Improved Detection and Control of Infectious Laryngotracheitis Virus on Poultry Farms

Abstract

Infectious laryngotracheitis virus (ILTV) is an economically important disease of chickens. The disease control relies on rapid and accurate diagnosis. Traditional ILTV diagnostic and PCR methods are either time consuming or less sensitive. Therefore, a TaqMan® labeled probe real-time PCR and a novel DNA detection method, loop-mediated isothermal amplification (LAMP), were developed for ILTV DNA detection. Primers and probe for the real-time PCR and LAMP assays were designed from the same region of the ILTV ICP4 gene. They detected both chicken-embryo origin (CEO) and tissue-culture origin (TCO) propagated vaccine viruses without cross-reaction. DNA plasmids were constructed from a partial sequence of the ILTV ICP4 gene. The sensitivity of real-time PCR was 10 copies/ µl and statistical analysis indicated excellent reproducibility. Six specific primers were used for the LAMP assay and the sensitivity was 60 copies/ µl. Although the sensitivity of LAMP assay was lower than real-time PCR, it was easier to perform, less expensive, and less time consuming. The LAMP assay can be optimized for the detection of ILTV in poultry diagnostic laboratories. Water lines in commercial chicken houses can transmit avian pathogens. Biofilms in water lines can protect microorganisms from physical and chemical damage and release them in the water. In the current study, biofilms in water lines were shown to harbor ILTV that was transmitted to susceptible birds. Four sanitizers were tested for their ability to remove and/or inactivate ILTV in the water lines. Results indicated that two routinely used sanitizers, sodium hypochlorite and citric acid, were unable to inactivate ILTV. In contrast, live ILTV was inactivated by two more expensive commercial sanitizers that contained either sodium hydrogen sulfate or hydrogen peroxide. Selecting appropriate sanitizers for water line disinfection is critical for ILTV prevention and control. Water line disinfection is important because many live viral and bacterial vaccines are administered in the drinking water and without proper sanitization, these vaccines can inadvertently infect subsequent flocks. Darkling beetles, their larvae (lesser mealworm), and rodents are ubiquitous in chicken farms. They are known to transmit avian bacteria and viruses. Beetles and their larvae from ILTV infected chicken farms were collected. ILTV was detected in the insects by real-time PCR and virus isolation. The lung of one rat, captured from an ILTV positive house, was positive for ILTV DNA by real-time PCR. Results indicated that live ILTV could be isolated from the insects at least 42 days after the disease occurred on the farms. The PCR restriction fragment length polymorphism (RFLP) showed that the viruses from the infected poultry farms were of vaccine origin. This was the first investigation to demonstrate that darkling beetles, their larvae, rats, and drinking water may be contaminated with ILTV. Therefore, they might be important sources for ILTV transmission. The incidence and severity of ILTV has been reduced in many commercial farms in the US. In house composting for 5 days, litter treatment, heating house at 38°C, and rigorous biosecurity can help reduce ILTV in infected houses. However, ILT is still prevalent in chicken industry intensive regions. These experiments developed rapid, accurate, and economical methods to improve ILTV DNA detection. They also provided evidence that water lines, beetles, and rodents can carry ILTV. Appropriate sanitizers for water line disinfection and reducing the population of insects and rodents are important for the prevention and control of ILTV transmission.