Duck Cells Research Articles

The Application of Duck Embryonic Fibroblasts CCL-141 as a Cell Model for Adipogenesis.

The duck embryo fibroblast cell line CCL-141, which is currently the only commercialized duck cell line, has been underexplored in adipogenesis research. (1) Background: This study establishes an experimental protocol to induce adipogenesis in CCL-141 cells, addressing the importance of understanding gene functions in this process. (2) Methods: Chicken serum, fatty acids, insulin, and all-trans retinoic acid were used to treat CCL-141 cells, with adipogenesis confirmed by Oil Red O staining and gene expression quantification. CRISPR/Cas9 technology was applied to knockout PPARγ, and the resulting adipogenic phenotype was assessed. (3) Results: The treatments promoted adipogenesis, and the knockout of PPARγ validated the cell line's utility for gene function studies. (4) Conclusions: CCL-141 cells are a suitable model for investigating duck adipogenesis, contributing to the understanding of regulatory factors in this biological process.

Effect of the LIF gene on the cell cycle and apoptosis of ovarian granulosa cells in white Muscovy ducks

Leukaemia inhibitory factor (LIF), a member of the interleukin-6 (IL-6) family, is a multifunctional cytokine. The maturation-to-ovulation process of poultry follicles is determined by granulosa cell proliferation and differentiation. Granulosa cell apoptosis and degeneration lead to follicular atresia, which reduces the number of normally developing follicles and leads to a decrease in the poultry egg production rate, thus affecting the large-scale development of poultry breeding. In this study, the LIF gene overexpression vector pCDH-CMV-LIF and a siRNA that inhibits LIF gene expression were transfected into primary granulosa cells from white Muscovy duck ovaries for functional study. Compared with that in the control group, LIF gene expression was confirmed to be significantly decreased or increased in the transfection groups (P < 0.01). After LIF overexpression, the expression of the cell cycle-related genes CCND1, CDK-1 and PCNA was decreased (P < 0.05); apoptosis was promoted; the proapoptotic genes Bax and caspase-3 were significantly upregulated (P < 0.01); and the antiapoptotic gene Bcl-2 was significantly downregulated (P < 0.01). After LIF interference, the expression of the cell cycle-related genes CCND1, CCNE1, CDK-1 and PCNA and the antiapoptotic gene Bcl-2 significantly increased (P < 0.01), whereas the expression of the proapoptotic genes Bax, caspase-3 and caspase-9 significantly decreased (P < 0.01). In summary, the LIF gene is involved in regulating the biological function of ovarian granulosa cells in white Muscovy ducks. LIF gene expression promotes granulosa cell apoptosis and inhibits cell cycle progression. These experimental results provide insights into the follicular development mechanism of white Muscovy ducks.

Duck STING mediates antiviral autophagy directing the interferon signaling pathway to inhibit duck plague virus infection

Migratory birds are important vectors for virus transmission, how migratory birds recognize viruses and viruses are sustained in birds is still enigmatic. As an animal model for waterfowl among migratory birds, studying and dissecting the antiviral immunity and viral evasion in duck cells may pave a path to deciphering these puzzles. Here, we studied the mechanism of antiviral autophagy mediated by duck STING in DEF cells. The results collaborated that duck STING could significantly enhance LC3B-II/I turnover, LC3B-EGFP puncta formation, and mCherry/EGFP ratio, indicating that duck STING could induce autophagy. The autophagy induced by duck STING is not affected by shRNA knockdown of ATG5 expression, deletion of the C-terminal tail of STING, or TBK1 inhibitor BX795 treatment, indicating that duck STING activated non-classical selective autophagy is independent of interaction with TBK1, TBK1 phosphorylation, and interferon (IFN) signaling. The STING R235A mutant and Sar1A/B kinase mutant abolished duck STING induced autophagy, suggesting binding with cGAMP and COPII complex mediated transport are the critical prerequisite. Duck STING interacted with LC3B through LIR motifs to induce autophagy, the LIR 4/7 motif mutants of duck STING abolished the interaction with LC3B, and neither activated autophagy nor IFN expression, indicating that duck STING associates with LC3B directed autophagy and dictated innate immunity activation. Finally, we found that duck STING mediated autophagy significantly inhibited duck plague virus (DPV) infection via ubiquitously degraded viral proteins. Our study may shed light on one scenario about the control and evasion of diseases transmitted by migratory birds.

