Genetic characterization, pathogenicity, and protection studies with an avian adenovirus isolate associated with inclusion body hepatitis.

An avian adenovirus (AAV) was isolated from liver samples of two 2-wk-old broiler-breeder flocks obtained from grandparents vaccinated at 10 and 17 wks of age with an autogenous inactivated vaccine containing the European AAV 8 (8565 strain) and 11 (1047 strain) serotypes (AAV8/11 vaccine). Affected broiler-breeders exhibited clinical signs and macroscopic and microscopic lesions associated with inclusion body hepatitis (IBH). The isolated adenovirus, identified as Stanford, was molecularly characterized as European serotype 9. The pathogenicity of the Stanford strain was confirmed after inoculation of specific-pathogen-free (SPF) chickens at 1-7 days of age, causing 100% and 20% mortality, respectively. The level of protection against IBH was evaluated in two broiler-breeder progenies from AAV 8/11-vaccinated grandparent flocks and a commercial broiler flock by challenge at 1 or 7 days of age with the AAV 8 and 11 serotypes and/or the Stanford strain. The broiler-breeder progenies and the commercial broiler flock exhibited protection against IBH after challenge. No significant differences in mean body weights were observed at 3 wk of age in any of the evaluated groups. We conclude that broiler-breeder progenies from 30- to 50-wk-old grandparents vaccinated with the AAV 8/11 vaccine were adequately protected against challenge with the AAV 8 and 11 serotypes and the Stanford strain.

Detection of Massachusetts and Arkansas serotypes of infectious bronchitis virus in broilers.

The objective of this study was to compare the presence of the Arkansas (Ark) and Massachusetts (Mass) serotypes of infectious bronchitis virus (IBV) in the tracheas and cecal tonsils of commercial broilers after vaccination at 1 day of age by coarse spray. When given as a single serotype vaccine, the Mass strain was detected by reverse transcriptase-polymerase chain reaction (RT-PCR)-restriction fragment length polymorphism (RFLP) only in the tracheas, whereas the Ark strain was detected in both the tracheas and cecal tonsils. By in situ hybridization, the Mass and Ark nucleocapsid (Nc) genes were detected only at 7 days in the tracheas. When both strains were given in the mixed vaccine, the Mass strain was more consistently detected by RT-PCR-RFLP in the tracheas and cecal tonsils at early stages of infection (up to 14 days) and the Arkansas strain was more consistently detected at late stages of infection (21 and 28 days). By in situ hybridization, the IBV Nc gene was more consistently detected in the trachea at early stages of infection (7, 14, and 21 days) and in the cecal tonsils at late stages of infection (21, 28, and 35 days). In general, the Mass strain was more frequently recovered from the tracheal and cecal tonsil tissues at earlier stages of infection and the Ark strain was recovered at later stages of infection.

Influence of orally administered CpG-ODNs on the humoral response to bovine serum albumin (BSA) in chickens.

Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have been reported to be effective mucosal adjuvants in mice when given orally. Studies on their effectiveness in chickens are currently very limited. This study investigated whether CpG-ODNs could adjuvant the immune response to BSA when given orally to a commercial line of SCWL chickens. In two experiments, performed over time, chickens were given selected concentrations of CpG-ODNs with BSA followed by 6 consecutive days of ad libitum access to drinking water containing 1.4 mg/ml BSA. Serum responses, and in some cases intestinal specific antibodies, were measured out to 33 days post-immunization. Birds receiving a single dose of CpG-ODN had consistently higher IgG, IgM, and IgA titers in the serum, dependent upon dose, and in specific areas of the intestine when compared to the non-immunized and BSA only groups. These findings suggest that a single oral CpG-ODN administration can accelerate the kinetics of antigen specific antibodies of all three isotypes in commercial-strain chickens immunized via the drinking water using common protein antigen.

Molecular characterization of infectious bursal disease virus from commercial poultry in the United States and Latin America.

