Synergistic effects of oral inoculation with a recombinant Lactobacillus plantarum NC8 strain co-expressing interleukin-2 and interleukin-17B on the efficacy of the infectious bronchitis vaccine in chickens.

Mucosal vaccination strategies are easier to implement than others in large-scale poultry farming. However, the adjuvants that are approved for veterinary use, which are predominantly aluminum- and oil-emulsion-based adjuvants, are not suitable for mucosal vaccination and carry a risk of adverse reactions. In this study, we engineered a novel Lactobacillus plantarum NC8 strain that co-expresses chicken interleukin-2 (IL-2) and IL-17B, which we designated NC8-ChIL2-17B, and evaluated its potential as an oral immunoadjuvant. The immunomodulatory properties of NC8-ChIL2-17B were evidenced by its ability to activate macrophages and inhibit the proliferation of infectious bronchitis virus (IBV) in vitro. We then confirmed its immunoadjuvant activity in vivo by orally administering NC8-ChIL2-17B along with a commercial IBV vaccine to chicks. The results indicated that NC8-ChIL2-17B enhanced the immune response elicited by the IBV vaccine and increased the levels of IBV-specific IgG and sIgA antibodies produced in response to IBV infection. Additionally, administration of NC8-ChIL2-17B promoted weight gain and beneficially modulated the gut microbiota, resulting in improved chicken performance. These findings suggest that oral administration of NC8-ChIL2-17B is a promising strategy to enhance the immune efficacy of the IBV vaccine in chickens, offering an efficacious alternative adjuvant.

Heterologous maternal antibodies derived from infectious bronchitis vaccines prevent the development of lesions associated with false layer syndrome.

Infectious bronchitis virus (IBV) strains of the Delmarva (DMV)/1639 genotype have been causing false layer syndrome (FLS) in the Eastern Canadian layer operations since the end of 2015. FLS is characterized by the development of cystic oviducts in layer pullets infected at an early age. Currently, there are no homologous vaccines for the control of this IBV genotype. Our previous research showed that a heterologous vaccination regimen incorporating Massachusetts (Mass) and Connecticut (Conn) IBV types protects layers against DMV/1639 genotype IBV. The aim of this study was to investigate the role of maternal antibodies conferred by breeders received the same vaccination regimen in the protection against the development of DMV/1639-induced FLS in pullets. Maternal antibody-positive (MA+) and maternal antibody-negative (MA-) female progeny chicks were challenged at 1 day of age and kept under observation for 16 weeks. Oviductal cystic formations were observed in 3 of 14 birds (21.4 %) in the MA- pullets, while the lesions were notably absent in the MA+ pullets. Milder histopathological lesions were observed in the examined tissues of the MA+ pullets. However, the maternal derived immunity failed to demonstrate protection against the damage to the tracheal ciliary activity, viral shedding, and viral tissue distribution. Overall, this study underscores the limitations of maternal derived immunity in preventing certain aspects of viral pathogenesis, emphasizing the need for comprehensive strategies to address different aspects of IBV infection.

Deciphering the Genetic Variation: A Comparative Analysis of Parental and Attenuated Strains of the QXL87 Vaccine for Infectious Bronchitis.

The QXL87 live attenuated vaccine strain for infectious bronchitis represents the first approved QX type (GI-19 lineage) vaccine in China. This strain was derived from the parental strain CK/CH/JS/2010/12 through continuous passage in SPF chicken embryos. To elucidate the molecular mechanism behind its attenuation, whole-genome sequencing was conducted on both the parental and attenuated strains. Analysis revealed 145 nucleotide mutations in the attenuated strain, leading to 48 amino acid mutations in various proteins, including Nsp2 (26), Nsp3 (14), Nsp4 (1), S (4), 3a (1), E (1), and N (1). Additionally, a frameshift mutation caused by a single base insertion in the ORFX resulted in a six-amino-acid extension. Subsequent comparison of post-translational modification sites, protein structure, and protein-protein binding sites between the parental and attenuated strains identified three potential virulence genes: Nsp2, Nsp3, and S. The amino acid mutations in these proteins not only altered their conformation but also affected the distribution of post-translational modification sites and protein-protein interaction sites. Furthermore, three potential functional mutation sites-P106S, A352T, and L472F, all located in the Nsp2 protein-were identified through PROVEAN, PolyPhen, and I-Mutant. Overall, our findings suggest that Nsp2, Nsp3, and S proteins may play a role in modulating IBV pathogenicity, with a particular focus on the significance of the Nsp2 protein. This study contributes to our understanding of the molecular mechanisms underlying IBV attenuation and holds promise for the development of safer live attenuated IBV vaccines using reverse genetic approaches.

