Chicken Anemia Research Articles

Síndrome da hepatite-hidropericárdio por Aviadenovirus e em reprodutores de corte

ABSTRACT: Fowl aviadenovirus (FAdV) is an important pathogen in the global poultry industry and the etiology of inclusion body hepatitis-hydropericardium syndrome (HHS) in chickens. Since the 1990s, several outbreaks of HHS have occurred in poultry producing areas, including South America. The coinfection of FAdV and chicken anemia virus (CAV) may markedly impact the incidence of HHS. This study describes an outbreak of HHS in coinfection with CAV in industrial broiler breeders and characterizes the FAdV isolate. The three-week-old male broiler breeders had pale bone marrow, enlarged and yellowish liver, splenomegaly, and atrophied thymus; one chicken was also found with hydropericardium. Virus isolation was performed in SPF chicken embryos of liver and thymus. Tissues of the naturally infected chickens and the inoculated embryos were evaluated by PCR and histopathology. All affected chickens and inoculated embryos were positive for FAdV and CAV. The inoculated embryos had enlarged, greenish and hemorrhagic livers, and 30% died within 7 days of inoculation. Phylogenetic analysis of the FAdV isolate hexon gene partial sequence enabled grouping with E species. The E species has recently become a relevant species in several countries. The association of FAdV with CAV in breeders is of further concern due to both being capable of vertical transmission. Within the last decade, a worldwide upsurge of HHS in broiler breeders owing to failed biosecurity has occurred. In this episode, the failure on biosecurity may have enabled challenge with both FAdV and CAV, with pathological synergism. The CAV-impaired adaptive immunity may have benefited the FAdV infection.

Investigation on the prevalence of chicken anaemia virus in poultry flocks of Kerala, India

Chicken anaemia virus (CAV) is the aetiological agent of chicken infectious anaemia (CIA), an immunosuppressive disease, which brings a huge economic burden to the poultry industry globally. Concurrent infections and vaccination failures further aggravate the impact of the disease on the sector. The epidemiology of the disease and virulence of the circulating strains is to be known while formulating prevention strategies for any infectious disease. So far, there are no reports regarding the presence of CAV among poultry flocks in Kerala. In this scenario, the present study contemplated the investigation of the chicken anaemia virus using ELISA and PCR techniques. Indirect ELISA revealed the presence of CAV antibodies in 80 samples out of the 92 serum samples screened. Among the 100 samples collected from suspected cases, 29 were found to be positive for CAV infection with detection primers targeting the VP2 gene. This implies widespread distribution of the virus in Kerala.

Development of a one-run real-time PCR detection system for pathogens associated with poultry infectious diseases.

Infectious diseases are an important issue in the poultry industry, requiring early diagnosis and countermeasures. To address this, we present a system based on TaqMan real-time PCR to detect pathogen genome in specimens collected from chickens. We designed 12 primer-probe sets for pathogens causing respiratory or systemic symptoms. In field samples, we detected three viruses, including DNA and RNA viruses, and three bacteria. The chicken anemia virus and Avibacterium paragallinarum were detected only in young and laying hens, respectively. Bacteria were detected only in throat swabs, and gallid alphaherpesvirus 2 was detected in different specimens at each developmental stage. Our novel TaqMan real-time PCR system effectively detects pathogen's gene in chickens, while taking age into account.

Assessment of pathogenicity and immunosuppressive effect of chicken anaemia virus in the embryonated chicken

Assessment of pathogenicity and immunosuppressive effect of chicken anaemia virus in the embryonated chicken

Apoptin causes apoptosis in HepG-2 cells via Ca2+ imbalance and activation of the mitochondrial apoptotic pathway.

