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.

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.

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.

Protection conferred by live infectious bronchitis vaccine viruses against variant Middle East IS/885/00-like and IS/1494/06-like isolates in commercial broiler chicks.

The ability of the infectious bronchitis H120 (a Massachusetts strain) and CR88 (a 793B strain) live attenuated vaccine viruses to protect from two Middle East infectious bronchitis virus isolates, IS/885/00-like (IS/885) and IS/1494/06-like (IS/1494) in broiler chicks was investigated. Day-old chicks were separated into three groups, (I) vaccinated with H120 at day-old followed by CR88 at 14 days-old, (II) vaccinated with H120 and CR88 simultaneously at day-old and again with CR88 at 14 days-old, (III) control unvaccinated. At 30 days-old, each of the groups was challenged with virulent IS/885 or IS/1494. Protection was evaluated based on the clinical signs, tracheal and kidney gross lesions and tracheal ciliostasis. Results showed that administering combined live H120 and CR88 vaccines simultaneously at day-old followed by CR88 vaccine at 14 days-old gave more than 80 per cent tracheal ciliary protection from both of the Middle East isolates. In addition, this programme conferred 100 per cent protection from clinical signs and tracheal or kidney lesions. The other vaccination programme, H120 at day-old followed by CR88 at 14 days-old, the tracheal ciliary protection conferred were 60 per cent and 80 per cent from IS/885/00-like and IS/1494/06-like, respectively.

Genotypes of infectious bronchitis viruses circulating in the Middle East between 2009 and 2014.

We are reporting on the infectious bronchitis virus (IBV) genotypes circulating within seven Middle East countries and the alterations in genotype distributions between 2009 and 2014. Tissue samples on FTA cards were received over the six-year period. Viral RNA was extracted using phenol chloroform and subjected to nested RT-PCR targeting a 393 bp region of the S1 gene before being followed by sequencing. From the 461 submitted samples, 363 were IBV positive by RT-PCR (77.01%). Of these, 355 (97.80%) gave sequences that can be genotyped. They belonged to six genotypes; 793B (43.66%), IS/1494/06 (18.31%), Massachusetts (Mass) (12.96%), IS/885/00 (11.27%), Q1 (11.27%) and D274 (2.25%). The prominence of 793B is not surprising, given that 793B vaccine strains are widely used in the Middle East. Sequence analysis demonstrated that the majority of 793B (67.13%) and Mass (81.13%) strains were closely related to vaccine strains based on 99-100% homology with the partial-S1 gene. Vaccinal strains belonging to the D274 genotype were present but only at a low level. Variable proportions of 793B, Mass, D274, IS/1494/06, IS/885/00 and Q1 field strains were identified in different countries. After 2012, the 793B field strain showed distinct clustering compared to strains from earlier years. Translated amino acid alterations were minimal but still may have played an important role in the persistence of this virus despite the use of live 793B vaccines. Huge challenges for an efficient protection against virulent IBVs and chicken production are posed by co-circulating793B, Mass and D274 viruses with less than 99% homology to the respective vaccine strains, along with the recently emerged variant IBVs, despite active IBV vaccination strategies in the Middle East, continuous surveillance of IBV genotypes is essential in formulating optimal control strategies, including the choice and development of new vaccine strains and formulation of vaccination programmes.

Mucosal, Cellular, and Humoral Immune Responses Induced by Different Live Infectious Bronchitis Virus Vaccination Regimes and Protection Conferred against Infectious Bronchitis Virus Q1 Strain.

The objectives of the present study were to assess the mucosal, cellular, and humoral immune responses induced by two different infectious bronchitis virus (IBV) vaccination regimes and their efficacy against challenge by a variant IBV Q1. One-day-old broiler chicks were vaccinated with live H120 alone (group I) or in combination with CR88 (group II). The two groups were again vaccinated with CR88 at 14 days of age (doa). One group was kept as the control (group III). A significant increase in lachrymal IgA levels was observed at 4 doa and then peaked at 14 doa in the vaccinated groups. The IgA levels in group II were significantly higher than those in group I from 14 doa. Using immunohistochemistry to examine changes in the number of CD4(+) and CD8(+) cells in the trachea, it was found that overall patterns of CD8(+) cells were dominant compared to those of CD4(+) cells in the two vaccinated groups. CD8(+) cells were significantly higher in group II than those in group I at 21 and 28 doa. All groups were challenged oculonasally with a virulent Q1 strain at 28 doa, and their protection was assessed. The two vaccinated groups gave excellent ciliary protection against Q1, although group II's histopathology lesion scores and viral RNA loads in the trachea and kidney showed greater levels of protection than those in group I. These results suggest that greater protection is achieved from the combined vaccination of H120 and CR88 of 1-day-old chicks, followed by CR88 at 14 doa.

Immune responses and interactions following simultaneous application of live Newcastle disease, infectious bronchitis and avian metapneumovirus vaccines in specific-pathogen-free chicks.

Interactions between live Newcastle disease virus (NDV), avian metapneumovirus (aMPV) and infectious bronchitis virus (IBV) vaccines following simultaneous vaccination of day old specific pathogen free (SPF) chicks were evaluated. The chicks were divided into eight groups: seven vaccinated against NDV, aMPV and IBV (single, dual or triple) and one unvaccinated as control. Haemagglutination inhibition (HI) NDV antibody titres were similar across all groups but were above protective titres. aMPV vaccine when given with other live vaccines suppressed levels of aMPV enzyme-linked immunosorbent assay (ELISA) antibodies. Cellular and local immunity induced by administration of NDV, aMPV or IBV vaccines (individually or together) showed significant increase in CD4+, CD8+ and IgA bearing B-cells in the trachea compared to the unvaccinated group. Differences between the vaccinated groups were insignificant. Simultaneous vaccination with live NDV, aMPV and IBV did not affect the protection conferred against aMPV or IBV.