Protection afforded by avian influenza vaccination programmes consisting of a novel RNA particle and an inactivated avian influenza vaccine against a highly pathogenic avian influenza virus challenge in layer chickens up to 18 weeks post-vaccination.

The efficacies of an oil adjuvanted-inactivated reverse genetics-derived H5 avian influenza virus (AIV) vaccine and an alphavirus replicon RNA particle (RP) AIV vaccine were evaluated in commercial Leghorn chickens. Challenge utilized A/turkey/MN/12582/2015, an isolate representing the U.S. H5N2 Clade 2.3.4.4 responsible for the 2015 highly pathogenic avian influenza (HPAI) epornitic in commercial poultry the United States. As part of a long-term, 36-week study, chickens were challenged at seven weeks of age after receiving a single vaccination, at 18 weeks of age following a vaccine prime-single boost, and at 36 weeks of age after a prime- double-boost. All vaccine programmes reduced virus oropharyngeal and cloacal shedding and mortality compared to the non-vaccinated control birds; however, chickens receiving at least one administration of the RP vaccine generally had diminished viral shedding especially from the cloacal swabbings. A detectable serum antibody response and protection were observed through 18 weeks post-vaccination. Our data suggest that, in conjunction with a comprehensive eradication, enhanced biosecurity and controlled marketing plan, vaccination programmes of commercial layer chickens with novel RP vaccines may represent an important tool for preventing HPAI-related mortalities and decreasing viral load during a catastrophic influenza outbreak. RESEARCH HIGHLIGHTS Immunization of poultry following a vaccination schedule consisting of inactivated and RNA particle vaccines offered significant protection against lethal disease following HPAIV challenge. Virus shedding was significantly (P < 0.05) reduced in chickens vaccinated with either inactivated and/or recombinant vaccines. Serum antibody titres were not a reliable indicator of protection. An inactivated vaccine containing 384 HAU of the homologous antigen was unable to induce complete protection.

Transcriptional analysis of the innate immune response of ducks to different species-of-origin low pathogenic H7 avian influenza viruses.

BACKGROUND: Wild waterfowl, including ducks, represent the classic reservoir for low pathogenicity avian influenza (LPAI) viruses and play a major role in the worldwide dissemination of AIV. AIVs belonging to the hemagglutinin (H) 7 subtype are of epidemiological and economic importance due to their potential to mutate into a highly pathogenic form of the virus. Thus far, however, relatively little work has been conducted on elucidating the host-pathogen interactions of ducks and H7 LPAIVs. In the current study, three H7 LPAIVs isolated from either chicken, duck, or turkey avian species were evaluated for their comparative effect on the transcriptional innate immune response of ducks. RESULTS: Three H7 LPAIV isolates, chicken-origin (A/chicken/Maryland/MinhMa/2004), duck-origin (A/pintail/Minnesota/423/1999), and turkey-origin (A/turkey/Virginia/SEP-67/2002) were used to infect Pekin ducks. At 3 days post-infection, RNA from spleen tissue was used for transcriptional analysis using the Avian Innate Immune Microarray (AIIM) and quantitative real-time RT-PCR (qRT-PCR). Microarray analysis revealed that a core set of 61 genes was differentially regulated in response to all three LPAIVs. Furthermore, we observed 101, 135, and 628 differentially expressed genes unique to infection with the chicken-, duck-, or turkey-origin LPAIV isolates, respectively. qRT-PCR results revealed significant (p<0.05) induction of IL-1ß, IL-2, and IFNγ transcription, with the greatest induction observed upon infection with the chicken-origin isolate. Several key innate immune pathways were activated in response to LPAIV infection including the toll-like receptor and RIG-I-like receptor pathways. CONCLUSIONS: Pekin ducks elicit a unique innate immune response to different species-of-origin H7 LPAIV isolates. However, twelve identifiable genes and their associated cell signaling pathways (RIG-I, NOD, TLR) are differentially expressed regardless of isolate origin. This core set of genes are critical to the duck immune response to AI. These data provide insight into the potential mechanisms employed by ducks to tolerate AI viral infection.