Apoptin is derived from the chicken anemia virus and exhibits specific cytotoxic effects against tumor cells. Herein, we found that Apoptin induced a strong and lasting endoplasmic reticulum (ER) stress response, Ca2+ imbalance, and triggered the mitochondrial apoptotic pathway.The aim of this study was to explore the mechanisms by which Apoptin exhibited anti-tumor effects in HepG-2 cells. The intracellular levels of calcium (Ca2+ ) were induced by ER stress and determined by electron microscopy, flow cytometry, and fluorescence staining. The mitochondrial injury was determined by mitochondrial membrane potential and electron microscopy. Western blotting was used to investigate the levels of key proteins in ER stress and the apoptotic pathway in mitochondria. The relationship between Ca2+ levels and apoptosis in Apoptin-treated cells was analyzed using a Ca2+ chelator (BAPTA-AM), flow cytometry, and fluorescence staining. We also investigated the in vivo effects of Ca2+ imbalance on the mitochondrial apoptotic pathway using tumor tissues xenografted on nude mice. This study showed that Apoptin induced a strong and long- lastingERstressandinjury,whichsubsequentlyledtoanimbalanceofcellularCa2+ levels, a reduction in the mitochondrial membrane potential, a significant extent image in the mitochondrial structure, and an increase in the expression levels of Smac/Diablo and Cyto-C. In summary, Apoptin induced apoptosis in HepG-2 cells via Ca2+ imbalance and activation of the mitochondrial apoptotic pathway. This study provided a new direction for antitumor research in Apoptin.

Chicken anemia virus VP1 negatively regulates type I interferon via targeting interferon regulatory factor 7 of the DNA-sensing pathway

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway plays a vital role in sensing viral DNA in the cytosol, stimulating type I interferon (IFN) production and triggering the innate immune response against DNA virus infection. However, viruses have evolved effective inhibitors to impede this sensing pathway. Chicken anemia virus (CAV), a nonenveloped ssDNA virus, is a ubiquitous pathogen causing great economic losses to the poultry industry globally. CAV infection is reported to downregulate type I IFN induction. However, whether the cGAS-STING signal axis is used by CAV to regulate type I IFN remains unclear. Our results demonstrate that CAV infection significantly elevates the expression of cGAS and STING at the mRNA level, whereas IFN-β levels are reduced. Furthermore, IFN-β activation was completely blocked by the structural protein VP1 of CAV in interferon stimulatory DNA (ISD) or STING-stimulated cells. VP1 was further confirmed as an inhibitor by interacting with interferon regulatory factor 7 (IRF7) by binding its C-terminal 143–492 aa region. IRF7 dimerization induced by TANK binding kinase 1 (TBK1) could be inhibited by VP1 in a dose-dependent manner. Together, our study demonstrates that CAV VP1 is an effective inhibitor that interacts with IRF7 and antagonizes cGAS-STING pathway-mediated IFN-β activation. These findings reveal a new mechanism of immune evasion by CAV.

Copper chitosan nanocomposite as antiviral and immune-modulating effect in broiler experimentally infected with chicken anemia virus

In the last few years, inorganic nanosystems, or nanometals, were of great interest to conventional therapy. In this study, Copper Chitosan Nanocomposite (CuCNP) was monitored for its antiviral, immune-stimulant, and lowering agent of Cu residue roles by using Chicken Anemia Virus (CAV) as a model. CuCNP is a metallic oxide nanocomposite with specific properties, such as sphere shape, no aggregation, and narrow size distribution 24.71±1.68 nm PdI: 0.691±0.02. We grouped 100 broiler chicks into four groups. Group 1 served as a regular negative control group. In drinking water, G2 was treated with CuCNP 1 mg/ml for five days. The G3 was infected with 0.2 ml I/M of CAV strain (MN339532) at one day old with CuCNP 1 mg/ml in drinking water for five days. G4 virus-positive control group with viral 3.987×106 virus copies/ml. Different serum and organ tissue homogenate samples were collected at different time intervals to measure residues, CAV viral concentration in organs, and serum to monitor cellular and humoral immunity. The excellent results of CuCNP are improving the innate immune response phagocytic activity, lysozyme, and NO, also cytokine levels mRNA of IFN-γ, IL-6, and IL-10 in G2 and G3 and elevating CAV antibody titers with decreases the CAV viral load in organs with a noticeable decrease of its residues in G2 and G3. The current study provides evidence of the immunostimulatory effect of CuCNP on CAV infection. It clarifies a constant reduction of CuCNP residues in broilers muscle and liver tissues, keeping its levels below Cu maximum residual limits (MRLs).