Circular RNAs are associated with the resistance to Newcastle disease virus infection in duck cells.

Newcastle disease virus (NDV) is prevalent worldwide with an extensive host range. Among birds infected with velogenic NDV strains, chickens experience high pathogenicity and mortality, whereas ducks mostly experience mild symptoms or are asymptomatic. Ducks have a unique, innate immune system hypothesized to induce antiviral responses. Circular RNAs (circRNAs) are among the most abundant and conserved eukaryotic transcripts. These participate in innate immunity and host antiviral response progression. In this study, circRNA expression profile differences post-NDV infection in duck embryo fibroblast (DEF) cells were analyzed using circRNA transcriptome sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to reveal significant enrichment of differentially expressed (DE) circRNAs. The circRNA-miRNA-mRNA interaction networks were used to predict the related functions of circRNAs. Moreover, circ-FBXW7 was selected to determine its effect on NDV infection in DEFs. NDV infection altered circRNA expression profiles in DEF cells, and 57 significantly differentially expressed circRNAs were identified post-NDV infection. DEF responded to NDV by forming circRNAs to regulate apoptosis-, cell growth-, and protein degradation-related pathways via GO and KEGG enrichment analyses. circRNA-miRNA-mRNA interaction networks demonstrated that DEF cells combat NDV infection by regulating cellular pathways or apoptosis through circRNA-targeted mRNAs and miRNAs. circ-FBXW7 overexpression and knockdown inhibited and promoted viral replication, respectively. DEF cells mainly regulated cell cycle alterations or altered cellular sensing to combat NDV infection. These results demonstrate that DEF cells exert antiviral responses by forming circRNAs, providing novel insights into waterfowl antiviral responses.

Primary culture and endocrine functional analysis of Leydig cells in ducks (Anas platyrhynchos).

Testicular Leydig cells (LCs) are the primary known source of testosterone, which is necessary for maintaining spermatogenesis and male fertility. However, the isolation, identification, and functional analysis of testosterone in duck LCs are still ambiguous. The aim of the present study was to establish a feasible method for isolating highly purified primary duck LCs. The highly purified primary duck LCs were isolated from the fresh testes of 2-month-old ducks via the digestion of collagenase IV and Percoll density gradient centrifugation; hematoxylin and eosin (H&E), immunohistochemistry (IHC) staining, ELISA, and radioimmunoassay were performed. Results revealed that the LCs were prominently noticeable in the testicular interstitium of 2-month-old ducks as compared to 6-month-old and 1-year-old ducks. Furthermore, IHC demonstrated that the cultured LCs occupied 90% area of the petri dish and highly expressed 3β-HSD 24h after culture (hac) as compared to 48 and 72 hac. Additionally, ELISA and radioimmunoassay indicate that the testosterone level in cellular supernatant was highly expressed in 24 and 48 hac, whereas the testosterone level gradually decreased in 72 and 96 hac, indicating the primary duck LCs secrete testosterone at an early stage. Based on the above results, the present study has effectively developed a technique for isolating highly purified primary duck LCs and identified its biological function in synthesizing testosterone.

Study on Propagation and Adaptation of EDS-76 Avian Adenovirus in Duck and SPF Primary Embryonic Chicken Cell Culture Comparison to Duck and SPF Embryonated Chicken Eggs.

