Pros and Cons on Use of Live Viral Vaccines in Commercial Chicken Flocks.

The poultry industry is the largest source of meat and eggs for the growing human population worldwide. Key concerns in poultry farming are nutrition, management, flock health, and biosecurity measures. As part of the flock health, use of live viral vaccines plays a vital role in the prevention of economically important and common viral diseases. This includes diseases and production losses caused by Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, infectious bursal disease virus, Marek's disease virus, chicken infectious anemia virus, avian encephalomyelitis virus, fowlpox virus, and avian metapneumovirus. These viruses cause direct and indirect harms, such as financial losses worth millions of dollars, loss of protein sources, and threats to animal welfare. Flock losses vary by type of poultry, age of affected animals, co-infections, immune status, and environmental factors. Losses in broiler birds can consist of high mortality, poor body weight gain, high feed conversion ratio, and increased carcass condemnation. In commercial layers and breeder flocks, losses include higher than normal mortality rate, poor flock uniformity, drops in egg production and quality, poor hatchability, and poor day-old-chick quality. Despite the emergence of technology-based vaccines, such as inactivated, subunit, vector-based, DNA or RNA, and others, the attenuated live vaccines remain as important as before. Live vaccines are preferred in the global veterinary vaccine market, accounting for 24.3% of the global market share in 2022. The remaining 75% includes inactivated, DNA, subunit, conjugate, recombinant, and toxoid vaccines. The main reason for this is that live vaccines can induce innate, mucosal, cellular, and humoral immunities by single or multiple applications. Some live vaccine combinations provide higher and broader protection against several diseases or strains of viruses. This review aimed to explore insights on the pros and cons of attenuated live vaccines commonly used against major viral infections of the global chicken industry, and the future road map for improvement.

Estudio recapitulativo- Pros y contras del uso de vacunas virales vivas en parvadas de pollos comerciales. La industria avícola es la mayor fuente de carne y huevos para la creciente población humana en todo el mundo. Las principales preocupaciones en la avicultura son la nutrición, el manejo, la salud de las parvadas y las medidas de bioseguridad. Como parte de la salud de las parvadas avícolas, el uso de vacunas virales vivas juega un papel vital en la prevención de enfermedades virales comunes y de importancia económica. Esto incluye enfermedades y pérdidas en la producción causadas por el virus de la enfermedad de Newcastle, el virus de la bronquitis infecciosa, el virus de la laringotraqueítis infecciosa, el virus de la enfermedad infecciosa de la bolsa, el virus de la enfermedad de Marek, el virus de la anemia infecciosa del pollo, el virus de la encefalomielitis aviar, el virus de la viruela aviar y el metapneumovirus aviar. Estos virus causan daños directos e indirectos, como pérdidas financieras valoradas en millones de dólares, pérdida de fuentes de proteínas y amenazas al bienestar animal. Las pérdidas en las parvadas avícolas varían según el tipo de aves, la edad de los animales afectados, las coinfecciones, el estado inmunológico y los factores ambientales. Las pérdidas en aves de engorde pueden consistir en una alta mortalidad, un pobre aumento de peso corporal, un alto índice de conversión alimenticia y un mayor decomiso de las canales. En las gallinas de postura comerciales y en las parvadas de reproductoras, las pérdidas incluyen una tasa de mortalidad superior a la normal, una escasa uniformidad de la parvada, caídas en la producción y calidad de los huevos, una pobre incubabilidad y una mala calidad de los pollitos de un día. A pesar de la aparición de vacunas de base tecnológica, como las inactivadas, subunitarias, vectoriales, de ADN o ARN, entre otras, las vacunas vivas atenuadas siguen siendo tan importantes como antes. Las vacunas vivas son las preferidas en el mercado mundial de vacunas para uso veterinario y representaron el 24.3% de la cuota de mercado mundial en el año 2022. El 75% restante incluye vacunas inactivadas, de ADN, de subunidades, conjugadas, recombinantes y toxoides. La razón principal de esto es que las vacunas vivas pueden inducir inmunidad innata, de mucosas, celular y humoral mediante aplicaciones únicas o múltiples. Algunas combinaciones de vacunas vivas brindan una protección mayor y más amplia contra varias enfermedades o cepas de virus. Esta revisión tuvo como objetivo explorar ideas sobre los pros y los contras de las vacunas vivas atenuadas comúnmente utilizadas contra las principales infecciones virales de la industria avícola mundial, y las rutas futuras para mejorar.

