Abstract
Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens. In this study, a real-time PCR method was developed for fast and accurate detection and quantitation of ILTV DNA of chickens experimentally infected with ILTV strain LJS09 and naturally infected chickens. The detection lower limit of the assay was 10 copies of DNA. There were no cross reactions with the DNA and RNA of infectious bursal disease virus, chicken anemia virus, reticuloendotheliosis virus, avian reovirus, Newcastle disease virus, and Marek's disease virus. The real-time PCR was reproducible as the coefficients of variation of reproducibility of the intra-assay and the inter-assay were less than 2%. The real-time PCR was used to detect the levels of the ILTV DNA in the tissues of specific pathogen free (SPF) chickens infected with ILTV at different times post infection. ILTV DNA was detected by real-time PCR in the heart, liver, spleen, lung, kidney, larynx, tongue, thymus, glandular stomach, duodenum, pancreatic gland, small intestine, large intestine, cecum, cecal tonsil, bursa of Fabricius, and brain of chickens in the infection group and the contact-exposure group. The sensitivity, specificity, and reproducibility of the ILTV real-time PCR assay revealed its suitability for detection and quantitation of ILTV in the samples from clinically and experimentally ILTV infected chickens.
Highlights
Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens, which was first described in the USA in 1925 [1]
DNA was extracted from the specimen infected chicken embryo kidney (CEK) cells for polymerase chain reaction (PCR) analysis using a pair of gB Forward/Reverse primers designed based on the infectious laryngotracheitis virus (ILTV) gB gene
Isolation and identification of ILTV Larynx tissue of the ILTV infected chicken was full of caseous exudates (Fig. 1), which was in agreement with the clinical signs before death
Summary
Infectious laryngotracheitis (ILT) is an acute, highly contagious upper-respiratory infectious disease of chickens, which was first described in the USA in 1925 [1]. The symptoms of ILT are nasal discharge, conjunctivitis, reduced egg production, gasping, coughing, expectoration of bloody mucus, and marked dyspnea that may lead to suffocation. This disease causes economic losses in poultry industries worldwide [2]. Virus isolation has been used to detect ILTV, it is time consuming. DNA detection by conventional polymerase chain reaction (PCR) or real-time PCR has become a preferred method of virus diagnosis [8,9,10,11]. Real-time PCR has gained wide acceptance due to its improved rapidity, sensitivity, reproducibility, and the reduced risk of carryover contamination [12]
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