Apoptosis In Chickens Research Articles

Effect of Glucose Supplementation on Apoptosis in the Pectoralis major of Chickens Raised under Thermoneutral or Heat Stress Environment.

Reduced feed intake during heat stress (HS) disrupts glucose homeostasis, thereby resulting in endoplasmic reticulum (ER) stress and triggering apoptosis in chickens. We hypothesize that glucose supplementation could reduce apoptosis in chickens raised under HS. This study comprised 456 28-day-old broiler chickens randomly assigned to four treatment combinations under glucose supplementation and HS. The treatments were TN0, TN6, HS0, and HS6 with two glucose levels (0% and 6%) and two temperature levels (25 °C (thermoneutral-TN) and 35 °C (8.00 AM to 8.00 PM, (HS)). After 7 days post-HS, the blood glucose level for the HS6 group was higher than for TN0, TN6, and HS0. We studied the mRNA expression of genes and caspase-3 activity in the four experimental groups. The expressions of GCN2, ATF4, CHOP, and FOXO3a increased during HS regardless of glucose supplementation, while PERK and MAFbx increased only under HS with glucose supplementation. We show that under TN conditions, glucose supplementation led to a significant increase in cellular apoptosis in the Pectoralis (P.) major. However, under HS with glucose, the level of apoptosis was similar to that of chickens raised under TN conditions with no glucose supplementation. The utility of glucose to curtail apoptosis under HS should be tested under other intense models of HS.

Exposure to copper induces endoplasmic reticulum (ER) stress-mediated apoptosis in chicken (Gallus gallus) myocardium.

Copper (Cu), an omnipresent environmental pollutant, can cause potential harm to the public and ecosystems. In order to study the cardiotoxicity caused by Cu, molecular biology techniques were used to analyze the effect of Cu on ER stress-mediated cardiac apoptosis. In vivo investigation, 240 1-day-old chickens were fed with Cu (11, 110, 220, and 330mg/kg) diet for 7 weeks. The consequence showed that high-Cu can induce ER stress and apoptosis in heart tissue. The vitro experiments, the Cu treatment for 24h could provoke ultrastructural damage and upregulate the apoptosis rate. Meanwhile, GRP78, GRP94, eIF2α, ATF6, XBP1, CHOP, Bax, Bak1, Bcl2, Caspase-12 and Caspase-3 genes levels, and GRP78, GRP94 and Caspase-3 proteins levels were increased, which indicated that ER stress and apoptosis in cardiomyocytes. But the mRNA level of Bcl2 were decreased after Cu exposure. Conversely, Cu-induced ER stress-mediated apoptosis can be alleviated by treatment with 4-PBA. These findings generally showed that Cu exposure can contribute to ER stress-mediated apoptosis in chicken myocardium, which clarifies the important mechanism link between ER stress and apoptosis, and provides a new perspective for Cu toxicology.

Exposure to copper activates mitophagy and endoplasmic reticulum stress-mediated apoptosis in chicken (Gallus gallus) cerebrum.

A large amount of copper (Cu) used in production activities can lead to the enrichment of Cu in the environment, which can cause toxicity to animals. However, the toxicity mechanism of Cu on the cerebrum is still uncertain. Hence, a total of 240 chickens were separated into four groups in this study to reveal the potential connection between mitophagy and endoplasmic reticulum (ER) stress-mediated apoptosis in the chicken cerebrum in the case of excess Cu exposure. The cu exposure situation was simulated by diets containing various levels of copper (11 mg/kg, control group; 110 mg/kg, group I; 220 mg/kg, group II and 330 mg/kg, group III) for 49 days. The results of histology showed that vacuolar degeneration was observed in the treated groups, and the mitochondria swell and autophagosomes formation were found under excess Cu treatment. Additionally, the expression of mitophagy (PINK1, Parkin, LC3I, LC3II and p62) and ER stress (GRP78, PERK, ATF6, IRE1α, XBP1, CHOP, and JNK) indexes were significantly upregulated under excess Cu exposure. Furthermore, the mRNA and protein expression of Bcl-2 were decreased, while Bak1, Bax, Caspase12, and Caspase3 were increased compared to the control group. In summary, this study demonstrated that an overdose of Cu could induce mitophagy and ER stress-mediated apoptosis in the chicken cerebrum. These findings revealed an important potential connection between Cu toxicity and cerebrum damage, which provided a new insight into Cu neurotoxicity.

