Chick Kidney Cells Research Articles

Copper exposure promotes ferroptosis of chicken (Gallus gallus) kidney cells and causes kidney injury

PurposeWhile copper (Cu) is essential for biological organisms, excessive Cu can be harmful. Ferroptosis is a programmed cell death pathway, but the role of ferroptosis in renal injury induced by Cu is limited. The aim of this study was to investigate the role of ferroptosis in kidney injury in chickens and the molecular mechanism by which Cu promotes renal ferroptosis. Materials and methodsChicken were subjected to Cu treatment by artificially adding excess Cu to the basal diet (the Cu concentration in the diet was supplemented to 110–330 mg/kg), and the impact on kidney fibrosis, tissue structure, and ferroptosis-related molecular markers was studied. Then, the expression levels of genes and proteins related to ferroptosis, iron metabolism and ferroautophagy were detected to explore the promoting effect of Cu on ferroptosis in chicken kidney. Main findingsCu treatment resulted in significant fibrosis and tissue structure damage in chicken kidneys. Molecular analysis revealed a significant upregulation of LC3Ⅱ, P62, ATG5, and NCOA4, along with a decrease in FTH1 and FTL protein levels. Additionally, crucial markers of ferroptosis, including the loss of GPX4, SLC7A11, and FSP1, and an increase in PTGS2 and ACSL4 protein levels, were observed in chicken kidneys after Cu exposure. ConclusionOur study showed that dietary Cu excess caused kidney injury in brochickens and exhibited ferroptosis-related features, including lipid peroxidation, reduction of ferritin, and downregulation of FSP1 and GPX4. These results indicate that excess Cu can induce renal ferroptosis and lead to kidney injury in chickens. This study highlights the complex interplay between Cu ions and ferroptosis in the context of renal injury and provides a new perspective for understanding the mechanism of Cu-induced renal injury.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry and ultra-performance liquid chromatography-mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titter of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titter of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry and ultra-performance liquid chromatography-mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titter of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titter of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry and ultra-performance liquid chromatography-mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titter of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titter of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

CATH-2-derived antimicrobial peptide inhibits multidrug-resistant Escherichia coli infection in chickens.

Avian colibacillosis (AC), caused by infection with Escherichia coli (E. coli), is a major threat to poultry health, food safety and public health, and results in high mortality and significant economic losses. Currently, new drugs are urgently needed to replace antibiotics due to the continuous emergence and increasing resistance of multidrug-resistant (MDR) strains of E. coli caused by the irrational use of antibiotics in agriculture and animal husbandry. In recent years, antimicrobial peptides (AMPs), which uniquely evolved to protect the host, have emerged as a leading alternative to antibiotics in clinical settings. CATH-2, a member of the antimicrobial cathelicidin peptide family, has been reported to have antibacterial activity. To enhance the antimicrobial potency and reduce the adverse effects on animals, we designed five novel AMPs, named C2-1, C2-2, C2-3, C2-4 and C2-5, based on chicken CATH-2, the secondary structures of these AMPs were consistently α-helical and had an altered net charge and hydrophobicity compared to those of the CATH-2 (1-15) sequences. Subsequently, the antimicrobial activities of CATH-2 (1-15) and five designed peptides against MDR E. coli were evaluated in vitro. Specifically, C2-2 showed excellent antimicrobial activity against either the ATCC standard strain or veterinary clinical isolates of MDR E. coli, with concentrations ranging from 2-8 μg/mL. Furthermore, C2-2 maintained its strong antibacterial efficacy under high temperature and saline conditions, demonstrating significant stability. Similarly, C2-2 retained a high level of safety with no significant hemolytic activity on chicken mature red blood cells or cytotoxicity on chicken kidney cells over the concentration range of 0-64 μg/mL. Moreover, the administration of C2-2 improved the survival rate and reduced the bacterial load in the heart, liver and spleen during MDR E. coli infection in chickens. Additionally, pathological damage to the heart, liver and intestine was prevented when MDR E. coli infected chickens were treated with C2-2. Together, our study showed that C2-2 may be a promising novel therapeutic agent for the treatment of MDR E. coli infections and AC.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry, and ultra-performance liquid chromatography–mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titer of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titer of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry, and ultra-performance liquid chromatography–mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titer of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titer of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry, and ultra-performance liquid chromatography–mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulence titer of 10−5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. Results Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p<0.05). Conclusions QLCs from flavonoids from the leaves of BD have in vitro antiviral bioactivity against NDV at a virulence titer of 10-5 Tissue Culture Infectious Dose 50% (TCID50) in chicken kidney cell culture. QLCs may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease.

