigeon adenovirus 2 using a TaqMan real-time PCR assay.

Pigeons infected with aviadenoviruses have been found worldwide. Recently, pigeon adenovirus 2 (PiAdV-2) has been widely distributed in racing pigeons in Germany. However, the epidemiology of this virus remains unclear due to the lack of a specific detection platform for PiAdV-2. In this study, we first detected PiAdV-2 positivity in racing pigeons (designated FJ21125 and FJ21128, which share 100% nucleotide identity with each other based on the fiber 2 gene) in Fujian, Southeast China. These genes shared 99.8% nucleotide identity with PiAdV-2 (GenBank No. NC_031501) but only 54.1% nucleotide identity with PiAdV-1 (GenBank No. NC024474). Then, the TaqMan-qPCR assay for the detection of PiAdV-2 was established based on fiber 2 gene characterization. The established assay had a correlation coefficient of 1.00, with an amplification efficiency of 99.0%. The minimum detection limit was 34.6 copies/µL. Only PiAdV-2 exhibited a positive fluorescent signal, and no signal was detected for other pathogens (including PiCV, FAdV-4, FAdV-8a, EDSV, PPMV-1, RVA and PiHV). The assay has good reproducibility, with a coefficient of variation less than 2.42% both intragroup and intergroup. The distributions of PiAdV-2 in fecal samples from YPDS (35 samples) and healthy (43 samples) racing pigeons from different geographical areas were investigated and were 37.14% (YPDS) and 20.93% (healthy), respectively. In summary, we developed a TaqMan-qPCR platform for the detection of PiAdV-2 infection with high sensitivity, specificity, and reproducibility. We confirmed the presence of PiAdV-2 in China, and our data suggested that there is no indication of a correlation between YPDS and PiAdV-2. This study provides more information on the pathogenesis mechanism and epidemiological surveillance of PiAdV-2.

Whole-genome resequencing revealed the population structure and selection signal of 4 indigenous Chinese laying ducks.

The assessment of animal genetic structure had significant importance for the preservation and breeding of animal germplasm resources. Selection signals are genotype markers generated during the process of biological evolution, and the detection of selection signals could reveal the direction of species evolution. The aim of this study was to generate a whole-genome resequencing data from Jinding duck, Shanma duck, Youxian Partridge duck, and Taiwan Brown tsaiya duck to reveal their population structure and selection signals. The population structure analysis revealed significant genetic differences among the 4 indigenous laying ducks, indicating their independent lineage. Specifically, Shanma duck and Youxian partridge duck were closely and likely originated from a common ancestor. In addition, selection sweep analysis was performed using the population genetic differentiation coefficient (Fst) and nucleotide diversity ratio (π ratio). The top 5% was used as the threshold for the Fst and π ratio, and the 2 thresholds were combined to identify selected genomic regions. In the selected regions of the 3 comparison groups, 136, 143, and 268 candidate genes were detected. Further screening of all candidate genes revealed that 35 candidate genes appeared simultaneously in 3 comparative groups, with 16 genes annotated. The 16 genes were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The results revealed 5 functional genes (AQP3, PIK3C3, NOL6, RPP25, and DCTN3) that may be related to important economic traits in laying ducks and involved mainly invasopressin-regulated water reabsorption, ribosome biogenesis, and the PI3K signaling pathway. The results provide insights into the protection and exploitation of genetic resources of Chinese indigenous laying ducks.

Rapid detection of goose megrivirus using TaqMan real-time PCR technology.

The aim of this study was to develop an efficient and accurate platform for the detection of the newly identified goose megrivirus (GoMV). To achieve this goal, we developed a TaqMan real-time PCR technology for the rapid detection and identification of GoMV. Our data showed that the established TaqMan real-time PCR assay had high sensitivity, with the lowest detection limit of 67.3 copies/µL. No positive signal can be observed from other goose origin viruses (including AIV, GPV, GoCV, GHPyV, and GoAstV), with strong specificity. The coefficients of variation of repeated intragroup and intergroup tests were all less than 1.5%, with excellent repeatability. Clinical sample investigation data from domestic Minbei White geese firstly provided evidence that GoMV can be transmitted both horizontally and vertically. In conclusion, since the TaqMan real-time PCR method has high sensitivity, specificity, and reproducibility, it can be a useful candidate tool for GoMV epidemiological investigation.

The network regulation mechanism of the effects of heat stress on the production performance and egg quality of Jinding duck was analyzed by miRNA‒mRNA.