Design of a Multiepitope Vaccine against Chicken Anemia Virus Disease.

Chicken anemia virus (CAV) causes severe clinical and sub-clinical infection in poultry globally and thus leads to economic losses. The drawbacks of the commercially available vaccines against CAV disease signal the need for a novel, safe, and effective vaccine design. In this study, a multiepitope vaccine (MEV) consisting of T-cell and B-cell epitopes from CAV viral proteins (VP1 and VP2) was computationally constructed with the help of linkers and adjuvant. The 3D model of the MEV construct was refined and validated by different online bioinformatics tools. Molecular docking showed stable interaction of the MEV construct with TLR3, and this was confirmed by Molecular Dynamics Simulation. Codon optimization and in silico cloning of the vaccine in pET-28a (+) vector also showed its potential expression in the E. coli K12 system. The immune simulation also indicated the ability of this vaccine to induce an effective immune response against this virus. Although the vaccine in this study was computationally constructed and still requires further in vivo study to confirm its effectiveness, this study marks a very important step towards designing a potential vaccine against CAV disease.

Long read sequencing revealed proventricular virome of broiler chicken with transmission viral proventriculitis

BackgroundTransmissible viral proventriculitis (TVP) causes significant economic loss to the poultry industry. However, the exact causative agents are obscure. Here we examine the virome of proventriculus from specified pathogen free (SPF) chickens that reproduced by infection of proventricular homogenate from broiler chicken with TVP using long read sequencing of the Pacific Biosciences RSII platform. The normal SPF chickens were used as control.ResultsOur investigation reveals a virome of proventriculitis, including three Gyrovirus genera of the Aneloviridae: Gyrovirus homsa1 (GyH1) (also known as Gyrovirus 3, GyV3) (n = 2662), chicken anemia virus (CAV) (n = 482) and Gyrovirus galga1 (GyG1) (also known as avian Gyrovirus 2, AGV2) (n = 11); a plethora of novel CRESS viral genomes (n = 26) and a novel genomovirus. The 27 novel viruses were divided into three clusters. Phylogenetic analysis showed that the GyH1 strain was more closely related to the strains from chicken (MG366592) than mammalian (human and cat), the GyG1 strain was closely related to the strains from cat in China (MK089245) and from chicken in Brazil (HM590588), and the CAV strain was more closely related to the strains from Germany (AJ297684) and United Kingdom (U66304) than that previously found in China.ConclusionIn this study, we revealed that Gyrovirus virome showed high abundance in chickens with TVP, suggesting their potential role in TVP, especially GyH1. This study is expected to contribute to the knowledge of the etiology of TVP.

Overview of Infectious Anemia In chicken and Role of PCR in disease Diagnose

Chicken Infectious Anemia is a viral disease in poultry caused by CAV chicken Anemia virus usually effecting chicken and other species of poultry birds. This review will show the role of PCR which is a device used to diagnose disease. In this review I also brief some sign and symptom of the disease which will help a lay men to diagnose the disease. In this review I also discuss the control method of disease by which we can control the disease and save the farmer from big loss. The objective of that review was to aware the people from risk of CIA and give possible solution to combat with disease. The review will help out scholars to understand CIA. This review also bring light toward the use of PCR as in Pakistan farmer are not using PCR and in result there is big difficulty in diagnose of disease. This study will also aware about use of vaccine to the people so people will know the importance of Vaccine.

Genomic Sequence and Pathogenicity of the Chicken Anemia Virus Isolated From Chicken in Yunnan Province, China.

