SPF Chickens Research Articles

Generation and evaluation of a recombinant Newcastle disease virus strain R2B with an altered fusion protein cleavage site as a vaccine candidate

Newcastle disease (ND) is a highly contagious and fatal disease of chickens. Newcastle disease virus (NDV) strain R2B is an Indian mesogenic strain used for secondary vaccination in chickens. Mesogenic strains have increased virulence and immunogenicity but may cause disease in vaccinated birds, thus rendering them ineffective for use. In this study, we generated a recombinant NDV by changing the fusion protein cleavage site of mesogenic rNDV-R2B from a polybasic amino acid motif RRQKRF to a dibasic amino acid motif GRQGRL leading to generation of an attenuated virus, rNDV-R2B-FPCS. The modified recombinant virus had similar growth characteristics as rNDV-R2B, but was less virulent in susceptible chickens. Immunization of the recombinant attenuated virus to one week of age SPF chickens generated a protective immune response with a substantial reduction in virus shed after challenge with virulent NDV. The results of the study indicate that the modified rNDV-R2B-FPCS virus can be used for primary immunization in birds without any adverse reactions.

Molecular Analysis, Biochemical Characterization, Antimicrobial Activity, and Immunological Analysis of Proteus mirabilis Isolated from Broilers.

Proteus mirabilis, a Gram-negative bacterium, is ubiquitous in the environment and is considered as the normal microflora in the human gastrointestinal tract. However, this bacterium is an opportunistic pathogen in humans, often causing urinary tract infections. Moreover, Proteus has been frequently isolated from food animals, including poultry. Whether this bacterium contributes to the foodborne illness in humans is unclear. In this report, P. mirabilis isolates recovered from broilers during housing in the units were characterized, their antimicrobial activity was assayed, and broiler immune response to the soluble proteins was determined. Cecal contents and fecal droppings were treated according to the standard protocol for isolation. Speciation based on biochemical reactions and the antimicrobial activity of the isolates were carried out using commercial kits. Immunoblot was assayed to determine immune status of broilers against P. mirabilis. A total of 10 isolates of P. mirabilis were selected for further characterization. These isolates could grow in pH 6.0 and 1% NaCl conditions. They were resistant to sodium lactate, troleandomycin, rifamycin SV, vancomycin, but sensitive to nalidixic acid, cefotaxime and novobiocin. Moreover, the CTX, ACC, CMY-1, BIC, NDM, VEB, qnrB and qnrD genes were detected by PCR amplification in all isolates. Sera from broilers harboring this bacterium reacted to the P. mirabilis soluble proteins, but not from litter- and age-matched P. mirabilis negative and SPF chickens, indicating that this bacterium infected chickens that could have humoral immune response against P. mirabilis. This study provides a rationale for further monitoring P. mirabilis during poultry production to determine whether this bacterium poses potential threats to public health.

Cleavage site of Newcastle disease virus determines viral fitness in persistent infection cells

Newcastle disease, caused by infection with virulent strains of Newcastle disease virus (NDV), poses a risk for the poultry industry. The virulence of NDV is mainly determined by the cleavage site of F protein. Lentogenic NDV can become velogenic after passages in SPF chicken brain and air sac based on some strains isolated from water birds, because the proportion of virulent-related strains gradually increases. In contrast, this proportion remains unchanged if NDV is passaged via 10-day-old SPF chicken embryos. This information suggests that environmental conditions rather than mutation affect NDV fitness in quasispecies. However, it is unknown how the environment selects virulent-related strains from a viral population. In this study, velogenic and lentogenic NDV marked by green or red fluorescence were used to establish persistent infection (PI) in BHK-21 cells. Monitoring viruses by different methods, we found that, without competition, persistently infected cells harbored lentogenic and velogenic NDV strains similarly in terms of viral release, viral spread and the period of persistent viral infection. In contrast, under competitive co-infection, velogenic NDV became dominant in quasispecies from the fifth passage of PI cells, which resulted in the progressive disappearance of the lentogenic NDV strain. This domination was concomitant with a short-term reduction in the superinfection exclusion and supernatant interference in PI cells resulting in a velogenic virus rebound. We concluded that virulent-related F protein cleavage site facilitates the spread and replication of NDV in conditions under which cells do not secret trypsin-like proteases and do not inhibit free virus infection, resulting in a gradual increase in virulent strains in quasispecies with the number of passages.