From June 1999 to September 2001, 216 bursal samples from broiler farms in the United States and from countries of Latin America were submitted to the Poultry Diagnostic and Research Center at the University of Georgia for the purpose of genotyping field infectious bursal disease viruses (IBDVs). The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify a 248-bp product, encompassing the hypervariable region of VP2 gene. The genotyping was conducted by restriction fragment length polymorphism (RFLP) analysis with six restriction endonucleases, DraI, SacI, TaqI, Sty, BstNI, and SspI. For the 150 samples received from the United States, 125 samples (83.3%) were RT-PCR positive for the presence of IBDV. One hundred positive samples (80%) had RFLP identical to the variant Delaware E strain, whereas 10 samples (8.0%) exhibited a RFLP pattern similar to this antigenic variant. Other IBDV strains such as Grayson Laboratory strain (GLS), Lukert, PBG-98, Delaware A, and the vaccine strains Sal-1 and D-78 were also detected. Two samples exhibited a pattern similar to the standard challenge (STC) strain, and seven strains (5.6%) were not classified by RFLP. Sixty-six bursal samples previously inactivated with phenol were received from Latin American countries. IBDV strains with analyzed genotypes similar to the Lukert strain were predominantly detected in Mexico. IBDV strains similar to variant E were detected in Colombia and Ecuador. Peru and Venezuela exhibited a higher heterogeneity of IBDV strains due to the detection of classic Delaware type as well as GLS variant strains. IBDV strains detected from Brazil and Dominican Republic exhibited RFLP patterns identical to very virulent IBDV strains prevalent in several countries in Europe, Asia, and Africa.

Evaluation of the protection conferred by commercial vaccines against the California 99 isolate of infectious bronchitis virus.

An infectious bronchitis virus (IBV) was isolated from commercial broilers from the state of California exhibiting respiratory distress, inflamed tracheas, airsaculitis, and edematous lungs. After reverse transcriptase-polymerase chain reaction (RT-PCR), the California isolate exhibited an identical restriction fragment length polymorphism (RFLP) pattern to some isolates obtained from California, known as California 99 isolates. Commercial Mass-Conn and Mass-Ark vaccines were used to vaccinate commercial broiler chickens via eye drop once at 1 or 10 days of age or twice at 1 and 10 days of age. At 27 days of age the birds were challenged via eye drop with the isolated IBV California 99 strain. Protection was measured by failure to reisolate the challenge virus from tracheas 5 days postchallenge and complemented withthe tracheal and epithelium thickness scores. When the Mass-Ark vaccine was included in the vaccination programs, there was protection against challenge with the IBV California 99 isolate. The Mass-Conn vaccine conferred protection when used once at 1 day of age and twice at 1 and 10 days of age. However, no total protection was achieved when used as the only vaccine at 10 days of age, since one of the replicates was positive for virus isolation. Significant differences (P < 0.05) in the epithelium thickness and tracheal scores were observed between the unvaccinated-unchallenged group and the groups vaccinated once or twice with the Mass-Conn vaccine. Based on these results, all chickens were protected against the California 99 isolate when the IBV Arkansas type was used as a vaccine.

Molecular characterization of Spanish infectious bursal disease virus field isolates.

Nine Spanish isolates of infectious bursal disease virus (IBDV) were characterized and classified after reverse transcriptase-polymerase chain reaction of a 248-bp fragment of the VP2 gene hypervariable region and restriction fragment length polymorphism (RFLP). The restriction endonucleases (REs) used were BstNI, Sad, SspI, TaqI, DraI, and StyI. Sequencing of the amplified product and further comparison of these sequences with published sequence data from other IBDV strains were also performed. Very virulent and classic strains were identified. None of the strains identified had molecular characteristics similar to that of the American variant strains. Four very virulent strains (VG-248, 5939, 6145, and 7333) were digested by the TaqI, SspI, and StyI enzymes. The sequences of these strains were closely related to other European and Japanese very virulent IBDV (vvIBDV) strains. Strains VG-311, VG-262, and VG-208 were digested by the BstNI and Sad REs and were classified as classic strains. Strains VG-276 and VG-313 had unique RFLP patterns. VG-276 exhibited the SspI RE site, which has been reported as a characteristic of vvIBDV strains, whereas the VG-313 strain exhibited a Sad and StyI RE site indicative of the classic IBDV Edgar and 52-70 strains. However, nucleotide sequence analysis of the amplified hypervariable region strain VG-276 revealed a higher identity with the classic strains STC, 52/70, and 9109 IBDV strains, whereas strain VG-313 exhibited a higher identity with the vvIBDV strains.

