Specific Pathogen Free Chicken Embryos Research Articles

Quercetin attenuates lipopolysaccharide-induced hepatic inflammation by modulating autophagy and necroptosis

Lipopolysaccharide (LPS) from Gram-negative bacteria initially induces liver inflammation with proinflammatory cytokines expressions. However, the underlying hepatoprotective mechanism of quercetin on LPS-induced hepatic inflammation remains unclear. Specific pathogen-free chicken embryos (n = 120) were allocated control vehicle, PBS with or without ethanol vehicle, LPS (125 ng/egg) with or without quercetin treatment (10, 20, or 40 nmol/egg, respectively), quercetin groups (10, 20, or 40 nmol/egg). Fifteen-day-old embryonated eggs were inoculated abovementioned solutions via the allantoic cavity. At embryonic d 19, the livers of the embryos were collected for histopathological examination, RNA extraction, real-time polymerase chain reaction, and immunohistochemistry investigation. We found that the liver presented inflammatory response (heterophils infiltration) after LPS induction. The LPS-induced mRNA expressions of inflammation-related factors (TLR4, TNFα, IL-1β, IL-10, IL-6, MYD88, NF-κB1, p38, and MMP3) were upregulated after LPS induction when compared with the PBS group, while quercetin could downregulate these expressions as compared with the LPS group. Quercetin significantly decreased the immunopositivity to TLR4 and MMP3 in the treatment group when compared with the LPS group. Quercetin could significantly downregulate the mRNA expressions of autophagy-related genes (ATG5, ATG7, Beclin-1, LC3A, and LC3B) and necroptosis-related genes (Fas, Bcl-2, Drp1, and RIPK1) after LPS induction. Quercetin significantly decreased the immunopositivity to LC3 in the treatment group when compared with the LPS group; meanwhile, quercetin significantly decreased the protein expressions of LC3-I, LC3-II, and the rate of LC3-II/LC3-I. In conclusions, quercetin can alleviate hepatic inflammation induced by LPS through modulating autophagy and necroptosis.

Genomic characterization and pathogenicity of a novel fowl adenovirus serotype 11 isolated from chickens with inclusion body hepatitis in China

Fowl adenovirus serotype 11 (FAdV-11) is one of the primary causative agents of inclusion body hepatitis (IBH), which causes substantial economic losses in the world poultry industry. In this study, we characterized the genome of the fowl adenovirus serotype 11 (FAdV-11) isolate FJSW/2021. The full genome of FJSW/2021 was 44, 154 base pairs (bp) in length and had a similar organization to that of previously reported FAdV-11 isolates. Notably, compared with those of other reported FAdV-11 strains, the preterminal protein (pTP) of FAdV-11 FJSW/2021 has six amino acid (aa) insertions (S-L-R-I-I-C) between 470 and 475 and one aa mutation of L476F; moreover, the tandem repeat (TR) regions of TR1 and TR2 were 33 bp (1 repeat) and 1,080 bp (8 repeats) shorter than those of the Canadian nonpathogenic isolate ON NP2, respectively. The pathogenicity of FJSW/2021 was studied in 10-day-old specific pathogen-free chicken embryos following allantoic cavity inoculation and in 1-day-old, 1-wk-old and 2-wk-old SPF chickens following intramuscular inoculation with 107 TCID50 of the virus. The results showed that FJSW/2021 can induce typical severe IBH in chicks less than 2 wk old. These findings highlighted the genetic differences between the pathogenic and non-pathogenic FAdV-11 isolates. The data will provide guidance for identifying the virulence factors of FAdV-11 strains. The animal challenge model developed in our study will allow precise evaluation of the efficacy of potential FAdV-11 vaccine candidates.