Characterization of nephropathogenic infectious bronchitis virus DMV/1639/11 recovered from Delmarva broiler chickens in 2011.

A limited outbreak of nephropathogenic infectious bronchitis (NIB) occurred in three Delmarva (DMV) commercial broiler chicken flocks in 2011. Isolates of NIB virus (NIBV)--DMV/1639/11, DMV/3432/11, and DMV/3902/11--were characterized by sequence analysis of the N-terminal subunit (S1) of the spike (S) gene. Findings indicated that the isolates were identical to each other and to PA/9579A/10, a 2010 isolate from poultry in Pennsylvania. The 2010 and 2011 isolates appear to have originated from a 1997-2000 NIB outbreak in Pennsylvania. DMV/1639/11 and PA/9579A/10 were determined to be nephropathogenic in susceptible chickens, yielding virus reisolations from kidney and inducing characteristic interstitial nephritis microscopic lesions. In a controlled laboratory study, 40% of chickens vaccinated with a combination live vaccine containing infectious bronchitis virus (IBV) strains Massachusetts (Mass) + Connecticut (Conn) were positive on virus isolation attempts after challenge with DMV/1639/11, compared with only 13% of Mass + Arkansas (Ark) vaccinates. Both combination vaccines gave partial protection against the development of DMV/1639/11-induced renal lesions. Although numerically fewer chickens vaccinated with Mass + Conn had interstitial nephritis compared with those vaccinated with Mass + Ark, neither vaccine combination offered greater protection (P < 0.05) than observed in unvaccinated chickens challenged with DMV/1639/11. Mass + Ark vaccinations, applied under commercial conditions in the hatchery (spray) and on-farm (spray), did not protect the trachea or kidney from DMV/1639/11 challenge. Serologic testing of broiler flocks found < 3% (2 of 69) tested to possess specific antibodies to DMV/1639/11, indicating the virus had not become established in the region.

Comparison of pooling 11 or 5 oropharyngeal swabbings for detecting avian influenza virus by real-time reverse transcription-PCR in broiler chickens.

The effect of pooling 11 or 5 oropharyngeal (O/P) swabbings on detecting avian influenza virus (AIV) by real-time reverse transcription (RRT)-PCR was evaluated. The model used for the evaluation was designed to minimize viral load and, thus, assess the effect of the pooling on detection. Two-week-old broiler chickens were inoculated via the intranasal route with the low pathogenicity chicken/Maryland/Minh Ma/04 H7N2 strain or remained uninoculated. On days 2, 3, 4, 5, 7, 9, 11, and 14 postinoculation (PI), O/P swabbings were collected from individual infected birds and pooled with either 10 or 4 O/P swabs from uninfected broilers to produce 10 replicate pools of 11 or 5 swabbings, respectively. AIV was readily detected (80%-100%) by RRT-PCR in the pools of 11 and pools of 5 swabbings from days 2 through 5 PI. Detection in pools of both types decreased to similar levels on day 7 (40% for the pools of 11 and 50% for the pools of 5). AIV was not detected on day 9, 11, and 14 PI in pools of either size. On a given sample day PI, mean cycle threshold (Ct) values were consistently higher (lower genome levels) in the pools of 11 compared to the pools of 5. These differences were statistically significant on days 3 and 5 PI, yet Ct values associated with both types of pools were clearly interpretable as AIV positive.

Coordination of SARS-CoV-2 wastewater and clinical testing of university students demonstrates the importance of sampling duration and collection time.

