Specific Pathogen-free Chickens Research Articles

Influenza A Virus Subtype H9N2 Infection Induces Respiratory Microbiota Dysbiosis in Chickens via Type-I Interferon-Mediated Mechanisms

Abstract Avian influenza virus (AIV) poses significant threats to poultry and human health. This study investigates the impact of H9N2 AIV infection on the respiratory microbiota of chickens using 16S rRNA gene sequencing. Forty-eight one-day-old specific pathogen-free (SPF) chickens were assigned to six groups: a control and five post-infection groups (days 1, 3, 5, 7, and 9). After a 15-day microbiota stabilization period, the infected chickens received a viral inoculum (10^7 TCID50/mL) via ocular, intra-nasal, and intra-tracheal routes. Tracheal and broncho-alveolar lavage samples were analyzed. Significant reductions in microbiota diversity were observed on days 5, 7, and 9 post-infection, compared to d0 controls. PERMANOVA confirmed significant beta diversity differences (P = 0.001) between infected and uninfected groups. The microbial shifts from d5 to d9 were marked by increased Proteobacteria, decreased Actinobacteria and Firmicutes, and a rise in Dickeya. Elevated type I interferon (IFN-β) and viperin gene expression at d5 coincided with reduced microbiota diversity, highlighting the respiratory microbiota's role in modulating host responses to AIV H9N2 infection and suggesting potential biomarkers for respiratory dysbiosis.

Determination of the Pathogenicity of the Variant UPM 1432/2019 IBDV in SPF Chicken Eggs and Chicken Fibroblast Cell Line

The infectious bursal disease virus (IBDV) is a nuisance to chicken productivity in the global poultry industry due to its high pathogenicity. In this study, the pathogenicity of the UPM 1432/2019 variant was evaluated using specific pathogen-free (SPF) chicken eggs and fibroblast cell lines. This viral strain was isolated from chickens displaying symptoms of watery diarrhoea and ruffled feathers. Following inoculation, the SPF chicken eggs, and cell lines were meticulously monitored for indicators of viral replication and the onset of cytopathic effects (CPE). The results showed that the virus could replicate in the SPF eggs and cell lines due to the presence of CPE in some cells. In addition, virus-induced weight loss in infected embryonated chicken eggs was evaluated. The UPM 1432/2019 and UPM 1219/2019 strains demonstrated similar mortality rates and CPE features. These findings indicate that the UPM 1432/2019 variant is pathogenic in SPF chicken eggs and fibroblast cell lines. Furthermore, the new UPM 1432/2019 variant caused IBD with pathogenic characteristics similar to the traditional UPM 1219/2019 variant. The emergence of the UPM 1432/2019 variant poses a risk to the economy and poultry production and is potentially resistant to the existing bursal disease vaccines. Consequently, the UPM 1432/2019 variant may significantly contribute to economic losses for chicken farm operators. Moreover, this study provides essential information on the potential virulence of the UPM 1432/2019 variant of IBDV and its potential impact on the poultry industry.

The Generation of a H9N2 Avian Influenza Virus with HA and C3d-P29 Protein Fusions and Vaccine Development Applications

Background: Maternal-derived antibody (MDA) interferes with immune responses, leading to the failure of H9N2 avian influenza vaccinations in poultry. So far, none of the commercially available H9N2 avian influenza vaccines used in poultry have been able to overcome MDA interference. Methods: To develop a vaccine that can overcome MDA interference, one or multiple copies of the minimum-binding domain (P29) from the complement protein C3d were inserted in between the signal peptide and the head domain of the hemagglutinin (HA) protein on a H9N2 avian influenza virus (A/Chicken/Shanghai/H514/2017, named H514). Results: The HA proteins containing P29 stimulated stronger type I interferences than wild-type HA proteins in vitro. The modified viruses with the HA proteins containing one copy of P29 (rH514-P29.1) and two copies of P29.2 (rH514-P29.2) were successfully rescued using reverse genetics. The inactivated vaccines developed with rH514-P29.1 or rH514-P29.2 induced higher and faster humoral immune responses than the vaccine developed with rH514 in specific pathogen-free (SPF) chickens. To evaluate the vaccines’ efficacy in the presence of MDA and to ensure a uniform level of MDA, passively transferred antibody (PTA) was used as a model to mimic MDA in 1-day-old SPF chickens. Our results showed that the rH514-P29.2 inactivated vaccine induced significantly higher HI titers than the rH514 inactivated vaccine in the presence of PTA. More importantly, it reduced viral shedding after being challenged with H514 in the presence of PTA. Conclusions: Our results suggest that vaccine antigens fused with two copies of P29 can decrease the interference of MDA on immunity in chickens. Overall, our results provide a new strategy for overcoming MDA interference.