Prevalence, Genotype Diversity, and Distinct Pathogenicity of 205 Gammacoronavirus Infectious Bronchitis Virus Isolates in China during 2019-2023.

Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious disease in chickens and seriously endangers the poultry industry. The emergence and co-circulation of diverse IBV serotypes and genotypes with distinct pathogenicity worldwide pose a serious challenge to the development of effective intervention measures. In this study, we report the epidemic trends of IBV in China from 2019 to 2023 and a comparative analysis on the antigenic characteristics and pathogenicity of isolates among major prevalent lineages. Phylogenetic and recombination analyses based on the nucleotide sequences of the spike (S) 1 gene clustered a total of 205 isolates into twelve distinct lineages, with GI-19 as a predominant lineage (61.77 ± 4.56%) exhibiting an overall increasing trend over the past five years, and demonstrated that a majority of the variants were derived from gene recombination events. Further characterization of the growth and pathogenic properties of six representative isolates from different lineages classified four out of the six isolates as nephropathogenic types with mortality rates in one-day-old SPF chickens varying from 20-60%, one as a respiratory type with weak virulence, and one as a naturally occurring avirulent strain. Taken together, our findings illuminate the epidemic trends, prevalence, recombination, and pathogenicity of current IBV strains in China, providing key information for further strengthening the surveillance and pathogenicity studies of IBV.

Glycerides of lauric acid supplementation in the chicken diet enhances the humoral and cellular immune response to infectious bronchitis virus.

Controlling pathogenic infections while reducing antibiotic usage is an important challenge during poultry production. In addition to vaccination strategies, several solutions to enhance the immune response against pathogens are evaluated. In this study, we aim to determine the effects of the glycerides of lauric acid (GLA) supplementation in chickens' diets on humoral and cellular immune response pathogenic infections, using an in vivo model of infectious bronchitis virus (IBV). One-day-old Ross 308 broilers were vaccinated with live attenuated IBV and fed diets supplemented with or without GLA at 3 kg/ton. The levels of early (day 7) specific anti-IBV in sera were significantly increased in broilers fed GLA, compared to the control groups (P<0.05), showing a stronger primary humoral response. The secretion levels of main cytokines remained similar in spleens of all the experimental groups. However, the splenocytes from broilers fed GLA showed higher activation and effector abilities when measured by IFN-γ ELISpot in presence of N-261-280 IBV peptide or Concanavalin A (Con A), a pan T lymphocytes mitogen. In response to N-261-280 peptide, GLA group showed a 2-fold increase of spot numbers (P < 0.05) and 3-fold increase of spot surfaces (P < 0.01) compared to the control groups. Similarly, Con A stimulation showed a 2-fold increases in spot surfaces and numbers in the GLA supplemented group compared to the control group (P < 0.01). In summary, GLA supplementation in chicken feed enhances the primary humoral immune response and strengthen the T lymphocytes mediated cellular immune response. These findings demonstrate how GLA can improve chicken resilience against pathogenic challenges by enhancing their immune responses.

Protection Against Infectious Bronchitis Virus Vaccine Recombinants and Chicken-Selected Vaccine Subpopulations.

Outbreaks of infectious bronchitis (IB) continue to occur from novel variants of IB virus (IBV) emerging from selection of vaccine subpopulations and/or naturally occurring recombination events. S1 sequencing of Arkansas (Ark) -type viruses obtained from clinical cases in Alabama broilers and backyard chickens shows both Ark Delmarva Poultry Industry (ArkDPI) vaccine subpopulations as well as Ark vaccine viruses showing recombination with other IB vaccine viruses. IB Ark-type isolates AL5, most similar to an ArkDPI vaccine subpopulation selected in chickens, AL4, showing a cluster of three nonsynonymous changes from ArkDPI subpopulations selected in chickens, and AL9, showing recombination with Massachusetts (Mass) -type IBV, were examined for pathogenicity and ability to break through immunity elicited by vaccination with a commercial ArkDPI vaccine. Analysis of predicted S1 protein structures indicated the changes were in regions previously shown to comprise neutralizing epitopes. Thus, they were expected to contribute to immune escape and possibly virulence. Based on clinical signs, viral load, and histopathology, all three isolates caused disease in naïve chickens, although AL9 and AL5 viral loads in trachea were statistically significantly higher (30- and 40-fold) than AL4. S1 gene sequencing confirmed the stability of the relevant changes in the inoculated viruses in the chickens, although virus in some individual chickens exhibited additional S1 changes. A single amino acid deletion in the S1 NTD was identified in some individual chickens. The location of this deletion in the predicted structure of S1 suggested the possibility that it was a compensatory change for the reduced ability of AL4 to replicate in the trachea of naïve chickens. Chickens vaccinated with a commercial ArkDPI vaccine at day of hatch and challenged at 21 days of age showed that vaccination provided incomplete protection against challenge with these viruses. Moreover, based on viral RNA copy numbers in trachea, differences were detected in the ability of the vaccine to protect against these IBV isolates, with the vaccine protecting the most poorly against AL4. These results provide additional evidence supporting that IBV attenuated vaccines, especially ArkDPI vaccines, contribute to perpetuating the problem of IB in commercial chickens.