Variations in Pathogenicity and Molecular Characterization of Infectious Bursal Disease Virus (IBDV) In Egypt

Infectious bursal disease consider a vital threat to poultry industry worldwide and It has special potentiality in Egypt as the country become endemic with several clades from avian influenza from both types highly pathogenic (H5N1 and H5N8) and low pathogenic one (H9N2). The Egyptian government depends on the inactivated vaccine strategy to control avian influenza and any infection with IBD in broiler flocks alter the inactivated vaccine response in addition to other economic loss from the IBD infections. The present study was conducted to investigate the variations in pathogenicity and molecular characterization of IBDV in Egypt. Twenty-six bursal samples were collected from different chicken farms during the period of June 2015 to April 2016; the virus was isolated by inoculation of bursal suspension into 10-11 days ECE via CAM. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was applied for IBD targeting Hypervariable Region (HVR) of VP2 gene. Out of 26 tested isolates, 11 isolates were positive by RT-PCR. Sequence analysis of PCR products of 11 positive isolates were carried out and revealed that all +ve isolates were characterized as very virulent (vvIBDV) strains and they showed 80.6-97.5% identity with Giza 2008 IBD strain. Six selected positive vvIBDV isolates were used to study the pathogenicity of IBDV in 105 one-day-old SPF chickens. At day 211st the SPF chickens were divided into 7 groups 15 bird/each, the groups 1-6 were experimentally infected with 0.1 mL of EID50 of 6 selected positive IBDV field isolates via naso-ocular route. Group 7 kept as non-infected control. Experimental infection caused clinical signs and gross lesions typical for IBDV and mortalities ranged from 26.7 to 80% as well as bursal/body weight index lower than 0.7 indicating bursal atrophy. The Microscopical examination of bursal samples collected at days 3 and 7 post-infection revealed different degrees of histopathological bursal changes as lymphoid depletion with proliferating interfollicular connective tissue, atrophy of the follicles, stromal edema and associated with inflammatory response. The circulating strains were highly related to vvIBDV strains belonging to serotype-1 isolates and indicating that they are highly pathogenic to 21-day-old SPF chickens and that vvIBDV strains succeeded in surviving in the Egyptian farms despite of the application of intensive vaccination programs.

Immunogenicity and protective efficacy of recombinant fiber-2 protein in protecting SPF chickens against fowl adenovirus 4

Since a novel hyper-virulent fowl adenovirus serotype 4 (FAdV-4) infection occurred in 2015, the novel FAdV-4 has been widely spreading across China, causing significant economic losses to the poultry industry. As the urgency of the issue calls for effective and efficient solutions, the present study investigated the possibility of the fiber-2 protein of the FAdV-4 to serve as a vaccine candidate against the novel FAdV-4. In the research, fiber-2 proteins were expressed in Escherichia coli, and then purified. To evaluate the immunogenicity of the recombinant fiber-2 protein, we investigated both the humoral and cellular immune responses in chickens immunized with fiber-2. The humoral immunity was assessed by detecting IgY antibodies and virus-neutralizing antibodies in chicken serum at 7, 14, 21 days post-immunization (dpi). We examined cellular immune responses by detecting CD3+CD4+ and CD3+CD8+ changes in chickens’ peripheral blood through using flow cytometry at 7, 14, 21 dpi. The cytokine production in the serum of the immunized chickens was detected by ELISA at 7, 14, 21 dpi to further explore the impact of the recombinant protein on the regulation of cytokines. The protective efficacy was determined by the survival rate of the immunized chickens challenged with the novel FAdV-4. The results show that the level of IgY antibodies of the chickens immunized with fiber-2 protein was significantly higher than that of the chickens immunized with an inactivated vaccine against FAdV-4. Moreover, 7 days after immunization, the CD4+ T-cell proliferative response of the chickens immunized with fiber-2 was significantly higher than that of the chickens immunized with the inactivated vaccine. Challenge experiment showed that the fiber-2 protein could provide full protection and the inactivated vaccine could provide 90 percent protection against the FAdV-4. These results suggest that the recombinant fiber-2 protein can be an ideal candidate for subunit vaccines against the disease.