Integrated analysis of miRNA and mRNA expression profiles in the bursa of Fabricius of specific pathogen-free chickens infected with avian reticuloendotheliosis virus strain SNV.

Reticuloendotheliosis virus (REV) is a gamma retrovirus that can cause immunosuppression, dwarf syndrome and acute reticulocytoma in poultry. The molecular mechanism by which REV infection leads to immunosuppression and tumorigenesis is poorly understood. In this study, we elucidated the regulatory network of miRNA-mRNA and the major signaling pathways involved in REV-SNV infection. Therefore, we used the spleen necrosis virus (SNV) model of REV to inoculate one-day-old specific pathogen-free (SPF) chickens and then performed global miRNA and mRNA expression profiling by conducting high-throughput sequencing of 18 bursa of Fabricius samples collected at 7, 14, and 21 dpi. In total, 213 differentially expressed miRNAs (DEMs) and 3311 differentially expressed genes (DEGs) were identified. In the miRNA-mRNA network constructed based on the association analysis of these DEMs and DEGs, 1376 negatively correlated miRNA-mRNA pairs were identified; among them, 82 pairs were identified at 7 dpi, 203 pairs were identified at 14 dpi, and 873 pairs were identified at 21 dpi. Moreover, the results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the mRNAs in the network revealed greater enrichment of immune-related pathways, such as the immune system, signal transduction, cell growth and death, and signaling molecules and interactions. We confirmed the selected immune-related DEMs and their DEGs by conducting quantitative RT-PCR (qRT-PCR) analysis. These findings increased our understanding of the interactions of miRNAs and their target genes during infection with REV-SNV, and contributed to the understanding of host-virus interactions.

Combination Adjuvants Enhance Recombinant H5 Hemagglutinin Vaccine Protection Against High-Dose Viral Challenge in Chickens.

Recombinant avian influenza subunit vaccines often require adjuvants to enhance immune responses. This study aims to evaluate the immune-enhancing potential of seven combination adjuvants in specific pathogen-free (SPF) chickens. SPF chickens were vaccinated with combinations of ISA78VG and adjuvants, including Quil-A, CpG, and monophosphoryl lipid A (MPLA). Their immune responses were assessed using a vaccination and viral challenge protection model. The combinations of ISA78VG with Quil-A, CpG&MPLA or CpG&Quil-A significantly enhanced antibody responses and provided cross-protection against the H5N8-20135 strain. The ISA78VG&MPLA and ISA78VG&CpG&MPLA combinations induced the stronger IFN-γ production, with CpG further amplifying the immune response. The ISA78VG&Quil-A formulation, in particular, stimulated rapid antibody responses, achieving a 100% seroconversion by day 14 and high titers of hemagglutination inhibition (HI) antibodies against both the recombinant HA antigen and the H5N6-20053 virus. The ISA78VG&Quil-A combination is an ideal adjuvant for enhancing the immunogenicity of avian influenza rHA subunit vaccines, offering a promising strategy for H5 subtype vaccine development.

Dihydromyricetin Suppresses Lipopolysaccharide-Induced Intestinal Injury Through Reducing Reactive Oxygen Species Generation and NOD-Like Receptor Pyrin Domain Containing 3 Inflammasome Activation.