The pathogenicity and immune effects of different generations of Mycoplasma synoviae on chicken embryos

ABSTRACT 1. Mycoplasma synoviae (MS) is the primary causative agent of synovitis in avian species. In order to investigate the pathogenicity and immunological responses associated with MS in specific pathogen-free chicken embryos, a series of generations (F1, F95, F120, F160 and F200) of MS were introduced into 7-day-old SPF chicken embryos and subsequent mortality rates were recorded and analysed 2. Reverse transcription-quantitative polymerase chain reaction was performed to detect expression of heat shock proteins HSP27, HSP40, HSP60, HSP70 and HSP90 and inflammatory factors interleukin (IL)-1β, caspase-1 and IL-18 in the tracheal tissue. 3. The results showed that the mortality rate of SPF chicken embryos decreased with an increase in the number of passages, with the highest being 80% (8/10) for F1 generation and the lowest being 10% (1/10) for F200. The expression of HSP27, IL-1β, HSP40, caspase-1, HSP70 and HSP90 showed a significant downregulation trend with an increase in the generation (except IL-18; P < 0.05). The HSP60 expression was significantly upregulated with increasing generations (P < 0.05). 4. A relationship between pathogenicity and the number of passages was observed and the decrease in pathogenicity appeared to be associated with HSP and genes related to inflammatory factors. The present work offers a scientific foundation for screening potential MS strains that might be employed to develop attenuated vaccines.

Circulation of the reassorted very virulent genotype of infectious bursal disease virus isolated from vaccinated broiler chickens in Bangladesh

Objective: The methodology employed in this research was designed to identify and characterize the infectious bursal disease virus (IBDV) at the molecular level, originating from recent outbreaks in Bangladesh. Materials and Methods: The IBDV outbreak farm was investigated, and bursa of Fabricius (BF) specimens were acquired from infected chickens. Initially, viruses in the processed samples were detected in chicken embryo fibroblast (CEF) cells, and the RT-PCR method was used to confirm IBDV. The positive samples were injected through chorioallantoic membrane route into the embryo of a 10-day-old specific pathogen-free (SPF) egg for virus isolation and pathogenicity testing. Finally, we sequenced the VP2 gene to identify phylogenetic relationships and detect mutations. Results: From the 77 collected samples, 42.85% (33/77) were found positive for cytopathic effects in CEF cells, and IBDV was detected in 31.16% (24/77) of the samples by RT-PCR. IBDV was isolated in SPF chicken embryos. In the pathogenicity test, infectious bursal disease was evident in seronegative chickens with visible signs of disease. Sequence analysis shows that the broiler-isolated viruses clustered with genotype A3B2 and backyard chickens with genotype A1B1. The presence of amino acid motifs for virulence markers was revealed in the partially sequenced VP2 gene with a mutation at S254G in four IBDV isolates from broilers. However, amino acids for virulence markers were absent in two isolates from backyard chickens, which shows sequence homology with IBDV classic strains. Conclusion: In this study, we identified and characterized circulating reassorted IBDV from vaccinated broilers, which may be one of the major causes of vaccination failure in broilers.

A Novel Variant of Avian Reovirus Is Pathogenic to Vaccinated Chickens.

Avian reovirus (ARV) infections, characterized by severe arthritis, tenosynovitis, pericarditis, and poor weight gain, have become increasingly serious in recent years. The economic impact is significant as it causes growth inhibition and immunosuppression. Some commercial poultry in China have been widely vaccinated with available ARV vaccines; however, infections continue to occur even after vaccination. This study aimed to isolate a novel variant, ARV-SD19/11103, from the joint tissues of infected broiler chickens vaccinated with ARV vaccines in Shandong Province. Genetic evolution analysis of the major protective antigen σC gene in ARVs showed that ARV-SD19/11103 was located in the genotype cluster I but not in the same sub-cluster as the S1133 vaccine strain. The amino acid sequence similarity between SD19/11103 and vaccine strains S1133, 1733, and 2408 was <80%. After analyzing the amino acid sequences of the σC protein, 33 amino acid differences were found between the new variant isolate and the vaccine strains. This novel variant showed obvious pathogenicity in specific pathogen-free chicken embryos and chicks and could cause serious disease in chickens vaccinated with commercially available ARV vaccines. Cross-neutralization experiments further demonstrated a significant antigenic difference between the novel variant and genotype cluster I ARV strains. The novel variant strain isolated in this study provides an important theoretical basis for understanding the prevalence and genetic evolutionary characteristics of ARV variant strains in our country. This study identified the causes of ARVs circulating and emphasizes the needs for developing new vaccines against novel ARV variants.