Evaluation of protection and immunity induced by infectious bronchitis vaccines administered by oculonasal, spray or gel routes in commercial broiler chicks.

Broiler chicks' responses following combined IBV live attenuated Massachusetts and 793B strains through gel, spray or oculonasal (ON) vaccination routes were cross-compared. Subsequently, the responses following IBV M41 challenge of the unvaccinated and vaccinated groups were also assessed. Post-vaccination humoral and mucosal immune responses, alongside viral load kinetics in swabs and tissues, were determined using commercial ELISA assays, monoclonal antibody-based IgG and IgA ELISA assays and qRT-PCR respectively. After challenged with IBV-M41 strain, humoral and mucosal immune responses, ciliary protection, viral load kinetics, and immune gene mRNA transcriptions between the three vaccination methods were examined and compared. Findings showed that post-vaccinal humoral and mucosal immune responses were similar in all three vaccination methods. Post vaccinal viral load kinetics is influenced by method of administration. The viral load peaked in the ON group within the tissues and the OP/CL swabs in the first and third weeks respectively. Following M41 challenge, ciliary protection and mucosal immune responses were not influenced by vaccination methods as all three methods offered equal ciliary protection. Immune gene mRNA transcriptions varied by vaccination methods. Significant up-regulation of MDA5, TLR3, IL-6, IFN-α and IFN-ß genes were recorded for ON method. For both spray and gel methods, significant up-regulation of only MDA5 and IL-6 genes were noted. The spray and gel-based vaccination methods gave equivalent levels of ciliary protection and mucosal immunity to M41 virulent challenge comparable to those provided by the ON vaccination. Analysis of viral load and patterns of immune gene transcription of the vaccinated-challenged groups revealed high similarity between turbinate and choanal cleft tissues compared to HG and trachea. With regards to immune gene mRNA transcription, for all the vaccinated-challenged groups, similar results were found except for IFN-α, IFN-ß and TLR3, which were up-regulated only in ON compared to gel and spray vaccination methods.

Comparison of Immune-Related Gene Expression in Two Chicken Breeds Following Infectious Bronchitis Virus Vaccination.

This study aims to identify the immune-related genes and the corresponding biological pathways following infectious bronchitis virus vaccination in Taiwan Country and White Leghorn chicken breeds. Transcriptomic analyses of the spleen of these two breeds were conducted by next-generation sequencing. Compared to White Leghorn chicken, Taiwan Country chicken showed a significantly higher level of anti-infectious bronchitis virus (IBV) antibodies at 14 and 21 days pos vaccination. At 7 days post vaccination, in the Taiwan Country chicken, higher expression of mitogen-activated protein kinase 10, Major histocompatibility complex class 1, and V-set pre-B cell surrogate light chain 3 were found. In contrast, the White Leghorn chicken had a high expression of interleukin 4 induced 1, interleukin 6, and interleukin 22 receptor subunit alpha 2. These findings have highlighted the variations in immune induction between chickens with distinct genetic background and provided biological pathways and specific genes involved in immune responses against live attenuated IBV vaccine.

Avian metapneumovirus subtype B vaccination in commercial broiler chicks: heterologous protection and selected host transcription responses to subtype A or B challenge.

Avian metapneumovirus (aMPV) causes respiratory disease and drops in egg production in chickens, and is routinely controlled by vaccination. However, the host's immune response to virulent challenge in vaccinated or unvaccinated broiler chickens is poorly characterized. We show that subtype B vaccination offers heterologous (subtype A challenge) and homologous (subtype B challenge) protection. Subtype B challenge caused significantly greater humoral antibody titres in vaccinated and unvaccinated chickens. In turbinate and lung tissues of unvaccinated-challenged chickens, IgA and IgY mRNA transcription was significantly up-regulated after subtype B challenge compared to subtype A. Cellular immunity (CD8-α and CD8-ß) gene transcripts were significantly up-regulated during early and later stages of infection from subtype B or subtype A, respectively. Immune gene transcriptional responses (IL-1ß, IL-6 and IL-18) were significantly up-regulated after challenge. Gene transcription results showed that mRNA expression levels of CD8-α, CD8-ß, TLR3 and IL-6, particularly in turbinate and trachea tissues, are useful parameters to include in future aMPV vaccination-challenge studies.

Route of infectious bronchitis virus vaccination determines the type and magnitude of immune responses in table egg laying hens.