Egg drop syndrome (EDS) is a major viral infectious poultry disease with severe economic losses in laying hens. The disease is caused by an adenovirus and can be transmitted horizontally and vertically. This study investigated the EDS virus (EDSV) infection in duck embryo fibroblasts (DEF), specific pathogen-free (SPF) embryo fibroblasts, and SPF egg embryos using different methods. The results were compared to the virus culture in duck and SPF chicken eggs. Duck and chicken fibroblast cells were used as the primary cell culture in Dulbecco's Modified Eagle Medium, and the low-pathogenic duck adenovirus was used to infect the ducks and SPF fibroblasts primary cell cultures, as well as the duck and SPF eggs. The titer of the virus was measured by hemagglutination assay, ECID50, plaque-forming unit, and TCID50 methods. The results revealed that EDSV could proliferate in the chorioallantoic membrane of DEF cells and duck eggs, compared to the chorioallantoic membrane of chicken embryo fibroblasts (CEF) and SPF chicken eggs. The findings showed that duck egg embryos and primary DEF cell lines are more appropriate for EDSV replication, compared to CEF and SPF chicken eggs. This suggests that the use of DEF culture for producing avian adenovirus EDS-76 is a suitable alternative for the embryonic egg culture.

BX795, a kinase inhibitor, inhibit duck plague virus infection via targeting US3 kinase

Duck plague virus (DPV) is a typical DNA virus of waterfowl, it causes huge economic losses to the duck industry due to the higher mortality and lower egg production. The disease is one of the frequent epidemics and outbreaks on duck farms because present vaccines could not provide complete immunity and there are no specific antiviral drugs available. Therefore, the development of antiviral drugs is urgently needed. In this study, we evaluated the antiviral activity of BX795, a specific kinase inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1), protein kinase B (AKT) and Tank binding kinase 1 (TBK1), against DPV in different duck cells. Our study demonstrated that BX795 reveals prominent antiviral activity in a dose-dependent manner in different types of duck cells. Time-addition and antiviral duration analysis uncovered that BX795 inhibits viral infection therapeutically and its antiviral activity lasts longer than 96 h. Further studies have shown that BX795 prevents cell-to-cell spread of the DPV rather than affects other stage of viral life cycle. Mechanistically, BX795 can inhibit DPV US3 kinase activity, reduce the phosphorylation of US3 substrates, and prevent the interaction between US3 and UL47. Taking together, our study demonstrated BX795, which disrupts the viral kinase activity, is a candidate antiviral agent for DPV.

Ubiquitin-specific protease 18 in mallard (Anas platyrhynchos) interferes with type I interferon-mediated inhibition of high pathogenicity avian influenza virus replication

Ubiquitin-specific protease 18 (USP18) is a well-established innate immune factor in vertebrates. Although Anatidae birds rarely exhibit distinctive clinical signs during high pathogenicity avian influenza virus (HPAIV) infections, some virus strains cause deadly diseases. Here, we investigated the association between USP18 expression and pathogenicity during HPAIV infections in the Anatidae mallard Anas platyrhynchos. First, mallard USP18 gene (duUSP18) was cloned, and its transcriptional variants, with three different open reading frames, were characterized. Experimental infections with two different pathogenic strains, Miyazaki and Takeo, demonstrated an early induction of duUSP18 mRNA upon HPAIV infection in a bird's whole body in vivo and in primary duck cells in vitro, which was positively associated with pathogenicity in mallards. In addition, duUSP18 knockdown under interferon-β stimulation attenuated viral replication, regardless of pathogenicity. These results indicate a role for duUSP18 in favoring viral replication and virus resistance to type I interferon immunity in mallards.

Duck Plague Virus Negatively Regulates IFN Signaling to Promote Virus Proliferation via JNK Signaling Pathway.

Duck plague virus (DPV), a member of the alphaherpesvirus subfamily, can cause severe damage and immunosuppression in ducks and geese in China. Since lacking an available cell model, the antiviral signal transduction pathways induction and regulation mechanisms related to DPV infection in duck cells are still enigmatic. Our previous study developed a monocyte/macrophages cell model, which has been applied to study innate immunity with DPV. In the present study, we compared and analyzed transcriptome associated with the DPV infection of CHv (virulent strain) and CHa (avirulent strain) at 48hpi based on the duck monocyte/macrophages cell model and RNA-seq technology. Differentially expressed genes (DEGs) analysis showed 2,909 and 2,438 genes altered in CHv and CHa infected cells compared with control cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the DEGs were mainly involved in biological processes such as metabolic pathways, viral infectious diseases, immune system, and signal transduction. The CHv and CHa virus differentially regulated MAPK, NF-κB, and IFN signaling pathways based on transcriptome sequencing data and RT-qPCR results. The JNK inhibitor SP600125 enhanced the IFN signaling, but potentially reduced the VSV and DPV titers in the cell culture supernatant, indicating that JNK negatively regulates the IFN pathway and the inflammatory pathway to promote virus proliferation. The research results may provide promising information to understand the pathogenesis of DPV and provide a novel mechanism by which DPV modulates antiviral signaling and facilitate virus proliferation through hijacking the JNK pathway, which provides a new means for the prevention and control of DPV infection.