An Experimental Study on Apoptosis in Lymphoid Tissues of SPF Chicken after Avian Influenza Serotypes H9N2 Infection

Background and purpose: Influenza virus induces cell death in animals and human through necrosis and apoptosis. The present study is an attempt to experimentally examine the type of cell death in lymphoid tissue of chicken infected by avian influenza serotype H9N2 (A/chicken/Iran/772/2000). Methodology: Twenty SPF three weeks old chickens were grouped in two identical groups. The treatment group was infected by influenza virus (0.2ml, dilution 1:10, titration EID 50 107.5) and the control group received saline serum of the same volume through intranasal inoculation. Spleen, thymus, bursa of fabricius tissue specimens were collected after 72hrs. Afterward, microscopic intersections (5-6µm) stabilized in buffer formalin (10%) and marked by hematoxylin-eosin were prepared from the specimens.Findings: Histopathological examination of lymphoid tissues of the treatment and control chickens showed necrosis, apoptosis, and lymphoid depletion in lymphoid tissues of the treatment group. The difference between treatment and control groups in terms of apoptosis in spleen tissue was significant (p<0.001); however, the difference in thymus and bursa of fabricius tissues was not significant. As to necrosis and lymphoid depletion, there was a significant difference between the two groups with regard to spleen, thymus, and bursa fabricius tissues. (p<0.001)Conclusion: The survey showed that avian influenza serotype H9N2 can cause damages to lymphoid tissues through inducing apoptosis and necrosis

Detection of Salmonella Infection in Chickens by an Indirect Enzyme-Linked Immunosorbent Assay Based on Presence of PagC Antibodies in Sera.

The outcomes of infection of humans and animals with Salmonella range from a persistent asymptomatic carrier state to temporal mild gastroenteritis or severe systemic infection. A rapid and accurate diagnostic test would help formulate strategies for effective prevention of their infections in the animal population. Current sequencing data predict that the outer membrane protein, PagC, is present in all common Salmonella serovars with sequence similarities of more than 98%. PagC sequences in other bacterial species are less than 65% similarity at the amino acid level to those of Salmonella PagC. We hypothesized that PagC could be immunogenic and detection of antibodies to this protein could be an accurate indicator of Salmonella infection. The pagC gene from Salmonella enterica serovar Typhimurium CVCC542 was expressed in Escherichia coli. The purified recombinant PagC protein was immobilized in microtiter plate wells. Sera from SPF chickens infected with Salmonella or other non-Salmonella pathogens by injection were added and binding of PagC protein was detected by the horseradish peroxidase (HRP)-labeled goat anti-chicken antibody. Sera from Salmonella-infected chickens showed high specificity in contrast to the sera from chickens infected with other bacteria. When 87 Salmonella antibody-positive sera from Salmonella Pullorum orally infected SPF chicken and 93 negative sera from uninfected SPF chicken were tested, 98.3% agreement was detected. The rPagC enzyme-linked immunosorbent assay (ELISA) and agglutination had 80.6% agreement in detecting 252 clinical chicken sera samples. These results suggest that PagC antibody-based indirect ELISA can serve as a convenient and novel method for the diagnosis of Salmonella infection.

Adaptation of velogenic Newcastle disease virus in Vero cells: velogenicity of virus unaltered after adaptation

In the present experiment, velogenic Newcastle disease virus (NDV) was adapted to Vero cells without the addition of exogenous trypsin. The cytopathic effect (CPE) in NDV-infected Vero cells was observed from passage 3 (P 3) onwards with concomitant increase in the haemagglutination (HA) and virus titres. The CPE was characterized by rounding of cells, their detachment from the surface and syncytia formation. The Vero-adapted NDV was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), which detected the amplification of haemagglutinin neuraminidase (HN) gene from virus-infected cells. The mean death time (MDT) of 58 hours upon inoculation to embryonated chicken eggs and the intravenous pathogenicity index (IVPI) of 2.12 following inoculation to SPF chicken suggested that Vero adapted virus retained the velogenicity even after ten passages. Furthermore, the nervous signs and haemorrhages in the pro-ventriculus of birds that died at day 5 post inoculation with PIO virus confirmed that the Vero-adapted NDV was a neurotropic viscerotropic velogenic strain.