Lnc90386 Sponges miR-33-5p to Mediate Mycoplasma gallisepticum-Induced Inflammation and Apoptosis in Chickens via the JNK Pathway.

Mycoplasma gallisepticum (MG) is one of the most important pathogens, that causes chronic respiratory disease (CRD) in chickens. Long non-coding RNAs (lncRNAs) are emerging as new regulators for many diseases and some lncRNAs can function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we found that miR-33-5p was significantly up-regulated both in MG-infected chicken embryonic lungs and chicken embryo fibroblast cells (DF-1), and Lnc90386 negatively correlated with miR-33-5p. miR-33-5p, as a new regulator for MG infection, repressed apoptosis, inflammatory factors in DF-1 cells by targeting JNK1. Further analyses showed that Lnc90386 sponged miR-33-5p to weaken its inhibitory effect on JNK1, forming the ceRNA regulatory network. Furthermore, knockdown of Lnc90386 significantly inhibited apoptosis and inflammatory factors, and promoted DF-1 cells proliferation. However, co-treatment with miR-33-5p inhibitor and Lnc90386 siRNA showed that knockdown of Lnc90386 could partially eliminate the inhibiting effect of miR-33-5p inhibitor on inflammation, cell apoptosis and proliferation. In conclusion, Lnc90386 sponges miR-33-5p to defend against MG infection by inhibiting the JNK signaling pathway.

Exposure to Copper Activates Mitophagy and Endoplasmic Reticulum (Er) Stress-Mediated Apoptosis in Chicken (Gallus Gallus) Cerebrum

Exposure to Copper Activates Mitophagy and Endoplasmic Reticulum (Er) Stress-Mediated Apoptosis in Chicken (Gallus Gallus) Cerebrum

Investigation of the Crosstalk between GRP78/PERK/ATF-4 Signaling Pathway and Renal Apoptosis Induced by Nephropathogenic Infectious Bronchitis Virus Infection.

This study aims to explore the crosstalk between GRP78/PERK/ATF-4 signaling pathway and renal apoptosis induced by nephropathogenic infectious bronchitis virus (NIBV). Hy-Line brown chickens were divided into two groups (Con, n = 100 and Dis, n = 200). At 28 days of age, each chicken in the Dis group was intranasally injected with SX9 strain (10-5/0.2 ml). Venous blood and kidney tissues were collected at 1, 5, 11, 18 and 28 days postinfection. Our results showed that NIBV infection upregulated the levels of creatinine, uric acid, and calcium (Ca2+) levels. Histopathological examination revealed severe hemorrhage and inflammatory cell infiltration near the renal tubules. Meanwhile, NIBV virus particles and apoptotic bodies were observed by ultramicro electron microscope. In addition, RT-qPCR and Western blot showed that NIBV upregulated the expression of GRP78, PERK, eIF2α, ATF-4, CHOP, Caspase-3, Caspase-9, P53, Bax, and on the contrary, downregulated the expression of Bcl-2. Furthermore, immunofluorescence localization analysis showed that the positive expression of Bcl-2 protein was significantly decreased. Correlation analysis indicated that endoplasmic reticulum (ER) stress gene expression, apoptosis gene expression, and renal injury were potentially related. Taken together, NIBV infection can induce renal ER stress and apoptosis by activating of GRP78/PERK/ATF-4 signaling pathway, leading to kidney damage. IMPORTANCE Nephropathogenic infectious bronchitis virus (NIBV) induced renal endoplasmic reticulum stress in chickens. NIBV infection induced kidney apoptosis in chickens. GRP78/PERK/ATF-4 signaling pathway is potentially related to renal apoptosis induced by NIBV.

Antagonistic effect of selenium on lead-induced neutrophil apoptosis in chickens via miR-16-5p targeting of PiK3R1 and IGF1R.