Wastewater surveillance has been a useful tool complementing clinical testing during the COVID-19 pandemic. However, transitioning surveillance approaches to small populations, such as dormitories and assisted living facilities poses challenges including difficulties with sample collection and processing. Recently, the need for reliable and timely data has coincided with the need for precise local forecasting of the trajectory of COVID-19. This study compared wastewater and clinical data from the University of Delaware (Fall 2020 and Spring 2021 semesters), and evaluated wastewater collection practices for enhanced virus detection sensitivity. Fecal shedding of SARS-CoV-2 is known to occur in infected individuals. However, shedding concentrations and duration has been shown to vary. Therefore, three shedding periods (14, 21, and 30 days) were presumed and included for analysis of wastewater data. SARS-CoV-2 levels detected in wastewater correlated with clinical virus detection when a positive clinical test result was preceded by fecal shedding of 21 days (p< 0.05) and 30 days (p < 0.05), but not with new cases (p = 0.09) or 14 days of shedding (p = 0.17). Discretely collected wastewater samples were compared with 24-hour composite samples collected at the same site. The discrete samples (n = 99) were composited examining the influence of sampling duration and time of day on SARS-CoV-2 detection. SARS-CoV-2 detection varied among dormitory complexes and sampling durations of 3-hour, 12-hour, and 24-hour (controls). Collection times frequently showing high detection values were between the hours of 03:00 to 05:00 and 23:00 to 08:00. In each of these times of day 33% of samples (3/9) were significantly higher (p < 0.05) than the control sample. The remainder (6/9) of the collection times (3-hour and 12-hour) were not different (p > 0.05) from the control. This study provides additional framework for continued methodology development for microbiological wastewater surveillance as the COVID-19 pandemic progresses and in preparation for future epidemiological efforts.

The pathogenesis of low pathogenicity H7 avian influenza viruses in chickens, ducks and turkeys.

BACKGROUND: Avian influenza (AI) viruses infect numerous avian species, and low pathogenicity (LP) AI viruses of the H7 subtype are typically reported to produce mild or subclinical infections in both wild aquatic birds and domestic poultry. However relatively little work has been done to compare LPAI viruses from different avian species for their ability to cause disease in domestic poultry under the same conditions. In this study twelve H7 LPAI virus isolates from North America were each evaluated for their comparative pathogenesis in chickens, ducks, and turkeys. RESULTS: All 12 isolates were able to infect all three species at a dose of 106 50% egg infectious doses based on seroconversion, although not all animals seroconverted with each isolate-species combination. The severity of disease varied among isolate and species combinations, but there was a consistent trend for clinical disease to be most severe in turkeys where all 12 isolates induced disease, and mortality was observed in turkeys exposed to 9 of the 12 viruses. Turkeys also shed virus by the oral and cloacal routes at significantly higher titers than either ducks or chickens at numerous time points. Only 3 isolates induced observable clinical disease in ducks and only 6 isolates induced disease in chickens, which was generally very mild and did not result in mortality. Full genome sequence was completed for all 12 isolates and some isolates did have features consistent with adaptation to poultry (e.g. NA stalk deletions), however none of these features correlated with disease severity. CONCLUSIONS: The data suggests that turkeys may be more susceptible to clinical disease from the H7 LPAI viruses included in this study than either chickens or ducks. However the severity of disease and degree of virus shed was not clearly correlated with any isolate or group of isolates, but relied on specific species and isolate combinations.

Identification of Type A Influenza Viruses from Wild Birds on the Delmarva Peninsula, 2007-10.