As an integral component of the gram-negative bacterial cellular envelope, excess production of lipopolysaccharide (LPS) regularly precipitates causing intestinal damage and barrier dysfunction in avian species. Dihydromyricetin (DHM), a naturally occurring constituent in rattan tea, exhibits protective characteristics against various tissue injuries. However, the intervention mechanism of DHM on intestinal injury induced by LPS in chickens has not been determined. Consequently, this study aimed to elucidate the mechanisms through which DHM mitigates LPS-induced intestinal damage in chickens through the reactive oxygen species (ROS)-NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome. Primary intestinal epithelial cells (IECs) were isolated and cultured from 14-day-old specific pathogen free (SPF) chicken embryos, and DHM ranging from 20 to 320 μmol/L increased cell survival rates. Additionally, DHM at 20 and 40 μmol/L demonstrated reduction in oxidative stress and ROS accumulation, mirroring the impact of ROS inhibitor (2.5 mmol/L NAC). DHM efficiently regulated ROS production, thereby augmenting ZO-1, occludin and claudin-1 expression to enhance barrier function; upregulating bcl-2 expression and downregulating bax and caspase-3 expression to regulate apoptosis and suppressing inflammation in IECs. Suppression of ROS subsequently attenuates NLRP3 inflammasome activation, leading to a remarkable downregulation of IL-1β, IL-18 and lactate dehydrogenase (LDH) secretion, consistent with direct inactivation of NLRP3 inflammasome (10 μmol/L MCC950). Notably, DHM diminished IL-1β and IL-18 levels and LDH activity via suppression of ROS-regulated NLRP3 and caspase-1 expression and activation. In summary, DHM prevents LPS-induced intestinal impairment by modulating ROS generation and NLRP3 inflammasome activation.

The highly pathogenic avian influenza H5N8 sublineage 2.3.4.4b virus: A critical analysis of genetic steadiness based on full HA gene sequencing in Egypt

The highly pathogenic avian influenza virus (HPAIV) continues to be a significant threat to the poultry industry despite the implementation of vaccination programs. The main goal of this study was to explore the incidence of avian influenza viruses (AIVs) and the stability of the hemagglutinating (HA) gene sequence. For this drive, oropharyngeal swabs (n=425) were collected from various wild and domesticated bird species from 13 provinces in Egypt from 2019 to 2021. Viral isolation in specific pathogen-free chicken embryos revealed positive HA activity in 9.88% (n=32) of the wild and 36.37% (n=64) of domesticated species. Only eight H5N8 avian influenza viruses were delivered (4/each category) in mixed infection with Newcastle disease virus (NDV). Clinically, domesticated examined birds exhibited clinical findings suggesting avian influenza, but AIVs were delivered from both apparently healthy and sick wild birds. A/chicken-cobb/Egypt/55/2019(H5N8) sequence generated in this study has been submitted to GenBank under the accession number ON724339, and its in vivo pathogenicity was evaluated in four-week-old chickens, which revealed 90% mortality and typical clinical findings of HPAIV. Based on the full HA gene sequence, the strain has a Furin-sensitive cleavage site in the HA protein, which is an HPAIV. It has been clustered in clade 2.3.4.4b within AIVs from 2016 to 2023 and coexistence of genetically stable viruses 2016-2019 with 99.8-100% similarity. The existing strain is fully identical to three strains: A/green-winged teal/Egypt/877/2016(H5N8), A/northern shoveler/Egypt/MB-D-816OP/2016(H5N8), and A/duck/Egypt/N13736E/2017 (H5N8), with the only point of mutation at position 284 (G284E). In our analysis, the majority of H5NX AIV strains detected in Egypt since 2016 possessed 156T. As a result, there is evidence suggesting that H5N8 has been introduced sequentially through the migration of European wild birds along the Black Sea–Mediterranean and East Asia–West Africa flyways. This raises the possibility of future adaptation to mammals due to spillover from avian-origin strains, especially in native aquatic birds such as geese and ducks.

Development of High-Production Bacterial Biomimetic Vesicles for Inducing Mucosal Immunity Against Avian Pathogenic Escherichia coli.