Contribution of mutation I142M in fusion protein and Q44R in matrix protein of Newcastle disease virus to virulence in ducks.

Although verogenic Newcastle disease viruses (NDVs) generally cause subclinical infection in waterfowls such as ducks, NDVs with high virulence in waterfowl have been sporadically reported. We previously reported that the NDV d5a20b strain, which is obtained by serial passaging of the velogenic 9a5b strain in domestic ducks, showed increased virulence in ducks (Hidaka et al., 2021). The d5a20b strain had 11 amino acid substitutions in its P/V, M, F, HN, and L proteins as compared to 9a5b. In the present study, we generated a series of recombinant (r) NDVs with these amino acid substitutions to identify the molecular basis of virulence of NDV in ducks, and evaluated their influences on virulence and in vitro viral properties. Each of the single amino acid substitutions in either the F protein I142M or the M protein Q44R contributed to the enhancement of intracerebral and intranasal pathogenicity in domestic ducks. The cell-cell fusion activity of the virus with F I142M was five times higher than that of the parental r9a5b. The virus with M Q44R rapidly replicated in duck embryo fibroblasts. Additionally, the rM+F+HN strain, which has the same amino acid sequences as d5a20b in M, F, and HN proteins, showed the highest level of virulence and replication efficiency among the generated recombinant viruses, nearly comparable to rd5a20b. These results suggest that multiple factors are involved in the high growth ability of NDV in duck cells, leading to increased virulence in vivo.

Orbiviruses in biting midges and mosquitoes from the Zambezi region, Namibia.

The genus Orbivirus includes a variety of pathogenic viruses that are transmitted by biting midges, mosquitoes and ticks. Some of the economically most relevant orbiviruses are endemic to Namibia, like the livestock-pathogenic Bluetongue and African horse sickness viruses. Here, we assessed the diversity of orbiviruses circulating in the Zambezi region of north-eastern Namibia. A total of 10 250 biting midges and 10 206 mosquitoes were collected and screened for orbivirus infections. We identified Palyam virus (PALV) in a pool of biting midges (Culicoides sp.) sampled in the Wuparo Conservancy and three strains of Corriparta virus (CORV) in Culex sp. mosquitoes sampled in Mudumu National Park and the Mashi Conservancy. This is, to our knowledge, the first detection of PALV and CORV in Namibia. Both viruses infect vertebrates but only PALV has been reported to cause disease. PALV can cause foetal malformations and abortions in ruminants. Furthermore, a novel orbivirus, related to Kammavanpettai virus from India and Umatilla virus from North America, was discovered in biting midges (Culicoides sp.) originating from Mudumu National Park and tentatively named Mudumu virus (MUMUV). Complete genomes of PALV, CORV and MUMUV were sequenced and genetically characterized. The Namibian CORV strain showed 24.3 % nucleotide divergence in its subcore shell gene to CORV strains from Australia, indicating that African CORV variants vary widely from their Australian relatives. CORV was isolated in cell culture and replicated to high titres in mosquito and duck cells. No growth was found in rodent and primate cells. The data presented here show that diverse orbiviruses are endemic to the Zambezi region. Further studies are needed to assess their effects on wildlife and livestock.

Duck Tembusu virus induces stronger cellular responses than Japanese encephalitis virus in primary duck neurons and fibroblasts.

