Characterization of infectious bursal disease viruses isolated in 2007 from Delmarva commercial broiler chickens.

A study was performed in 2007 to isolate and characterize infectious bursal disease viruses (IBDVs) in commercial broilers grown in the Delmarva (DMV) Peninsula region of the United States. Bursae of Fabricius were collected weekly from 1 to 4 wk of age from broilers on 10 farms with a history of poor performance. Microscopic pathology was used to determine the infectious bursal disease (IBD) status of the broilers. Bursae from 1- and 2-wk-old broilers did not show IBD microscopic lesions. Moreover, broilers on 1 of the 10 farms were IBD lesion free at 3 and 4 wk of age. However, 3 of 9 and 9 of 9 farms yielded broilers with IBD-affected bursae from 3- and 4-wk-old commercial broilers, respectively. Ten IBDV isolates were recovered from 3 of 3 lesion-positive bursal pools at 3 wk of age and 7 of 9 lesion-positive bursal pools at 4 wk of age. Analysis of the viral protein (VP) 2 genes identified all isolates as serotype 1 Delaware (Del) variant viruses. Five field isolates, each representing different molecular clades of the Delaware variant viruses, were selected for further study. Experimental infection of specific-pathogen-free white leghorn chickens with isolates DMV/4813/07, DMV/4947/07, DMV/4955/07, DMV/5038/07, and DMV/5041/07 produced gross and microscopic pathology of the bursa consistent with Delaware variant infection. Monoclonal antibody testing showed DMV/4813/07, DMV/4947/07, DMV/ 4955/07, and DMV/5041/07 to be similar to previous recognized variant viruses. However, DMV/5038/07 was found to be unreactive with the monoclonal antibodies that typically recognize reference strains STC, Del E, GLS, RS593, and AL2. In a challenge of immunity study, 10-day-old progeny from breeders immunized with a commercially available inactivated IBDV vaccine containing the Del E and classic strains were protected to a lesser degree against isolate DMV/5038/07 compared to Del E challenge based on microscopic lesion scores (P < 0.01) of the bursa. This result suggests the virus is antigenically different from the Del E strain contained in the vaccine. Collectively, the monoclonal antibody and progeny challenge of immunity findings suggest DMV/5038/07 is antigenically different from the Del E strain contained in the vaccine.

Potential of low pathogenicity avian influenza viruses of wild bird origin to establish experimental infections in turkeys and chickens.

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 10(6.0) 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.

Inactivation of avian influenza virus using four common chemicals and one detergent.

Five disinfectant chemicals were tested individually for effectiveness against low pathogenic avian influenza virus (LPAIV), A/H7N2/Chick/MinhMa/04, on hard, nonporous surfaces. The tested agents included acetic acid, calcium hydroxide, sodium carbonate, sodium hydroxide, and a powdered laundry detergent without bleach. Multiple common chemicals including acetic acid (1 and 3%), sodium hydroxide (2%), and calcium hydroxide (1%) effectively inactivated LPAIV on a metal surface. The laundry detergent without bleach, sodium carbonate (4%), and the lower concentration of sodium hydroxide (1%) were not able to consistently inactivate LPAIV on hard, nonporous surfaces.

Massachusetts live vaccination protects against a novel infectious bronchitis virus S1 genotype DMV/5642/06.

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 (> or = 99.6% nucleotide identity; > or = 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.

Inactivation of avian influenza virus using common detergents and chemicals.

Six disinfectant chemicals were tested individually for effectiveness against low pathogenic avian influenza virus (LPAIV) A/H7N2/Chick/MinhMa/04. The tested agents included acetic acid (C2H4O2), citric acid (C6H8O7), calcium hypochlorite (Ca(ClO)2), sodium hypochlorite (NaOCl), a powdered laundry detergent with peroxygen (bleach), and a commercially available iodine/acid disinfectant. Four of the six chemicals, including acetic acid (5%), citric acid (1% and 3%), calcium hypochlorite (750 ppm), and sodium hypochlorite (750 ppm) effectively inactivated LPAIV on hard and nonporous surfaces. The conventional laundry detergent was tested at multiple concentrations and found to be suitable for inactivating LPAIV on hard and nonporous surfaces at 6 g/L. Only citric acid and commercially available iodine/acid disinfectant were found to be effective at inactivating LPAIV on both porous and nonporous surfaces.