To evaluate the immunoprotective effect of bacterial biomimetic vesicles (BBVs) against avian pathogenic Escherichia coli (APEC), a ΔtolA J11 mutant strain was generated by deleting the tolA gene in the low pathogenic O78 serotype J11 strain. The total protein content of outer membrane vesicles (OMVs) derived from the ΔtolA J11 strain exhibited a sevenfold increase compared to the wild-type strain. Additionally, high-pressure homogenization technology was employed to produce BBVs, resulting in a sixfold increase in total protein content compared to spontaneously secreted OMVs from ΔtolA J11. The immunogenicity of both OMVs and BBVs was assessed through intranasal or intramuscular immunization in specific pathogen-free (SPF) chickens. Results demonstrated that intranasal immunization with OMVs or BBVs in chickens elicited specific IgY antibodies against APEC outer membrane proteins and specific sIgA antibodies in the nasal cavity and trachea, as well as a significant increase in the proliferation response of chicken peripheral blood lymphocytes. The bacterial load in the blood and various organs of the challenged chickens were significantly reduced, resulting in a 66.67% and 58.30% survival rate against a high pathogenic serotype O78 strain challenge, while the control group exhibited only a 16.67% survival rate. The intramuscular immunization with OMVs or BBVs in chickens only induced specific IgY antibodies, with a survival rate of only 33.33% for challenged chickens during the same period. Therefore, intranasal vaccination of the highly productive BBVs is capable of eliciting an immune response similar to that of OMVs and providing protection against APEC infection, thus offering innovative insights for the advancement of APEC vaccines.

The inhibitory effect of Hypericum japonicum on H9N2 avian influenza virus

The H9N2 subtype of avian influenza virus (AIV) causes severe immunosuppression and high mortality in view of its frequent co-infection with other pathogens, resulting in significant economic losses in the poultry industry. Current vaccines provide suboptimal immune protection against H9N2 AIV owing to antigenic variations, highlighting the urgent need for safe and effective antiviral drugs for the prevention and treatment of this virus. This study aimed to investigate the inhibitory effects of Hypericum japonicum extract on H9N2 AIV. Our findings revealed that the extract obtained through resin column separation using 60% ethanol (S06-60%) inhibited H9N2 AIV replication in Madin-Daby canine kidney cells in a dose-dependent manner. The maximum safe concentration of the water-soluble S06-60% extract was determined to be 0.05 mg/mL. Time-course experiments indicated that S06-60% primarily exerted its antiviral effects during the viral pretreatment and adsorption stages. Furthermore, in vivo experiments conducted on specific pathogen-free chickens confirmed the effectiveness of S06-60% in inhibiting H9N2 AIV infection and mitigating associated damage to tracheal and lung tissues. Overall, our study highlights the therapeutic potential of Hypericum japonicum extract S06-60% as a viable antiviral candidate against H9N2 AIV, offering promising implications for its application in poultry health management to reduce the economic impact on the poultry industry.

Development and Evaluation of a Novel Chimeric Genotype VII Newcastle Disease Vaccine: Overcoming Maternal Antibody Interference and Spray Administration.

Newcastle disease virus (NDV) poses a significant threat to the poultry industry, with the emergence of genotype VII NDV leading to extensive outbreaks and economic losses. Vaccination is the primary means of controlling NDV, but the presence of maternal antibodies (MDAs) can interfere with the immunological effect of live virus vaccines. Thus, we constructed a chimeric NDV live virus vaccine, LX-OAI4S, by replacing the extracellular regions of the F and HN genes of the NDV LX strain with the corresponding regions of the A-VII vaccine strain. The chimeric vaccine LX-OAI4S demonstrated high genetic stability, good safety, and strong reproductive capacity in chicken embryos. The LX-OAI4S vaccine induced rapid antibody production in specific pathogen-free (SPF) and commercial chickens via the intranasal and intraocular (IN/IO) routes, with hemagglutination inhibition (HI) antibody titers reaching 4.71 ± 1.03 log2 at 7 days post-vaccination (dpv), significantly higher than those of the two classical vaccine strains La Sota and VG/GA. The LX-OAI4S vaccine group provided effective protection against the challenge of genotype VII NDV virulent strain JS2/06 and inhibited viral shedding. When administered via spray, the LX-OAI4S vaccine elicited high systemic immunity against NDV in both SPF and commercial chickens, effectively protecting against clinical disease and reducing viral shedding. The chickens were exposed to high-dose vaccine for spray vaccination, and no adverse reactions were observed after vaccination. Despite the presence of anti-NDV MDAs in chickens, the NDV-specific antibody titers were significantly greater in the vaccinated groups than in the unvaccinated group. The vaccine exhibited high immunogenicity and the potential to overcome maternal antibody interference. The LX-OAI4S vaccine is a promising candidate for an ND vaccine. Its administration via spray can effectively prevent the occurrence of ND, making it a valuable tool for the poultry industry.

