Development of a SYBR Green quantitative polymerase chain reaction assay for rapid detection and quantification of infectious laryngotracheitis virus.

Infectious laryngotracheitis is an acute viral respiratory disease of chickens with a worldwide distribution. Sensitive detection of the causative herpesvirus is particularly important because it can persist in the host at a very low copy number and be transmitted to other birds. Quantification of viral genome copy number is also useful for clinical investigations and experimental studies. In the study presented here, a quantitative polymerase chain reaction (qPCR) assay was developed using SYBR Green chemistry and the viral gene UL15a to detect and quantify infectious laryngotracheitis virus (ILTV) in ILTV-inoculated chicken embryos or naturally infected birds. The specificity of the assay was confirmed using a panel of viral and bacterial pathogens of poultry. The sensitivity of the assay was compared with two conventional PCR assays, virus titration and an antigen-detecting enzyme-linked immunosorbent assay. The qPCR developed in this study was highly sensitive and specific, and has potential for quantification of ILTV in tissues from naturally and experimentally infected birds and embryos.

A polymerase chain reaction-coupled high-resolution melting curve analytical approach for the monitoring of monospecificity of avian Eimeria species.

Coccidiosis is a significant disease of poultry caused by different species of Eimeria. Differentiation of Eimeria species is important for the quality control of the live attenuated Eimeria vaccines derived from monospecific lines of Eimeria spp. In this study, high-resolution melting (HRM) curve analysis of the amplicons generated from the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2) was used to distinguish between seven pathogenic Eimeria species of chickens, and the results were compared with those obtained from the previously described technique, capillary electrophoresis. Using a series of known monospecific lines of Eimeria species, HRM curve analysis was shown to distinguish between Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox and Eimeria tenella. Computerized analysis of the HRM curves and capillary electrophoresis profiles could detect the dominant species in several specimens containing different ratios of E. necatrix and E. maxima and of E. tenella and E. acervulina. The HRM curve analysis identified all of the mixtures as "variation" to the reference species, and also identified the minor species in some mixtures. Computerized HRM curve analysis also detected impurities in 21 possible different combinations of the seven Eimeria species. The PCR-based HRM curve analysis of the ITS-2 provides a powerful tool for the detection and identification of pure Eimeria species. The HRM curve analysis could also be used as a rapid tool in the quality assurance of Eimeria vaccine production to confirm the purity of the monospecific cell lines. The HRM curve analysis is rapid and reliable and can be performed in a single test tube in less than 3 h.

Classification of fowl adenovirus serotypes by use of high-resolution melting-curve analysis of the hexon gene region.

Identification of fowl adenovirus (FAdV) serotypes is of importance in epidemiological studies of disease outbreaks and the adoption of vaccination strategies. In this study, real-time PCR and subsequent high-resolution melting (HRM)-curve analysis of three regions of the hexon gene were developed and assessed for their potential in differentiating 12 FAdV reference serotypes. The results were compared to previously described PCR and restriction enzyme analyses of the hexon gene. Both HRM-curve analysis of a 191-bp region of the hexon gene and restriction enzyme analysis failed to distinguish a number of serotypes used in this study. In addition, PCR of the region spanning nucleotides (nt) 144 to 1040 failed to amplify FAdV-5 in sufficient quantities for further analysis. However, HRM-curve analysis of the region spanning nt 301 to 890 proved a sensitive and specific method of differentiating all 12 serotypes. All melt curves were highly reproducible, and replicates of each serotype were correctly genotyped with a mean confidence value of more than 99% using normalized HRM curves. Sequencing analysis revealed that each profile was related to a unique sequence, with some sequences sharing greater than 94% identity. Melting-curve profiles were found to be related mainly to GC composition and distribution throughout the amplicons, regardless of sequence identity. The results presented in this study show that the closed-tube method of PCR and HRM-curve analysis provides an accurate, rapid, and robust genotyping technique for the identification of FAdV serotypes and can be used as a model for developing genotyping techniques for other pathogens.

Relationship between mortality, clinical signs and tracheal pathology in infectious laryngotracheitis.

Previous studies in our laboratory using a combination of polymerase chain reaction and restriction fragment length polymorphism have identified at least five different genotypes of infectious laryngotracheitis virus (ILTV). However, the virulence of these classes of ILTV was not investigated. In this study, five groups (16 birds each) of 3-week-old specific pathogen free chickens were inoculated via the intratracheal route with 10(3) median embryo infected dose of five different strains of ILTV. Three further groups of chickens were inoculated similarly with the vaccine strains SA2 and A20 or with sterile phosphate-buffered saline (PBS) for comparison. Four days post-inoculation, clinical signs were monitored for scoring, and eight chickens from each group were subsequently euthanized, weighed and subjected to pathological and histopathological examinations. The remaining birds were monitored for clinical signs and mortality until 21 days post-inoculation. All groups inoculated with ILTV strains showed moderate to severe clinical signs 4 days after inoculation. The strain Q1-96 caused only minimal breathing symptoms with a median score that was not significantly different to that of the group inoculated with PBS, but was significantly different to those of the groups inoculated with other ILTV strains. The strain Q1-96 caused severe photophobia and conjunctivitis with a median score that was significantly higher than those of all other groups except for the group inoculated with the strain N3-04. All ILTV strains caused a significant reduction in weight gain when compared with the group inoculated with PBS. The strain Q1-96 caused an average weigh loss of 14% that was significantly higher than those of other ILTV strains. The strains S2-04 and Q1-96 induced only minor microscopic tracheal lesions while all the other ILTV strains, including the vaccine strains A20 and SA2, induced moderate to severe microscopic tracheal lesions. Median scores for microscopic tracheal lesions were well correlated with the number of viral genomes detected in trachea. The results revealed that there is considerable variation among ILTV strains in their tropism for trachea or conjunctiva. In addition it was revealed that ILTV strains with high affinity for conjunctiva can severely affect weigh gain. The ILTV numbers and microscopic lesions in trachea were not found to be reliable indicators of virulence since they are not necessarily correlated with mortality rate in ILT.

Differentiation of infectious laryngotracheitis virus isolates by restriction fragment length polymorphic analysis of polymerase chain reaction products amplified from multiple genes.

Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.