Brian S. Ladman

S1 gene characteristics and efficacy of vaccination against infectious bronchitis virus field isolates from the United States and Israel (1996 to 2000).

The S1 genes of isolates of avian coronavirus infectious bronchitis virus (IBV) from commercial chickens in the US and Israel (20 isolates from each country) were studied using reverse transcription-polymerase chain reaction restriction fragment length polymorphism and sequencing. Partial sequences spanning the amino terminus region of S1 from amino acid residues 48 to 219, based on the Beaudette strain, were used for analysis. Phylogenetic clustering and high-sequence identity values were used to identify isolates that appeared to be derived from live IBV vaccines used in the two countries. Novel variant strains, unrelated by S1 sequencing and restriction fragment length polymorphism analyses to reference and vaccine strains, were also identified. Based on S1 sequence identity to available vaccines, the potential to use vaccination to control IBV infections was evaluated. Vaccination with commercial live strains Massachusetts (Mass), Arkansas (Ark) or DE/072/92, generally produced immunity against vaccine-related field isolates displaying high S1 sequence similarities (≥90%) to the respective vaccine strains. Immunization with a bivalent vaccine containing the Mass and Ark strains provided good cross-protection, averaging 81% against challenge with five variant isolates from the US having amino acid identity values ranging from 62 to 69% to Mass and from 68 to 83% to Ark, respectively. In contrast, the H120 vaccine strain induced low levels of protection, ranging from 25 to 58% against variant field isolates from Israel with amino acid identity values from 65 to 67%.

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

BackgroundAvian 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.ResultsAll 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.ConclusionsThe 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.

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

BackgroundInfectious 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.ResultsInfectious 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.ConclusionPeriodical evaluation of cross-protective capabilities of IBV vaccine(s) versus recently recovered field isolates should be performed to ensure optimum control of IBV.

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

ABSTRACT 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%).

Virulence of Low Pathogenicity H7N2 Avian Influenza Viruses from the Delmarva Peninsula for Broiler and Leghorn Chickens and Turkeys

Abstract The virulence of low pathogenicity (LP) type A H7N2 avian influenza virus (AIV) isolates recovered from chickens in Delaware and the eastern shore of Maryland in 2004 was evaluated. Three-week-old leghorn- and broiler-type chickens and turkeys were inoculated via the conjunctival sac with 103.5–104.0 50% embryo infectious dose (EID50) of virus per bird with A/chicken/Delaware/Viva/04, A/chicken/Delaware/Hobo/04, and A/chicken/Maryland/Minh Ma/04. In broilers, the viruses produced respiratory signs, airsacculitis, and microscopic lesions in the trachea and lung. In contrast, signs and lesions were less severe in turkeys, and they were rarely observed in specific-pathogen-free (SPF) leghorns. In broilers and SPF leghorns, AIV peaked on day 3 postinoculation (PI), based on virus isolation and real-time reverse transcription-polymerase chain reaction, and antigen capture testing. Infection in turkeys peaked on day 7 PI. Serum antibodies generally were detected earlier in broilers (day 7 PI) than in turkeys or SPF leghorns (day 14 PI) using agar gel immunodiffusion, hemagglutination-inhibition, and the enzyme-linked immunosorbent assay. A second trial was performed to further examine the disease susceptibility of the leghorn chicken given the comparatively mild responses noted in the first trial. A 10-fold higher dose of 104.5–105.0EID50 per chick given via the conjunctival sac was used. In addition, commercial-type leghorns were tested as were chicks from the SPF leghorn source. The higher AIV dose resulted in more rapid and consistent rates of infection and higher serum antibody responses in both types of leghorn chickens. However, as observed in the first trial, clinical signs and microscopic lesions in both types of leghorns were infrequent and very mild. These findings indicate leghorn-type chickens, which are commonly used for pathogenicity assessments because of their availability, may not be the most suitable host for evaluating the virulence potential of LP AIV. Abbreviations: AGID = agar gel immunodiffusion; AI = avian influenza; AIV = avian influenza virus; CAS = chorioallantoic sac; Ct = cycle threshold; EID50 = embryo infectious dose50; ELD50 = embryo lethal dose50; ELISA = enzyme-linked immunosorbent assay; HA = hemagglutination; HI = hemagglutination-inhibition; HP = high pathogenicity; LP = low pathogenicity; NVSL = National Veterinary Service Laboratory; PBS = phosphate-buffered saline; PI = postinoculation; RRT-PCR = real-time reverse transcription-polymerase chain reaction; SPF = specific-pathogen-free Virulencia en pollos de engorde, aves leghorn y pavos, de aislados de virus H7N2 de influenza aviar de baja patogenicidad provenientes de la península de Delmarva. Se evaluó la virulencia de aislamientos del virus de influenza aviar tipo A H7N2 aislados en el año 2004 a partir de aves domésticas en los estados de Delaware y la costa este de Maryland. Aves de la línea de postura (leghorn), pollos de engorde y pavos de tres semanas de edad fueron inoculados por vía ocular con una dosis infecciosa 50% para embrión de pollo de 103.5–104.0 de los siguientes virus: A/Pollo/Delaware/Viva/04, A/Pollo/Delaware/Hobo/04 y A/Pollo/Maryland/Minh Ma/04. En los pollos de engorde los virus produjeron signos respiratorios, aerosaculitis y lesiones microscópicas en la tráquea y pulmón. En contraste, las lesiones fueron menos severas en los pavos y fueron escasamente observadas en las aves leghorn libres de patógenos específicos. Mediante aislamiento viral, la prueba de reacción en cadena por la polimerasa transcriptasa reversa en tiempo real y pruebas de captura de antígenos, se determinó que en los pollos de engorde y en las aves libres de patógenos específicos los virus de influenza aviar alcanzaron un título máximo al tercer día post inoculación. La infección en los pavos alcanzó un título máximo siete días posteriores a la infección. Utilizando la prueba de inmunodifusión en agar, la prueba de inhibición de la hemoaglutinación y la prueba de inmunoensayo asociado a enzimas, los anticuerpos séricos se detectaron más temprano en pollos de engorde (día siete post inoculación) que en los pavos o las aves libres de patógenos (14 días de edad). Se realizó un segundo experimento con la finalidad de examinar más a profundidad la susceptibilidad de las aves leghorn a los virus de influenza aviar debido a las reacciones comparativamente leves observadas en el primer experimento. Para el segundo experimento se administró en la conjuntiva una dosis 10 veces más alta (104.5–105.0dosis infecciosa 50% para embrión de pollo) por ave. Adicionalmente, se evaluaron aves tipo leghorn comerciales, así como las aves leghorn libres de patógenos específicos. La dosis mas alta del virus de influenza aviar resultó en tasas de infección más rápidas y sostenidas, así como en mayores títulos de anticuerpos séricos en ambos tipos de aves leghorn. Sin embargo, como se observó en el primer experimento, los signos clínicos y las lesiones microscópicas fueron poco comunes y muy leves en ambos tipos de aves leghorn. Estos hallazgos indican que las aves tipo leghorn, que son comúnmente utilizadas debido a su disponibilidad para las evaluaciones de patogenicidad, tal vez no sean el huésped mas adecuado para evaluar el potencial de virulencia de los virus de influenza aviar de baja patogenicidad.