Transcription analysis of the interaction between chicken thymus and recombinant avian leukosis virus isolate FJ15HT0

Avian leukosis virus (ALV) induces multiple avian tumors, growth decrease and immune suppression. Previously, a novel natural recombinant ALV isolate FJ15HT0 was proven to be associated with significant body weight decrease, immune suppression and lymphocytoma in infected SPF chickens. In order to uncover the interaction between virus and host, we compared differences in the transcriptomes of the thymuses from the mock chickens and simulated congenitally infected chickens at 5days (d), 13d and 21d of age by RNA-seq analysis of the thymuses. Signaling pathways including cytokine–cytokine receptor interactions, peroxisome proliferator-activated receptor (PPAR) signaling pathway, Janus tyrosine kinase/signal transducers and activators of transcription (Jak-STAT) signaling pathway and fatty acid degradation were involved in the interaction between FJ15HT0 and SPF chickens. Interestingly, fold change of ciliary neurotrophic factor receptor α (CNTFRα) in infected donor collected from 2d to 21d showed a significant positive correlation with the corresponding expression of the viral gp85 gene in thymuses (r=0.656, P<0.01) and in livers (r=0.525, P<0.05). It will provide new insights for the molecular pathogenesis of ALV infection.

Comparative transcriptome analysis reveals induction of apoptosis in chicken kidney cells associated with the virulence of nephropathogenic infectious bronchitis virus

Avian infectious bronchitis virus (IBV) that causes respiratory and nephritic diseases in chicken is a major poultry pathogen leading to serious economic loss worldwide. The nephropathogenic IBV strains cause nephritis and kidney lesions intrinsically and the pathogenic mechanism is still unclear. In the present study, SPF chicks were infected with three nephropathogenic IBVs of different virulence and their gene expression profiles in chicken kidney were compared at transcriptome level. As a result, 1279 differentially expressed (DE) genes were found in very virulent SCDY2 inoculated group, 145 in virulent SCK2 group and 74 in non-virulent LDT3-A group when compared to mock infected group. Gene Ontology (GO) and KEGG pathway enrichment analysis on SCDY2 group displayed that the up-regulated DE genes were mainly involved in cell apoptosis, and the down-regulated genes were involved in metabolic processes and DNA replication. Protein-Protein Interaction (PPI) analysis showed that DE genes in SCDY2 group formed a network, and the core of the network was composed by cell apoptosis and immune response proteins. The clustering of gene expression profile among the three virus inoculated groups indicated that the majority of up-regulated DE genes on apoptosis in very virulent SCDY2 group were up-regulated more or less in virulent SCK2 group and those down-regulated on innate immune response in SCDY2 group were also down-regulated differently in SCK2 group. In addition, the number of apoptotic cells detected experimentally in kidney tissue were very different among the three virus inoculated groups and were positively accordant with the viral titer, kidney lesions and viral virulence of each group. Taken all together, the present study revealed that virulent nephropathogenic IBV infection modified a number of gene expression and induction of apoptosis in kidney cells may be a major pathogenic determinant for virulent nephropathogenic IBV.

Co-infection of Chlamydia psittaci with H9N2, ORT and Aspergillus fumigatus contributes to severe pneumonia and high mortality in SPF chickens

Since 2007, most areas of China have seen outbreaks of poultry airsacculitis, which causes hugely economic losses to the poultry industry. However, there are no effective measures to combat the problem. In this study, 105 rations were collected to isolate Aspergillus spp. from the diseased farms. In subsequent experiments, SPF chickens were inoculated with Ornithobacterium rhinotracheale (ORT), Chlamydia psittaci (C. psittaci) and Aspergillus fumigatus (A. fumigatus), and mortality rate, body weight gain and lesion score were evaluated. Of these ration samples, 63 (60.0%) were A. fumigates, 21 (20.0%) were Aspergillus niger (A. niger) and 11 (10.5%) were Aspergillus candidus (A. candidus). Furthermore, SPF birds infected with C. psittaci, ORT, H9N2 virus and A. fumigatus conidia exhibited a mortality rate of 40%, while simultaneous co-infection with C. psittaci, ORT and A. fumigatus resulted in a mortality rate of 20%. The avian airsacculitis was manifested in the C. psittaci + ORT/A. fumigatus, C. psittaci + H9N2 + ORT/A. fumigatus and C. psittaci + H9N2/A. fumigatus groups while others had transient respiratory diseases without mortality. Our survey indicates that feed-borne A. fumigatus is prevalent in poultry rations. The combination of C. psittaci, ORT, H9N2 and A. fumigatus conidia contributes to the replication of avian airsacculitis by aggravating the severe damage to the air sacs and lungs of chickens.