Fowl adenovirus 8a isolated from chickens with runting and stunting syndrome induces inclusion body hepatitis and hepatitis-hydropericardium syndrome in chicken embryos.

Fowl adenovirus (FAdV) is the etiological agent of inclusion body hepatitis (IBH) and hepatitis-hydropericardium syndrome (HHS) in poultry. It is also detected in chickens with runting and stunting syndrome (RSS). FAdV has been detected worldwide, and genotypes 8a, 8b, and 11 have been identified in chickens with enteric problems in Brazil. Nevertheless, none of them have been isolated; therefore, these viruses propagate; thus, the viral behavior and pathogenicity are unknown in Brazil. This study aimed to isolate FAdV from the enteric content of chickens affected by RSS. Enteric content samples from chickens affected with RSS and a positive polymerase chain reaction (PCR) test for FAdV were inoculated into specific pathogen-free chicken embryonated eggs (CEEs) through the yolk and chorioallantoic membrane routes on 8 and 10 days of age, respectively and the eggs were incubated for five days for viral isolation. The embryos exhibited dwarfism, beak atrophy, and pale claws. In addition, some embryos displayed edema and gelatin-like characteristics. The liver exhibited hepatomegaly and multiple necrotic foci, resembling the appearance of nutmeg. In addition, the kidneys appeared enlarged and pale. After 8 days of incubation, the hearts of the inoculated embryos showed hydropericardium. Microscopic evaluation revealed the presence of hepatitis, which was characterized by the presence of intranuclear inclusion bodies and cellular necrosis. Viral FAdV particles were observed in kidney cells using electron microscopy. Viral DNA was detected in the embryos in all three passages, and viral gene copies were also measured in some organs, with high FAdV gene copies detected in the spleen and bursa beyond the liver. The molecular characterization of FAdV revealed that the isolated strain belonged to genotype 8a of FAdV. Here, FAdV-8a from chickens infected with RSSs produced IBH/HHS in CEE, and FAdV-8a detected in RSS outbreaks, in addition to producing IBH/HHS in chicken embryos, could be a possible viral agent that causes IBH/HHS in chickens.

Efficacy of the H7N9 vaccine as a candidate for the Korean avian influenza antigen bank

The avian influenza A virus (H7N9), first detected in China in 2013, is a zoonotic virus that remains persistent in bird populations despite a decline in human cases owing to control measures. Therefore, this study aimed to develop a vaccine as one preventive strategy in anticipation of the potential entry of H7N9 into Korea. Using the hemagglutinin and neuraminidase consensus sequences of H7N9 from 2018–2019, a recombinant H7N9 vaccine, rgAPQAH7N9, was developed, and its protective efficacy in specific pathogen-free chickens was evaluated. The rgAPQAH7N9 vaccine exhibited proliferation in eggs and demonstrated high immunogenicity, with a hemagglutination inhibition titer of 9.3 log2. Furthermore, the vaccine provided complete protection, as vaccinated chickens did not exhibit clinical signs or viral shedding. Moreover, when the rgAPQAH7N9 vaccine was boosted, the resulting immunity was long-lasting, with hemagglutination inhibition titers > 7 log2 persisting after 6 months. Therefore, the rgAPQAH7N9 vaccine virus may be considered a potential candidate for inclusion in the avian influenza antigen bank to ensure preparedness for emergency vaccination in poultry.