Duck Tembusu virus (DTMUV) and Japanese encephalitis virus (JEV) are mosquito-borne flaviviruses. These two viruses infect ducks; however, they show different neurological outcomes. The mechanism of DTMUV- and JEV-induced neuronal death has not been well investigated. In the present study examined the differences in the mechanisms involved in virus-induced cell death and innate immune responses between the DTMUV KPS54A61 strain and the JEV JaGAr-01 strain using primary duck neurons (DN) and duck fibroblasts (CCL-141). DN and CCL-141 were permissive for the infection and replication of these two viruses, which up-regulated the expression of innate immunity genes. Both DTMUV and JEV induced cell death via a caspase-3-dependent manner; however, DTMUV triggered more cell death than did JEV in both CCL-141 and DN. These findings suggest that DTMUV infection causes apoptosis in duck neurons and fibroblasts more strongly than JEV. The levels of the mRNA expression of innate immunity-related genes after DTMUV infection were generally higher than the levels after JEV infection, suggesting that DTMUV-induced immune response in duck cells may exhibit toxic effects rather than protective effects.

Primary Chicken and Duck Endothelial Cells Display a Differential Response to Infection with Highly Pathogenic Avian Influenza Virus.

Highly pathogenic avian influenza viruses (HPAIVs) in gallinaceous poultry are associated with viral infection of the endothelium, the induction of a ‘cytokine storm, and severe disease. In contrast, in Pekin ducks, HPAIVs are rarely endothelial tropic, and a cytokine storm is not observed. To date, understanding these species-dependent differences in pathogenesis has been hampered by the absence of a pure culture of duck and chicken endothelial cells. Here, we use our recently established in vitro cultures of duck and chicken aortic endothelial cells to investigate species-dependent differences in the response of endothelial cells to HPAIV H5N1 infection. We demonstrate that chicken and duck endothelial cells display a different transcriptional response to HPAI H5N1 infection in vitro—with chickens displaying a more pro-inflammatory response to infection. As similar observations were recorded following in vitro stimulation with the viral mimetic polyI:C, these findings were not specific to an HPAIV H5N1 infection. However, similar species-dependent differences in the transcriptional response to polyI:C were not observed in avian fibroblasts. Taken together, these data demonstrate that chicken and duck endothelial cells display a different response to HPAIV H5N1 infection, and this may help account for the species-dependent differences observed in inflammation in vivo.

LncRNA lnc_13814 promotes the cells apoptosis in granulosa cells of duck by acting as apla-miR-145-4 sponge

ABSTRACT Follicle development is a vital factor which determines the reproductive performance of poultry. Long noncoding RNAs (lncRNAs) have been reported to maintain animal reproductive function and play key roles in ovarian development and hormone secretion. But the regulatory mechanism of lncRNAs in duck follicle development has seldom been reported. In this study, to better explore the molecular mechanism of follicle development in ducks, the follicular lncRNA was sequenced and analyzed. A total of 9,551 lncRNAs were predicted in the duck follicles. Four hundred and forty-five lncRNAs were differentially expressed between the white follicles and yellow follicles. The results of our studies showed that lnc_13814 promoted cell apoptosis in duck GCs. Furthermore, the bioinformatics analysis results demonstrated that lnc_13814 was involved in a lncRNA-miRNA-mRNA coexpression network and it was observed to sponge two follicle-related miRNAs by a luciferase activity assay. Moreover, we found that overexpression of lnc_13814 significantly increased DNA damage inducible transcript 3 (DDIT3) expression and downregulated GCs apoptosis. Finally, we found that lnc_13814 directly binds to and inhibits apla-mir-145-4; then, lnc_13814 increases the expression of DDIT3 and up-regulates GCs apoptosis. Taken together, our findings demonstrate that lncRNAs have potential effects on duck ovarian follicles and lncRNAs may represent a new approach to understand follicular development.

Simultaneous circulation of two West Nile virus lineage 2 clades and Bagaza virus in the Zambezi region, Namibia.