A novel multi-variant epitope ensemble vaccine against avian leukosis virus subgroup J.

The hypervariable antigenicity and immunosuppressive features of avian leukosis virus subgroup J (ALV-J) has led to great challenges to develop effective vaccines. Epitope vaccine will be a perspective trend. Previously, we identified a variant antigenic neutralizing epitope in hypervariable region 1 (hr1) of ALV-J, N-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-C. BLAST analysis showed that the mutation of A, E, T and H in this epitope cover 79% of all ALV-J strains. Base on this data, we designed a multi-variant epitope ensemble vaccine comprising the four mutation variants linked with glycine and serine. The recombinant multi-variant epitope gene was expressed in Escherichia coli BL21. The expressed protein of the variant multi-variant epitope gene can react with positive sera and monoclonal antibodies of ALV-J, while cannot react with ALV-J negative sera. The multi-variant epitope vaccine that conjugated Freund's adjuvant complete/incomplete showed high immunogenicity that reached the titer of 1:64,000 at 42 days post immunization and maintained the immune period for at least 126 days in SPF chickens. Further, we demonstrated that the antibody induced by the variant multi-variant ensemble epitope vaccine recognized and neutralized different ALV-J strains (NX0101, TA1, WS1, BZ1224 and BZ4). Protection experiment that was evaluated by clinical symptom, viral shedding, weight gain, gross and histopathology showed 100% chickens that inoculated the multi-epitope vaccine were well protected against ALV-J challenge. The result shows a promising multi-variant epitope ensemble vaccine against hypervariable viruses in animals.

Transmission and immunopathology of the avian influenza virus A/Anhui/1/2013 (H7N9) human isolate in three commonly commercialized avian species.

H7N9 virus infection is a global concern, given that it can cause severe infection and mortality in humans. However, the understanding of H7N9 epidemiology, animal reservoir species and zoonotic risk remains limited. This work evaluates the pathogenicity, transmissibility and local innate immune response of three avian species harbouring different respiratory distribution of α2,6 and α2,3 SA receptors. Muscovy ducks, European quails and SPF chickens were intranasally inoculated with 105 embryo infectious dose (EID)50 of the human H7N9 (A/Anhui/1/2013) influenza isolate. None of the avian species showed clinical signs or macroscopic lesions, and only mild microscopic lesions were observed in the upper respiratory tract of quail and chickens. Quail presented more severe histopathologic lesions and avian influenza virus (AIV) positivity by immunohistochemistry (IHC), which correlated with higher IL-6 responses. In contrast, Muscovy ducks were resistant to disease and presented higher IFNα and TLR7 response. In all species, viral shedding was higher in the respiratory than in the digestive tract. Higher viral shedding was observed in quail, followed by chicken and ducks, which presented similar viral titres. Efficient transmission was observed in all contact quail and half of the Muscovy ducks, while no transmission was observed between chicken. All avian species showed viral shedding in drinking water throughout infection.

Heterologous prime-boost immunization of Newcastle disease virus vectored vaccines protected broiler chickens against highly pathogenic avian influenza and Newcastle disease viruses.

Avian Influenza virus (AIV) is an important pathogen for both human and animal health. There is a great need to develop a safe and effective vaccine for AI infections in the field. Live-attenuated Newcastle disease virus (NDV) vectored AI vaccines have shown to be effective, but preexisting antibodies to the vaccine vector can affect the protective efficacy of the vaccine in the field. To improve the efficacy of AI vaccine, we generated a novel vectored vaccine by using a chimeric NDV vector that is serologically distant from NDV. In this study, the protective efficacy of our vaccines was evaluated by using H5N1 highly pathogenic avian influenza virus (HPAIV) strain A/Vietnam/1203/2004, a prototype strain for vaccine development. The vaccine viruses were three chimeric NDVs expressing the hemagglutinin (HA) protein in combination with the neuraminidase (NA) protein, matrix 1 protein, or nonstructural 1 protein. Comparison of their protective efficacy between a single and prime-boost immunizations indicated that prime immunization of 1-day-old SPF chicks with our vaccine viruses followed by boosting with the conventional NDV vector strain LaSota expressing the HA protein provided complete protection of chickens against mortality, clinical signs and virus shedding. Further verification of our heterologous prime-boost immunization using commercial broiler chickens suggested that a sequential immunization of chickens with chimeric NDV vector expressing the HA and NA proteins following the boost with NDV vector expressing the HA protein can be a promising strategy for the field vaccination against HPAIVs and against highly virulent NDVs.