Environmental contamination by heavy metals, such as lead (Pb), can lead to severe immune dysfunction. MicroRNAs (miRNAs) are involved in regulating immunity. Whether Pb can regulate neutrophil apoptosis through miRNA, and whether selenium (Se) can antagonize this response are still unknown. We treated neutrophils with 12.5μM (CH3OO)2Pb and 1μM Na2SeO3 for 3h, after which apoptosis was evaluated using acrideine orange/ethidium bromide (AO/EB) dual fluorescent staining and flow cytometry. The results showed that neutrophil apoptosis was significantly increased following Pb exposure, and that this response was prevented upon Se addition. Pb up-regulates miR-16-5p and leads to the subsequent down-regulation of the target genes phosphoinositide-3-kinase regulatory subunit 1 (PiK3R1), insulin-like growth factor 1 receptor (IGF1R), and phosphatidylinositol 3 kinase (Pi3K)-protein kinase B (AKT), followed by activation of the tumor protein P53 (P53)-B-cell lymphoma-2 (Bcl-2)/Bcl-2-Associated X protein (Bax)-cytochrome c (Cytc)-Caspase 9 (mitochondrial apoptotic pathway) and the tumor necrosis factor receptor superfamily member 6 (Fas)-Fas-associated death domain protein (Fadd)-Caspase 8 (death receptor pathway). Pb also triggered oxidative stress and indirectly activated the mitochondrial apoptotic pathway. We conclude that miR-16-5p plays a key role in the apoptosis of neutrophils exposed to Pb by down-regulating the expression of PiK3R1 and IGFR1, thereby activating the mitochondrial apoptotic pathway and death receptor pathway. Se can prevent Pb-induced apoptosis.

Antagonistic Effects Of Baicalin On Mycoplasma gallisepticum-Induced Inflammation And Apoptosis By Restoring Energy Metabolism In The Chicken Lungs.

BackgroundBaicalin possesses potential anti-inflammatory, anti-tumor and anti-oxidant activities. In the present study, we attempted to investigate the preventive effects of baicalin against Mycoplasma gallisepticum (MG)-induced inflammation, apoptosis and energy metabolism dysfunction in chicken lungs.MethodsExperimental chickens were randomly divided into 1) control group, 2) MG infection group, 3) MG-infected group treated with baicalin at a dose of 450 mg/kg and 4) baicalin alone treated group (450 mg/kg). After 7 days of post-treatment, serum and lung tissues were collected for different experimental analyses. The hallmarks of inflammation, apoptosis and energy metabolism dysfunction were detected by histological and ultrastructural examination, qRT-PCR, Western blotting and terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) assay.ResultsThe level of serum inflammatory markers were increased with MG infection. Histological and ultrastructural analysis showed excessive inflammatory cells infiltrates, alveolar wall thickening, hemorrhages, mitochondrial and nuclear damage, including mitochondrial swelling and condensation of DNA in the lungs of chickens infected with MG. TUNEL assay positive-stained nuclei were significantly increased in MG infection group. In addition, the mRNA and protein expression level of energy metabolism-related genes and ATPase activities were significantly reduced. Meanwhile, MG-induced morphological and ultrastructural changes were partially disappeared with baicalin-treatment, and the level of serum inflammatory markers were significantly reduced. It has been noted that baicalin significantly attenuated MG-induced inflammation and apoptosis in the chicken lungs through the suppression of nuclear factor-kappa B and reduced extensive positive-stained apoptotic nuclei. More importantly, ATPase activities and mRNA and protein expression level of energy metabolism-related genes were significantly improved with baicalin-treatment in the lungs of chickens infected with MG.ConclusionConclusively, it has been suggested from these results that baicalin-treatment efficiently prevented MG-induced inflammation, apoptosis and energy metabolism dysfunction in the chicken lungs and provide basis for new therapeutic targets to control MG infection.

Copper-Mediated Mitochondrial Fission/Fusion Is Associated with Intrinsic Apoptosis and Autophagy in the Testis Tissues of Chicken.