Flaviviruses include a great diversity of mosquito-borne arboviruses with epidemic potential and high global disease burden. Several flaviviruses are circulating in southern Africa affecting humans and livestock, among them West Nile virus (WNV) and Wesselsbron virus. Despite their high relevance, no arbovirus surveillance study has been conducted for more than 35 years in Namibia. In this study we assessed the diversity of flaviviruses circulating in mosquitoes in the densely populated, semi-tropical Zambezi region of north-eastern Namibia. In total, 10,206 mosquitoes were sampled in Bwabwata and Mudumu national parks and Mashi and Wuparo conservancies and screened for flavivirus infections. A high infection rate with insect-specific flaviviruses was found with 241 strains of two previously known and seven putative novel insect-specific flaviviruses. In addition, we identified ten strains of WNV in the main vector Cx. univittatus sampled in the Mashi conservancy. Surprisingly, the strains fell into two different clades of lineage 2, 2b and 2d. Further, three strains of Bagaza Virus (BAGV) were found in Cx. univittatus mosquitoes originating from Mudumu national park. Assessment of BAGV growth in different cell lines showed high replication rates in mosquito and duck cells and about 100,000fold lower replication in human, primate and rodent cells. We demonstrate a wide genetic diversity of flaviviruses is circulating in mosquitoes in the Zambezi region. Importantly, WNV and BAGV can cause outbreaks including severe disease and mortality in humans and birds, respectively. Future studies should focus on WNV and BAGV geographic distribution, as well as on their potential health impacts in and the associated social and economic implications for southern Africa.

SC75741 antagonizes vesicular stomatitis virus, duck Tembusu virus, and duck plague virus infection in duck cells through promoting innate immune responses

Duck Tembusu virus (DTMUV) and duck plague virus (DPV) are typical DNA and RNA viruses of waterfowl, causing drastic economic losses to the duck farm industry in terms of high mortality and decreased egg production. These 2 viruses reappear from time to time because the available vaccines fail to provide complete immunity and no clinical antiviral drugs are available for them. In the present study, we evaluated the antiviral activity of SC75741 for DTMUV, DPV, and the model virus, vesicular stomatitis virus infection in duck cells. SC75741, a nuclear factor-kappa B (NF-κB)-specific inhibitor in mammal cells, revealed the highest antiviral activity among the inhibitors specific to c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (p38), and NF-κB signaling. The antiviral activity of SC75741 was dose-dependent and showed effects in different duck cell types. Time-addition and duration assay demonstrated that SC75741 inhibited virus infection in the middle of and after virus infection at least for 72 h in duck embro fibroblast cells. The DPV viral adsorption and genomic copy number were reduced, indicating that SC75741 blocks the phase of the virus life cycle at viral entry and genomic replication. In addition, SC75741 enhanced the expression of interferon only when stimulator of interferon genes (STING) was overexpressed or pre-activated by the virus infection, suggesting that SC75741 acts as a STING agonist. In conclusion, SC75741 is a candidate antiviral agent for DTMUV and DPV.

In Vitro and In Ovo Host Restriction of Aquatic Bird Bornavirus 1 in Different Avian Hosts.

Aquatic bird bornavirus 1 (ABBV-1) is associated with chronic meningoencephalitis and ganglioneuritis. Although waterfowl species act as the natural host of ABBV-1, the virus has been sporadically isolated from other avian species, showing the potential for a broad host range. To evaluate the host restriction of ABBV-1, and its potential to infect commercial poultry species, we assessed the ability of ABBV-1 to replicate in cells and embryos of different avian species. ABBV-1 replication was measured using multi- and single-step growth curves in primary embryo fibroblasts of chicken, duck, and goose. Embryonated chicken and duck eggs were infected through either the yolk sac or chorioallantoic cavity, and virus replication was assessed by immunohistochemistry and RT-qPCR in embryonic tissues harvested at two time points after infection. Multi-step growth curves showed that ABBV-1 replicated and spread in goose and duck embryo fibroblasts, establishing a population of persistently infected cells, while it was unable to do so in chicken fibroblasts. Single-step growth curves showed that cells from all three species could be infected; however, persistence was only established in goose and duck fibroblasts. In ovo inoculation yielded no detectable viral replication or lesion in tissues. Data indicate that although chicken, duck, and goose embryo fibroblasts can be infected with ABBV-1, a persistent infection is more easily established in duck and goose cells. Therefore, ABBV-1 may be able to infect chickens in vivo, albeit inefficiently. Additionally, our data indicate that an in ovo model is inadequate to investigating ABBV-1 host restriction and pathogenesis.