Protección contra los virus de la bronquitis infecciosa vacunales recombinantes y las subpoblaciones de vacunas seleccionadas en pollos. Los brotes de la bronquitis infecciosa aviar continúan presentándose a partir de nuevas variantes de dicho virus, que surgen de la selección de subpoblaciones de vacunas y/o eventos de recombinación que ocurren naturalmente. La secuenciación del gene S1 de virus tipo Arkansas (Ark) obtenidos de casos clínicos en pollos de engorde y de traspatio de Alabama muestra que tanto las subpoblaciones de la cepa vacunal Arkansas Delmarva Poultry Industry (ArkDPI) así como los virus de la vacuna Arkansas muestran recombinación con otros virus vacunales de la bronquitis infecciosa. Los aislamientos del virus de la bronquitis infecciosa Arkansas tipo "AL5", más similares a una subpoblación de vacuna ArkDPI seleccionada en pollos, "AL4", que muestra un grupo de tres cambios no sinónimos de subpoblaciones de ArkDPI seleccionadas en pollos y el tipo "AL9", que muestra recombinación con el serotipo Massachusetts, se examinaron para determinar su patogenicidad y capacidad para traspasar la inmunidad generada por la vacunación con una vacuna comercial ArkDPI. El análisis de las estructuras predichas de la proteína S1 indicó que los cambios se produjeron en regiones que previamente se había demostrado comprendían epítopos neutralizantes. Por lo tanto, se esperaba que contribuyeran al escape inmunológico y posiblemente a la virulencia. Con base en los signos clínicos, la carga viral y la histopatología, los tres aislados causaron enfermedad en pollos sin exposición previa, aunque las cargas virales de AL9 y AL5 en la tráquea fueron estadísticamente significativamente mayores (30 y 40 veces) en comparación con AL4. La secuenciación del gene S1 confirmó la estabilidad de los cambios relevantes en los virus inoculados en los pollos, aunque el virus en algunos pollos individuales exhibió cambios adicionales en el gene S1. Se identificó una deleción de un solo aminoácido en el dominio terminal N del gene S1 (NTD S1) en algunos pollos individuales. La ubicación de esta eliminación en la estructura predicha del gene S1 sugirió la posibilidad de que se tratara de un cambio compensatorio por la capacidad reducida de AL4 para replicarse en la tráquea de pollos sin exposición previa. Los pollos vacunados con una vacuna comercial ArkDPI el día de la eclosión y desafiados a los 21 días de edad mostraron que la vacunación proporcionó una protección incompleta contra el desafío con estos virus. Además, basándose en el número de copias del ARN viral en la tráquea, se detectaron diferencias en la capacidad de la vacuna para proteger contra estos aislados del virus de la bronquitis infecciosa, siendo la vacuna con la protección más deficiente contra AL4. Estos resultados proporcionan evidencia adicional que respalda que las vacunas atenuadas contra el virus de la bronquitis infecciosa, especialmente las vacunas ArkDPI, contribuyen a perpetuar esta enfermedad en los pollos comerciales.

Infectious bronchitis virus: an overview of the "chicken coronavirus".

Infectious bronchitis virus (IBV) is a highly contagious avian Gammacoronavirus that affects mainly chickens (Gallus gallus) but can circulate in other avian species. IBV constitutes a significant threat to the poultry industry, causing reduced egg yield, growth and mortality levels that can vary in impact. The virus can be transmitted horizontally by inhalation or direct/indirect contact with infected birds or contaminated fomites, vehicles, farm personnel and litter (Figure 1). The error-prone viral polymerase and recombination mechanisms mean diverse viral population results, with multiple genotypes, serotypes, pathotypes and protectotypes. This significantly complicates control and mitigation strategies based on vigilance in biosecurity and the deployment of vaccination.

The efficacy of simultaneous successive classic and variant infectious bronchitis virus vaccines versus circulating variant II Egyptian field virus.