Application of lentinan in suppression of Marek's disease virus infection

Marek's disease virus (MDV) is an extremely widespread avian immunosuppressive virus. In recent years, many reports have shown that there are still cases of MDV infection and immunosuppression after immunization with the vaccine. Consequently, there is a need to develop alternative complementary approaches for strengthening the efficacy of MDV prevention and control measures. Lentinan (LNT) is a macromolecular compound with immune-enhancing activity extracted from shiitake mushrooms. To explore the value and effectiveness of administering LNT through drinking water in the prevention and control of MDV, this study first observed the effects of high and low doses of LNT on weight gain, organ development, viral replication, and antibody titer of an avian influenza virus subtype H9 (AIV-H9) inactivated vaccine in specific pathogen-free (SPF) chicks infected with the wild strain of MDV. The results showed that both high and low doses of LNT significantly alleviated the weight gain retardation and liver and spleen enlargement caused by MDV infection, and significantly inhibited the replication of MDV in SPF chicks (P < 0.05), compared with the MDV-positive control group, both high and low doses of LNT significantly increased the antibody titer of AIV-H9 after immunization with inactivated AIV-H9 vaccine (P < 0.0001). On this basis, we also observed the effects of a chicken Marek's disease meq gene deletion live vaccine (SC9-1 strain), administered alone or in combination with LNT, on MDV infections of varying virulence in Hy-Line Brown chicks. The results showed that combined administration of LNT and the SC9-1 vaccine resulted in a significant alleviation of weight gain retardation and liver and spleen enlargement due to MDV infection (P < 0.05), as well as a significant inhibition of MDV replication and release in Hy-Line Brown chicks compared to the vaccine alone (P < 0.0001). These findings suggest that LNT not only alleviates the adverse effects of MDV infection in chickens but also enhances the efficacy of MDV vaccination, offering a potential auxiliary measure for controlling MDV infection.

First detection and biological characterization of an avian metaavulavirus 8 isolated from a migratory swan goose in Qinghai Lake, Northwest China.

Avian metaavulavirus 8 (AMAV-8), formerly known as avian paramyxovirus 8 (APMV-8), has been detected sporadically in wild birds worldwide since it was first identified in a Canadian goose in 1976. However, the presence of AMAV-8 in birds has never been reported in China. To understand the epidemiological situation of AMAV-8 and its ability to infect chickens, we conducted a surveillance study and in vivo analysis of the AMAV-8 isolate identified in total of 14,909 clinical samples collected from wild and domestic birds from 2014 to 2022 in China. However, in 2017, only one AMAV-8 virus (Y7) was successful isolated from the fresh droppings of a migratory swan goose in Qinghai Lake in Northwest China. Thereafter, we report the complete genome sequence of the Y7 strain with a genome length of 15,342 nucleotides and the Y7 isolate was genetically closely-related to wild bird-origin AMAV-8 viruses previously circulated in the United States, Japan, and Kazakhstan. Furthermore, AMAV-8 infections of one-day-old specific pathogen-free (SPF) chicks did not induce any clinical signs over the entire observation period but was associated with viral shedding for up to 8 days. Interestingly, although all birds infected with the Y7 strain seroconverted within the first week of infection, virus replication was only detected in the trachea but not in other tissues such as the brain, lung, or heart. Here, we report the complete genome, genetic and biological characterization, replication and pathogenicity analysis in vivo and first detection of AMAV-8 in China.

Recombinant Marek’s disease virus type 1 provides full protection against H9N2 influenza A virus in chickens

The H9N2 subtype of the avian influenza virus (AIV) poses a significant threat to the poultry industry and human health. Recombinant vaccines are the preferred method of controlling H9N2 AIV, and Marek’s disease virus (MDV) is the ideal vector for recombinant vaccines. During this study, we constructed two recombinant MDV type 1 strains that carry the hemagglutinin (HA) gene of AIV to provide dual protection against both AIV and MDV. To assess the effects of different MDV insertion sites on the protective efficacy of H9N2 AIV, the HA gene of H9N2 AIV was inserted in UL41 and US2 of the MDV type 1 vector backbone to obtain recombinant viruses rMDV-UL41/HA and rMDV-US2/HA, respectively. An indirect immunofluorescence assay showed sustained expression of HA protein in both recombinant viruses. Additionally, the insertion of the HA gene in UL41 and US2 did not affect MDV replication in cell cultures. After immunization of specific pathogen-free chickens, although both the rMDV-UL41/HA and rMDV-US2/HA groups exhibited similar levels of hemagglutination inhibition antibody titers, only the rMDV-UL41/HA group provided complete protection against the H9N2 AIV challenge, and also offered complete protection against challenge with MDV. These results demonstrated that rMDV-UL41/HA could be used as a promising bivalent vaccine strain against both H9N2 avian influenza and Marek’s disease in chickens.