To explore the differential regulation mechanism of heat stress on the egg production performance and egg quality of Jinding ducks, 200 Jinding ducks (360-day-old) in good health and with similar body weights and a normal appetite were selected and randomly divided into a control (normal temperature [NT]) group (20°C-25°C) and a heat stress (HS) group (32°C-36°C), with 4 replicates in each group and 25 ducks in each replicate. The pretrial period was 1 wk, and the formal trial period was 4 wk. At the end of the 4th wk, 12 duck eggs were collected from each replicate to determine egg quality. Pituitary and ovarian tissues of Jinding ducks were collected, transcriptome sequencing was performed to screen differentially expressed miRNAs and mRNAs related to high temperature and heat stress, and a competitive endogenous RNA regulatory network was constructed. The sequencing data were verified by qRT‒PCR method. The following results were obtained: (1) Compared with the NT group, the HS group had a significantly lower laying rate, total egg weight, average egg weight, total feed intake, and feed intake per duck (P < 0.01), an extremely significantly higher feed-to-egg ratio (P < 0.01), and a higher mortality rate. (2) Compared with the NT group, the HS group had an extremely significantly lower egg weight, egg yolk weight, eggshell weight, and eggshell strength (P < 0.01) and an extremely significantly lower yolk ratio and eggshell thickness (P < 0.01, P < 0.05); however, there was no significant difference in the egg shape index, Haugh unit or protein height (P > 0.05). (3) A total of 1,974 and 1,202 genes were identified in the pituitary and ovary, respectively, and there were 5 significantly differentially expressed miRNAs. The differentially expressed genes were involved in the arginine and proline metabolism pathways, ether lipid metabolism pathway, and drug metabolism-cytochrome P450 pathway, which are speculated to be related to the egg production performance of Jingding ducks under high-temperature heat stress. (4) Novel_221 may target the PRPS1 gene to participate in egg production performance; novel_168 and novel_289 may target PIGW; novel_289 may target Q3MUY2; and novel_289 and novel_208 may target PIGN or genes that may be related to high-temperature heat stress. (5) In pituitary tissue, upregulated novel_141 (center of the network) formed a regulatory network with HSPB1 and HSP30A, and downregulated novel_366 (center of the network) formed a regulatory network with the JIP1 gene. In ovarian tissue, downregulated novel_289 (center of the network) formed a regulatory network with the ZSWM7, ABI3, and K1C23 genes, novel_221 formed a regulatory network with the IGF1, BCL7B, SMC6, APOA4, and FARP2 genes, and upregulated novel_40 formed a regulatory network with the HA1FF10 gene. In summary, heat stress affects the production performance and egg quality of Jinding ducks by regulating the secretion of endocrine-related hormones and the release of neurotransmitters as well as the expression of miRNAs and mRNAs in pituitary and ovarian tissues. The miRNA‒mRNA regulatory network provides a theoretical basis for the molecular mechanism that regulates the stress response in pituitary and ovarian tissues, egg quality, and production performance under heat stress.

Analysis of circRNA-miRNA-mRNA regulatory network of embryonic gonadal development in Mulard duck.

The aim of the study was to explore the regulatory mechanism of differences in embryonic gonadal development between intergeneric distance hybrid offspring Mulard ducks and parent ducks. The morphological differences gonadal tissues of Muscovy ducks, Pekin ducks and Mulard ducks at 12.5-day embryonic age were observed by sectioning and hematoxylin-eosin (HE) staining. Then followed by transcriptome sequencing to screen for gonadal development-related differentially expressed circRNAs and mRNAs to construct a competitive endogenous RNA (ceRNA) regulatory network. Finally, qRT-PCR and luciferase reporter system were used to verify the sequencing data and targeting relationship of ceRNA pairs. The results showed that the seminiferous tubule lumen of Mulard ducks was not obvious, while there were obvious seminiferous tubules and tubular structures in testis of Pekin ducks and Muscovy ducks, with number and shape indicating maturity. There were 18 upregulated circRNAs and 16 downregulated circRNAs in Mulard ducks and Pekin ducks, respectively, and 39 upregulated circRNAs and 1 downregulated circRNA in Mulard ducks and Muscovy ducks, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis found that genes involves in dorso-ventral axis formation, for example, neurogenic locus notch homolog protein 1 (NOTCH1), were significantly enriched (P < 0.05). The novel_circ_0002265-gga-miR-122-5p-PAFAH1B2 regulatory network was constructed. The qRT-PCR results showed that the sequencing results were reliable. The dual-luciferase reporter assay showed that gga-miR-122-5p exists binding site of circ_0002265 and PAFAH1B2, indicating circ_0002265-gga-miR-122-5p-PAFAH1B2 targeting relationship. In summary, the embryonic gonadal development of intergeneric hybrid Mulard ducks may be regulated by differentially expressed circRNAs and genes, such as novel_circ_0000519, novel_circ_0003537, NOTCH1, FGFR2, PAFAH1B1, and PAFAH1B2, among which circ_0002265-gga-miR-122-5p-PAFAH1B2 may participate in the targeted regulation of gonadal development in Mulard ducks. The findings of this study are helpful for analyzing the mechanism of embryonic gonadal development differences in avians.