Quercetin Alleviates Inflammation and Energy Deficiency Induced by Lipopolysaccharide in Chicken Embryos.

Energy deficiency causes multiple organ dysfunctions after LPS induction. Quercetin is a phenolic compound found in herbal medicines. However, the effects of quercetin in alleviating LPS-induced energy deficiency remain unclear. In the present study, an in vivo LPS-induced inflammation model was established in chicken embryos. Specific pathogen-free chicken embryos (n = 120) were allocated to control, PBS with or without ethanol, quercetin (10, 20, or 40 nmol, respectively), and LPS (125 ng/egg) with or without quercetin groups. Fifteen day old embryonated eggs were injected with the abovementioned solutions via the allantoic cavity. On embryonic day 19, the tissues of the embryos were collected for histopathological examination using frozen oil red O staining, RNA extraction, real-time quantitative polymerase chain reaction, and immunohistochemical investigations. The glycogen and lipid contents in the liver increased after LPS stimulation as compared with the PBS group, whereas quercetin decreased the accumulation as compared with the LPS group. The mRNA expressions of AMPKα1 and AMPKα2 in the duodena, ceca, and livers were upregulated after LPS induction as compared with the PBS group, while quercetin could downregulate these expressions as compared with the LPS group. The immunopositivity of AMPKα2 in the villus, crypt, lamina propria, tunica muscularis, and myenteric plexus in the duodena and in the cytoplasms of hepatocytes significantly increased after LPS induction when compared with the PBS group (p < 0.01), whereas the immunopositivity to AMPKα2 in the quercetin treatment group significantly decreased when compared with the LPS group (p < 0.01 or p < 0.05). The LPS-induced high expressions of transcription factor PPARα and glucose transporter (SGLT1) were blocked by quercetin in the duodena, ceca, and livers. Quercetin treatment improved the LPS-induced decrease in APOA4 in the duodena, ceca, and livers. The mRNA expression of PEPT1 in the duodena and ceca increased after LPS challenge, whereas quercetin could downregulate PEPT1 gene expression. These data demonstrate that quercetin improved the energy deficiency induced by LPS in chicken embryos. The LPS-induced inflammation model was established to avoid the effect of LPS exposure from the environment and intestinal flora. The results form the basis the administration of quercetin pretreatment (in ovo infection) to improve the energy state of chicken embryos and improve the inflammation response.

Synthesis and antioxidant, antimicrobial, and antiviral activity of some pyrazole-based heterocycles using a 2(3H)-furanone derivative

Some pyrazole-based heterocycles such as pyrrolone, pyridazinone, and imidazole derivatives were synthesized utilizing the pyrazolyl-2(3H)-furanone derivative 3, which was obtained in a good yield via Perkin condensation of 5-chloro-4-formyl-3-methyl-1-phenylpyrazole with 3-(4-methylbenzoyl)propionic acid in the presence of cyclo-dehydrating agent (sodium acetate and acetic anhydride). Also, the acid hydrazide obtained was reacted with some carbonyl reagents such as acetic anhydride, benzoyl chloride, 4-chlorobenzaldehyde, and 1,3-diphenyl-4-formylpyrazole, aiming to achieve new pyrrolone derivatives. The antioxidant, antimicrobial, and antiviral activity screening of some synthesized compounds demonstrated that some of them offered strong potencies. An experimental trial was undertaken to explore the effect of different substances and applied against avian influenza HPAI-H5N1 (AIV) to evaluate the antiviral replication in specific pathogen-free chicken embryos. All the compounds were screened for their antimicrobial activities, and most of tested compounds showed potent inhibition growth activity toward Haemophilus (Gram-negative bacteria), Staphylococcus aureus (Gram-positive bacteria), and Candida albicans fungus. Upon antimicrobial screening, it was observed that the majority of the compounds were found to be active against Staphylococcus aureus, Haemophilus, and Candida albicans as compared to standard drugs. This experiment shows the potential usage of these compounds as antiviral agents and can be considered as a viable means to control the economically important avian influenza of poultry. These compounds can thus be recommended for their antiviral, antibacterial, and antifungal property and can very well be used as immunostimulants.