The aim of this study is to investigate whether copper (Cu) could induce testicular poisoning and influence the mitochondrial dynamics, apoptosis, and autophagy in chickens. For this purpose, thirty-six 1-day-old male Hy-line chickens were divided into control group (C group) and test group (Cu group). The chickens were exposed to 0 (C group) or 300mg/kg (Cu group) of copper sulfate (CuSO4) for 30, 60, and 90days. CuSO4 was added into the basal diet to make supplements. Testis tissues were subjected to observation of ultrastructure and detection of testis-related indexes. The results indicated that in the test group, the levels of the pro-apoptotic genes were up-regulated and the levels of the anti-apoptotic genes were down-regulated; the levels of mitochondrial fission-related genes markedly increased, and the levels of mitochondrial fusion-related genes were highly decreased; autophagy-related gene (autophagy-associated gene 4B (ATG4B), dynein, microtubule-associated protein 1 light chain 3 beta (LC3-II), ATG5, and beclin-1) levels were increased, while mammalian target of rapamycin (mTOR) and LC3-I levels were declined. The results of transmission electron microscopy (TEM) demonstrated that Cu induced mitochondrial fragmentation, which induced autophagy and apoptosis in chicken testes. In conclusion, CuSO4 exposure can influence the mitochondrial dynamics balance and lead to mitochondria-initiated intrinsic pathway of apoptosis and autophagy, which triggers the testicular poisoning in chickens. What is more, there is a correlation among mitochondrial dynamics, apoptosis, and autophagy.

Ameliorative effects of nano-elemental selenium against hexavalent chromium-induced apoptosis in broiler liver

The current study examined the ameliorative effects of nano-elemental selenium (Nano-Se) against chromium-VI (K2Cr2O7)-induced apoptosis in chickens. The expression of apoptosis-related genes was evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. A total of 60, one-day-old broiler chickens allotted to six equal groups, i.e., control group (standard diet), Cr(VI)-exposed group (K2Cr2O7 via drinking water), Nano-Se group (Nano-Se at 0.5mg/kg via diet), protection group (K2Cr2O7 + Nano-Se), cure group (K2Cr2O7 for initial 2weeks and then Nano-Se), and prevention group (opposite to the cure group) and were detected by the activities of pro-apoptosis (Bax, Caspase-3) and anti-apoptosis (Bcl-2) genes expression at day 35 of the experiment. Intense apoptosis was observed in liver tissues of chickens exposed to K2Cr2O7. The Nano-Se supplementation caused a significant decrease (P < 0.01) in the mRNA expression levels of Bax and Caspase-3 genes, while significantly elevated (P< 0.05) mRNA expression level of Bcl-2 gene was observed in Nano-Se experimental groups as compare to control and Cr(VI)-exposed group. The results quantified by the RT-qPCR were further confirmed by the western blot analysis. Altogether, these results suggest anti-apoptotic effects of Nano-Se in the chicken liver, which is interesting for further study. The present findings suggested that Nano-Se has protective effects against K2Cr2O7-induced apoptosis in broilers liver and can serve a key role as a protective agent against apoptosis.

Involvement of mitochondrial pathway in environmental metal pollutant lead-induced apoptosis of chicken liver: perspectives from oxidative stress and energy metabolism.

This study aimed to investigate the possible mechanisms of environmental metal pollutant lead (Pb)-induced apoptosis in chicken. Forty 8-day-old healthy chickens were randomly assigned to two groups (n=20/group) after raising standard commercial diet and drinking water for 1week: including control group and Pb group ((CH3COO)2Pb 350mg/L of drinking water); the chickens were given euthanasia and collected livers at 90days. A significant increase of apoptosis rate were found in Pb group and Pb induced obvious ultrastructural changes of chicken liver. The mRNA levels of glycometabolism key enzymes were significantly lower in Pb group than those in controls. Higher levels of malondialdehyde (MDA) and nitric oxide (NO) were observed in Pb group; the activities of antioxidant enzymes and ATPases were significantly lower in Pb group than those in controls, while the inducible nitric oxide synthase (iNOS) activity was on the contrary. The mRNA and protein levels of pro-apoptotic genes were all lower in Pb group than those in controls. Altogether, Pb-induced mitochondrial swelling and nuclear chromatin condensation, oxidative stress, energy metabolism disorder, thereby lead to apoptosis via mitochondrial pathway in chicken liver, suggesting that Pb-induced mitochondrial pathway apoptosis plays an important role in the mechanisms of Pb cytotoxicity to chicken liver.

Selenium protects against cadmium-induced kidney apoptosis in chickens by activating the PI3K/AKT/Bcl-2 signaling pathway.