Background: Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves ( Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods: This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry, and ultra-performance liquid chromatography-mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulency titer of 10 -5 Tissue Culture Infectious Dose 50% (TCID50) and in chicken kidney cell culture. Results: Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10 -5 dilution (p<0.05). Conclusions: QLCs from flavonoids from the leaves of BD have antiviral bioactivity against NDVs and may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

In vitro analysis of quercetin-like compounds from mistletoe Dendrophthoe pentandra (L.) Miq as a potential antiviral agent for Newcastle disease

Background: Recent evidence suggests that some flavonoid compounds obtained from crude methanol extract of mistletoe leaves (Dendrophthoe pentandra L. Miq), also known as Benalu Duku (BD), have antimicrobial effects. Thus, the plant has the potential to eliminate viruses that may cause outbreaks in chicken farms. This study aimed to prove the in vitro ability of flavonoid compounds, namely quercetin-like compounds (QLCs), to eliminate field viruses, specifically the Newcastle disease virus (NDV). Methods: This research was performed in two stages. An in vitro test was used with a post-test of the control groups designed at a significance of 0.05. BD leaves (5 kg) were extracted using a maceration method with methanol and then separated into hexane, chloroform, ethyl acetate, and methanol fractions. The final extracted products were separated using semi-preparative high-performance liquid chromatography (HPLC) to obtain QLCs. The QLCs were identified and compared with quercetin using HPLC, proton and carbon nuclear magnetic resonance spectrometry, Fourier transform infrared spectrophotometry, and ultra-performance liquid chromatography–mass spectrometry. The activity of QLCs was tested in vitro against the NDV at a virulency titer of 10−5 Tissue Culture Infectious Dose 50% (TCID50) and in chicken kidney cell culture. Results: Solutions of 0.05% (w/v) QLCs were discovered to have antiviral activity against NDVs, with an average cytopathogenic effect antigenicity at a 10−5 dilution (p&lt;0.05). Conclusions: QLCs from flavonoids from the leaves of BD have antiviral bioactivity against NDVs and may have the potential to be developed as medicinal compounds for the treatment of other human or animal viral infections.

Reconstitution and Mutagenesis of Avian Infectious Laryngotracheitis Virus from Cosmid and Yeast Centromeric Plasmid Clones.

The genomes of numerous herpesviruses have been cloned as infectious bacterial artificial chromosomes. However, attempts to clone the complete genome of infectious laryngotracheitis virus (ILTV), formally known as Gallid alphaherpesvirus-1, have been met with limited success. In this study, we report the development of a cosmid/yeast centromeric plasmid (YCp) genetic system to reconstitute ILTV. Overlapping cosmid clones were generated that encompassed 90% of the 151-Kb ILTV genome. Viable virus was produced by cotransfecting leghorn male hepatoma (LMH) cells with these cosmids and a YCp recombinant containing the missing genomic sequences - spanning the TRS/UL junction. An expression cassette for green fluorescent protein (GFP) was inserted within the redundant inverted packaging site (ipac2), and the cosmid/YCp-based system was used to generate recombinant replication-competent ILTV. Viable virus was also reconstituted with a YCp clone containing a BamHI linker within the deleted ipac2 site, further demonstrating the nonessential nature of this site. Recombinants deleted in the ipac2 site formed plaques undistinguished from those viruses containing intact ipac2. The 3 reconstituted viruses replicated in chicken kidney cells with growth kinetics and titers similar to the USDA ILTV reference strain. Specific pathogen-free chickens inoculated with the reconstituted ILTV recombinants succumbed to levels of clinical disease similar to that observed in birds inoculated with wildtype viruses, demonstrating the reconstituted viruses were virulent. IMPORTANCE Infectious laryngotracheitis virus (ILTV) is an important pathogen of chicken with morbidity of 100% and mortality rates as high as 70%. Factoring in decreased production, mortality, vaccination, and medication, a single outbreak can cost producers over a million dollars. Current attenuated and vectored vaccines lack safety and efficacy, leaving a need for better vaccines. In addition, the lack of an infectious clone has also impeded understanding viral gene function. Since infectious bacterial artificial chromosome (BAC) clones of ILTV with intact replication origins are not feasible, we reconstituted ILTV from a collection of yeast centromeric plasmids and bacterial cosmids, and identified a nonessential insertion site within a redundant packaging site. These constructs and the methodology necessary to manipulate them will facilitate the development of improved live virus vaccines by modifying genes encoding virulence factors and establishing ILTV-based viral vectors for expressing immunogens of other avian pathogens.