Potential of Low Pathogenicity Avian Influenza Viruses of Wild Bird Origin to Establish Experimental Infections in Turkeys and Chickens

Abstract The potential of low pathogenicity (LP) avian influenza virus (AIV) isolates of wild bird origin to establish infection in commercial turkeys and broiler chickens was studied. Isolates, representing subtypes H5N1, H7N3, H6N2, and H3N6, were recovered in 2005 and 2006 from waterfowl and shorebirds in the Delmarva Peninsula region of the east coast of the United States. The LP AIV isolates were not pathogenic for 2-wk-old meat-type turkeys and broiler chickens. No mortality, clinical signs, or gross lesions were observed following intratracheal and conjunctival sac routes of exposures with 106.0 EID50 (embryo infectious dose) per bird. Isolates resulting in an established infection based on virus isolation were: A/mallard/Maryland/1159/2006 (H5N1) in the upper respiratory tract of turkeys; A/mallard/Delaware/418/2005 (H7N3) in the upper respiratory and intestinal tracts of turkeys and chickens; and A/shorebird-environment/Delaware/251/2005 (H3N6) in the upper respiratory and intestinal tracts of chickens. Infections were also confirmed by production of AIV-specific serum antibodies detected by hemagglutination inhibition.

Comparison of Pooling 11 or 5 Oropharyngeal Swabbings for Detecting Avian Influenza Virus by Real-Time Reverse Transcription–PCR in Broiler Chickens

SUMMARY. 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.

Massachusetts Live Vaccination Protects Against a Novel Infectious Bronchitis Virus S1 Genotype DMV/5642/06

Abstract Four infectious bronchitis virus (IBV) isolates were recovered from commercial broiler chicken flocks located on the Delmarva Peninsula (east coast of the United States) in the spring of 2006. Sequence analysis of the S1 subunit of the spike glycoprotein gene showed the four isolates were highly related to each other (≥99.6% nucleotide identity; ≥98.9% amino acid identity). Basic local alignment search tool analysis indicated the highest S1 amino acid identity of isolate DMV/5642/06, typical of the four Delmarva (DMV) isolates, was to CA/1737/04, an isolate obtained from broilers in California in 2004. A pathogenicity study conducted, using two-week-old commercial broilers, showed that DMV/5642/06 caused respiratory but not renal (kidney) disease. A vaccination–challenge study in three-week-old specific-pathogen-free leghorn chickens demonstrated that a commercial live attenuated IBV vaccine containing the Massachusetts strain conferred protection against challenge with DMV/5642/06 based on virus reisolation attempts and microscopic pathology.

Complete Genome Sequence of an Avian Paramyxovirus Type 4 from North America Reveals a Shorter Genome and New Genotype

ABSTRACT 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.

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

SUMMARY 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.