Background: Being a ubiquitous, highly contagious virus with a continuous mutation and a large number of evolutions worldwide, the infectious bronchitis virus (IBV) continues to wreak problems among Egyptian chickens and generate economic losses. The commonly applied IBV vaccination protocols in broilers include alternatives to classic and/or variant attenuated live virus vaccines. Aim: The current study targeted to assess the protective efficacy of concurrent and successive Ma5 and 4/91 vaccine strain regimens against the field variant II IBV strain (IBV-EGY-ZU/Ck-127/2021) in chickens. Methods: Commercial broiler chickens were vaccinated with Ma5 and 4/91 strains simultaneously at 1 and 14 days of age. The evaluation parameters included clinical protection and humoral and early innate immunity aspects in the renal tissues of vaccinated and infected birds. Results: The vaccine regimen ameliorated the clinical and histopathological lesions against variant II IBV and enhanced body gain as well as succeeded in preventing tracheal shedding and minimizing cloacal shedding of the field virus. The IL-1ß mRNA gene expression was evident as early as 24 hours, with highly significant upregulation at 48 hours post vaccination and 24 hours post challenge (PC) in vaccinated birds. Remarkable upregulation was observed in oligoadenylate synthetases (OAS) expression 48 hours PC in vaccinated and unvaccinated infected birds. The vaccinated birds developed a significant antibody titer of 704.0 ± 111.98 at 28 days of age, with a consistent antibody titer increase after the challenge. Conclusion: Overall, a combination of heterologous protectotype commercial vaccines achieved good protection against the Egyptian variant II IBV strain. This vaccine program could be an effective protocol against the threat posed by IBV viruses circulating in the Egyptian field.

Infectious bronchitis virus (IBV) triggers autophagy to enhance viral replication by activating the VPS34 complex.

Autophagy plays an important role in the lifecycle of viruses. However, there is currently a lack of systematic research on the relationship between Infectious Bronchitis Virus (IBV) and autophagy. This study aims to investigate the impact of IBV on autophagy and the role of autophagy in viral replication. We observed that IBV infection increased the expression of microtubule-associated protein 1 light chain 3, a marker of autophagy, decreased the expression of sequestosome 1, and led to elevated intracellular LC3 puncta levels. These findings suggest that IBV infection activates the autophagic process in cells. To investigate the impact of autophagy on the replication of IBV, we utilized rapamycin as an autophagy activator and 3-methyladenine as an autophagy inhibitor. Our results indicate that IBV promotes viral replication by inducing autophagy. Further investigation revealed that IBV induces autophagosome formation by inhibiting the mTOR-ULK1 pathway and activating the activity of vacuolar protein sorting 34 (VPS34), autophagy-related gene 14, and the Beclin-1 complex. VPS34 plays a crucial role in this process, as inhibiting VPS34 protein activity enhances cell proliferation after IBV infection. Additionally, inhibiting VPS34 significantly improves the survival rate of IBV-infected chicks, suppresses IBV replication in the kidney, and alleviates tracheal, lung, and kidney damage caused by IBV infection. In summary, IBV infection can induce autophagy by modulating the mTOR/ULK1 signaling pathway and activating the VPS34 complex, while autophagy serves to promote virus replication.

The Effect of Global Spread, Epidemiology, and Control Strategies on the Evolution of the GI-19 Lineage of Infectious Bronchitis Virus.

The GI-19 lineage of infectious bronchitis virus (IBV) has emerged as one of the most impactful, particularly in the "Old World". Originating in China several decades ago, it has consistently spread and evolved, often forming independent clades in various areas and countries, each with distinct production systems and control strategies. This study leverages this scenario to explore how different environments may influence virus evolution. Through the analysis of the complete S1 sequence, four datasets were identified, comprising strains of monophyletic clades circulating in different continents or countries (e.g., Asia vs. Europe and China vs. Thailand), indicative of single introduction events and independent evolution. The population dynamics and evolutionary rate variation over time, as well as the presence and intensity of selective pressures, were estimated and compared across these datasets. Since the lineage origin (approximately in the mid-20th century), a more persistent and stable viral population was estimated in Asia and China, while in Europe and Thailand, a sharp increase following the introduction (i.e., 2005 and 2007, respectively) of GI-19 was observed, succeeded by a rapid decline. Although a greater number of sites on the S1 subunit were under diversifying selection in the Asian and Chinese datasets, more focused and stronger pressures were evident in both the European (positions 2, 52, 54, 222, and 379 and Thai (i.e., positions 10, 12, 32, 56, 62, 64, 65, 78, 95, 96, 119, 128, 140, 182, 292, 304, 320, and 323) strains, likely reflecting a more intense and uniform application of vaccines in these regions. This evidence, along with the analysis of control strategies implemented in different areas, suggests a strong link between effective, systematic vaccine implementation and infection control. However, while the overall evolutionary rate was estimated at approximately 10-3 to 10-4, a significant inverse correlation was found between viral population size and the rate of viral evolution over time. Therefore, despite the stronger selective pressure imposed by vaccination, effectively constraining the former through adequate control strategies can efficiently prevent viral evolution and the emergence of vaccine-escaping variants.