Chicken anemia virus (CAV), which has been reported in many countries, causes severe anemia and immunosuppression in chickens. In this study, a CAV strain YN04 belonging to genotype A was first identified from infected chickens in Yunnan province, China. Moreover, the animal infection experiments further confirmed that the strain YN04 is a highly pathogenic strain, which can cause 86.67% mortality in chickens in the infection group. The mean death time of infected chickens was 13.1 days post infection (dpi). CAV infection induced severe anemia with significant decrease in packed cell volume (PCV), and serious atrophy and lesion of thymus and bursa with high viral load at 14 dpi. Besides, CAV infection caused a sharp decrease in chicken body weight and immune organ indices including the ratio of thymus or bursa to body weight at 21 dpi, which displayed the potential immunosuppression state at this stage. These findings enrich the epidemiological data on CAV and may provide information for preventing its further spread in Yunnan province, China.

Rate of Multiple Viral and Bacterial CoInfection(s) in Influenza A/H9N2-Infected Broiler Flocks.

Repeated cases of low pathogenic influenza A/H9N2 virus (IAV/H9N2) have been reported in commercial chickens since its emergence in 1998 in Pakistan. However, recently increased mortality and severe respiratory complications under field conditions have been noticed, suggesting concomitant influenza infections with respiratory viral and/or bacterial pathogens. Therefore, the present study aimed to investigate the presence of IAV/H9N2 coinfecting with multiple viral and bacterial pathogens in broiler chicken flocks. We surveyed 60 broiler flocks with respiratory signs from March through July 2019 in Punjab, Pakistan. Suspected flocks were screened for the presence of IAV using a lateral-flow device. Tracheal, cloacal, and bone marrow samples were collected and further tested for seven viral agents (chicken anemia; Newcastle disease; infectious bronchitis; infectious laryngeotracheitis [ILT]; and IAV subtypes H9, H7, and H5) and three bacterial agents (Mycoplasma gallisepticum; Mycoplasma synovae; Ornithobacterium rhinotracheale [ORT]) using PCR assays. Upon initial screening for IAV, 35/60 (58.3%) flocks tested positive. The coinfection of IAV/H9N2 with other pathogens was detected in 25 (71.4%) flocks and only IAV/H9N2 was detected in 10 (28.6%) flocks out of total positive IAV flocks (n = 35). IAV subtypes H5 and H7, ILT, and ORT were not detected throughout the study period. The detection rate of double, triple, and quadruple combinations of coinfections with IAV/H9N2 were 37% (13 flocks), 26% (9 flocks), 9% (3 flocks), respectively. Higher average mortality (28.5%) was found in broiler chicken flocks coinfected with viral and/or bacterial pathogens than in flocks where only H9 low pathogenic IAV/H9N2 was detected (20.8%). In conclusion, higher circulation of IAV/H9N2 with other viral and bacterial pathogens may contribute to higher production and economic losses at the farm level.

Cross-species transmission potential of chicken anemia virus and avian gyrovirus 2.

The Gyrovirus genus consists of nonenveloped, icosahedral viruses with small circular single-stranded DNA genomes. Gyroviruses have been detected in diverse hosts, including humans, chickens, rodents, and cats. Two Gyroviruses were detected in canine serum samples using PCR in this study. The results indicated that four serum samples were positive for CAV (0.28%, 2/700) or AGV2 (0.28%, 2/700). Additionally, recombination analysis showed that AGV2 and CAV might have originated from the recombination of viruses similar to those detected in chickens and humans. We detected a total of 14 mutations in CAV VP1 amino acid sequences and identified new mutations at positions 31, 388, 390, 399, and 421 for the first time. The identification of T390C, C912T, T1230C, and T1297C mutations in AGV2 VP1, R93C mutations in AGV2 VP2, and R58C mutations AGV2 VP3 indicated that the differences might be related to a transboundary movement among hosts, which requires further elucidation. To the best of our knowledge, this study is the first report of an AGV2-infected dog in China, suggesting that the cross-species transmission of viruses with circular single-stranded DNA genomes is a public health concern.

Genomic Characterization of Chicken Anemia Virus in Broilers in Shandong Province, China, 2020-2021.