Ultrastructural studies on the pancreatic acini in duck (Anas boscas) and pigeon (Columba livia)

The aim of this research work was to study the histological structure of the pancreatic acini by transmission electron microscope in two avian species, duck and pigeon. The specimens were collected and processed for electron microscopic study. The results showed that the acini of the two avian species were two types; the first one was an electron dense and the second one an electron lucent. The light acinar cells were larger in size than the dark cells. These cells contained centrally located ovoid nuclei with prominent nucleoli and abundant euchromatin. The cytoplasm was electron lucent, with many rough endoplasmic reticulum, polymorphic mitochondria. Numerous zymogen granules were distributed in the basal part and around the nucleus, so these cells considered active cells. The dark acinar cells were characterized by an electron dense cytoplasm. The most prominent cell organelle in these cells were the zymogen granules that appeared in different sizes while other organelles as mitochondria, and rough endoplasmic reticulum were inconspicuous or few, so these cells were considered as inactive cells. The nucleus with indented nuclear membrane located centrally with prominent nucleoli and abundant heterochromatin. Prominent intercellular spaces between the individual acinar cells, as well as well-developed basement membrane separating the electron dense cells and the lumen contained the secretion between acinar cells. It could be concluded that the acinar cells in ducks and pigeons were divided into two types, that is, light and dark acinar cells which mainly attributed to the activity of these cells.

Cadmium induces cytotoxicity through oxidative stress-mediated apoptosis pathway in duck renal tubular epithelial cells

Cadmium (Cd) is a well studied nephrotoxic metal element. To investigate the effects of Cd-induced cytotoxicity on oxidative stress-mediated apoptosis in primary renal tubular epithelial cells of duck. Shaoxing duck (Anas platyrhyncha) renal tubular epithelial cells were cultured in medium in absence and presence of 3CdSO4·8H2O (1.25, 2.5, 5.0 μM Cd), in N-acetyl-l-cysteine (NAC) (100 μM), and the combination of Cd and NAC for 12 h. After 12 h exposure, morphologic observation and function, reactive oxygen species (ROS) level, antioxidant indices, the activity of ATPase, intracellular pH and [Ca2+]i, mitochondrial membrane potential (MMP), and apoptosis-related genes mRNA were determined. The results showed that Cd exposure could induce release of intracellular lactate dehydrogenase (LDH), simultaneously, enhance the ROS generation, acidification, malondialdehyde (MDA) and [Ca2+]i, decrease glutathione (GSH), Na+, K+-ATPase, Ca2+-ATPase, catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) activities as well as MMP, upregulated Bak-1, Bax and Caspase-3 mRNA expression, inhibited Bcl-2 mRNA expression, and induced cell apoptosis. The toxicity of Cd to cells showed a dose-dependent manner. Antioxidant NAC could efficiently alleviate Cd-induced the cytotoxicity. Taken together, these results suggest that Cd exposure cause cytotoxicity through oxidative stress-mediated apoptosis pathway in duck renal tubular epithelial cells.

Spread of Highly Pathogenic Avian Influenza (HPAI) H5N5 Viruses in Europe in 2016-2017 Appears Related to the Timing of Reassortment Events.

During the epizootic of highly pathogenic avian influenza (HPAI) H5N8 virus in Europe in 2016–2017, HPAI viruses of subtype H5N5 were also isolated. However, the detection of H5N5 viruses was limited compared to H5N8. In this study, we show that the genetic constellation of a newly isolated H5N5 virus is different from two genotypes previously identified in the Netherlands. The introduction and spread of the three H5N5 genotypes in Europe was studied using spatiotemporal and genetic analysis. This demonstrated that the genotypes were isolated in distinguishable phases of the epizootic, and suggested multiple introductions of H5N5 viruses into Europe followed by local spread. We estimated the timing of the reassortment events, which suggested that the genotypes emerged after the start of autumn migration. This may have prevented large-scale spread of the H5N5 viruses on wild bird breeding sites before introduction into Europe. Experiments in primary chicken and duck cells revealed only minor differences in cytopathogenicity and replication kinetics between H5N5 genotypes and H5N8. These results suggest that the limited spread of HPAI H5N5 viruses is related to the timing of the reassortment events rather than changes in virus pathogenicity or replication kinetics.