Lack of interaction between avian leukosis virus subgroup J and fowl adenovirus (FAV) in FAV-antibody-positive chickens.

Unfounded field speculation has suggested that avian leukosis virus subgroup J (ALV-J) predisposes young meat-type chickens to inclusion body hepatitis caused by fowl adenovirus (FAV). To address this hypothesis, we infected 1-day-old grandparent meat-type chickens carrying maternal antibodies against FAV with a field isolate of FAV associated with inclusion body hepatitis in broilers, ALV-J, or both FAV and ALV-J. We examined the effects of FAV alone or in combination with ALV-J on the basis of clinical signs, overall mortality, growth rate, and gross and microscopic lesions. With such criteria for evaluating possible interactions, we found no significant differences in the dually infected birds in comparison with chickens that received a monovalent challenge with either FAV or ALV-J.

Molecular characterization of seven field isolates of infectious bursal disease virus obtained from commercial broiler chickens.

Specific-pathogen-free sentinel birds were used as an initial biological system to isolate infectious bursal disease virus (IBDV) field isolates from commercial broiler farms exhibiting recurrent respiratory problems and poor performance. Reverse transcription (RT)-polymerase chain reaction (PCR) was used to amplify a 248-bp product encompassing the hypervariable region of the IBDV VP2 gene. Restriction fragment length polymorphism (RFLP) analysis of the RT-PCR products was performed with the restriction endonucleases DraI, SadI, TaqI, StyI, BstNI, and SspI. Two isolates (619 and 850) exhibited a RFLP pattern characteristic of Delaware variant E IBDV. Restriction enzyme digestion for four isolates (625, 849, 853, and 11,153) revealed unmatched RFLP patterns when compared with reference IBDV strains. Nucleotide and deduced amino acid sequence analyses of the VP2 hypervariable region for these six isolates revealed identity (96.3% up to 98%) with Delaware E variant IBDV strain. However, serine at position 254, which is characteristic of Delaware variant strains, was substituted by asparagine in these six isolates. The seventh IBDV isolate (9109) also exhibited a unique RFLP pattern, which included the SspI restriction site, which is characteristic of very virulent (vv) IBDV strains. Nucleotide and amino acid sequence analyses of the hypervariable region for this isolate revealed identity (90%) with the standard challenge strain. However, the leucine residue at position 294 was substituted by isoleucine. This substitution corresponds to one of the amino acids that are conserved in the vvIBDV strains. Antigenic index studies of the predicted amino acid sequence of the hypervariable region of VP2 from isolates 619, 625, 849, 850, 853, and 11,153 exhibited a profile almost identical to variant E, whereas the isolate 9109 exhibited a profile characteristic of standard IBDV strains.

Molecular characterization of Brazilian infectious bursal disease viruses.

A reverse transcriptase-polymerase chain reaction (RT-PCR) procedure was used to amplify a VP2 gene fragment (248 bp) from infectious bursal disease virus (IBDV). The procedure allowed the detection of known IBDV strains from the United States, along with field isolates and commercial vaccines produced in Brazil. Amplified VP2 fragments were further characterized by restriction fragment length polymorphism (RFLP) analysis. From 55 Brazilian commercial flocks, 48 field samples were IBDV positive by RT-TCR. Vaccine RFLP patterns were found in 12 flocks, a pattern compatible with classic IBDV in one flock, four new patterns in 31 flocks, and a pattern compatible with very virulent (vv) IBDV in four flocks. Sequence analysis showed that the vvIBDV RFLP patterns were closely related to the vvIBDVs described in Europe and Asia. Phylogenetic analysis of the four new RFLP patterns showed that they were closely related to but distinct from other classic, variant, and vvIBDVs, suggesting a high prevalence of different IBDV strains in Brazilian commercial flocks.

Adenovirus pathogenicity in immature ostriches.

Two group I avian adenoviruses implicated as the possible cause of "fading chick syndrome" in ostriches less than 8 wk of age were isolated in primary chicken embryo liver cells. These viruses were identified by virus neutralization and further characterized by a pathogenicity trial in immature ostriches. The results showed that these isolates were noninfectious in ostrich chicks.

