Protection conferred by coarse spray vaccination against challenge with infectious bursal disease virus in commercial broilers.

The efficacy of coarse spray vaccination against pathogenic infectious bursal disease virus (IBDV) in commercial broilers was evaluated. Different coarse spray vaccination schedules using a commercial 2512 strain vaccine were compared with single or double drinking water application at 1 and/or 10 days of age. At 29 days of age, the chickens were challenged with the virulent Edgar strain of IBDV. Seven days postchallenge, severe gross bursal atrophy was observed in the unvaccinated-challenged birds. After challenge and regardless of the method of vaccination used, moderate-to-severe lymphoid depletion was observed, indicating challenge virus replication, later confirmed by reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analysis. Coarse spray and drinking water vaccination induced protection against body weight loss. Significant differences (P < 0.05) were observed between the unvaccinated-challenged group (1483 g) and the birds vaccinated at 10 days of age by coarse spray (1812 g). The coarse spray vaccination also induced protection against challenge-induced gross bursal atrophy, as determined by bursal index values. After challenge, significant bursal atrophy was observed in the birds orally vaccinated at 1 day (0.61), 10 days (0.66), and 1 and 10 days (0.63) as well as the unvaccinated-challenged birds (0.62), but not in the coarse-spray-vaccinated groups that exhibited bursal indexes above 0.70 and did not differ from the unvaccinated-unchallenged control group. These results suggest that coarse spray vaccination can be considered as another tool to control IBDV in the field.

The VG/GA strain of Newcastle disease virus: mucosal immunity, protection against lethal challenge and molecular analysis.

The Villegas-Glisson/University of Georgia (VG/GA) strain of Newcastle disease virus (NDV) isolated from the intestine of healthy turkeys has been proposed to replicate in the respiratory and intestinal tract of chickens. In the present study, the virus distribution, the mucosal and systemic immune response, the efficacy against lethal challenge and the full genome sequence of the VG/GA strain were compared with the La Sota strain of NDV. The VG/GA strain was detected at different time points in the respiratory and intestinal tract of chickens with a preferential tropism for the latter. Both the VG/GA and La Sota strains induced NDV-specific immunoglobulin A (IgA) at the upper respiratory tract. IgA levels were higher in the trachea for the La Sota strain, while they were higher in the bile and intestine for the VG/GA strain. Positive correlation between virus distribution of the viruses and IgA production was observed. Despite the presence of the maternal antibodies in broilers, early vaccination with the VG/GA strain afforded 95% to 100% protection against lethal challenge, equivalent to the protection conferred by the La Sota strain. Full genome sequence analysis classified the VG/GA strain within class II, genotype II viruses, which also include most of the respirotropic vaccine strains. Differences with the La Sota strain at the nucleotide and amino acid levels that may explain the differential phenotype of the VG/GA were observed; however, verification of the significance of those changes is required. Taken together, these results validate field observations on the efficacy of VG/GA vaccination and demonstrated the unique characteristics of the strain.

Evaluation of FTA paper and phenol for storage, extraction and molecular characterization of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is an important poultry pathogen and is distributed world wide that can cause immune suppression and lesions of the bursa of Fabricius. The main component of the virus, VP2, is not only responsible for the bird's immune response, but is important for the molecular identification of this virus as well. The nucleic acid of the virus must be adequately preserved to be analyzed by reverse-transcriptase PCR (RT-PCR) and sequenced for the molecular characterization of the field strain. Phenol inactivation has been the standard for IBDV tissue collection and international shipment; however, there have been some reports of interference with molecular detection capabilities when using phenol. Phenol is also a hazardous chemical and must be handled and shipped carefully. The ability to use the Flinders Technology Associates filter paper (FTA card) for inactivation of several avian pathogens has been proven previously, however no work has been published on its use in IBDV nucleic acid detection. Bursas from experimentally infected birds was imprinted on FTA cards, and then placed in phenol. Samples were evaluated and compared based on molecular detection capabilities between the two inactivation methods. The nucleic acid of the virus was detected in 85% of the FTA card inactivated samples compared to 71% in the phenol inactivated samples. Sequence analysis was performed on samples inactivated by both methods and no differences were found. When comparing the RNA stability at different temperatures, euthanized IBDV infected birds were held at two different temperatures before sampling. No differences were detected for FTA sampling; however, for tissues in phenol the nucleic acid was only detectable up to 2 h post-mortem in the tissues held at 4 degrees C prior to sampling. These findings indicate that the FTA card is an efficient and reliable alternative collection method for molecular detection and characterization of IBDV.

Use of FTA filter paper for the molecular detection of Newcastle disease virus.

The feasibility of using Flinders Technology Associates filter papers (FTA cards) to collect allantoic fluid and chicken tissue samples for Newcastle disease virus (NDV) molecular detection was evaluated. Trizol RNA extraction and one-step reverse transcriptase-polymerase chain reaction (RT-PCR) were used. FTA cards allowed NDV identification from allantoic fluid with a titre of 10(5.8) median embryo lethal doses/ml. The inactivated virus remained stable on the cards for 15 days. NDV was detected from FTA imprints of the trachea, lung, caecal tonsil and cloacal faeces of experimentally infected birds. RT-PCR detection from FTA cards was confirmed by homologous frozen-tissue RT-PCR and virus isolation. Direct nucleotide sequence of the amplified F gene allowed prediction of NDV virulence. No virus isolation was possible from the FTA inactivated samples, indicating viral inactivation upon contact. The FTA cards are suitable for collecting and transporting NDV-positive samples, providing a reliable source of RNA for molecular characterization and a hazard-free sample.