Adaptation of the infectious bronchitis virus H120 vaccine strain to Vero cell lines

Infectious bronchitis virus (IBV) has restricted cell and tissue tropism. IBVs, except the Beaudette strain, can infect and replicate in chicken embryos, primary chicken embryo kidneys, and primary chicken kidney cells, only. The limited viral cell tropism of IBV substantially hinders in vitro cell-based research on pathogenic mechanisms and vaccine development. Herein, the parental H120 vaccine strain was serially passaged for five generations in chicken embryos, 20 passages in CK cells and 80 passages in Vero cells. This passaging yielded a Vero cell-adapted strain designated HV80. To further understand viral evolution, serial assessments of infection, replication, and transmission in Vero cells were performed for the viruses obtained every tenth passage. The ability to form syncytia and the replication efficiency significantly after the 50th passage (strain HV50). HV80 also displayed tropism extension to DF-1, BHK-21, HEK-293 T, and HeLa cells. Whole genome sequencing of viruses from every tenth generation revealed a total of 19 amino acid point mutations in the viral genome by passage 80, nine of which occurred in the S gene. The second furin cleavage site appeared in viral evolution and may be associated with cell tropism extension of HV80.

The Genetic Stability, Replication Kinetics and Cytopathogenicity of Recombinant Avian Coronaviruses with a T16A or an A26F Mutation within the E Protein Is Cell-Type Dependent.

The envelope (E) protein of the avian coronavirus infectious bronchitis virus (IBV) is a small-membrane protein present in two forms during infection: a monomer and a pentameric ion channel. Each form has an independent role during replication; the monomer disrupts the secretory pathway, and the pentamer facilitates virion production. The presence of a T16A or A26F mutation within E exclusively generates the pentameric or monomeric form, respectively. We generated two recombinant IBVs (rIBVs) based on the apathogenic molecular clone Beau-R, containing either a T16A or A26F mutation, denoted as BeauR-T16A and BeauR-A26F. The replication and genetic stability of the rIBVs were assessed in several different cell types, including primary and continuous cells, ex vivo tracheal organ cultures (TOCs) and in ovo. Different replication profiles were observed between cell cultures of different origins. BeauR-A26F replicated to a lower level than Beau-R in Vero cells and in ovo but not in DF1, primary chicken kidney (CK) cells or TOCs. Genetic stability and cytopathic effects were found to differ depending on the cell system. The effect of the T16A and A26F mutations appear to be cell-type dependent, which, therefore, highlights the importance of cell type in the investigation of the IBV E protein.

The antagonistic effect of selenium on lead-induced apoptosis and necroptosis via P38/JNK/ERK pathway in chicken kidney

Lead (Pb), as a toxic heavy metal pollutant, has been paid much attention. Pb is often discharged into the environment through the soot, wastewater and waste residue in industrial production, which poses a great threat to animal health. Selenium (Se) is a trace element known to antagonize the toxicity caused by heavy metals. However, the interaction between Se and Pb in chicken kidney and its specific biological mechanism are still unclear. So, we constructed chicken models of Pb exposure and Pb, Se co-exposure. Therefore, we used western blot and qRT-PCR to detect the expression of related genes. The results showed that Pb activated the MAPK signaling pathway by up-regulating the expression of MARK pathway genes to induce the expression of pro-apoptotic genes and necroptosis-related genes. Se can regulate the MARK signaling pathway and attenuated the expression of MAPK pathway genes altered by Pb to reduce apoptosis and necroptosis of chicken kidney cells. Our study gives new ideas for the specific mechanism of Pb nephrotoxicity and provides a reference for comparative medicine and clinical medication.