Wild waterfowl and shorebirds in the Delaware-Maryland-Virginia (Delmarva) Peninsula region within the Atlantic Flyway were sampled as part of the Early Detection of Highly Pathogenic H5N1 Avian Influenza (AI) in Wild Migratory Birds program. The U.S. Department of Agriculture (USDA) and state wildlife agencies submitted 7858 samples for AI virus (AIV) testing by real-time reverse transcription PCR (rRT-PCR) to the University of Delaware Poultry Health System from April 2007 to March 2011. Virus isolation attempts were performed on samples with matrix gene cycle threshold (Ct) values ≤33.9. Using rRT-PCR, AIV was detected in 14% (1091/7857) of the samples. In species with sample sizes >100, American black duck ( Anas rubripes ; 28%), ruddy turnstone ( Arenaria interpres ; 27%), American green-winged teal ( Anas crecca ; 21%), semipalmated sandpiper ( Calidris pusilla ; 27%), greater snow goose ( Chen caerulescens atlanticus; 12%), mallard ( Anas platyrhynchos ; 10%), and northern pintail ( Anas acuta ; 14%) showed the highest rates of AIV detection. Forty-two AIVs were recovered from eight species: American black duck, mallard, ruddy turnstone, American green-winged teal, greater snow goose, Canada goose ( Branta canadensis ), ring-necked duck ( Aythya collaris ), and mallard × American black duck ( Anas platyrhynchos × Anas rubripes ). Recovered H5 (n = 2) and H7 (n = 2) viruses were found to be low pathogenicity by the USDA National Veterinary Services Laboratory. Additional AIVs represented a diversity of subtype combinations: H1-H4, H6, and H10 and H11 and N subtypes N1-N9 and N6-N9. The rate of AIV recovery from swabbings was inversely related to Ct value, ranging from 50% for Ct values of 16.0-18.9 to 5.1% for Ct values of 31-33.9.

S1 gene sequence analysis of a nephropathogenic strain of avian infectious bronchitis virus in Egypt.

BACKGROUND: Infectious bronchitis is highly contagious and constitutes one of the most common and difficult poultry diseases to control. IBV is endemic in probably all countries that raise chickens. It exists as dozens of serotypes/genotypes. Only a few amino acid differences in the S1 protein of vaccine and challenge strains of IBV may result in poor protection. Tropism of IBV includes the respiratory tract tissues, proventriculus and caecal tonsils of the alimentary tract, the oviduct and the kidney. RESULTS: Infectious bronchitis virus (IBV) strain closely related to Massachusetts (Mass) serotype was isolated from broiler chickens suffering from severe renal and respiratory distresses. The isolate was serologically identified by Dot-ELISA and further characterized by RT-PCR then genotyped using S1 gene sequence analysis. Alignment of the S1 sequence of the isolate with 16 IBV strains revealed high homology to isolates related to Mass serotype. Inoculation with the strain reproduced the disease in experimental 1-day-old chickens and resulted in 20% mortality, severe renal and moderate respiratory distresses. Marked histopathological changes in both kidney and trachea were observed in experimentally infected chickens. A protection study using the H120 live attenuated vaccine showed low protection rate in spite of high S1 sequence homology (97%). Protection based criteria were: virus re-isolation attempts from trachea, tracheal and renal histopathology as well as IBV antigens detection by immunofluorescent antibody technique in kidney sections. CONCLUSION: Periodical evaluation of cross-protective capabilities of IBV vaccine(s) versus recently recovered field isolates should be performed to ensure optimum control of IBV.

Efficacy of Recombinant HVT-IBD Vaccines Administered to Broiler Chicks from a Single Breeder Flock at 30 and 60 Weeks of Age.