Cadmium (Cd) is a toxic heavy metal that can induce apoptosis. Selenium (Se) is a necessary trace element and can antagonize the toxicity of many heavy metals, including Cd. PI3K/AKT/Bcl-2 is a key survival signaling pathway that regulates cellular defense system against oxidative injury as well as cell proliferation, survival, and apoptosis. The antagonistic effects of Se on Cd-induced toxicity have been reported. However, little is known about the effect of Se on Cd-induced apoptosis in chicken kidneys via the PI3K/AKT/Bcl-2 signaling pathway. In the present study, we fed chickens with Se, Cd, or both Se and Cd supplements, and after 90days of treatment, we detected the related index. The results showed that the activity of inducible nitric oxide synthase (iNOS) and concentration of nitric oxide (NO) were increased; activities of the mitochondrial respiratory chain complexes (complexes I, II, and V) and ATPases (the Na+-K+-ATPase, the Mg2+-ATPase, and the Ca2+-ATPase) were decreased; expression of PI3K, AKT, and Bcl-2 were decreased; and expression of Bax, Bak, P53, Caspase-3, Caspase-9, and cytochrome c (Cyt c) were increased. Additionally, the results of the TUNEL assay showed that the number of apoptotic cells was increased in the Cd group. By contrast, there was a significant improvement of the correlation indicators and occurrence of apoptosis in the Se+Cd group compared to the Cd group. In conclusion, our results confirmed that Se had a positive effect on ameliorating Cd-induced apoptosis in chicken kidney tissue by activating the PI3K/AKT/Bcl-2 signaling pathway.

Aflatoxin B1 invokes apoptosis via death receptor pathway in hepatocytes.

The fungal metabolites produced by Aspergillus flavus and Aspergillus parasiticus cause detrimental health effects on humans and animals. Particularly aflatoxin B1 (AFB1) is the most studied and a well-known global carcinogen, producing hepatotoxic, genotoxic and immunotoxic effects in multiple species. AFB1 is shown to provoke liver dysfunctioning by causing hepatocytes apoptosis and disturbing cellular enzymatic activities. In liver, AFB1 causes apoptosis via extrinsic mechanism because of high expression of death receptor pathway. The detailed mechanism of AFB1 induced hepatocytes apoptosis, via death receptor pathway still remains elusive. So the present study was conducted to explore apoptotic mechanism initiated by death receptors and associated genes in aflatoxin B1 induced liver apoptosis in chickens fed with AFB1 for 3 weeks. Results from the present study displayed histopathological and ultrastructural changes in liver such as hydropic degeneration, fatty vacuolar degeneration and proliferation of bile duct in hepatocytes in AFB1 group, along with imbalance between reactive oxygen species (ROS) and antioxidant defense system upon AFB1 ingestion. Moreover, AFB1 intoxicated chickens showed upregulation of death receptors FAS, TNFR1 and associated genes and downregulation of inhibitory apoptotic proteins XIAP and BCL-2. The results obtained from this novel and comprehensive study including histopathological, ultrastructural, flow cytometrical and death receptor pathway gene expression profiles, will facilitate better understanding of mechanisms and involvement of death receptor pathway in hepatocytes apoptosis induced by AFB1 and ultimately may be helpful in bringing down the toxigenic potential of AFB1.

Relationship between Eimeria tenella development and host cell apoptosis in chickens

Coccidiosis causes considerable economic losses in the poultry industry. At present, the pathology of coccidiosis is preventable with anticoccidials and vaccination, although at considerable cost to the international poultry industry. The purpose of the present study was to elucidate the relationship between Eimeria tenella development and host cell apoptosis in chickens, which provides a theoretical basis for further study of the injury mechanism of E. tenella and the prevention and treatment of coccidiosis. Cecal epithelial cells from chick embryo were used as host cells in vitro. In addition, flow cytometry, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate (dUTP) nick-end labeling, and histopathological assays were used to detect the dynamic changes in E. tenella infection rates, DNA injury rates, and apoptosis rates in groups treated with and without the caspase-9 inhibitor Z-LEHD-FMK. Following E. tenella infection, we demonstrated that untreated cells had less apoptosis at 4 h and, inversely, more apoptosis at 24 to 120 h compared with control cells. Furthermore, after the application of Z-LEHD-FMK, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assays, and translation of phosphatidyl serines to the host cell plasma membrane surface, the treated group chick embryo cecal epithelial cells exhibited decreased apoptosis and DNA injuries (P < 0.01) at 24 to 120 h. However, light microscopy showed that E. tenella infection rates of treated cells were higher (P < 0.01) than untreated cells during the whole experimental period. Together, these observations suggest that E. tenella can protect host cells from apoptosis at early stages of development but can promote apoptosis during the middle to late stages. In addition, the inhibition of host cell apoptosis can be beneficial to the intracellular growth and development of E. tenella.