The first whole genome sequence and pathogenicity characterization of a fowl adenovirus 4 isolated from ducks associated with inclusion body hepatitis and hydropericardium syndrome

ABSTRACTIn June 2015, an infectious disease with high prevalence causing severe hydropericardium syndrome (HPS) first appeared in duck farms of northeast China. The disease showed high morbidity of 35% and mortality of 15% in a commercial duck farm with 200,000 45-day-old ducks. One strain of hypervirulent fowl adenovirus serotype 4 was identified and designated as HLJDAd15. The whole genome of the duck isolate was sequenced and found to contain the same large deletions as a genotype that has become prevalent in chickens in China recently, indicating that this disease might be transmitted from chickens to ducks. The pathogenicity of HLJDAd15 was evaluated in SPF chickens and ducks. The results showed that chickens were more susceptible to this new genotype of fowl adenovirus, and it was more difficult to infect ducks than chickens with the duck origin virus. Thus, it appears that this severe HPS in ducks is far more likely to have been transmitted from chickens to ducks than from ducks to ducks. Therefore, transmission from chickens to ducks constitutes a threat to the duck farming industry, and this transmission route is a very important consideration for the prevention and control of the new genotype of fowl adenovirus. This is the first whole genome sequence of a FAdV-4 isolated from ducks, and this information is important for understanding the molecular characteristics and evolution of aviadenoviruses. The potential risks of infection with this new hypervirulent FAdV-4 genotype in chickens and ducks urgently require an effective vaccine.

Single gene reassortment of highly pathogenic avian influenza A H5N1 in the low pathogenic H9N2 backbone and its impact on pathogenicity and infectivity of novel reassortant viruses.

Avian influenza A H5N1 and H9N2 viruses have been extensively circulating in various avian species and frequently infect mammals, including humans. The synchronous circulation of both viruses in Egypt provides an opportunity for possible genetic assortment, posing a probable threat to global public health. To assess the potential risk of the IAV reassortants derived from co-circulation of these two AI subtypes, reverse genetics technology was used to generate a set of IAV reassortants carrying single genetic segments of clade 2.2.1.2 virus A/duck/Egypt/Q4596D/2012 (H5N1), a representative of the most prevalent H5N1 clade in Egypt, in the genetic backbone of A/chicken/Egypt/S4456B/2011 (H9N2), a representative of G1-like H9N2 lineage which is widely circulating in Egypt. Furthermore, the genetic compatibility, growth kinetics and virulence were evaluated in vitro in mammalian systems using the MDCK cell line and avian system using SPF embryonated chicken eggs. Pathogenicity and virus shedding were further tested using SPF chickens. Out of the eight desired H9-reassortants, we could rescue only 5 reassortant viruses, either due to difficulty in cloning (PB1 of H5N1 virus) or genetic incompatibility (NP-H5/H9 and NA-H5/H9). Results revealed higher replication rates for the H9N2 virus having the NS segment of H5N1 virus. The lowest survival rate in both SPF eggs and SPF chickens was associated with the H5N1 parent virus infection, followed by the HA-H5/H9 virus. Our findings also suggest that all other reassortant viruses were of lower pathogenicity than the wild type H5N1 virus.