Chicken immune responses to infectious bronchitis virus (IBV) vaccination can depend on route of administration, vaccine strain and bird age. Typically for layer chickens, IBV vaccinations are administered by spray in the hatchery at day-old and boosted at intervals with live vaccines via drinking water (DW). Knowledge of live attenuated IBV vaccine virus kinetics and the immune response in egg-laying hens is exceptionally limited. Here, we demonstrated dissemination of vaccine viruses and differences in hen innate, mucosal, cellular and humoral immune responses following vaccination with Massachusetts or 793B strains, administered by DW or oculonasal (ON) routes. Detection of IBV in the Mass-vaccinated groups was greater during early time-points, however, 793B was detected more frequently at later timepoints. Viral RNA loads in the Harderian gland and turbinate tissues were significantly higher for ON-Mass compared to all other vaccinated groups. Lachrymal fluid IgY levels were significantly greater than the control at 14 days post-vaccination (dpv) for both vaccine serotypes, and IgA mRNA levels were significantly greater in ON-vaccinated groups compared to DW-vaccinated groups, demonstrating robust mucosal immune responses. Cell mediated immune gene transcripts (CD8-α and CD8-ß) were up-regulated in turbinate and trachea tissues. For both vaccines, dissemination and vaccine virus clearance was slower when given by DW compared to the ON route. For ON administration, both vaccines induced comparable levels of mucosal immunity. The Mass vaccine induced cellular immunity to similar levels regardless of vaccination method. When given either by ON or DW, 793B vaccination induced significantly higher levels of humoral immunity.

Host immune response to infectious bronchitis virus Q1 in two commercial broiler chicken lines.

This study investigated the pathogenesis of infectious bronchitis virus (Gammacoronavirus) strain Q1 in two commercial broiler chicken lines, and the host immune response to infection. Chicks from each line were grouped into either infected or control. Following Q1 infection at day-old, fast (Line-A) and slow (Line-B) growing chicks were monitored for clinical signs and body weights. At 3, 7, 9, 14, 21 and 28 days post infection (dpi), five birds were humanely euthanised, and trachea, kidney and proventriculus tissues were collected for quantitative RT-PCR and histopathology. Blood was collected weekly to determine IBV-specific ELISA antibody titres. Q1 infection significantly reduced the body weights of Line-A chicks at 14 and 21 dpi, but there were no significant differences in Line-B. Through qRT-PCR, significantly higher viral loads were found in the trachea, proventriculus and kidney tissues of Line-A chicks at 7-9 dpi. At day-old and at 28 dpi, the mean antibody titre in Line-B was notably higher than Line-A. Significant IFN-α mRNA expression was noted in the trachea and kidneys of Line-A, whereas no change occurred in Line-B. Chicks in Line-B, compared to those in Line-A, demonstrated a tissue-dependent increase of IFN-ß, TLR3, IL-1ß and IL-6 and LITAF gene transcription responses to IBV Q1. It appears that the level of maternal antibodies, growth rates, and other inherent host genetic factors could have influenced the differences in viral loads and immune responses.

Backyard poultry cases in UK small animal practices: Demographics, health conditions and pharmaceutical prescriptions.

BACKGROUND: Backyard poultry ownership is of keen interest in the United Kingdom. However, despite this, little is known about veterinary care engagement and outcomes of visits in this group of species. METHODS: This study described and characterised veterinary practice-visiting backyard poultry, utilising electronic health record data supplied by veterinary practices voluntarily participating in the Small Animal Veterinary Surveillance Network between 1st April 2014 and 31st March 2019. RESULTS: In total, 4424 recorded poultry consultations originating from 197 veterinary practices (352 sites) were summarised. Chicken consultation (n = 3740) peak incidence was in early summer (April-June), relative to all recorded species. More chickens resided in rural (incident rate ratio = 2.5, confidence interval [CI] 2.3-2.6, p <0.001) or less deprived areas. Non-specific clinical signs were commonly recorded (17.6% of chicken consultations, CI 15.9-19.2), as were those indicative of advanced disease. This latter finding was reflected in prescribed management strategies, with euthanasia comprising 29.8% (CI 27.0-32.6) of consultations. Antimicrobials were commonly prescribed (33.0% of consultations, CI 29.8-36.2), 43.8% of which included antimicrobials considered 'highest priority critically important' by the World Health Organisation. CONCLUSION: Our findings indicate a need to tailor antimicrobial prescription guidance to the backyard poultry setting. In addition, late presentation of disease, vague clinical descriptions in clinical narratives and high euthanasia rates show that disease identification, management and knowledge of poultry health and welfare among owners and veterinary surgeons can be improved.