Molecular and Serological Investigation of Infectious Bronchitis Virus in the East Shewa, Central Ethiopia.

Background: Infectious bronchitis (IB) is an economically important disease in poultry with worldwide distribution. The occurrence of IB has been reported both in commercial and backyard poultry in Ethiopia, although comprehensive information lacks available prevalence of the disease and the circulating serotypes. Methods: A cross-sectional study was conducted from November 2021 to June 2022 in seven commercial farms found in East Shewa, Central Ethiopia. Serological assay using indirect ELISA, virus isolation techniques in embryonated eggs, and molecular techniques such as one-step reverse transcriptase polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (PCR) targeting a 466 bp S1 gene were employed. Results: A total of 196 blood samples, 7 pools (35) of swab samples, and 5 pools of tracheal samples were investigated. The results of serological analysis revealed that 97.96% (192/196; 95% CI: 94.86-99.44) of the sera samples were found to be positive for antibodies against IBV. Out of the 7 pools of swab and 5 pools of tracheal tissue samples analyzed using RT-PCR 33.3% (4/12) of them gave positive results all from swab samples. The RT-PCR-positive samples were subjected to a nested PCR yielding 295bp and 154bp indicating the circulation of Mass and 793/B (4/91) strains of IBV, respectively. The 12 pools of samples inoculated into embryonated egg showed cytopathic changes such as congestion, bleeding, and deformation only after three passages. Conclusion: Two serotypes of IBV are circulating in Ethiopian chickens, and molecular identification of the Massachusetts serotype is the first report in Ethiopia. Further epidemiological investigation is needed in order to devise effective control measures.

Baicalin Protects Broilers against Avian Coronavirus Infection via Regulating Respiratory Tract Microbiota and Amino Acid Metabolism.

An increasing amount of evidence indicates that Baicalin (Bai, a natural glycosyloxyflavone compound) exhibits an antiviral effect against avian viruses. However, it remains unclear if the antiviral effect of Bai against infectious bronchitis virus (IBV) is exerted indirectly by modulating respiratory tract microbiota and/or their metabolites. In this study, we investigated the protection efficacy of Bai in protecting cell cultures and broilers from IBV infection and assessed modulation of respiratory tract microbiota and metabolites during infection. Bai was administered orally to broilers by being mixed in with drinking water for seven days. Ultimately, broilers were challenged with live IBV. The results showed that Bai treatment reduced respiratory tract symptoms, improved weight gain, slowed histopathological damage, reduced virus loads and decreased pro-inflammation cytokines production. Western blot analysis demonstrated that Bai treatment significantly inhibited Toll-like receptor 7 (TLR7), myeloid differentiation factor 88 (MyD88) and nuclear factor kappa-B (NF-κB) expression both in cell culture and cells of the trachea. Bai treatment reversed respiratory tract microbiota dysbiosis, as shown by 16S rDNA sequencing in the group of broilers inoculated with IBV. Indeed, we observed a decrease in Proteobacteria abundance and an increase in Firmicutes abundance. Metabolomics results suggest that the pentose phosphate pathway, amino acid and nicotinamide metabolism are linked to the protection conferred by Bai against IBV infection. In conclusion, these results indicated that further assessment of anti-IBV strategies based on Bai would likely result in the development of antiviral molecule(s) which can be administered by being mixed with feed or water.

Differential analysis of IBV-infected primary chicken embryonic fibroblasts and immortalized DF-1.

