Evaluating two approaches for using positive control in standardizing the avian influenza H5 reverse transcription recombinase polymerase amplification assay.

Highly pathogenic avian influenza H5N1 virus causes heavy losses in poultry farms worldwide. Molecular diagnostic techniques like RT-PCR and real-time RT-PCR are considered the gold standard for identification of H5 influenza viruses in clinical samples. These techniques are hampered by the need of well-equipped laboratories, large space requirement, and relatively long time-to-result. Recombinase polymerase amplification (RPA) assay represents an excellent alternative to PCR since it is more simple, rapid, economic, and portable. Reverse transcription RPA (RT-RPA) assay was recently developed for sensitive and specific detection of H5N1 virus in 6-10 min. To ensure the accuracy of the developed assay, two approaches for using a positive control were evaluated in this study. These approaches included: 1) all-in-one (internal positive control; IPC), 2) two-tubes-per-one-sample (external positive control; EPC). Sigma virus (SIGV) RNA and turkey mitochondrial DNA were tested as positive controls in both approaches. For all-in-one approach, both targets (H5 and IPC) were strongly inhibited. In contrast, very good amplification signals were obtained for the two types of EPC with no effect on the analytical sensitivity and specificity of H5 RT-RPA assay in two-tubes-per-one-sample approach. The performance of EPC-based H5 RT-RPA was further validated using 13 tracheal swabs. The results were compared to real-time RT-PCR and proved superior specificity in detecting H5N1 but not H5N8 viruses. Inclusion of EPC did not affect the aptitude of both assays in terms of sensitivity, specificity and reproducibility. In conclusion, the two-tubes-per-one-sample approach was more reliable to control the false negative results in H5 RT-RPA assay.

Oral administration of hyperimmune IgY: an immunoecological approach to curbing acute infectious bursal disease virus infection.

Infectious bursal disease (IBD) is one of the most important viral diseases of poultry. Hygienic management and proper vaccination are currently the only economic approach for control of this disease. Attempts have been made to control the disease after the onset of an outbreak using parenteral administration of hyperimmune IgY preparations. Such attempts are usually cumbersome and time consuming with an overall reduced economic return. We investigated the use of oral administration of hyperimmune chicken IgY to control IBDV outbreaks early after their discovery in poultry farms. Our approach attempted to change the environmental viral load around susceptible birds and, to modify the host's initial immune-contact with the virulent virus and the subsequent balance of the immune response to that virus. An experimental exposure/protection model that simulates a natural infection in susceptible populations was developed. IBDV hyperimmune yolk was orally administered to a group of IBDV-exposed susceptible layer chicks via drinking water. Disease patterns and mortality rates were monitored up to 10 days post exposure and compared to that in the exposed-untreated group of the same breed and age. Mortality rates dropped by 66.6% in the exposed-treated group compared to the control exposed-untreated group. Similarly, the morbidity shifted towards a milder syndrome in the exposed-treated group as compared to the control exposed-untreated group. To our knowledge, this is the first report of a successful control of an experimental IBDV infection in susceptible poultry populations using oral administration of hyperimmune yolk preparations.

The immune response to bovine viral diarrhea virus: a constantly changing picture.

Bovine viral diarrhea virus (BVDV) is one of the major immuno-suppressive viruses of cattle. The effect on the innate and acquired immune system is unique and results in dramatic immune dysfunction. BVDV infection also has the ability to cause persistent infection (PI) in the developing fetus. This Pl syndrome creates a requirement for high levels of BVDV immunity from vaccines to prevent these infections. BVDV vaccines and their future development continue to be an enigma in the control of BVDV.