Potentiation of Apoptin-Induced Apoptosis by Cecropin B-Like Antibacterial Peptide ABPs1 in Human HeLa Cervical Cancer Cell Lines is Associated with Membrane Pore Formation and Caspase-3 Activation

Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in chicken or human tumor cells, localizing in their nuclei as opposed to the cytoplasm of non-transformed cells. The present study was undertaken to investigate whether ABPs1 could potentiate apoptininduced apoptosis in HeLa cells. ABPs1 and the apoptin genes were successfully cloned into pIRES2-EGFP expression vector and expressed in HeLa cells. We report that ABPs1 augments apoptin cell growth inhibition in a concentration- and time-dependent manner. The DAPI staining and scanning electron microscopy observations revealed apoptotic bodies and plasma membrane pores, which were attributed to apoptin and ABPs1, respectively. Further, ABPs1 in combination with apoptin was found to increase the expression of Bax and to decrease the expression of survivin compared with either agent alone or the control. The apoptotic rate of HeLa cells treated with ABPs1 and apoptin in combination for 48 h was 53.95%. The two-gene combination increased the caspase-3 activity of HeLa cells. Taken together, our study suggests that ABPs1 combined with apoptin significantly inhibits HeLa cell proliferation, and induces cell apoptosis through membrane defects, up-regulation of Bax expression, down-regulation of survivin expression, and activation of the caspase-3 pathway. Thus, the combination of ABPs1 and apoptin could serve as a means to develop novel gene therapeutic agents against human cervical cancer.

Transforming growth factor-β1-induced satellite cell apoptosis in chickens is associated with β1 integrin-mediated focal adhesion kinase activation

Transforming growth factor-β1 (TGF-β1) induces apoptosis in many types of cells. The cell adhesion receptor, β1 integrin subunit, prevents apoptosis and may be involved in TGF-β1-induced muscle cell apoptosis. In the current study, chicken primary satellite cells, myogenic precursors, were used to investigate the apoptotic effect of TGF-β1 on muscle cells. The data from the current study showed that the addition of exogenous TGF-β1 reduced β1 integrin expression and altered its localization. Treatment of the satellite cells with TGF-β1 increased the number of apoptotic cells indicated by annexin-V using flow cytometry. The number of caspase-positive cells was increased in the TGF-β1-treated immunostained cells, which supported that TGF-β1 induced satellite cell apoptosis. It has been shown that β1 integrin is involved in muscle cell survival. In response to the activation of β1 integrin, focal adhesion kinase (FAK) phosphorylates tyrosine at residue 397 and activates cell survival signal transduction. The phosphorylation of FAK was significantly reduced from 30 min to 4 h after TGF-β1 treatment during both satellite cell proliferation and differentiation. These data suggested that the apoptotic effect of TGF-β1 on satellite cells is likely associated with a β1 integrin-mediated FAK signaling pathway during satellite cell proliferation and differentiation.

Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells

The ability of infectious bursal disease virus (IBDV) serotypes 1 and 2, and the role of VP4 of both serotypes as well as the capacity of three IBDV intermediate serotype 1-specific vaccine strains to induce apoptosis in a chicken B-lymphocyte cell line, DT40, were investigated using the TUNEL technique. It was observed that IBDV serotype 1 infected the DT40 cell line and directly induced apoptosis. In contrast, the non-pathogenic serotype 2 neither infected nor induced apoptosis, but was able to reduce the serotype 1-induced apoptosis when the two viruses were present in combination. VP4 of both serotypes did not induce apoptosis. IBDV VP2 of serotype 2 induced apoptosis in the same proportion and intensity as VP2 of serotype 1. IBDV intermediate vaccines varied in their ability to induce apoptosis in the DT40 cell line, which was also decreased-delayed in presence of serotype 2 IBDV. We hypothesize that both serotypes compete for the same receptor in DT-40 cells, and suggest that IBDV-induced apoptosis is a multistep process involving virus replication, protein expression, and release of virions.