Metabolomic characterization of Liancheng white and Cherry Valley duck breast meat and their relation to meat quality.

Liancheng white duck is a typical local duck breed in Fujian Province famous for its meat traits. To better understand how meat quality varies with breed, the chemical composition of breast meats of Liancheng white ducks (LD) and Cherry Valley ducks (CD) were examined using ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS).The correlation between meat quality and the differential metabolites was further analyzed. The results showed that the effects of breed on duck breast meat were significant for pH, color, cooking loss, and shear force. Liancheng white duck breast meat exhibited a higher shear force and pH, and lower cooking loss and lightness (L*24), redness (a*24), and yellowness (b*24) than CD. Metabolomic analysis revealed significant differences between the meat extracts from the 2 duck breeds. A total of 49 and 57 significantly different metabolites were identified in positive and negative ion modes, respectively. These differentially accumulated metabolites (DAMs) could be divided into 28 classes, of which the 4 main categories were carbohydrates, amino acids, fatty acids, and eicosanoids. Liancheng white duck might have better nutritional and medicinal value considering the higher content of (4Z,7Z,10Z,13Z,16Z,19Z)-4,7,10,13,16,19-docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), and prostaglandinF3α (PGF3α), having anti-inflammatory orantioxidant effects. Carbohydrate concentration negatively correlated with pH24. The 4 metabolites positively correlated with the shear force. These results provide an overall perspective for bridging the gap between variation of duck meat quality and metabolites with respect to breed.

Comparative transcriptome and histomorphology analysis of testis tissues from mulard and Pekin ducks.

Testicular transcriptomes were analyzed to characterize the differentially expressed genes between mulard and Pekin ducks, which will help establish gene expression datasets to assist in further determination of the mechanisms of genetic sterility in mulard ducks. Paraffin sections were made to compare the developmental differences in testis tissue between mulard and Pekin ducks. Comparative transcriptome sequencing of testis tissues was performed, and the expression of candidate genes was verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). In mulard ducks, spermatogonia and spermatocytes were arranged in a disordered manner, and no mature sperm were observed in the testis tissue. However, different stages of development of sperm were observed in seminiferous tubules in the testis tissue of Pekin ducks. A total of 43.84 Gb of clean reads were assembled into 193 535 UniGenes. Of these, 2131 transcripts exhibited differential expression (false discover rate < 0.001 and fold change ≥ 2 ), including 997 upregulated and 1134 downregulated transcripts in mulard ducks as compared to those in Pekin duck testis tissues. Several upregulated genes were related to reproductive functions, including ryanodine receptor 2 (RYR2), calmodulin (CALM), argininosuccinate synthase and delta-1-pyrroline-5-carboxylate synthetase ALDH18A1 (P5CS). Downregulated transcripts included the testis-specific serine/threonine-protein kinase 3, aquaporin-7 (AQP7) and glycerol kinase GlpK (GK). The 10 related transcripts involved in the developmental biological process were identified by GO (Gene Ontology) annotation. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways indicated that peroxisome proliferator-activated receptors (PPARs) and calcium signaling pathways were significantly ( P < 0.001 ) associated with normal testis physiology. The differential expression of select genes implicated in reproductive processes was verified by qRT-PCR, which was consistent with the expression trend of transcriptome sequencing (RNA-seq). Differentially expressed candidate genes RYR2, CALM, P5CS, AQP7 and GK were identified by transcriptional analysis in mulard and Pekin duck testes. These were important for the normal development of the male duck reproductive system. These data provide a framework for the further exploration of the molecular and genetic mechanisms of sterility in mulard ducks. Highlights. The mulard duck is an intergeneric sterile hybrid offspring resulting from mating between Muscovy and Pekin ducks. The transcriptomes of testis tissue from mulard and Pekin ducks were systematically characterized, and differentially expressed genes were screened, in order to gain insights into potential gonad gene expression mechanisms contributing to genetic sterility in mulard ducks.