A Comparative Study of Using Poly (D, L, Lactide-co-Glycolic Acid) and Chitosan Nanoparticle as Vaccine Delivery System for a Recombinant Fusion Protein of Newcastle Disease Virus

Introduction: The more effective method of preventing many infectious diseases is vaccination. Numerous infectious diseases that affect both humans and animals have significantly decreased as a result of routine immunization. Aim: The present study aimed to compare the efficacy of in-house built chitosan and Polylactide coglycolic acid (PLGA) nanoparticles coupled with Pichia pastoris expressed immunogenic fusion (F) protein of Newcastle disease (ND). Objectives: Synthesis of biodegradable nanoparticles such as PLGA and chitosan offers a promising opportunity as a vaccine delivery system. Methods: Chitosan nanoparticles and PLGA nanoparticles were synthesized by ionic gelation, and double emulsion solvent evaporation, respectively, and the size was 38.6± 0.84 nm and 320 ±1.5nm, respectively. They demonstrated good epitope integrity of recombinant fusion protein and in vitro release kinetics studies have proved consistent release profile of protein Results: In vivo pathogenicity assay of separately injected nanoparticles has proved no abnormal signs and mortality in chickens. Specific pathogen-free (SPF) chicks were vaccinated with chitosan and PLGA nanoparticles and a recombinant fusion protein of the ND virus. It was demonstrated that PLGA nanoparticles coupled with a fusion protein of Newcastle disease virus conferred a marginally better immune response than chitosan nanoparticles. Comparative study-based results showed that PLGA-based nanoparticles proved a better vaccine delivery vehicle and generated an effective immune response without needing further adjuvants. Conclusion: The present study is a scientific platform for developing the PLGA-based vaccine delivery vehicle to improve immune responses against many infectious diseases.

Molecular characteristic, evolution, and pathogenicity analysis of avian infectious bronchitis virus isolates associated with QX type in China

Infectious bronchitis virus (IBV) is one of the major avian pathogens plaguing the global poultry industry. Although vaccination is the primary preventive measure for IBV infection, the emergence of virus variants with mutations and recombination has resulted in IBV circulating globally, presenting a challenge for IB control. Here, we isolated 3 IBV strains (CZ200515, CZ210840, and CZ211063) from suspected sick chickens vaccinated with IBV live attenuated vaccines (H120, 4/91, or QXL87). Phylogenetic analysis of the S1 gene sequence of the spike (S) revealed that the 3 isolates belonged to the QX-type (GI-19 lineage). Whole genome sequencing and recombination analysis indicated that CZ200515 and CZ210840 contained genetic material from 4/91 and Scyz3 (QX-type), possibly due to recombination between the circulating strain and the 4/91 vaccine strain, while no evidence of recombination was found in CZ211063. Pathogenicity analysis in 1-day-old specific pathogen-free (SPF) chickens demonstrated that all 3 isolates caused severe tissue damage and varying degrees of mortality. Virus cross-neutralization assay revealed decreased antigen relatedness between the isolates and the QX-type vaccine strain (QXL87). Amino acid sequence homology analysis of S1 revealed 5%-6.5% variances between the isolates and QXL87. Analysis of the S1 subunit structure revealed that mutations of amino acid residues in the hypervariable region (HVR) and the neutralizing epitope region resulted in antigenic variation in isolates by changing the antigen conformation. Our data indicate antigenicity variances between QX isolates and QXL87 vaccine strains, potentially resulting in immune evasion occurrences. Overall, these results offer crucial insights into the epidemiology and pathogenicity of QX-type IBV, facilitating improved selection and formulation of vaccines for disease management.

Very virulent infectious bursal disease virus infection triggered microscopic changes, apoptosis, and inflammatory cytokines imbalance in chicken spleen and thymus

ABSTRACT Infectious bursal disease virus (IBDV) can cause a highly contagious disease, resulting in severe damage to the immune system that causes immunosuppression in young chickens. Both spleen and thymus are important immune organs, which play a key role in eliciting protective immune responses. However, the effects of very virulent IBDV (vvIBDV) strain LJ-5 infection on chicken spleen and thymus are still unknown. In the present study, 3-week-old specific pathogen-free chickens were infected with vvIBDV for 1–5 days. The vvIBDV infection significantly increased the spleen index and decreased the thymus index. Microscopic analysis indicated necrosis, depletion of the lymphoid cells, and complete loss of structural integrity in spleen and thymus. Ultrastructural analysis displayed mitochondrial and nuclear damage, including mitochondrial cristae breaks, and deformation of nuclear membrane in vvIBDV-infected spleen and thymus tissues. Cytokine levels increased in the spleen and thymus after IBDV infection, promoting inflammation and causing an inflammatory imbalance. Moreover, the mRNA expression of apoptosis-related genes was significantly upregulated in the vvIBDV-infected group compared to the control group. Meanwhile, the mRNA expression of mitochondrial dynamics was altered in the spleen and thymus of vvIBDV-infected chickens. These results suggested that vvIBDV infection triggers an imbalance of inflammatory cytokines, and apoptosis in the spleen and thymus, resulting in immune injury in chickens. This study provides basic data for the further study of vvIBDV pathogenesis.