Chicken infectious anemia (CIA), caused by chicken anemia virus (CAV), is an immunosuppressive disease characterized by growth retardation, aplastic anemia, lymphoid depletion, and immunodepression in young chickens. In this study, 33 CAV strains were isolated from broilers in Shandong Province during 2020–2021. Phylogenetic analysis of full-length genome sequences showed that most CAV strains isolated in this study were scattered across different branches, but mainly clustered in two genotypes, indicating a certain regional characteristic. Analysis of VP1 protein identified several amino acid substitutions which were relevant with the virulence and virus spread efficiency. Interestingly, four putative DNA recombination events were detected in the genomes of novel isolated CAV strains. In summary, this study demonstrated a genomic diversity of CAV in broilers isolated in Shandong Province during 2020–2021, and provided information for the further study of CAV molecular epidemiology and viral evolution.

A sensitive triple nanoparticle-assisted PCR assay for detection of fowl adenovirus, infectious bursal disease virus and chicken anemia virus

Fowl adenovirus (FAdV) infections in chickens have resulted in global economic losses in the poultry industry. Infectious bursal disease virus (IBDV) and chicken anemia virus (CAV) infections lead to immunosuppression in chickens, and concomitant co- infection with FAdV usually produces severe and lethal infections. These co-infections are common occurrences on chicken farms and affect large number of chickens. Thus, a rapid, sensitive and specific diagnostic test for these viruses becomes a prerequisite to effective control and isolation measures. We developed a triplex nanoparticle-assisted PCR (nano-PCR) assay that can simultaneously detect these 3 viruses in a single assay tube using PCR primers directed at respective specific genes of each virus. The assay was specific for FAdVs, CAV and IBDV, and it did not amplify Newcastle disease virus, infectious bronchitis virus, egg drop syndrome virus or Marek’s disease virus. The minimum detection limit was 27.2 femtogram (fg) for all three viruses and was 1000-fold more sensitive than multiplex PCR using identical primers. Screening of 69 clinical samples from 40 to 50 days old chickens with obvious lesions in liver using the nano-PCR compared with a multiplex PCR yielded identical results. Of the 69 samples, 13 were detected positive including 4 for FAdV, 4 for IBDV and 6 for CAV single virus infections, respectively, as well as 5 for FAdV/CAV, 2 for FAdV/IBDV and 3 for IBDV/CAV co-infections. The triple nano-PCR assay developed in our laboratory is a sensitive, specific and simple method that can be used for detection of FAdV, CAV and IBDV as single or mixed infections.

Molecular and pathological characterization of natural co-infection of poultry farms with the recently emerged Leucocytozoon caulleryi and chicken anemia virus in Egypt.

In the summers of 2018 and 2019, a disease outbreak stroke 25 broiler chicken farms and 3 broiler breeder farms in different Governorates in Egypt. The disease caused a mortality rate ranging from 3.2 to 9%. Postmortem examination showed petechial hemorrhage in the breast and thigh muscles, thymus gland, and peritoneal cavity and extensive hemorrhages in the kidneys. A total of 140 liver, kidney, lung, skeletal muscles, thymus, and spleen samples were collected. Twenty-eight pooled samples were created and examined by PCR and histopathological examination to identify the causative pathogens. All collected samples were PCR-negative to Newcastle disease virus (NDV), avian influenza viruses (H5, H9, and H7), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and fowl adenovirus (FadV). Leucocytozoon caulleryi (L. caulleryi) genetic material was identified by PCR in 17 out of the 28 collected samples (61%). Five chicken farms (18%) showed positive PCR results for both L. caulleryi and chicken anemia virus (CAV). Histopathological examination revealed unilocular megaloschizonts in thymus, skeletal muscle, and lung as well as massive hemorrhages in parenchymatous organs. Nucleotide sequences of the identified pathogens were compared with other reference sequences available in the GenBank. The identified L. caulleryi has a close relationship with those previously detected in Asia, indicating potential transmission route of the parasite. The CAV has a close genetic relation with CAVs previously identified in Egypt. Furthermore, a real-time PCR for rapid, specific, and quasiquantitative detection of L. caulleryi was developed with a detection limit of 100 genome copies per reaction.