A recombinant oncolytic influenza virus expressing a PD-L1 antibody induces CD8+ T-cell activation via the cGas-STING pathway in mice with hepatocellular carcinoma

ObjectiveTo evaluate targeted killing of hepatocellular carcinoma (HCC) cells by a recombinant oncolytic influenza virus expressing a PD-L1 antibody (rgFlu/PD-L1) and to develop a novel immunotherapy for HCC. MethodsUsing influenza virus reverse genetics, a recombinant oncolytic virus was generated in the background of the A/Puerto Rico/8/34 (PR8) virus, then identified via screening and passage in specific pathogen-free chicken embryos. Hepatocellular carcinoma cell killing by rgFlu/PD-L1 was confirmed in vitro and in vivo. Transcriptome analyses were used to explore PD-L1 expression and function. Western blotting revealed that PD-L1 activated the cGas-STING pathway. ResultsrgFlu/PD-L1 expressed the PD-L1 heavy and light chain in PB1 and PA, respectively; PR8 served as the backbone. The hemagglutinin titer of rgFlu/PD-L1 was 29, and the virus titer was 9–10 logTCID50/mL. Electron microscopy revealed that the rgFlu/PD-L1 morphology and size were consistent with wild-type influenza virus. The MTS assay showed that rgFlu/PD-L1 induced significant killing of HCC cells but not normal cells. rgFlu/PD-L1 inhibited PD-L1 expression and induced apoptosis in HepG2 cells. Notably, rgFlu/PD-L1 controlled the viability and function of CD8+ T cells by activating the cGas-STING pathway. ConclusionrgFlu/PD-L1 activated the cGas-STING pathway in CD8+ T cells, causing them to kill HCC cells. This approach represents a novel immunotherapy for liver cancer.

Quercetin Ameliorates Lipopolysaccharide-Induced Duodenal Inflammation through Modulating Autophagy, Programmed Cell Death and Intestinal Mucosal Barrier Function in Chicken Embryos.

Diarrhea has been a global health problem for centuries, and the treatment has become increasingly difficult duo to the antibiotics overuse and resistance. Quercetin is a common flavonoid of extracts of vegetables, fruits, and traditional Chinese herbs, however, the mechanism of quercetin alleviating LPS-induced duodenal inflammation remains elusive. Specific pathogen-free chicken embryos (n = 120) were allocated to groups including control, PBS with or without alcohol, LPS (125 ng/egg) with or without quercetin (10, 20, or 40 nmol/egg, respectively), and quercetin groups (10, 20, or 40 nmol/egg). Fifteen day-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity. At embryonic day 19, the duodena of the embryos were collected for histopathological examination, RNA extraction and real-time polymerase chain reaction, immunohistochemical investigations, and Western blotting. The results demonstrated quercetin enhanced the inflammatory cell infiltration in the Peyer's patch of the intestinal mucosa after LPS induction. The LPS-induced expressions of these inflammation-related factors (TLR4, IL-1β, MMP3, MMP9, NFKB1, IFNγ, IL-8, IL-6) were completely blocked by quercetin. Quercetin also decreased the protein expression of TLR4, IL-1β, MMP3, and MMP9 after LPS induction. Quercetin could down-regulate autophagy gene expression (ATG5, LC3-1, LC3-2, and LKB1), and decreased the protein expression of ATG5, and LC3-1/LC3-2 after LPS induction. Quercetin treatment prevented LPS-induced increases of the gene expressions of programmed cell death factors (TNFα, Fas, CASP1, CASP3, CASP12, Drp1, and RIPK1); meanwhile, quercetin decreased the protein expression of CASP1 and CASP3 after LPS challenge. LPS reduced the gene expression of mucin 2, but upregulated the mRNA and protein expression of claudin 1, occludin, and ZO-1, and this was balanced by quercetin. This evidence suggests that quercetin can alleviate duodenal inflammation induced by LPS through modulating autophagy, programmed cell death, intestinal barrier function.