Infectious Bronchitis Virus Infection of Chicken: The Essential Role of Mucosal Immunity.

Infection with either virulent or vaccine strains of infectious bronchitis virus (IBV) elicits a complex interaction of nonspecific, innate, mucosal, cellular, and humoral immunity, thereby mounting an optimal defensive response in chickens. Through this process, mucosal immunity plays an essential role in preventing infection and clearing the virus. It also assists in the development of longer lasting local immunity against IBV, mainly in the respiratory tract but also in the oviduct and gastrointestinal mucosal linings. The head-associated lymphoid tissues, particularly the Harderian gland, have an important role in the synthesis of immunoglobulin A (IgA). Levels of this immunoglobulin in lachrymal fluid often reflect the degree of protection against IBV challenge. Beyond the head, the importance of mucosal immunity has predominantly been studied in the trachea. Though IgA has been the major focus of IBV mucosal immunity investigations, the role of mucosa-associated nonspecific, innate, and cellular immune responses may also be significant. Ciliary movements and nonspecific substances in the mucosa, such as mucins and peptides, assist in the entrapment and removal of living and nonliving antigens. Mucosa-associated innate immune responses determine cascades of downstream cellular and humoral immunity against IBV. Cellular immunity, particularly involving CD4+, CD8+, and other T-cell subsets, have been studied in mucosa-associated sites, especially the trachea. The strength of cellular immunity at mucosal sites has been associated with protection against IBV. Recently, the evaluation of mucosal immunity has shifted from traditional methods to quantitative assays of mRNA transcription of immune genes. This and other molecular-based approaches will likely boost our understanding of chicken mucosal immunity against virulent and vaccine strains of IBV. It has been well accepted that mucosal immunity plays an important role in pathogenicity, vaccinal immunity, and protection conferred against virulent IBV.

Estudio recapitulativo- Infección por el virus de la bronquitis infecciosa en el pollo: El papel esencial de la inmunidad de las mucosas. La infección con cepas virulentas o vacunales del virus de la bronquitis infecciosa provoca una interacción compleja de inmunidad no específica, innata, de mucosas, celular y humoral, lo que genera una respuesta de defensa óptima en los pollos. A través de este proceso, la inmunidad de las mucosas juega un papel esencial en la prevención de infecciones y en la eliminación del virus. También ayuda al desarrollo de una inmunidad local más duradera contra el virus de la bronquitis infecciosa, principalmente en el tracto respiratorio, pero también en el oviducto y en las mucosas gastrointestinales. Los tejidos linfoides asociados a la cabeza, en particular la glándula de Harder, tienen un papel importante en la síntesis de inmunoglobulina A (IgA). Los niveles de esta inmunoglobulina en el líquido lagrimal a menudo reflejan el grado de protección contra la exposición a este virus. Más allá de la cabeza, la importancia de la inmunidad de las mucosas se ha estudiado predominantemente en la tráquea. Aunque la IgA ha sido el foco principal de las investigaciones de la inmunidad de mucosas para el virus de la bronquitis infecciosa, el papel de las respuestas inmunitarias celulares, innatas e inespecíficas asociadas a las mucosas también puede ser importante. Los movimientos ciliares y las sustancias inespecíficas de la mucosa, como las mucinas y los péptidos, ayudan a atrapar y eliminar antígenos vivos o inanimados. Las respuestas inmunitarias innatas asociadas a las mucosas determinan las cascadas de inmunidad humoral y celular contra el virus de la bronquitis infecciosa. Se ha estudiado la inmunidad celular, que involucra particularmente a CD4 +, CD8 + y otras subpoblaciones de células T, en sitios asociados a mucosas, especialmente la tráquea. La fuerza de la inmunidad celular en los sitios de las mucosas se ha asociado con la protección contra el virus de la bronquitis. Recientemente, la evaluación de la inmunidad de la mucosa se ha desplazado de los métodos tradicionales a los ensayos cuantitativos de la transcripción de ARN mensajero de genes inmunes. Este y otros enfoques moleculares probablemente aumentarán el conocimiento de la inmunidad de mucosas del pollo contra las cepas virulentas y vacunales del virus de la bronquitis infecciosa. Está bien aceptado que la inmunidad de las mucosas juega un papel importante en la patogenicidad, la inmunidad vacunal y la protección conferida contra virus virulentos de la bronquitis infecciosa.