Spectrophotometric microplate assay for titration and neutralization of avian nephritis virus based on the virus cytopathicity

IntroductionPlaque assay (PA) is a gold standard for virus titration and neutralization of various cytopathic viruses, including avian nephritis virus (ANV), the etiological agent associated with kidney disorders in chickens. In this study, as an alternative to the labor-intensive PA, we developed a spectrophotometric microplate assay (MA) for ANV titration and neutralization based on the virus cytopathicity to primary chicken kidney (CK) cells. MethodsCK cells were infected with ANV in the presence or absence of chicken serum in a 96-well microplate, and the virus-induced cytolysis was quantified by measurement of neutral red uptake using a spectrophotometer. The absorbance values obtained were subjected to a sigmoidal four-parameter logistic regression analysis for the virus titer determination and serum neutralization assessment. Accuracy and reliability of the serum neutralization MA in comparison to the standard PA was statistically evaluated. ResultsThe ANV-MA was capable of quantifying infectious virus titers based on a virus dose-dependent cytolysis of CK cells, and serum neutralization could be assessed as an inhibition of the virus-induced cytolysis accordingly. Statistical evaluation using a 2 × 2 contingency table and receiver-operating characteristic analyses showed 82 % sensitivity, 99 % specificity and 0.97 area under the curve, supporting an overall diagnostic accuracy of the neutralization MA. ConclusionThe newly developed MA using simplified experimental procedures in the microplate format and direct spectophotometric data readout is readily applicable to general laboratories for high-throughput screening of serum neutralization of ANV.

Effects of compound probiotics and aflatoxin-degradation enzyme on alleviating aflatoxin-induced cytotoxicity in chicken embryo primary intestinal epithelium, liver and kidney cells

Aflatoxin B1 (AFB1) is one of the most dangerous mycotoxins for humans and animals. This study aimed to investigate the effects of compound probiotics (CP), CP supernatant (CPS), AFB1-degradation enzyme (ADE) on chicken embryo primary intestinal epithelium, liver and kidney cell viabilities, and to determine the functions of CP + ADE (CPADE) or CPS + ADE (CPSADE) for alleviating cytotoxicity induced by AFB1. The results showed that AFB1 decreased cell viabilities in dose-dependent and time-dependent manners. The optimal AFB1 concentrations and reactive time for establishing cell damage models were 200 µg/L AFB1 and 12 h for intestinal epithelium cells, 40 µg/L and 12 h for liver and kidney cells. Cell viabilities reached 231.58% (p < 0.05) for intestinal epithelium cells with CP addition, 105.29% and 115.84% (p < 0.05) for kidney and liver cells with CPS additions. The further results showed that intestinal epithelium, liver and kidney cell viabilities were significantly decreased to 87.12%, 88.7% and 84.19% (p < 0.05) when the cells were exposed to AFB1; however, they were increased to 93.49% by CPADE addition, 102.33% and 94.71% by CPSADE additions (p < 0.05). The relative mRNA abundances of IL-6, IL-8, TNF-α, iNOS, NF-κB, NOD1 (except liver cell) and TLR2 in three kinds of primary cells were significantly down-regulated by CPADE or CPSADE addition, compared with single AFB1 group (p < 0.05), indicating that CPADE or CPSADE addition could alleviate cell cytotoxicity and inflammation induced by AFB1 exposure through suppressing the activations of NF-κB, iNOS, NOD1 and TLR2 pathways.

Exogenous nitric oxide stimulates early egress of Eimeria tenella sporozoites from primary chicken kidney cells in vitro.

Egress plays a vital role in the life cycle of apicomplexan parasites including Eimeria tenella, which has been attracting attention from various research groups. Many recent studies have focused on early egress induced by immune molecules to develop a new method of apicomplexan parasite elimination. In this study, we investigated whether nitric oxide (NO), an immune molecule produced by different types of cells in response to cytokine stimulation, could induce early egress of eimerian sporozoites in vitro. Eimeria tenella sporozoites were extracted and cultured in primary chicken kidney cells. The number of sporozoites egressed from infected cells was analyzed by flow cytometry after treatment with NO released by sodium nitroferricyanide (II) dihydrate. The results showed that exogenous NO stimulated the rapid egress of E. tenella sporozoites from primary chicken kidney cells before replication of the parasite. We also found that egress was dependent on intra-parasitic calcium ion (Ca2+) levels and no damage occurred to host cells after egress. The virulence of egressed sporozoites was significantly lower than that of fresh sporozoites. The results of this study contribute to a novel field examining the interactions between apicomplexan parasites and their host cells, as well as that of the clearance of intracellular pathogens by the host immune system.

