Spatial and temporal variation of Marek's disease virus and infectious laryngotracheitis virus genome in dust samples following live vaccination of layer flocks.

Monitoring of Marek's disease virus (MDV) and infectious laryngotracheitis virus (ILTV) genome using poultry dust can be useful to monitor on-farm vaccination protocols but there are no set guidelines for collection of this sample type. This study assessed different dust collection methods for MDV and ILTV detection in a vaccinated layer flock (n = 1700) from day-old to 50 weeks of age. Birds were vaccinated against MDV at day-old, and ILTV by drinking water at week 6 and eye drop at week 12. Dust samples were collected weekly by settle plates (1-3 plates/15 m2) or by scraping surfaces in the poultry shed and tested for ILTV and MDV genomic copies (GC) by PCR. ILTV GC were detected 4 weeks post water vaccination, peaked at weeks 12-14 and became mostly undetectable after week 18. MDV was detected in dust on week 1, peaked at weeks 3-6, declined 3 logs by week 26 and remained detectable at this level until week 50. There was no difference in the detection rates of ILTV and MDV collected from settle plates in different locations of the shed (P > 0.10). There was no difference between settle plate and scraped samples in ILTV GC load but higher MDV GC were found in scraped samples. The settle plate method appears to reflect the current level of vaccine virus in the flock while the scrape method likely represents a cumulative record of shedding. Assessment of viral GC in dust samples is a good candidate for a practical method of estimating successful vaccine administration.

Development of a rapid and specific loop-mediated isothermal amplification detection method that targets Marek's disease virus meq gene.

A rapid, sensitive and specific loop-mediated isothermal amplification (LAMP) method was developed and evaluated for the detection of Marek's disease virus (MDV) by amplification of conserved MDV meq gene sequences. LAMP is an innovative technique that allows the rapid detection of targeted nucleic acid sequences under isothermal conditions without the need for complex instrumentation. In this study, meq gene sequences were amplified successfully from different MDV strains by LAMP within 60min and no cross-reactivity was observed in a panel of related viruses that were associated with diseases of chickens. The detection limit of LAMP was 3.2 copies/million cells compared with 320 copies/million cells required for conventional PCR. Positive detection rates were assessed using either LAMP or PCR by examination of feather follicles that were collected from chickens infected experimentally with either strain J-1 (n=20) or strain Md5 (n=17), In addition to these samples, three isolates that were suspected to have been infected in the clinic were also tested. Results showed that the positive detection rate for LAMP was 95% (38/40), compared with 87.5% (35/40) and 90% (38/40) for strains J-1 and Md5 by PCR, respectively. These results indicated that the LAMP assay was more sensitive, rapid and specific than conventional PCR for the detection of MDV. This easy-to-perform technique will be useful for the detection of MDV and will aid in the establishment of disease control protocols.

Phylogeny and recombination history of gallid herpesvirus 2 (Marek's disease virus) genomes.

Phylogenetic analyses based on concatenated amino acid sequences from orthologous loci from eight genomes of alpha herpesviruses infecting birds provided strong support for the following hypotheses: (1) gallid HV3 is a sister taxon to gallid HV2 but gallid HV1 is not closely related to the other two chicken herpesviruses; (2) meleagrid HV1 is closer to both gallid HV2 and gallid HV3 than is gallid HV1; (3) within gallid HV2, the virulent GA genome forms an outgroup to both the avirulent CVI988 genome and the highly virulent Md5 and Md11 genomes. Analysis of the pattern of synonymous nucleotide substitution between orthologous genes shared by four complete genomes of gallid HV2 showed strong evidence of past events of homologous recombination that homogenized certain loci between genomes. Eight of these loci represented cases of loci homogenized between the CVI988, on the one hand, and the Md5 and Md11 genomes, on the other hand. Two others represented loci where the GA genome was homogenized with those of Md5 and Md11. The two loci (UL49.5 and RLORF12) that were homogenized among the virulent genomes GA, Md5, and Md11 are candidates for contributing to viral virulence.

Absolute quantification using real-time polymerase chain reaction of Marek's disease virus serotype 2 in field dust samples, feather tips and spleens.

Methods for Taqman quantitative real-time PCR (qPCR) assays to detect the three serotypes of Marek's disease virus (MDV) are available, and absolute quantification has been developed for MDV serotype 1 and serotype 3. The development of a method for absolute quantification of Marek's disease virus serotype 2 (MDV2) is described in this paper. Using plasmid DNA, the lower detection limit of the MDV2 assay was determined to be 10 copies. Three independent assay runs showed highly reproducible Ct values and calculated copy numbers, with mean intra- and inter-assay coefficients of variation of less than 3% for Ct and less than 21.5% for calculated copy number. Absolute quantification of MDV2 was performed successfully on dust samples collected from poultry farms across Australia, material from infectious spleens and feather tips from chickens vaccinated with an attenuated strain of MDV2. Thus, it is now possible to use qPCR assays for absolute quantification of all three serotypes of MDV in a sample.