Infectious bronchitis virus (IBV), the causative agent of infectious bronchitis, is responsible for major economic losses in the poultry industry worldwide. While IBVs can usually be passaged in primary chicken embryonic fibroblasts (CEFs), most of the wild ones cannot adapt to passaged cell lines. In this study, the wild strain CK/CH/MY/2020 was used to infect primary CEF and immortalize DF-1 CEF cells. Results indicated that IBV was able to cause lesions and pass onto CEF, but not DF-1. Indeed, the virus could enter DF-1 cells and synthesize the associated structural gene but could not assemble into complete viral particles for release. Furthermore, transcriptome sequencing analysis showed significant differences in gene expression between CEF and DF-1 cells after viral infection, although the corresponding antiviral responses could be activated in both cell types. The biggest difference was in terms of the amino acid biosynthesis pathway and the cytokine receptor interaction pathway, which were significantly and specifically activated in CEF. This could actually explain why intact viruses can be assembled but not in DF-1. In addition, SLBP and P2RX7 affect the replication of IBV's structural genes to some extent. Overall, IBV can enter CEF and DF-1 cells, but the complex intracellular cytokine interactions affect the assembly and release of viral particles. The insight will be useful for the study of IBV through in vitro transmission and pathogenesis. IMPORTANCE: Infectious bronchitis virus (IBV) is responsible for high morbidity and mortality as well as substantial economic losses worldwide. Transcriptome sequencing of IBV-infected chicken embryonic fibroblast and DF-1 cells revealed that the virus elicits antiviral immunity in cells after viral infection, but IBV cannot activate DF-1 cells to produce sufficient amounts of viral structures to assemble into complete virions, which may be caused by the interactions between cytokines. The study of IBV cellular adaptations is important for vaccine development and investigation of the pathogenesis of IBV.

Comparison of quantitative PCR and digital PCR assays for quantitative detection of infectious bronchitis virus (IBV) genome.

The quantitative polymerase chain reaction (qPCR) technique is an extensively used molecular tool for the detection and quantification of viral genome load. However, since the qPCR assay is a relative quantification method that relies on an external calibration curve it has a lower assay precision and sensitivity. The digital PCR (dPCR) technique is a good alternative to the qPCR assay as it offers highly precise and direct quantification of viral genome load in samples. In this study, performance characteristics such as the quantification range, sensitivity, precision, and specificity of the dPCR technique was compared to qPCR technique for the detection and quantification of IBV genome loads in serial dilutions of IBV positive plasmid DNA, and IBV infected chicken tissue and swab samples. The quantification range of the qPCR assay was wider than that of the dPCR assay, however dPCR had a higher sensitivity compared to qPCR. The precision of quantification of DNA in plasmid samples in terms of repeatability and reproducibility of results was higher when using the dPCR assay compared to qPCR assay. The quantification results of IBV genome load in infected samples by the qPCR and dPCR assays displayed a high correlation. Hence, our findings suggest that dPCR could be used in avian virology research for improved precision and sensitivity in detection and quantification of viral genome loads.

Protection of laying chickens against the Canadian DMV/1639 infectious bronchitis virus infection through priming with heterologous live vaccine and boosting with heterologous or homologous inactivated vaccine.

The emergence of the Canadian Delmarva (DMV)/1639 infectious bronchitis virus (IBV) type strains was associated with egg production disorders in Eastern Canadian layer operations. While developing vaccines for novel IBV variants is not typically a reasonable approach, the consideration of an autogenous vaccine becomes more appealing, particularly when the new variant presents significant economic challenges. The current study aimed to compare the efficacies of two vaccination programs that included heterologous live priming by Massachusetts (Mass) and Connecticut (Conn) type vaccines followed by either a commercial inactivated Mass type vaccine or a locally prepared autogenous inactivated DMV/1639 type vaccine against DMV/1639 IBV challenge. The protection parameters evaluated were egg production, viral shedding, dissemination of the virus in tissues, gross and microscopic lesions, and immunological responses. The challenge with the DMV/1639 caused severe consequences in the non-vaccinated laying hens including significant drop in egg production, production of low-quality eggs, serious damage to the reproductive organs, and yolk peritonitis. The two vaccination programs protected the layers from the poor egg-laying performance and the pathology. The vaccination program incorporating the autogenous inactivated DMV/1639 type vaccine was more effective in reducing vial loads in renal and reproductive tissues. This was associated with a higher virus neutralization titer compared to the group that received the commercial inactivated Mass type vaccine. Additionally, the autogenous vaccine boost led to a significant reduction in the viral shedding compared to the non-vaccinated laying hens. However, both vaccination programs induced significant level of protection considering all parameters examined. Overall, the findings from this study underscore the significance of IBV vaccination for protecting laying hens.

Synergistic anti-infectious bronchitis virus activity of Phillygenin combined Baicalin by modulating respiratory microbiota and improving metabolic disorders.