The efficacy of commercially available recombinant herpesvirus of turkeys-infectious bursal disease (rHVT-IBD) virus vaccines was studied in broiler chickens derived from an IBDV-vaccinated breeder flock at 30 wk of age (Trial 1) and 60 wk of age (Trial 2). In parallel, specific-pathogen-free (SPF) white leghorn chickens were used to evaluate vaccine efficacy to control for the effects of maternally derived antibodies (MDA) associated with the broiler chickens. Broilers and SPF leghorns were vaccinated subcutaneously in the neck at 1 day of age with Vaxxitek® HVT+IBD or Vectormune® HVT-IBD vaccines and were placed in isolators. On 10, 14, 18, 22, and 26 days postvaccination (DPV), vaccinated and nonvaccinated broilers and SPF leghorns were bled prior to challenge via the oral-nasal route with infectious bursal disease (IBD) reference strains ST-C, Delaware variant E (Del E), or contemporary field isolates DMV/5038/07 or FF6. Microscopic lesion assessment of the bursa was useful for assessing IBDV challenge in both rHVT-IBD-vaccinated broiler and SPF leghorn chickens. In general, rHVT-IBD vaccines induced greater protection as the time between vaccination and challenge increased. Based on incidence of microscopic lesions (IML) of bursa tissue, Vaxxitek HVT+IBD vaccination of SPF leghorns induced protection by 18 DPV and continued to protect 22 DPV and 26 DPV in Trials 1 and 2. Vectormune HVT-IBD vaccine induced protection of SPF leghorns by 18 or 22 DPV in Trial 1, depending upon the IBDV challenge strain. However, the onset of protection was delayed until 22 or 26 DPV in Trial 2. With either commercial vaccine, rHVT-IBD vaccination of broiler chickens was not as effective as was observed in SPF leghorns, based on IML of bursa tissue. However, Vaxxitek HVT+IBD vaccination protected broilers following challenge with ST-C in both Trial 1 (30-wk-old breeder progeny) and Trial 2 (60-wk-old breeder progeny). Partial protection against FF6 (Trial 1) and DMV/5038/07 (Trial 2) challenges was observed. Vectormune HVT-IBD vaccination protected broilers vs. FF6 challenge in Trial 1. In Trial 2, the vaccine did not offer protection on the basis of IML of bursa tissue. The results indicate that 1) bursa/body weight ratios were not consistently useful as a tool for assessing IBDV challenge in broiler chickens with anti-IBDV MDA compared to assessment by IML of bursa tissue, though were useful for assessing protection in SPF leghorns; and 2) both vaccines may offer some protection to older broilers; however, a window of susceptibility exists between the waning of MDA and the development of vaccine-induced antibodies. The SPF studies showed that some vaccinated chickens were not protected from an IBDV challenge earlier than 14 DPV while broiler studies showed that MDA was not fully protective beyond 10 DPV. Because these vaccines did not protect chickens from an IBDV challenge during this window of susceptibility, our data show that breeder vaccination programs for IBDV must aim to maximize anti-IBDV MDA in progeny to protect against early IBDV challenge.

Complete genome sequence of an avian paramyxovirus type 4 from north america reveals a shorter genome and new genotype.

An avian paramyxovirus type 4 (APMV-4) was isolated from a duck in Delaware in 2010. Its genome is 15,048 nucleotides (nt) long, which is shorter by 6 nt than those for all previously reported strains. Phylogenetic analysis revealed that this strain formed a separate cluster within APMV-4 strains.

Infectious bronchitis virus S1 gene sequence comparison is a better predictor of challenge of immunity in chickens than serotyping by virus neutralization.

Five strains of infectious bronchitis virus isolated from commercial chickens from the state of Pennsylvania, USA during the years 1998 and 1999 were studied. The strains were selected for cross-challenge in specific pathogen free chickens and virus neutralization in chick embryos on the basis of partial S1 sequence amino acid identity values. The partial sequences analysed spanned the hypervariable amino terminus region of S1 from amino acid residues 48 to 219, based on the Beaudette strain. Using their S1 identity values, the strains represented a continuum of genetic, and thus antigenic, relationships. When compared with strain PA/5083/99, strain PA/Wolgemuth/98 had high sequence identity (96%) followed by PA/171/99 (85%), PA/5344/98 (70%) and PA/1220/98 (34%). The method of Archetti and Horsfall was used for calculating antigenic relatedness values of virus neutralization tests. The same formula was also applied to the percentage protection values of cross-challenge tests to derive protective relatedness values among the strains. The antigenic relatedness values, protective relatedness values, and the partial S1 sequence identity values were then analysed. The findings indicated partial S1 sequence identity values were more strongly correlated with protective relatedness values and than antigenic relatedness values.

Emergence of a nephropathogenic avian infectious bronchitis virus with a novel genotype in India.

We describe the emergence of a nephropathogenic avian infectious bronchitis virus (IBV) with a novel genotype in India. The Indian IBV isolate exhibited a relatively high degree of sequence divergence with reference strains. The highest homology was observed with strain 6/82 (68%) and the least homology with strain Mex/1765/99 (34.3%).