Minimal inhibitory concentration of seven antimicrobials to Mycoplasma gallisepticum and Mycoplasma synoviae isolates from six European countries.

Mycoplasma gallisepticum and Mycoplasma synoviae are bacterial pathogens that cause disease in poultry, adversely affecting their health and welfare, and are a financial burden on producers. This manuscript describes the results of the MycoPath project that is the first international antimicrobial susceptibility programme for mycoplasma pathogens isolated from poultry. Improved comparative analysis of minimal inhibitory concentration (MIC) results from participating countries was facilitated by using one laboratory determining all MICs. Chicken and turkey isolates were obtained from France, Germany, Great Britain, Hungary, Italy and Spain during 2014-2016. One isolate per farm was retained. The MIC of seven antimicrobial agents was determined using a broth microdilution method, with Friis Medium (M. gallisepticum) or Modified Chanock's Medium (M. synoviae). Of the 222 isolates recovered, 82 were M. gallisepticum and 130 were M. synoviae. M. gallisepticum MIC50/90 values were 0.12/0.5, 2/8, 0.5/4, 0.12/>64, 0.008/0.062, 0.008/32, 0.062/4 mg/l for doxycycline, enrofloxacin, oxytetracycline, spiramycin, tiamulin, tilmicosin and tylosin, respectively. For M. synoviae, the values were 0.5/1, 8/16, 0.5/1, 0.5/8, 0.25/0.5, 0.062/2 and 0.062/16 mg/l respectively. A bimodal MIC distribution for the fluoroquinolone (enrofloxacin) and the macrolides (spiramycin, tilmicosin and tylosin) indicate that both species have sub-populations that are less susceptible in vitro to those antimicrobials. Some differences in susceptibilities were observed according to host species, Mycoplasma species, and country of origin. This study provides a baseline of novel data for future monitoring of antimicrobial resistance in poultry Mycoplasma species. Additionally, this information will facilitate the selection of the antimicrobial agents most likely to be effective, thus ensuring their minimal use with targeted and correct therapeutic treatments.Highlights First large-scale pan-European collection of representative Mg and Ms isolates.MIC values assessed in central laboratory for Mg and Ms from chickens and turkeys.Range of MIC values for 82 Mg and 130 Ms isolates to seven licenced antibiotics shown.Data can be used to help determine Mg and Ms veterinary-specific breakpoints.

Influences of swab types and storage temperatures on isolation and molecular detection of Mycoplasma gallisepticum and Mycoplasma synoviae.

Routine diagnosis of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) is performed by collecting oropharyngeal swabs, followed by isolation and/or detection by molecular methods. The storage temperature, storage duration and the type of swab could be critical factors for successful isolation or molecular detection. The aim of this study was to compare the influence of different types of cotton-tipped swab stored at different temperatures, on the detection of MG and MS. To achieve this, combined use of traditional culture analysis (both agar and broth), with modern molecular detection methods was utilized. Performances of wooden and plastic shaft swabs, both without transport medium, were compared. Successful culture of M. gallisepticum was significantly more efficient from plastic swabs when compared to wooden, whereas no difference was seen for the re-isolation of M. synoviae. Storage at 4°C compared to room temperature also increased the efficiency of culture detection for both Mycoplasma species. When stored at room temperature, PCR detection limits of both MG and MS were significantly lower for wooden compared to plastic swabs. The qPCR data showed similar detection limits for both swab types when stored at both temperatures. The results suggest that swabs with a plastic shaft are preferred for MG and MS detection by both culture and PCR. While a lower storage temperature (4°C) is optimal for culture recovery, it seems that both temperatures investigated here are adequate for molecular detection and it is the swab type which carries a greater influence.

Comparative protective immunity provided by live vaccines of Newcastle disease virus or avian metapneumovirus when co-administered alongside classical and variant strains of infectious bronchitis virus in day-old broiler chicks.