Chicken anemia virus induced apoptosis: underlying molecular mechanisms

In 1990, the chicken anemia virus (CAV) genome was cloned by us and proven to be representative for CAV isolates worldwide. This genome contains unique promoter/enhancer replication elements and genes. Upon infection of its target cells, CAV replicates via a double-stranded (ds) DNA intermediate. From this ds CAV molecule, a single mRNA is transcribed, which encodes for three distinct proteins VP1, VP2, and VP3 or apoptin. Its capsid contains only the VP1 protein. However, for the production of the neutralizing epitope, co-synthesis of VP1 and VP2 are needed. CAV genomes with mutations in the 12 bp insert of the promoter/enhancer region were shown to produce immunogenic functional CAV particles. Mutations in these and other regulatory elements of CAV might also decrease its virus load resulting in a reduced pathogenic effect. CAV causes fatal cytopathogenic effects in e.g. chicken thymocytes via apoptosis. Under in vitro conditions, CAV replicates only in transformed chicken cell lines, which indicates that at least a part of the CAV life-cycle requires transformed-like cellular events. In these transformed cell lines, the synthesis of the apoptin protein alone mimics the CAV-induced apoptosis, whereas the VP2 protein also harbors some apoptotic activity. Extensive studies on apoptin resulted in the characterization of domains essential for its apoptotic activity and nuclear localization, which seems to be related with its ability to induce apoptosis. Therefore, both VP2 and apoptin are of interest in reducing the pathogenicity of CAV infections. A series of biomedical studies on apoptin have been carried out in human cell systems, which are informative about the mechanism of CAV-induced apoptosis in chicken (transformed) cells. Synthesis of apoptin alone induces apoptosis in various human transformed and/or tumorigenic cell lines, but not in normal human diploid cells. A striking difference in the cellular localization of apoptin was observed in human normal diploid cells versus tumor cells. In all tumor cells, apoptin is located mainly in the heterochromatic regions of the nucleus, whereas in normal cells it is present in peri-nuclear structures. Apoptin contains a bipartite nuclear localization signal, and one domain that resemble a nuclear export signal. Elucidation of parts of the apoptin-induced apoptotic pathway revealed unique characteristics: apoptin-induced apoptosis is independent of the tumor suppressor p53. The anti-apoptotic protein Bcl-2 does not inhibit but even accelerates apoptin-induced apoptosis in tumor cells, whereas over expression of Bcl-2 in normal cells has no effect on the apoptin activity. Upstream caspases are not involved, whereas downstream caspase 3 is, but seems not to be essential. A number of novel proteins were shown to interact with apoptin in transformed cells. Future studies of apoptin, VP2 and related cellular proteins in chicken cells will unravel the regulatory aspects of CAV-induced apoptosis.

548 Endosulfan-induced apoptosis in chickens

548 Endosulfan-induced apoptosis in chickens

Myonuclear apoptosis occurs during early posthatch starvation

Apoptosis is a naturally occurring process; it is important for the final shape and size of developing tissues, and it is characterized by some morphological features such as plasma membrane blebbing, nuclear breakdown, chromosomal fragmentation and apoptotic bodies followed by phagocytosis. The objective of the study was to evaluate the occurrence of apoptosis in chickens immediately posthatch under fed and starved conditions. Male broiler chickens were or were not provided feed for the first 3 days posthatch. Chickens were killed immediately after hatch, at 1 day of age, at 2 days of age and at 3 days of age. The Pectoralis thoracicus was removed, fixed, dehydrated, cleared and embedded in paraffin. Muscle sections were labeled using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL) for detection of apoptotic nuclei. Body weights were lower ( P<0.05) in the starved compared to the fed group at 2 and 3 days posthatch. Myofiber cross-sectional area was only smaller ( P<0.05) in the starved compared to the fed birds at 3 days posthatch. TUNEL-positive nuclei were present at all days for the fed and starved groups. The proportion of TUNEL-positive nuclei was higher ( P<0.05) for the starved group at day 2 and day 3 posthatch compared to the fed group at 3 days posthatch. Apoptosis is a mechanism that contributes to the smaller myofiber size observed at 3 days posthatch.