Virulence of low pathogenicity H7N2 avian influenza viruses from the Delmarva peninsula for broiler and leghorn chickens and turkeys.

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 10(3.5)-10(4.0) 50% embryo infections 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 10(4.5)-10(5.0)EID50 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.

Evaluating viral interference between infectious bronchitis virus and Newcastle disease virus vaccine strains using quantitative reverse transcription-polymerase chain reaction.

The potential for infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) replication interference was evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Fourteen-day-old broiler chickens were inoculated via eyedrop with live commercial vaccine strains of IBV and NDV alone or in combination to directly evaluate IBV and NDV replication in the trachea at 1, 3, and 5 days after vaccination. Commercial NDV vaccine strains used were B1, VG/GA, and C2. The vaccine strains of IBV tested were Massachusetts (Mass) and Arkansas (Ark). The NDV + Mass vaccines used were commercially manufactured combined products. The NDV + Ark vaccines used were commercial vaccines manufactured as single entity products that were administered by eyedrop to opposite eyes of each chicken. As measured by qRT-PCR, the replication of NDV strains B1, VG/GA, and C2 did not interfere with the growth of IBV Mass and Ark strain vaccines in the combined vaccine treatment groups. Combination vaccinations using B1 and VG/GA did not interfere with IBV immunity based on challenge or serum antibody production. In the C2 + Mass vaccination trial, IBV immunity after challenge was reduced, but it did not seem to be a result of reduced Mass vaccine growth or the ability of the Mass vaccine to induce serum IBV antibody. In contrast, the replication of IBV strains Mass and Ark interfered with the growth of NDV strains B1, VG/GA, and C2 as measured by qRT-PCR. However, interference with NDV replication was not reflected in a reduction in Newcastle disease challenge of immunity findings when combination Mass + NDV products manufactured by vaccine companies were tested. Moreover, NDV immunity was not compromised in two of three trials using single entity vaccines of NDV and Ark IBV vaccines manufactured separately but administered simultaneously. However, in one trial, NDV immunity was decreased where a NDV single entity product (C2) was given with an IBV single entity Ark vaccine. This finding emphasizes the importance of using manufactured combination vaccines whenever possible to avoid potential interference.

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 (> or = 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%.

Nephropathogenic infectious bronchitis in Pennsylvania chickens 1997-2000.

Nephropathogenic infectious bronchitis (NIB) was diagnosed in 28 infectious bronchitis virus (IBV)-vaccinated commercial chicken flocks in Pennsylvania from December 1997 to July 2000. Early dinical signs were increased flock mortality and urinary water loss (polyuria and pollakiuria) leading to wet litter. Daily mortality ranged from 0.01% in layers to 2.45% in broilers, with total broiler mortality as high as 23%. Severe renal swelling and accumulation of urates in the tubules were commonly seen. Visceral gout and urolithiasis were less frequently observed. Histopathologic changes included characteristic tubular epithelial degeneration and sloughing with lymphoplasmacytic interstitial nephritis. Minimal respiratory disease signs were noted in broilers. Egg production and shell quality declined in layers. Confirmatory diagnosis of NIB was made by IBV antigen-specific immunohistochemical staining of the renal tubular epithelium and virus isolation. Sequencing of the S1 subunit gene of 21 IBV isolates showed the NIB outbreak to be associated with two unique genotypes, PA/Wolgemuth/98 and PA/171/99. The cases from which the genotypes were isolated were clinically indistinguishable. The NIB viruses were unrelated to previously recognized endemic strains in Pennsylvania and were also dissimilar to each other. Genotype PA/Wolgemuth/98 was isolated almost exclusively during the first 14 mo of the outbreak, whereas PA/171/99 was recovered during the final 18 mo. The reason for the apparent replacement of PA/Wolgemuth/98 by PA/171/99 is not known.

Protection of chickens after live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus PA/Wolgemuth/98.