This study reports on the simultaneous administration of live NDV or aMPV subtype B vaccines alongside two live IBV (Massachusetts-H120 and 793B-CR88) vaccines in day-old maternal-antibody positive commercial broiler chicks. In the first experiment, chicks were divided into four groups; one unvaccinated and three groups vaccinated with live NDV VG/GA-Avinew, live H120 + CR88, or VG/GA-Avinew + H120 + CR88. In the second experiment, live aMPV subtype B vaccine was used in place of NDV. Clinical signs were monitored daily and oropharyngeal swabs were taken at regular intervals for vaccine virus detection. Blood was collected at 21 dpv for serology. 10 chicks from each group were challenged with virulent strains of M41 or QX or aMPV subtype B. For IBV, after 5 days post challenge (dpc), tracheal ciliary protection was assessed. For aMPV, clinical scores were recorded up to 10 dpc. For NDV, haemagglutination inhibition (HI) antibody titres were assayed as an indicator of protective immunity. In both experiments, ciliary protection for IBV vaccinated groups was maintained above 90%. The protection against virulent aMPV challenge was not compromised when aMPV, H120 and CR88 were co-administered. NDV HI mean titres in single and combined NDV-vaccinated groups remained above the protective titre (>3 log2). Both experiments demonstrated that simultaneous administration of live NDV VG/GA-Avinew or aMPV subtype B alongside H120 and CR88 vaccines does not interfere with protection conferred against NDV, IBV or aMPV.

Co-circulation of genetically diverse population of vaccine related and unrelated respiratory mycoplasmas and viruses in UK poultry flocks with health or production problems.

Respiratory diseases continue to have a major impact on poultry health, welfare and productivity. However, little information is available on their current status in UK poultry flocks. We investigated the presence of four economically important respiratory pathogens in healthy or problematic flocks; infectious bronchitis virus (IBV), avian metapneumovirus (aMPV), Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms). Samples from 131 UK poultry flocks were received during the 12 month study period. Oropharyngeal (OP) swabs were taken from eight birds per flock and accompanied with flock health information. The study included 118 chicken, 6 pheasant and 5 turkey flocks, and 1 quail and 1 partridge flock. Chicken flocks were of layers (n = 98), broilers (n = 15), breeders (n = 3) and undisclosed (n = 2). Flock ages ranged from 3 to 72 weeks old, and the average flock size was 17,633 birds. PCR detected 65 (49.6%), 59 (45%) and 8 (6.1%) flocks as positive for IBV, Mg/Ms and aMPV respectively. Analysis of the mgc2 gene of the Mg isolates revealed high similarities to Mg TS-11 and Mg 6/85. Further gene analysis found that the TS-11-like isolates were unrelated to the TS-11 vaccine. Multi-locus sequence typing (MLST) analysis identified the majority of positive Ms as ST21, along with ST2 (MS-H-like), ST6 and ST43. IBV S1 gene sequencing identified strains as 793B (66.7%), Arkansas (23.8%) and Massachusetts (9.5%). All aMPV positive samples belonged to subtype B. Findings indicate that over half of the flocks sampled were positive for at least one of the four vaccine or field strains of mycoplasmas or viruses.

Differential innate immune responses induced by classical and variant infectious bronchitis viruses in specific pathogen free chicks.

Avian infectious bronchitis virus (IBV) continues to cause serious economic losses in global chicken production. Concurrent circulation of both classic and variant IBVs have been identified in most parts of the world, raising major challenges to global prevention and control efforts. Therefore, immunopathogenesis, particularly early host responses, needs to be better understood for effective control of diseases caused by different strains of IBVs. We investigated differing immunopathogenesis in chickens following infection with IS/885/00-like (885), QX-like (QX) and M41 IBV strains. We confirmed that the histopathological changes, proinflammatory and innate immune gene responses were induced to different magnitudes, depending on the IBV strain. Results indicated that upregulation of proinflammatory cytokines (such as IL-6 and IL-1ß) and lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF) expression is induced by IBV M41 in the trachea and by IBV 885 and QX in the kidney, which mainly coincides with tracheal and renal histopathological lesions respectively caused by these strains. In addition, elevated levels of TLR3, MDA5 and IFN-ß expression occurred concurrently with greater lesion severity in IBV infected trachea and kidney tissues. Overall, this study reports marked differences in the activation of early host responses by pathogenic IBV strains.

Evaluation of full S1 gene sequencing of classical and variant infectious bronchitis viruses extracted from allantoic fluid and FTA cards.