Evaluation of Newcastle disease virus LaSota strain attenuated by codon pair deoptimization of the HN and F genes for in ovo vaccination

In ovo vaccination is an attractive immunization approach for the poultry industry. However, commonly used Newcastle disease virus (NDV) vaccines cannot be administered in ovo because of the reduced hatchability and embryo mortality. The codon pair deoptimization (CPD) approach has been used to efficiently and rapidly attenuate viruses by targeting the virulence genes. In this study, we aimed to attenuate the NDV LaSota (LS) vaccine strain for in ovo vaccination by CPD of the fusion (F) or/and hemagglutinin-neuraminidase (HN) genes with approximately 44 % suboptimal codon substitutions. Three NDV LS recombinants expressing codon deoptimized F (rLS/F-d), HN (rLS/HN-d), or both genes (rLS/F+HN-d) were generated using reverse genetics technology. Biological assays showed that the CPD viruses retained similar hemagglutination activity and growth ability to the parental rLS virus. The CPD of the HN gene slightly attenuated the rLS/HN-d and rLS/F+HN-d viruses, whereas the CPD of the F gene marginally increased the rLS/F-d virus pathogenicity compared to rLS. Nevertheless, all three CPD rLS viruses were still lethal to 10-day-old specific-pathogen-free (SPF) chicken embryos. In ovo inoculation of 18-day-old SPF chicken embryos with the CPD viruses severely reduced chicken’s hatch and survival rates. These results suggested that the CPD of the surface glycoprotein genes of the LS strain at the current level of suboptimal codon substitutions could not sufficiently attenuate the virus for use as an in ovo vaccine, and codon deoptimizing a greater proportion of the F and HN genes or additional gene(s) may be required for sufficient attenuation of the LS strain.

Genomic Characterization of CIAV Detected in Contaminated Attenuated NDV Vaccine: Epidemiological Evidence of Source and Vertical Transmission From SPF Chicken Embryos in China.

Live attenuated vaccines have been extensively used to prevent infectious disease in poultry flocks. Freedom from exogenous virus is a high priority for any veterinary vaccines. Recently, attenuated Newcastle disease virus (NDV) vaccines were detected to be contaminated with chicken infectious anemia virus (CIAV) in a routine screening for exogenous viruses. To investigate the possible source of the contamination, we conducted virological tests on a specific-pathogen-free (SPF) layer breeder flock that provide the raw materials for vaccines in this manufacturer. Firstly, CIAV antibodies in serum and egg yolks samples of the SPF laying hens were detected by ELISA assays. The results showed that CIAV antibodies in serum and egg yolks were 62% positive and 57% positive, respectively. Then, DNA was extracted from the NDV vaccines and SPF chicken embryonated eggs, and detected by molecular virology assays. The results showed that three assays for pathogens in embryonated eggs had similar positive rates (35.8%). And the sequences of CIAV from SPF embryos and NDV vaccines consisted of 2,298 nucleotides (nt) with 100% homology. The new full-length genome of CIAV was designated SDSPF2020 (Genbank accession number: MW660821). Data showed SDSPF2020 had the sequence similarities of 95.8–99.6% with reference strains, and shared the highest homology with the Chinese strain HLJ15125. These results strongly suggested that exogenous CIAV contamination is most likely caused by wild virus infection in SPF flocks and vertical transmission to chicken embryos. Collectively, this study illustrated that vertical transmission of CIAV from a SPF layer breeder flock to embryos was a non-neglible way for exogenous virus contamination in vaccine production.

Isolation, identification and genome analysis of an avian hepatitis E virus from white-feathered broilers in China

Avian hepatitis E virus (HEV) is the major causative pathogen of the big liver and spleen disease, hepatitis-splenomegaly syndrome, and hepatic rupture hemorrhage syndrome. Until now, there are 6 different avian HEV genotypes that infect chickens have been reported worldwide. Epidemiologic investigations of the avian HEV demonstrated that avian HEV has been widely spread in China in recent years. In this study, an avian HEV named YT-aHEV was obtained from white-feathered broilers using LMH cells by virus isolation assay in Shandong province, China. The complete genome consists of 6656-nt excluding the poly(A) tail. The isolate was highly similar to the CaHEV strain and segregated into the same branch belonging to avian HEV genotype 3. Indirect immunofluorescence using capsid protein-specific polyclonal antibodies confirmed that YT-aHEV could establish productive infection and replicate stably in LMH cells. Furthermore, an in vivo avian HEV infection model was established successfully in specific pathogen-free chicken embryos by intravenous experiments. In the present study, we demonstrate an avian HEV infection associated with liver lesions of hemorrhage and swelling by LMH cells for the first time in a white-feather broiler flock in China. This research also provides a new diagnosis method for detection of avian HEV, which laid a foundation for the understanding of pathogenicity and molecular biology of this virus for further study.