Phillygenin (PHI) and Baicalin (Bai) are the major chemical ingredients extracted from Forsythia suspensa and Scutellaria baicalensis, respectively. The mixture of Forsythia suspensa and Scutellaria baicalensis according to the theories of Traditional Chinese Veterinary Medicine, compounded formulation can effectively exert heat-clearing and detoxifying effect, but the synergistic anti-IBV activity of PHI combined with Bai was unclear. Here, the protection of PHI combined with Bai on avian infectious bronchitis virus (IBV) M41 infection and the change of respiratory microbiota and metabolomics profiles in broilers that infected with IBV were investigated. According to the experimental findings, the combination of PHI and Bai effectively alleviated broilers' slowing-growth weight and respiratory symptoms. This was accompanied by a reduction in viral copies and histopathological changes, as well as an increase of antiviral protein (G3BP1) level in tracheas and anti-IBV antibody levels in serum. In addition, 16s RNA sequencing revealed that IBV infection significantly changed respiratory microbiota composition at different taxonomic levels and respiratory metabolism composition in broilers. Interestingly, PHI combined with Bai modulated the composition of respiratory microfloras, especially the abundance of Firmicutes and Lactobacillaceae were upregulated, as well as the abundance of Proteobacteria was downregulated. The metabolomics results indicated that PHI combined with Bai involved in glucose, lipids, amino acids and nucleotide metabolism during IBV infection. In summary, PHI combined with Bai exhibited a synergistic effect on preventing infectious bronchitis (IB), with the protection being closely associated with the composition of respiratory microbiota and metabolites. Therefore, adding the mixture of PHI and Bai to the chicken drinking water is recommended to prevent and control IB in clinical.

Comparison of Infectious Bronchitis Virus (IBV) Pathogenesis and Host Responses in Young Male and Female Chickens.

Infectious bronchitis virus (IBV) is an avian coronavirus that causes a disease in chickens known as infectious bronchitis (IB). The pathogenesis of IBV and the host immune responses against it depend on multiple factors such as the IBV variant, breed and age of the chicken, and the environment provided by the management. Since there is limited knowledge about the influence of the sex of chickens in the pathogenesis of IBV, in this study we aim to compare IBV pathogenesis and host immune responses in young male and female chickens. One-week-old specific pathogen-free (SPF) White Leghorn male and female chickens were infected with Canadian Delmarva (DMV)/1639 IBV variant at a dose of 1 × 106 embryo infectious dose (EID)50 by the oculo-nasal route while maintaining uninfected controls, and these chickens were euthanized and sampled 4- and 11-days post-infection (dpi). No significant difference was observed between the infected male and female chickens in IBV shedding, IBV genome load in the trachea, lung, kidney, bursa of Fabricius (BF), thymus, spleen, and cecal tonsils (CT), and IBV-induced lesion in all the examined tissues at both 4 and 11 dpi. In addition, there was no significant difference in the percentage of IBV immune-positive area observed between the infected male and female chickens in all tissues except for the kidney, which expressed an increased level of IBV antigen in infected males compared with females at both 4 and 11 dpi. The percentage of B lymphocytes was not significantly different between infected male and female chickens in all the examined tissues. The percentage of CD8+ T cells was not significantly different between infected male and female chickens in all the examined tissues except in the trachea at 11 dpi, where female chickens had higher recruitment when compared with male chickens. Overall, although most of the findings of this study suggest that the sex of chickens does not play a significant role in the pathogenesis of IBV and the host immune response in young chickens, marginal differences in viral replication and host responses could be observed to indicate that IBV-induced infection in male chickens is more severe.

Virulent Newcastle disease virus genotypes V.3, VII.2, and XIII.1.1 and their coinfections with infectious bronchitis viruses and other avian pathogens in backyard chickens in Tanzania.