The V617I Substitution in Avian Coronavirus IBV Spike Protein Plays a Crucial Role in Adaptation to Primary Chicken Kidney Cells.

The naturally isolated avian coronavirus infectious bronchitis virus (IBV) generally cannot replicate in chicken kidney (CK) cells. To explore the molecular mechanism of IBV adapting to CK cells, a series of recombinant viruses were constructed by chimerizing the S genes of CK cell-adapted strain H120 and non-adapted strain IBYZ. The results showed that the S2 subunit determines the difference in cell tropism of the two strains. After comparing the amino acid sequences of S protein of CK cell-adapted strain YZ120, with its parental strain IBYZ, three amino acid substitutions, A138V, L581F, and V617I, were identified. Using YZ120 as the backbone, one or more of the above-mentioned substitutions were eliminated to verify the correlation between these sites and CK cell tropism. The results showed that the CK cell tropism of the YZ120 strain depends on the V617I substitution, the change of L581F promoted the adaptation in CK cells, and the change at 138 position was not directly related to the CK cell tropism. Further validation experiments also showed that V617I had a decisive role in the adaptation of IBV to CK cells, but other areas of the virus genome also affected the replication efficiency of the virus in CK cells.

Development of a rapid technique for extraction of viral DNA/RNA for whole genome sequencing directly from clinical liver tissues

Characterisation of the entire genome of Fowl aviadenoviruses (FAdV) requires isolation and propagation of the virus in chicken embryo liver or kidney cells, a process which is not only time consuming but may occasionally fail to result in viral growth. Furthermore, in a mixed infection, isolation in cell culture may result in the loss of viral strains. In this study, we optimised a FAdV DNA extraction technique directly from affected liver tissues using kaolin hydrated aluminium silicate treatment. The whole genome of FAdV was sequenced directly from extracted DNA without any targetted PCR based enrichment. The extraction method was also tested on avian liver tissues affected with the RNA virus Avian hepatitis E virus and demonstrated to yield sequencing grade RNA. Therefore, the method described here is a simple technique which is potentially useful for the extraction of sequencing grade DNA/RNA from tissues with high fat content.

Effects of the Soil-Derived Microorganism BX-1 on Chicken Newcastle Disease

In recent years, effective microorganisms (EMs) have been administered to humans and domestic animals, and their usefulness has been recognized for promoting health and enhancing immunity. For example, the preventative effects against flu are enhanced by ingestion of Lactobacillus by humans, and symptom relief of atopic dermatitis has been reported, with EMs actually used in commercial products. In addition, EM preparations are being used in livestock to prevent infections (e.g. Salmonella and Escherichia coli infection). In poultry, avian influenza and Newcastle disease are terrible and fatal infectious diseases that cause significant economic damage. Furthermore, countries designated as contaminated with these pathogens can experience major trade problems. Given the above, how to protect livestock from infections safely and at low cost without using disinfectants, antibiotics and vaccines is a major issue. In the present study, we examined whether or not Newcastle disease could be suppressed by feeding chickens BX-1 as an EM feed. A field strain of Newcastle virus was cloned from cloaca swabs of large numbers of dying chickens in a poultry farm in Indonesia by polymerase chain reaction (PCR) and hemaggregation assays. Chicken kidney cells and embryonated eggs were highly sensitive to this virus, and high titers of virus were able to be collected. The experimental viral inoculated to chickens showed a high mortality rate, with high pathogenicity in birds. Conventional chickens were also raised on a diet supplemented with BX-1 and directly infected with the Newcastle virus. The mortality was decreased in these infected birds. Even the low dose of BX-1 had an inhibitory effect on the lethality of the infection. These results suggest that BX-1 intake through an EM diet is effective in controlling Newcastle disease.