The inactivated vaccine of reassortant H3N2 canine influenza virus based on internal gene cassette from PR8 is safe and effective

Canine influenza (CI) is a contagious respiratory disease in dogs, which poses a threat to canine health. A safe, high-yield vaccine seed virus is critical for CI vaccine development. We developed a PR8-based reassortant H3N2 canine influenza virus (RT CIV) using the reverse genetic method and evaluated its yield in canine kidney epithelial (MDCK) cells, Vero cells, and specific pathogen-free (SPF) chicken embryos. Mice and dogs were infected with RT CIV, and the pathogenicity was evaluated. The viral titers of RT CIV increased in MDCK cells, Vero cells, and SPF chicken embryos; the HA yield in SPF chicken embryos increased 4-fold. However, RT CIV was not lethal to mice, and it showed similar virulence as wild-type CIV. RT CIV also showed minimal pathogenicity in dogs, which manifested as mild fever and rhinorrhea for the first two days post-infection. Thus, RT CIV carrying the internal gene cassette from PR8 showed almost no pathogenicity in dogs. And the reassortant virus inactivated vaccine could provide complete protection against H3N2 CIV. To our knowledge, this is the first report on the pathogenicity of PR8-based reassortant H3N2 CIV in dogs. These studies are relevant for developing a high-yield and safe CI vaccine.

Synergistic pathogenicity in sequential coinfection with fowl adenovirus type 4 and avian orthoreovirus

Hydropericardium hepatitis syndrome (HHS) is a fatal disease caused by fowl adenovirus serotype 4 (FAdV-4). Avian viral arthritis is an infectious disease characterized by movement disorders caused by avian orthoreovirus (ARV). In the early 2019, our epidemiologic survey on poultry diseases in eight commercial broiler farms in China showed that FAdV-4 and ARV have a high coinfection rate, accounting for 63 % of all ARV-positive samples. We designed chicken embryo and animal models to investigate the synergistic pathogenicity of FAdV-4 and ARV. Weakness and inappetence were observed in all specific-pathogen-free (SPF) chickens of the experimental group. FAdV-4 and ARV coinfection caused severe embryonic body and hepatic hemorrhage in SPF chicken embryos. Compared with the singular ARV-infected group, joint swelling was more severe in all coinfected groups. Compared with single virus infection, the coinfection of the two viruses increased the mortality of SPF chicken embryos and chickens. FAdV-4 and ARV coinfection resulted in significantly severe macroscopic and microscopic lesions of the liver, spleen, and kidney of SPF chickens. The detection results of viral load in allantoic fluid, liver, and cloacal swabs indicated that ARV enhanced FAdV-4 replication in SPF chicken embryos and chickens. Cytokine detection showed a significant change in interleukin-1 (IL-1), IL-6, and interferon-α (IFN-α) levels in coinfected groups compared with those in the single-infected groups. Additionally, FAdV-4 and ARV coinfection caused severe damage to the SPF chicken’s immune system. In summary, these findings provide insights into the pathology, prevention, and treatment of FAdV-4 and ARV coinfection.

Evaluation of Antiviral Activity of Different Medicinal Plants against Newcastle Disease Virus

Newcastle Disease (ND) is a highly contagious viral disease that has a tremendous negative impact on the poultry industry worldwide. Plant extracts were prepared from five different medicinal plants and applied against Newcastle Disease Virus (NDV) to evaluate the antiviral replication in Specific Pathogen-Free (SPF) chicken embryos. Three dilutions from each plant extract were prepared and mixed with a fixed titer (104 EID50/mL) of virulent NDV strain Herts 33. The mix was inoculated into nine-day-old SPF chicken embryos that were monitored for five days. Real-time PCR and hemagglutination tests were conducted to evaluate the activity of NDV and its viral RNA titer. Some plant extracts showed a complete inhibition of NDV evidenced by the absence of embryo deaths, the absence of HA titer and viral RNA in the allantoic fluid. These plant extracts were from Moringa peregrina (leaves), Acacia cyanophylla (leaves), Eucalyptus camaldulensis (fruits) and Pistacia atlantica (leaves and stems). Other plant extracts showed partial inhibition of NDV, such as Ceratonia siliqua (leaves) and Eucalyptus camaldulensis (leaves). This experiment shows the potential of using medicinal plants as antiviral agents.