Differential protein profiles in duck meat during the early postmortem storage period.

The present study was designed to investigate proteomic differences in duck breast muscle during the early postmortem storage period. The meat quality was evaluated at 0 hr and 24 hr postmortem at 4°C in Pekin ducks, black Muscovy ducks and Mule ducks. Differentially expressed proteins were detected by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS) at 0 hr and 24 hr postmortem in the three duck breeds. The results showed that 53 proteins spots were differentially expressed at 0 hr and 24 hr postmortem at 4°C in Pekin ducks, 75 spots in black Muscovy ducks, and 72 spots in Mule ducks. A total of 30 (10 spots for each breed) were selected for identification by mass spectrometry. Seven proteins were identified in Pekin ducks, eight in black Muscovy ducks and seven in Mule ducks. Moreover, the above results obtained by 2-DE and MALDI-TOF/TOF MS were confirmed by western blotting. To our knowledge, this study is the first to provide insights into the protein profiles of ducks during postmortem storage and provides a better understanding of the biochemical processes that contribute to duck meat quality.

Ovarian transcriptomic analysis of Shan Ma ducks at peak and late stages of egg production.

OBJECTIVE: To assess the differences in ovarian transcriptomes in Shan Ma ducks between their peak and late stages of egg production, and to obtain new transcriptomic data of these egg-producing ducks. METHODS: The Illumina HiSeq 2000 system was used for high throughput sequencing of ovarian transcriptomes from Shan Ma ducks at their peak or late stages of egg production. RESULTS: Greater than 93% of the sequencing data had a base quality score (Q score) that was not less than 20 (Q20). From ducks at their peak stage of egg production, 42,782,676 reads were obtained, with 4,307,499,083 bp sequenced. From ducks at their late stage of egg production, 45,316,166 reads were obtained, with 4,562,063,363 bp sequenced. A comparison of the two datasets identified 2,002 differentially expressed genes, with 790 upregulated and 1,212 downregulated. Further analysis showed that 1,645 of the 2,002 differentially expressed genes were annotated in the non-redundant (NR) database, with 646 upregulated and 999 downregulated. Among the differentially expressed genes with annotations in the NR database, 696 genes were functionally annotated in the clusters of orthologous groups of proteins database, involving 25 functional categories. One thousand two hundred four of the differentially expressed genes with annotations in the NR database were functionally annotated in the gene ontology (GO) database, and could be divided into three domains and 56 categories. The three domains were cellular component, molecular function, and biological process. Among the genes identified in the GO database, 451 are involved in development and reproduction. Analysis of the differentially expressed genes with annotations in the NR database against the Kyoto encyclopedia of genes and genomes database revealed that 446 of the genes could be assigned to 175 metabolic pathways, of which the peroxisome proliferator-activated receptor signaling pathway, insulin signaling pathway, fructose and mannose metabolic pathways, gonadotropin releasing hormone signaling pathway and transforming growth factor beta signaling pathway were significantly enriched. CONCLUSION: The differences in ovarian transcriptomes in Shan Ma ducks between their peak and late stages of egg production were elucidated, which greatly enriched the ovarian transcriptomic information of egg-producing ducks.

Chimeric influenza-virus-like particles containing the porcine reproductive and respiratory syndrome virus GP5 protein and the influenza virus HA and M1 proteins.

Both porcine reproductive and respiratory syndrome and swine influenza are acute, highly contagious swine diseases. These diseases pose severe threats for the swine industry and cause heavy economic losses worldwide. In this study, we have developed a chimeric virus-like particle (VLP) vaccine candidate for porcine reproductive and respiratory syndrome virus (PRRSV) and H3N2 influenza virus and investigated its immunogenicity in mice. The HA and M1 proteins from the H3N2 influenza virus and the PRRSV GP5 protein fused to the cytoplasmic and transmembrane domains of the NA protein were both incorporated into the chimeric VLPs. Analysis of the immune responses showed that the chimeric VLPs elicited serum antibodies specific for both PRRSV GP5 and the H3N2 HA protein, and they stimulated cellular immune responses compared to the responses to equivalent amounts of inactivated viruses. Taken together, the results suggested that the chimeric VLP vaccine represents a potential strategy for the development of a safe and effective vaccine to control PRRSV and H3N2 influenza virus.