Characterization of a hypervirulent fowl adenovirus 4 with the novel genotype newly prevalent in China and establishment of reproduction infection model of hydropericardium syndrome in chickens

Severe hydropericardium syndrome (HPS) has been present in layers in the northeast of China since June 2015, with mortality rates varying from 30 to 90%. Dead layers had severe hydropericardium with pericardial volumes of 5 to 20 mL, as well as inclusion body hepatitis. Laboratory investigations led to the isolation of a fowl adenovirus strain, HLJFAd15, from the liver tissue of dead layers. Natural deletions of ORF19 and ORF27 were found in this clinical strain by complete genome sequencing, which was identified with the novel genotype recently prevalent in China. The pathogenicity characterization was conducted in 35-day-old SPF chickens using HLJFAd15 with novel genotype of fowl adenovirus serotype 4 (FAdV-4). The reproduction disease cases of HPS with mortality rates of 76.9% by oral administration and 100% by intramuscular injection were induced successfully by challenging SPF chickens, respectively. Non-enveloped viral particles with a mean diameter of approximately 80 nm were found in the livers of virus-infected SPF chickens. Our study revealed that HLJFAd15 was identified with the novel genotype strains recently emerging in China by complete genome sequencing, and the strain was capable of causing HPS by the pathogenicity analysis. However, although there is currently no commercial vaccine against the novel genotype FAdV-4, the animal infection model established in this study was valuable for vaccine evaluation and development.

Cell-culture derived fowl adenovirus serotype 4 inactivated vaccine provides complete protection for virus infection on SPF chickens.

Inclusion body hepatitis and hepatitis-hydropericardium syndrome caused by high-pathogenic fowl adenovirus serotype 4 has recently plagued Chinese poultry industry and caused huge economic losses since 2013. So far, there is no commercial vaccine available to control this disease. In this study, we reported the development of both embryo-adapted and cell-culture derived inactivated FAdV-4 vaccines and evaluated their efficacies in chicken. Compared to embryo-adapted vaccine, cell-culture derived vaccine induced significantly earlier and higher serological response measured by AGP and ELISA. After virus challenge, chicken immunized with cell-culture derived vaccine did not showed any gross and histopathological lesions, whereas inclusion body hepatitis was observed in the liver of chicken vaccinated with embryo-adapted vaccine. No mortality was observed in both the vaccinated groups. The above results suggested that cell-culture derived FAdV-4 inactivated vaccine could be a better vaccine candidate than embryo-adapted vaccine to control FADV-4 infections in China.

A novel avian retrovirus associated with lymphocytoma isolated from a local Chinese flock induced significantly reduced growth and immune suppression in SPF chickens

Avian Leukosis Viruses (ALVs) are associated with neoplasias, immune suppression and reduced performance in chicken flocks. In the present study, a naturally occurring recombinant strain of ALV (FJ15HT0) was isolated from an infected flock of Chinese “Hetian” chickens, and was subsequently identified as an exogenous ALV by immuno-fluorescence assay (IFA), PCR and following entire proviral DNA nucleotide sequencing. This isolate is revealed as a novel recombinant virus, lacking viral oncogenes, with the gp85 (93.4%) of subgroup B, the U3 (92.1%) and R (95.2%) region of subgroup J, the U5 (93.8%) region and 5′UTR (95.7%) of subgroup C, as well as the gp37 (90.6%) and 3′ (92.2%) of ALV-E. The simulative congenital infection with this isolate in SPF chickens resulted in significant weight loss (P<0.05) and a significant reduction in the humoral immune response to the live NDV vaccine (P<0.05), but not to the inactive AIV-H5 vaccine (P>0.05). Foci of lymphocytomas were observed in tissues of congenitally infected chickens at 11 weeks post-hatch, demonstrating the acute oncogenicity of the isolate.

Cellular immune response in chickens infected with avian infectious bronchitis virus (IBV)

To understand the mechanistic basis of innate immunity against the infectious bronchitis virus (IBV), the gene transcription profile of pattern recognition receptors (PRRs) in SPF chicken tissues infected with an IBV-M41 strain was examined. IBV infection induced mRNA transcription of TLRs, RLRs, and NODs. TLR7, MyD88, TRAF6, MDA5, LGP2, and NLRC5 were stimulated, as well as mRNA activation of the downstream genes of NF-κB and IRF3. And mRNA for the pro-inflammatory cytokines of interferon-α (IFN)-α, IFN-β, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) showed over-expression. The IBV load in tissues gradually reduced. These results suggested that the three kinds of PRRs signaling pathways and innate immune cytokine were induced after IBV infection.