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Objective: To use specific real-time qPCR to determine (1) the vaccination success of Rispens CVI988 vaccine in feathers and dust; (2) persistence of Rispens infection in vaccinated layer chickens; (3) extent of co-infection with wild-type Marek's disease virus (MDV) in vaccinated layers; and (4) presence of Rispens virus in unvaccinated broiler flocks., Methods: Feather, dust and serum samples were collected from birds aged 3 days to 91 weeks from three layer farms. qPCR was used to detect MDV and Rispens in DNA extracted from dust and feathers. Previously tested MDV-positive dust samples from 100 broiler flocks were tested for the presence of Rispens using qPCR, while serum samples were used to detect anti-MDV antibody using ELISA., Results: Overall, 66% and 93% of feather and dust samples, respectively, from Rispens-vaccinated layers were Rispens-positive. Viral load in these samples varied between farms during early life, reaching readily detectable levels at 2-3 weeks of age. Vaccinated chickens maintained a high Rispens load in feathers and dust and high MDV antibody levels until 91 weeks of age. MDV infection was detected in 6.7% of feather samples from vaccinated chickens. Rispens virus was detected in 7% of samples from unvaccinated broiler flocks., Conclusion: Vaccine take can be measured effectively by Rispens-specific qPCR of feathers or dust from approximately 3 weeks post vaccination. Infection with Rispens is persistent, with lifelong shedding and serological response. The detectable infection rate of vaccinated chickens with MDV is low and there is preliminary evidence of escape of Rispens virus to unvaccinated flocks., (© 2016 Australian Veterinary Association.)

Objective: To evaluate the pathogenicity of Australian Marek's disease virus (MDV) isolate MPF23 (1985) against the reference strain MPF57 based on pathology, viral load and neuropathotyping on the basis of clinical signs., Procedure: Two MDV challenge isolates (MPF57 or MPF23) were administered to unvaccinated specific-pathogen free (SPF) layer chicks on day 5 after hatch at three challenge doses (500, 2000 or 8000 plaque-forming units (pfu)/chick). Mortality, body weight, immune organ weights, MDV load in peripheral blood lymphocytes (PBL) and clinical signs were measured to 56 days post challenge (dpc)., Results: MPF23 was the more pathogenic of the two viruses, inducing higher mortality (81% vs 62%) and incidence of MD lesions (100% vs 76%). MPF23 induced earlier, more sustained and more severe neurological signs in the period 26-56 dpc. However, there were few differences during the 0-23 dpc used in the neuropathotyping classification under test. The observed pattern during this earlier period classified both viruses as neuropathotype B, consistent with a very virulent pathotype. MDV load in PBL at 7 and 44 dpc did not differ between virus isolates, but the load at 7 dpc was significantly and negatively associated with time to euthanasia or death., Conclusion: MPF23 appears to be as, or more, virulent than the MDV strains isolated over the subsequent two decades. The neuropathotyping system developed in the USA did not clearly differentiate between the two isolates under test; however, extension of the period of assessment of clinical signs beyond 26 dpc did reveal clear differences., (© 2015 Australian Veterinary Association.)

Two real-time PCR assays were developed which enable quantitation and differentiation between pathogenic Australian isolates of Marek's disease virus (MDV) serotype 1 and the serotype 1 vaccine strain Rispens CVI988. The assays are based on a DNA sequence variation in the meq gene between pathogenic and vaccinal MDV1 which has been confirmed by sequencing of 20 Australian field strains of MDV. Complete specificity has been demonstrated in samples containing pathogenic MDV (n=20), Rispens (3 commercial vaccine strains), or both. The limit of detection of both the Rispens-specific and the pathogenic MDV1-specific assays was 10 viral copies/reaction. The tests successfully differentiated and quantified MDV in mixtures of pathogenic and vaccinal Rispens virus. A high resolution melt curve analysis targeting the same SNP used for the real-time PCR assays was also developed which successfully detected sequence variation between Md5, six Australian MDV1 isolates and the three Rispens vaccines. However it was ineffective at differentiating mixtures of pathogenic and vaccinal MDV1. The real-time PCR assays have both diagnostic and epidemiological applications as they enable differentiation and quantitation of Rispens CVI988 and pathogenic MDV1 in co-infected chickens in Australia., (Copyright © 2012 Elsevier B.V. All rights reserved.)