Protection provided by live and inactivated virus vaccination against challenge with the virulent nephropathogenic infectious bronchitis virus (NIBV) strain PA/Wolgemuth/98 was assessed. Vaccinations with combinations of live attenuated strains Massachusetts (Mass) + Connecticut (Conn) or Mass + Arkansas (Ark) were given by eyedrop to 2-wk-old specific-pathogen-free leghorn chickens. After live infectious bronchitis virus (IBV) vaccination, some chickens at 6 wk of age received an injection of either an oil emulsion vaccine containing inactivated IBV strains Mass + Ark or an autogenous vaccine prepared from NIBV PA/Wolgemuth/98. Challenge with PA/Wolgemuth/98 was given via eyedrop at 10 wk of age. Serum IBV enzyme-linked immunosorbent assay antibody geometric mean titers (GMTs) after vaccination with the combinations of live attenuated strains were low, ranging from 184 to 1,354, prior to NIBV challenge at 10 wk of age. Both inactivated vaccines induced an anamnestic response of similar magnitudes with serum GMTs of 6,232-12,241. Assessment of protection following NIBV challenge was based on several criteria virus reisolation from trachea and kidney and renal microscopic pathology and IBV-specific antigen immunohistochemistry (IHC). Live attenuated virus vaccination alone with combinations of strains Mass + Conn or Mass + Ark did not protect the respiratory tract and kidney of chickens after PA/Wolgemuth/98 challenge. Chickens given a live combination vaccination of Mass + Conn and boosted with an inactivated Mass + Ark vaccine were also susceptible to NIBV challenge on the basis of virus isolation from trachea and kidney butshowed protection on the basis of renal microscopic pathology and IHC. Live IBV-primed chickens vaccinated with an autogenous inactivated PA/Wolgemuth/98 vaccine had the highest protection against homologous virulent NIBV challenge on the basis of virus isolation.

Novel infectious bronchitis virus S1 genotypes in Mexico 1998-1999.

Seventeen infectious bronchitis virus (IBV) field isolates recovered from commercial broiler flocks in Mexico were identified by reverse transcription-polymerase chain reaction cycle sequencing of the S1 gene. The isolates were obtained from broilers on farms from the neighboring states of Queretaro and San Luis Potosi in 1998 and 1999. Flocks had an ongoing history of bacterial-complicated respiratory disease with mortality rates as high as 28% in spite of receiving live vaccinations for Massachusetts and Connecticut strains of IBV. Sequence analysis of the S1 gene identified two unique genotypes that have been described, as of this time, only in Mexico and thus appear to represent strains indigenous to the country. The Mex/1765/99 genotype was isolated from 64% (11/17) of the respiratory disease outbreaks. Three isolates (18%) were similar to the BL-56 genotype, a unique Mexican IBV strain observed initially in 1996. In addition to the two indigenous strains, three isolates (18%) were found to be the Connecticut genotype.

Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S-1 gene.

Direct automated cycle sequencing (DACS) of a reverse transcription-polymerase chain reaction (RT-PCR) product of the S-1 subunit of the spike peplomer gene was used to identify infectious bronchitis virus (IBV) serotypes. Degenerate primers CK4 and CK2, utilized previously in our laboratory, were selected for DACS because they successfully amplify a wide range of serotypes represented by various reference strains and field isolates and the resulting polymerase chain reaction (PCR) product contains diagnostically relevant S-1 sequences that can be used to identify the serotype of IBV. The S-1 nucleotide sequences generated by DACS were aligned and analyzed with commercial software to determine their relationship to the S-1 nucleotide sequences of IBV strains on deposit in the GenBank and EMBL databases. Reference strains Massachusetts (Mass) 41, Connecticut (Conn), Arkansas (Ark) DPI, JMK, and DE/072/92 were initially tested by DACS to establish the feasibility of the procedure. The DACS procedure was further evaluated with a panel of "unknowns" comprised of IBV reference strains, field isolates, and variant serotypes collected by our laboratory. The DACS procedure provided high-quality and reproducible S-1 sequence for all IBV serotypes tested, including variant serotypes that had not been sequenced previously. The S-1 nucleotide sequences for the amplified PCR products of reference strains Mass 41, Conn, Ark DPI, JMK, and DE/072/92 generated by DACS were highly homologous (>99% nucleotide identity) with their respective GenBank database sequences. In the unknown panel, the nucleotide identities of the DACS S-1 sequences of field isolates of serotypes previously identified by virus neutralization were also found to be very high (> or = 95.5%) after alignment with database sequences. In contrast, the nucleotide identities of S-1 sequences of variant serotypes 37, 3330, and PA/1220/98 and reference strain Clark 333, for which database sequences were not available, ranged from 27.7% to 73.8%, well below the identity values for a homologous serotype. With alignment software, the identities of strains in mixtures of RNAs of two different serotypes were not resolvable. DACS of IBV S-1 RT-PCR products will enable researchers to rapidly identify field strains, including new, previously unrecognized variant virus serotypes.