Sequence variability in the S1 gene determines the genotype of infectious bronchitis virus (IBV) strains. A single RT-PCR assay was developed to amplify and sequence the full S1 gene for six classical and variant IBVs (M41, D274, 793B, IS/885/00, IS/1494/06 and Q1) enriched in allantoic fluid (AF) or the same AF inoculated onto Flinders Technology Association (FTA) cards. Representative strains from each genotype were grown in specific-pathogen-free eggs and RNA was extracted from AF. Full S1 gene amplification was achieved using primer A and primer 22.51. Products were sequenced using primers A, 1050+, 1380+ and SX3+ to obtain short sequences covering the full gene. Following serial dilutions of AF, detection limits of the partial assay were higher than those of the full S1 gene. Partial S1 sequences exhibited higher-than-average nucleotide similarity percentages (79%; 352 bp) compared to full S1 sequences (77%; 1756 bp), suggesting that full S1 analysis allows greater strain differentiation. For IBV detection from AF-inoculated FTA cards, four serotypes were incubated for up to 21 days at three temperatures, 4°C, room temperature (approximately 24°C) and 40°C. RNA was extracted and tested with partial and full S1 protocols. Through partial sequencing, all IBVs were successfully detected at all sampling points and storage temperatures. In contrast, using full S1 sequencing it was not possible to amplify the gene beyond 14 days or when stored at 40°C. Data presented show that for full S1 sequencing, a substantial amount of RNA is needed. Field samples collected onto FTA cards are unlikely to yield such quantity or quality. ABBREVIATIONS: AF: allantoic fluid; CD50: ciliostatic dose 50; FTA: Flinders Technology Association; IB: infectious bronchitis; IBV: infectious bronchitis virus.

Infectious bronchitis vaccine virus detection and part-S1 genetic variation following single or dual inoculation in broiler chicks.

An investigation was undertaken of the extent of genetic variation occurring within infectious bronchitis virus (IBV) vaccine strains following vaccination of day-old broiler chicks. Chicks were divided into seven groups, with two groups receiving single Massachusetts (Mass) vaccinations while the other four were inoculated with combinations of different IBV serotypes; Mass, 793B, D274 and Arkansas (Ark). The remaining group was maintained as an unvaccinated control. Following vaccination, swabs and tissues collected at intervals were pooled and RNA was extracted for detection of IBV by reverse transcription polymerase chain reaction. Positive amplicons were sequenced for the part-S1 gene and compared to the original vaccine strain sequences. Single nucleotide polymorphisms, amino acid variations and hydrophobicity changes were identified and recorded for each sampling point. A total of 106 single nucleotide polymorphisms were detected within 28 isolates. The average single nucleotide polymorphism counts of swab isolates were greater than those found in tissue samples. This translated into 64 amino acid changes; however only six resulted in a change to the hydrophobicity properties. All hydrophobic alterations occurred within swab isolates and the majority were recovered at 3 days post vaccination suggesting such changes to be detrimental to early virus survival. Nucleotide deletions were seen only in the group given the combination of Mass and Ark. Of the 16 sequenced samples in this group, 13 contained the same AAT deletion at position 1033 1035 in the Ark strains. Findings presented in this study demonstrate alteration in the S1 nucleotide sequence following co-administration of live IBV vaccines.

Genetic mutations in live infectious bronchitis vaccine viruses following single or dual in vitro infection of tracheal organ cultures.

Despite regular co-vaccination of two different strains of live infectious bronchitis vaccine viruses, little is known about possible mutations in these viruses following vaccination. As an alternative to chicks, this study used an in vitro infection model to identify single-nucleotide polymorphisms (SNPs) within the part-S1 gene of two live infectious bronchitis virus vaccine strains (793B and Massachusetts) following single or dual inoculation onto tracheal organ cultures. Results indicate that viral titres reduced over the duration of the study; conversely, the amount of detected infectious bronchitis virus genome increased. Results demonstrate a greater number of non-synonymous SNPs in both vaccine strains when they are co-inoculated, compared with the single inoculations. The influence of the increased SNP and hydrophobic properties of the translated proteins on the vaccine viruses' virulence is unknown.

Experimental infection of IS/885/00-like infectious bronchitis virus in specific pathogen free and commercial broiler chicks.