Molecular Detection of Chicken Anemia Virus from Chickens in Yobe South, Nigeria

Molecular Detection of Chicken Anemia Virus from Chickens in Yobe South, Nigeria

Molecular characterization of chicken anemia virus in Guangxi Province, southern China, from 2018 to 2020.

BackgroundChicken anemia virus (CAV) causes chicken infectious anemia, which results in immunosuppression; the virus has spread widely in chicken flocks in China.ObjectivesThe aim of this study was to understand recent CAV genetic evolution in chicken flocks in Guangxi Province, southern China.MethodsIn total, 350 liver samples were collected from eight commercial broiler chicken farms in Guangxi Province in southern China from 2018 to 2020. CAV was detected by conventional PCR, and twenty CAV complete genomes were amplified and used for the phylogenetic analysis and recombination analysis.ResultsThe overall CAV-positive rate was 17.1%. The genetic analysis revealed that 84 CAVs were distributed in groups A, B, C (subgroups C1-C3) and D. In total, 30 of 47 Chinese CAV sequences from 2005-2020 belong to subgroup C3, including 15 CAVs from this study. There were some specific mutation sites among the intergenotypes in the VP1 protein. The amino acids at position 394Q in the VP1 protein of 20 CAV strains were consistent with the characteristics of a highly pathogenic strain. GX1904B was a putative recombinant.ConclusionsSubgroup C3 was the dominant genotype in Guangxi Province from 2018–2020. The 20 CAV strains in this study might be virulent according to the amino acid residue analysis. These data help improve our understanding of the epidemiological trends of CAV in southern China.

Rate of Transfer of Infectious Anaemia Maternal Antibodies from Broiler Breeders To the Progeny: a Field Evaluation

Clinical manifestation of the disease caused by the chicken anaemia virus (CAV) occurs when chicken chicks are vertically contaminated or before the second week of life. CAV control is based on the vaccination of broiler breeders in order to promote progeny protection through maternal antibodies. This work aims to evaluate, under field conditions, the antibody title in commercial broiler breeders at 28, 48, and 68 weeks of age, the rate of transference to the progeny, as well as the duration of antibodies in the progeny up to 21 days of age. Thus, a total of 92 sera samples from 93,000 broiler breeders vaccinated with a live vaccine for CAV at 14 weeks of age and 366 sera samples from their respective progeny were analyzed using ELISA. Breeders’ antibody title for CAV ranged between 5051 and 8660, and these titles may provide sufficient protection for their progeny. On average, 63% of the maternal antibodies were transferred to the progeny and lasted up to the second week of chick’s life. It is possible to conclude that the vaccine and the vaccination procedure used by this company for breeders against CAV seems to be effective in inducing high antibody levels in the breeders and transfering protective maternal antibodies to the progeny.

Apoptin induces pyroptosis of colorectal cancer cells via the GSDME-dependent pathway.

Apoptin is a small molecular weight protein encoded by the VP3 gene of chicken anemia virus (CAV). It can induce apoptosis of tumor cells and play anti-tumorigenic functions. In this study, we identified a time-dependent inhibitory role of apoptin on the viability of HCT116 cells. We also demonstrated that apoptin induces pyroptosis through cleaved caspase 3, and with a concomitant cleavage of gasdermin E (GSDME) rather than GSDMD. GSDME knockdown switched the apoptin-induced cell death from pyroptosis to apoptosis in vitro. Furthermore, we demonstrated that the effect of apoptin on GSDME-dependent pyroptosis could be mitigated by caspase-3 and caspase-9 siRNA knockdown. Additionally, apoptin enhanced the intracellular reactive oxygen species (ROS), causing aggregation of the mitochondrial membrane protein Tom20. Moreover, bax and cytochrome c were released to the activating caspase-9, eventually triggering pyroptosis. Therefore, GSDME mediates the apoptin-induced pyroptosis through the mitochondrial apoptotic pathway. Finally, using nude mice xenografted with HCT116 cells, we found that apoptin induces pyroptosis and significantly inhibits tumor growth. Based on this mechanism, apoptin may provide a new strategy for colorectal cancer therapy.