Pathogenicity of Fowl Adenovirus in Specific Pathogen Free Chicken Embryos

Inclusion body hepatitis (IBH) associated with fowl adenovirus (FAdV) infection has a worldwide distribution. The aim of the present study was to determine the pathogenicity of Malaysian FAdV serotype 9 (UPM04217) in specific pathogen free (SPF) embryonated chicken embryos. FAdV (titre 10(5.8)/ml) was inoculated into SPF embryonated chicken eggs (0.1 ml per egg) via the chorioallantoic membrane (CAM). There was 100% embryo mortality within 4-11 days post infection (dpi). The gross and microscopical lesions of the embryo were confined to the liver and were noted at 5, 7, 9 and 11 dpi. The liver was pale with multifocal areas of necrosis, fibrosis and haemorrhage. Microscopically, there was moderate to severe congestion and haemorrhage and severe and diffuse hepatocyte degeneration and necrosis, with intranuclear inclusion bodies (INIBs) and associated inflammation. Haemorrhage, congestion, degeneration, necrosis and hyperplasia of the CAM with INIBs were observed at 5, 7, 9 and 11 dpi. Varying degrees of congestion, haemorrhage, degeneration and necrosis were also observed in the yolk sac, kidney, spleen, heart and bursa of Fabricius. Ultrastructurally, numerous viral particles in the nucleus of hepatocytes were recorded at 7, 9 and 11 dpi, whereas at 5 dpi, fine granular and filamentous INIBs were observed. The INIBs in the CAM were present either as fine granular filamentous structures or as large viral inclusions. FAdV (UPM04217) is therefore highly pathogenic to SPF chicken embryos and the embryonic liver should be used for isolation and propagation of the virus.

Endogenous expression of ASLV viral proteins in specific pathogen free chicken embryos: relevance for the developmental biology research field

BackgroundThe use of Specific Pathogen Free (SPF) eggs in combination with RCAS retrovirus, a member of the Avian Sarcoma-Leukosis Virus (ASLV) family, is of standard practice to study gene function and development. SPF eggs are certified free of infection by specific pathogen viruses of either exogenous or endogenous origin, including those belonging to the ASLV family. Based on this, SPF embryos are considered to be free of ASLV viral protein expression, and consequently in developmental research studies RCAS infected cells are routinely identified by immunohistochemistry against the ASLV viral proteins p19 and p27. Contrary to this generally accepted notion, observations in our laboratory suggested that certified SPF chicken embryos may endogenously express ASLV viral proteins p19 and p27. Since these observations may have significant implications for the developmental research field we further investigated this possibility.ResultsWe demonstrate that certified SPF chicken embryos have transcriptionally active endogenous ASLV loci (ev loci) capable of expressing ASLV viral proteins, such as p19 and p27, even when those loci are not capable of producing viral particles. We also show that the extent of viral protein expression in embryonic tissues varies not only among flocks but also between embryos of the same flock. In addition, our genetic screening revealed significant heterogeneity in ev loci composition even among embryos of the same flock.ConclusionsThese observations have critical implications for the developmental biology research field, since they strongly suggest that the current standard methodology used in experimental studies using the chick embryo and RCAS vectors may lead to inaccurate interpretation of results. Retrospectively, our observations suggest that studies in which infected cells have been identified simply by pan-ASLV viral protein expression may need to be considered with caution. For future studies, they point to a need for careful selection and screening of the chick SPF lines to be used in combination with RCAS constructs, as well as the methodology utilized for qualitative analysis of experimental results. A series of practical guidelines to ensure research quality animals and accuracy of the interpretation of results is recommended and discussed.