Pathogenesis of an IS/885/00-like (IS/885) strain of variant infectious bronchitis virus (IBV) was examined in one day old specific pathogen free (SPF) and commercial broiler chicks. Chicks were humanely euthanized at 3, 6, 9, 12, 15, 21 and 28 days post infection (dpi) for necropsy examination, and tissues were collected for histopathology and virus detection by reverse transcription polymerase chain reaction (RT-PCR). Respiratory clinical signs and gross lesions consisting of tracheal caseous exudate and plugs, and swollen kidneys (with or without) urate deposits were observed in SPF and broiler chicks. The onset of disease developed more slowly and were of lesser severity in broiler compared to SPF chicks, reflecting the inhibitory effects of the IBV maternal-antibodies in the broiler chicks or genetic/strain susceptibility, or both. Head swelling was observed in one infected broiler chick at 15 dpi and the virus was recovered by RT-PCR and isolation. In the IS/885-infected SPF chicks, cystic oviducts were found in two female chicks. IS/885 was isolated from the cystic fluid. Using ELISA, low to moderate levels of the antibodies to IBV was detected in the SPF compared to broiler infected chicks.

Detection of variant infectious bronchitis viruses in Sri Lanka (2012-2015).

Poultry production is an important sector of agriculture in Sri Lanka; however, there is a lack of information regarding circulation of infectious bronchitis virus (IBV). RNA was extracted from chicken tissues, subjected to IBV S1 RT-PCR, and sequenced. Overall, 19 out of 34 (55.88 %) samples were IBV positive and contained the genotype 793B (n = 13; 68.42 %), D274 (n = 4; 21.05 %) or Massachusetts (n = 2; 10.53 %). All three genotypes contained at least one strain with less than 99 % nucleotide sequence identity to the corresponding vaccine strains. This report identified co-circulation of IBV strains 793B, Massachusetts and D274, in Sri Lanka that are divergent from the respective vaccine strains.

Heterologous live infectious bronchitis virus vaccination in day-old commercial broiler chicks: clinical signs, ciliary health, immune responses and protection against variant infectious bronchitis viruses.

Groups of one-day-old broiler chicks were vaccinated via the oculo-nasal route with different live infectious bronchitis virus (IBV) vaccines: Massachusetts (Mass), 793B, D274 or Arkansas (Ark). Clinical signs and gross lesions were evaluated. Five chicks from each group were humanely killed at intervals and their tracheas collected for ciliary activity assessment and for the detection of CD4+, CD8+ and IgA-bearing B cells by immunohistochemistry (IHC). Blood samples were collected at intervals for the detection of anti-IBV antibodies. At 21 days post-vaccination (dpv), protection conferred by different vaccination regimes against virulent M41, QX and 793B was assessed. All vaccination programmes were able to induce high levels of CD4+, CD8+ and IgA-bearing B cells in the trachea. Significantly higher levels of CD4+ and CD8+ expression were observed in the Mass2 + 793B2-vaccinated group compared to the other groups (subscripts indicate different manufacturers). Protection studies showed that the group of chicks vaccinated with Mass2 + 793B2 produced 92% ciliary protection against QX challenge; compared to 53%, 68% and 73% ciliary protection against the same challenge virus by Mass1 + D274, Mass1 + 793B1 and Mass3 + Ark, respectively. All vaccination programmes produced more than 85% ciliary protection against M41 and 793B challenges. It appears that the variable levels of protection provided by different heterologous live IBV vaccinations are dependent on the levels of local tracheal immunity induced by the respective vaccine combination. The Mass2 + 793B2 group showed the worst clinical signs, higher mortality and severe lesions following vaccination, but had the highest tracheal immune responses and demonstrated the best protection against all three challenge viruses.

Pathogenicity and tissue tropism of infectious bronchitis virus is associated with elevated apoptosis and innate immune responses.

To establish a characteristic host response to predict the pathogenicity and tissue tropism of infectious bronchitis viruses (IBV), we investigated innate immune responses (IIR) and apoptosis in chicken embryo kidney cells (CEKC) and tracheal organ cultures (TOC) infected with three IBV strains. Results showed nephropathogenic IBV strains 885 and QX induced greater apoptosis in CEKC than M41, which induced greater apoptosis in TOCs compared to 885 and QX. Elevated IIR is associated with tissue tropism of different IBV strains. Compared to M41, 885 and QX caused greater induction of toll like receptor 3 (TLR3), melanoma differentiation associated protein 5 (MDA5) and interferon beta (IFN-ß) in CEKC. In contrast, M41 infection caused greater expression of these genes than 885 or QX in TOCs. In summary, greater levels of apoptosis and elevated levels of TLR3, MDA5 and IFN-ß expression are associated with increased pathogenicity of IBV strains in renal and tracheal tissues.