Immune Responses in the Harderian Gland After Newcastle Disease Vaccination in Chickens with Maternal Antibodies.

Immune responses in the Harderian gland (HG) were characterized after Newcastle disease virus (NDV) LaSota ocular vaccination in antibody-naïve specific-pathogen-free (SPF) chickens and in chickens of commercial origin with NDV maternally derived antibodies (MDA). Ocular LaSota vaccination of 13-day-old white leghorn SPF chickens elicited serum antibody levels that consistently increased 15 days postvaccination, while the specific IgA response in lacrimal fluids was already detectable 10 days after vaccination. Eleven days postvaccination, the relative abundance of B cells, as well as T-helper cells (CD4+) and cytotoxic T cells (CD8+), in HGs was significantly increased, achieving maximum frequencies 16 days postvaccination. In a second experiment, chickens with MDA originating from NDV-vaccinated commercial white leghorn layer breeders, as well as white leghorn SPF chickens, were vaccinated with NDV LaSota. The LaSota virus successfully replicated in periocular tissues and in the trachea both in commercial and control SPF chickens after vaccination at 2 or 15 days of age (DOA). Vaccination at 2 DOA did not induce a serum NDV antibody response in chickens of commercial origin. In contrast, seroconversion was elicited in commercial chickens upon vaccination at 15 DOA, likely associated with waning of MDA. Unlike systemic IgG responses, vaccination at 2 or 15 DOA elicited strong specific IgA responses in lacrimal fluids in commercial chickens. The IgA response was highest 9 days after vaccination and showed a tendency to decline 15 days postvaccination. Commercial chickens vaccinated at 2 DOA showed increased B cells in HG 10 and 16 days postvaccination. The expansion of B cells in the HG in these chickens is consistent with increased IgA levels detected in lacrimal fluids. In contrast, control SPF chickens showed a more limited B-cell expansion in HG and lower IgA levels. Vaccination at 15 DOA also triggered a greater increase of B cells in HGs in commercial chickens than in control SPF chickens. The B-cell response was accompanied by T-helper (CD4+) cell expansion, occurring both in commercial and control SPF chickens. These cells expanded to a lesser extent when vaccination was performed at 2 DOA compared with vaccination at 15 DOA. CD8+ showed significant expansion irrespective of vaccination day and without differences detected between control SPF chickens and chickens with MDA. We conclude that NDV LaSota elicits vigorous humoral and cell immune responses in the HG. Furthermore, unlike the interference shown by MDA on vaccine-induced serum antibody responses, MDA do not interfere with the mucosal immune response of the HG.

Optimization of process parameters for specific pathogen-free chicken embryonic fibroblast cultivation for yellow fever vaccine production

The yellow fever (YF) vaccine is usually produced with egg-based methods, which has limitations, including potential adverse effects and low production yields. Alternatively, producing the vaccine using Vero cells or HEK 293 cells can overcome some of these issues, but these methods are significantly more expensive. In the current study, the YF vaccine candidate 17DD virus was produced in primary chicken embryo fibroblast (CEF) cells. The primary CEF cells isolation from eggs was optimized through a two-step process. In the first step, the important parameters that contribute to the development of the egg embryo, such as egg position, relative humidity (RH), and incubation time are optimized. In second step, primary CEF release parameters namely; trypsin volume and incubation temperature are optimized. Both steps were optimized using statistical methods. Further, the seeding cell density of isolated CEF was also optimized. It was observed that 5 x 104 cells/cm2 gave the highest virus titer of 3.89 million PFU/ml. The 17DD yields achieved in primary CEFs are much higher than egg-based production and it is an economically viable method.