Oropharyngeal (OP) and cloacal (CL) swabs from 2049 adult backyard chickens collected at 12 live bird markets, two each in Arusha, Dar es Salaam, Iringa, Mbeya, Morogoro and Tanga regions of Tanzania were screened for Newcastle disease virus (NDV) using reverse transcription real-time PCR (rRT-PCR). The virus was confirmed in 25.23% of the birds (n = 517; rRT-PCR CT ≤ 30), with the highest positivity rates observed in birds from Dar es Salaam region with higher prevalence during the dry season (September-November 2018) compared to the rainy season (January and April-May 2019). Next-generation sequencing of OP/CL samples of 20 out of 32 birds that had high amounts of viral RNAs (CT ≤ 25) resulted in the assembly of 18 complete and two partial genome sequences (15,192 bp and 15,045-15,190 bp in length, respectively) of NDV sub-genotypes V.3, VII.2 and XIII.1.1 (n = 1, 13 and 4 strains, respectively). Two birds had mixed NDV infections (V.3/VII.2 and VII.2/XIII.1.1), and nine were coinfected with viruses of families Astroviridae, Coronaviridae, Orthomyxoviridae, Picornaviridae, Pneumoviridae, and Reoviridae. Of the coinfecting viruses, complete genome sequences of two avastroviruses (a recombinant chicken astrovirus antigenic group-Aii and avian nephritis virus genogroup-5) and two infectious bronchitis viruses (a turkey coronavirus-like recombinant and a GI-19 virus) were determined. The fusion (F) protein F1/F2 cleavage sites of the Tanzanian NDVs have the consensus motifs 112 RRRKR↓F 117 (VII.2 strains) and 112 RRQKR↓F 117 (V.3 and XIII.1.1 strains) consistent with virulent virus; virulence was confirmed by intracerebral pathogenicity index scores of 1.66-1.88 in 1-day-old chicks using nine of the 20 isolates. Phylogenetically, the complete F-gene and full genome sequences regionally cluster the Tanzanian NDVs with, but distinctly from, other strains previously reported in eastern and southern African countries. These data contribute to the understanding of NDV epidemiology in Tanzania and the region.

Study of coinfection with local strains of infectious bursal disease virus and infectious bronchitis virus in specific pathogen-free chickens.

Immunosuppressive diseases cause great losses in the poultry industry, increasing the susceptibility to infections by other pathogens and promoting a suboptimal response to vaccination. Among them, infectious bursal disease virus (IBDV) arises as one of the most important around the world. IBDV infects immature B lymphocytes, affecting the immune status of birds and facilitating infections by other pathogens such as avian infectious bronchitis virus (IBV). Although it has been reported that the interaction between these viruses increases IBV clinical signs, there are no actual studies about the interaction between regional circulating isolates that validate this statement. In this context, the objective of our work was to evaluate the effect of the interaction between local isolates of IBDV (belonging to genogroup 4) and IBV (lineage GI-16) in chickens. Thus, specific pathogen-free chickens were orally inoculated with IBDV genogroup (G) 4 or with PBS at 5 d of age. At 14-days postinoculation (dpi) the animals were intratracheally inoculated with a GI-16 IBV or with PBS. At multiple time points, groups of birds were euthanized and different parameters such as histological damage, viral load, lymphocyte populations and specific antibodies were evaluated. The success of IBDV infection was confirmed by the severity of bursal atrophy, viral detection, and presence of anti-IBDV antibodies. In IBV-infected animals, the presence of viral genome was detected in both kidney and bursa. The coinfected animals showed higher degree of lymphocyte infiltration in kidney, higher rate of animals with IBV viral genome in bursa at 28 dpi, and a clear decrease in antibody response against IBV at 28, 35, and 40 dpi. The results indicate that the infection with the local isolate of IBDV affects the immune status of the chickens, causing major severe damage, in response to IBV infection, which could consequently severely affect the local poultry industry.

Predominance of low pathogenic avian influenza virus H9N2 in the respiratory co-infections in broilers in Tunisia: a longitudinal field study, 2018-2020.

Respiratory diseases are a health and economic concern for poultry production worldwide. Given global economic exchanges and migratory bird flyways, respiratory viruses are likely to emerge continuously in new territories. The primary aim of this study was to investigate the major pathogens involved in respiratory disease in Tunisian broiler poultry and their epidemiology. Between 2018 and 2020, broilers farms in northeastern Tunisia were monitored, and 39 clinically diseased flocks were sampled. Samples were screened for five viral and three bacterial respiratory pathogens using a panel of real-time PCR assays. The reemergence of H9N2 low pathogenic avian influenza virus (LPAIV) in commercial poultry was reported, and the Northern and Western African GI lineage strain was typed. The infectious bronchitis virus (IBV) GI-23 lineage and the avian metapneumovirus (aMPV) subtype B also were detected for the first time in broilers in Tunisia. H9N2 LPAIV was the most detected pathogen in the flocks tested, but rarely alone, as 15 of the 16 H9N2 positive flocks were co-infected. Except for infectious laryngotracheitis virus (ILTV), all of the targeted pathogens were detected, and in 61% of the respiratory disease cases, a combination of pathogens was identified. The major combinations were H9N2 + aMPV (8/39) and H9N2 + IBV (6/39), showing the high contribution of H9N2 LPAIV to the multifactorial respiratory diseases. This field survey provided evidence of the emergence of new respiratory viruses and the complexity of respiratory disease in Tunisia. A comprehensive and continuous surveillance strategy therefore is needed to better control respiratory pathogens in Tunisia.