N-halamine incorporated antimicrobial nonwoven fabrics for use against avian influenza virus.

Airborne pathogens are one of the most common avenues leading to poultry diseases. Preventing the avian influenza (AI) virus from entering the chicken hatchery house is critical for reducing the spread and transmission of AI disease. Many studies have investigated the incorporation of antimicrobials into air filters to prevent viruses from entering the indoor environment. N-halamines are one of the most effective antimicrobial agents against a broad spectrum of microorganisms. In this study, 1-chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC, a variety of N-halamine) was coated on nonwoven fabrics to give the fabric antimicrobial activity against the AI virus. Results showed that MC exhibited potent antiviral activity either in suspension or in the air. Higher concentrations of MC completely inactivated AI viruses and disrupted their RNA, preventing them from being detected by the real time reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Coating the fabrics with MC resulted in remarkably reduced presence of AI virus on the MC-treated fabric in a short period of time. Furthermore, aerosolized AI viruses were completely inactivated when they passed through filters coated with the MC compound. In addition, MC is not volatile and does not release any gaseous chlorine. The active chlorine in the MC compound is stable, and the coating procedure is straightforward and inexpensive. Therefore, this study validates a novel approach to reducing airborne pathogens in the poultry production environment.

Detection and isolation of infectious laryngotracheitis virus on a broiler farm after a disease outbreak.

A broiler farm in North Alabama suffered a mild infectious laryngotracheitis (ILT) outbreak, as determined by clinical disease and PCR. The poultry integrator sought help to control further outbreaks in subsequent flocks. Samples were collected from various areas of the poultry houses on the farm over an 8-wk period. The first sampling was conducted 8 days after the infected farm was depopulated; the second was conducted 2 days prior to subsequent flock placement; and the third was conducted when the new flock was 5 wk of age. Samples were examined for ILT virus (ILTV) DNA by real-time PCR and virus isolation in embryos. The infected houses were cleaned, disinfected, heated, litter composted, and curtains replaced after the first sampling and prior to placement of the next flock. Samples from all periods were positive for ILTV DNA. However, the number of positive samples and crossing point values indicated a decrease in the amount of viral DNA, while virus isolation in embryos was successful only on the first sampling. The subsequent flock was vaccinated against ILTV by in ovo route using a commercial recombinant vaccine. Cleaning and sanitation after the disease outbreak reduced the amount of ILTV on the farm and together with in ovo vaccination of the new flock may have prevented a recurrence of another ILT outbreak.

Development of TaqMan real-time RT-PCR for detection of avian reoviruses.

Avian reoviruses (ARVs) are an important cause of economic losses in commercial poultry. A TaqMan real-time RT-PCR assay for detecting of ARVs was developed. The primer-probe set was from the conserved region of ARV S4 genome segment. Real-time RT-PCR detected ARV strains including CO8 and ss412 strains, which belonged to different serological subgroups, and the test had no cross-reaction with other avian viruses. The detection limit of this assay was 5 ARV genome copies per 5 µl and was 150 times more sensitive than traditional RT-PCR. Statistical analyses indicated excellent reproducibility. For ARV strain 2408, a titer of 50% embryo infection dose and 50% tissue culture infectious dose equivalent to 3.9 ± 0.8, and 2.9 ± 0.3 ARV genome copies, respectively. This test was rapid, specific, and sensitive for the detection of ARVs and will be useful in veterinary diagnostic laboratories and for the quantitation of vaccine viruses for pharmaceutical companies.

Evaluation of the immune response and detection of infectious bursal disease viruses by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay after in ovo vaccination of commercial broilers.

Maternal immunity can interfere with infectious bursal disease virus (IBDV) vaccine efficacy. The effect of maternal antibody (MacAb) on the immune response and detection of two vaccine viruses, an IBDV immune complex (cx) and IBDV-2512, was investigated. Vaccines were administered in ovo at 100 mean embryo infectious dose to commerical broiler embryos derived from young (29 wk) or old (63 wk) flocks. Chickens with low MatAb were challenged with an antigenic standard IBDV strain at 21 and 35 days post in ovo vaccination (PIOV). At 28 and 42 days PIOV, birds were euthanatized and bursa weight: body weight ratios were determined. MatAb of progeny from the 29-wk-old flock was higher (P < 0.05) than that from progeny of the 63-wk-old flock. Progeny titers declined by day 21 PIOV. Birds with MatAb vaccinated with either the IBDV-2512 or IBDV-Icx vaccine did not mount an antibody response by 21 days PIOV. The IBDV-Icx vaccination protected chickens against bursal atrophy when they were challenged at 21 and 35 days PIOV (83% and 77%, respectively). Resistance to challenge in the absence of antibody implies that cellular immunity plays a role in IBDV protection. The IBDV-2512 vaccine caused atrophy of the bursae, and, therefore, we could not examine its protection by these criteria. Neither vaccine could be detected by antigen-capture chemiluminescent enzyme-linked immunosorbent assay. Therefore, total RNA was extracted and reverse transcription (RT)-polymerase chain reaction (PCR) and nested PCR were conducted to amplify the VP2 (hypervariable) region on viral RNA collected at 3, 6, 9, 15, and 21 days PIOV. Nested PCR detected the VP2 region of both IBDV vaccine viruses at day 3 PIOV. Only IBDV-2512 RNA was detected on day 6 PIOV. However, the IBDV-Icx RNA was detected on days 9 and 15 PIOV but not on day 21 PIOV. The IBDV-Icx RNA was evident in tissues and could induce protection against challenge. This work gives further insight into the mechanism of the IBDV-Icx vaccine.