Detection of infectious bursal disease virus in experimentally infected chickens by in situ hybridization.

In situ hybridization was used in a pathogenesis study of three vaccine pathotypes (Delaware variant A, D78, and BursaVac) of infectious bursal disease virus (IBDV). Tissues were excised (bursa, thymus, spleen, proventriculus, and cecal tonsils), fixed in formalin, and paraffin embedded at 12, 24, 48, 72, and 120 hr postinoculation (HPI). With an antisense VP2 gene probe, viral nucleic acid was detected in bursas from both D78- and BursaVac-infected chickens at 24, 48, 72, and 120 HPI. However, viral RNA was detected only in the Delaware variant A-infected birds at 72 HPI. Thymus and spleen were positive in the D78-infected birds at 48 HPI and in the BursaVac-inoculated group at 72 HPI. Viral nucleic acid was not present in detectable levels among any of the tissues tested at 12 HPI. However, by 24 hr, scattered positive lymphoid cells were visualized in the bursal follicles of chickens infected with D78 and BursaVac. In addition, low levels of viral nucleic acids were detected in the thymus and spleen among the D78- and BursaVac-infected birds. The sites of viral replication were consistent between the two vaccine-infected groups (D78 and BursaVac), whereas the chickens infected with Delaware variant A had limited IBDV replication in the bursa.

Genomic identification and characterization of avian adenoviruses associated with inclusion body hepatitis.

Four pathogenic avian adenovirus isolates associated with inclusion body hepatitis and mortality in commercial broiler chicks and chickens were characterized and identified. These group I avian adenovirus isolates were classified as group E (serotypes 6, 7, 8, and 9) avian adenoviruses on the basis of the restriction enzyme patterns of their viral DNA. Isolate 3718 was neutralized by a serotype 6 reference avian adenovirus antiserum and isolates 8193, 8380, and 8565 were all neutralized by a serotype 8 reference avian adenovirus antiserum by virus neutralization assays. Infectivity and virulence such as mortality, hemorrhages, enlarged green livers with intranuclear inclusion bodies, stunting, intestinal sloughing, and poor feathering were observed in specific-pathogen-free chicken embryos and were identical for all four isolates when embryos were inoculated via the yolk sac and/or chorioallantoic membrane. Complete mortality was observed within 72 hr postinoculation in specific-pathogen-free (SPF) chickens inoculated intramuscularly for all four avian adenovirus isolates.

Efficacy comparisons of disinfectants used by the commercial poultry industry.

Several commercially available disinfectants used by the poultry industry were evaluated for their effectiveness against selected bacteria and viruses. When tested in the absence of organic matter, most disinfectant products were effective at the manufacturer's recommended level within 10 min of contact time. However, when organic matter was present, longer contact times and/or higher disinfectant dosages were needed to maintain effectiveness. Pseudomona aeruginosa and infectious laryngotracheitis virus were very resistant organisms in the presence of organic matter. Evaluation of disinfectant efficacy against several microbials in the absence or presence of organic matter was highly practical, flexible, and reproducible.

A rapid-plate hemagglutination assay for the detection of infectious bronchitis virus.

A rapid-plate hemagglutination (HA) test to detect infectious bronchitis virus (IBV) in allantoic fluid of embryonated eggs was introduced into routine procedures for IBV identification. This system was tested in 468 diagnostic cases received by the Poultry Diagnostic and Research Center at the University of Georgia. Allantoic fluids from inoculated embryos were harvested and treated with commercially available neuraminidase enzyme. IBV in neuraminidase-treated allantoic fluid was identified by clear and consistent HA of chicken red blood cells within 1 min of incubation. The specificity of the neuraminidase rapid-plate HA assay was examined with other avian viruses in individual and dual embryonic infections. Sensitivity of this test was compared with embryo lesions and reverse transcriptase-polymerase chain reaction (RT-PCR). The rapid-plate HA assay of neuraminidase-treated allantoic fluid correlated with the RT-PCR during the early stages of IBV detection, identification, and isolation in embryonated eggs.