Pathogenicity of Mycoplasma lipofaciens strain ML64, isolated from an egg of a Northern Goshawk (Accipiter gentilis), for chicken embryos

Some Mycoplasma species are well-known avian pathogens and are of importance in poultry breeder flocks due to their pathogenic potential for embryos. Mycoplasmas are regularly detected in birds of prey, and a strain of Mycoplasma lipofaciens that was isolated from an egg of a Northern Goshawk (Accipiter gentilis) was examined for its pathogenicity in specific pathogen free chicken embryos since birds of prey eggs were not available for this purpose. The strain was found to be pathogenic, causing a high mortality as well as dwarfing, curled toes and infiltrations of heterophils in the liver, kidney, intestine and chorioallantoic membrane.

Isolation and Characterization of H3N2 Influenza A Virus from Turkeys

Five 34-wk-old turkey breeder layer flocks in separate houses of 2550 birds each in a single farm in Ohio experienced a drop in egg production from late January to early February 2004. Tracheal swabs (n = 60), cloacal swabs (n = 50), and convalescent sera (n = 110) from the flocks were submitted to the laboratory for diagnostics. Virus isolation was attempted in specific-pathogen free embryonating chicken eggs and Vero and MDCK cells. Virus characterization was performed using agar gel immunodiffusion, the hemagglutination test, the hemagglutination inhibition test, the virus neutralization test, reverse transcription-polymerase chain reaction, sequencing, and phylogenetic analysis. A presumptive influenza virus was successfully propagated and isolated on the first passage in MDCK cells, but initially not in Vero cells or specific-pathogen free chicken embryos. After two passages in MDCK cells, it was possible to propagate the isolate in specific-pathogen free chicken embryos. Preliminary sequence analysis of the isolated virus confirmed that it was influenza A virus with almost 100% (235/236) identity with the matrix gene of a swine influenza A virus, A/Swine/Illinois/100084/01 (H1N2). However, it was not possible to subtype the virus using conventional serotyping methods. The results of genetic characterization of the isolated virus showed that it was the H3N2 subtype and was designated as A/Turkey/OH/313053/04 (H3N2). Phylogenetic analysis of the eight gene segments of the virus showed that A/Turkey/OH/313053/04 (H3N2) isolate was most closely related to the triple-reassortant H3N2 swine viruses [A/Swine/WI/14094/99 (H3N2)] that have been circulating among pigs in the United States since 1998, which contains gene segments from avian, swine, and human viruses. The A/Turkey/OH/313053/04 (H3N2) isolated from turkeys in this study was classified as a low pathogenic avian influenza A virus because it only caused a drop in egg production with minor other clinical signs and no mortality.

Gastric gross and microscopic lesions caused by the UNAM-97 variant strain of infectious bronchitis virus after the eighth passage in specific pathogen-free chicken embryos

Herein we report a description of gross and microscopic lesions found in specific pathogen-free chicken embryos caused by UNAM-97 infectious bronchitis virus (IBV) variant strain after the eighth passage. Embryos were divided into three groups and were inoculated in the chorioallantoic sac with 0.2 mL of UNAM-97, Mass 41 IBV (positive control), or sterile PBS (negative control). Forty-eight hours later the allatoic fluid was taken and used to start a cycle of eight passages through 9-d-old embryos. Seven days after the last passage, embryos were harvested and macroscopic lesions in all organs were recorded. Proventriculus and gizzard samples were obtained from all embryos and routinely processed for microscopic and ultrastructural examinations. The UNAM-97 IBV variant strain caused two macroscopic lesions uncommon for Mexican strains: thin-walled proventriculus and gizzard, as well as urate accumulation within an extra-embryonic peritoneal sac, leaving the body through the umbilical duct and accompanied by the yolk sac. At microscopic level, two relevant findings were observed to be produced by this variant. In the proventriculus, there was a decrease in the gland papillary branching, while the gizzard showed a significant reduction in mucosa thickness and tubular-to-proliferative-cell ratio, as well as an absence of hyaline secretion in the lumen. Electrodense material scattered in proventricular and gizzard cells was observed, with a structure consistent with that of coronaviruses. These pathological chicken embryo findings have not been reported as being caused by other IBV strains in Mexico.