Chickens can durably clear herpesvirus vaccine infection in feathers while still carrying vaccine-induced antibodies.

Marek's disease (MD) is a major disease of chickens induced by Marek's disease virus (MDV) associated to lethal lymphomas. Current MD vaccines protect against lymphomas, but fail to prevent infection and shedding. The control of MDV shedding is crucial in order to eradicate this highly contagious virus. Like pathogenic MDV, MD vaccines infect the feather follicles of the skin before being shed into the environment. MD vaccines constitute excellent models to study virus interaction with feathers, the unique excretion source of these viruses. Herein we studied the viral persistence in feathers of a MD vaccine, the recombinant turkey herpesvirus (rHVT-ND). We report that most of the birds showed a persistent HVT infection of feathers over 41 weeks with moderate viral loads. Interestingly, 20% of the birds were identified as low HVT producers, among which six birds cleared the infection. Indeed, after week 14-26, these birds named controllers had undetectable HVT DNA in their feathers through week 41. All vaccinated birds developed antibodies to NDV, which lasted until week 41 in 95% of the birds, including the controllers. No correlation was found between HVT loads in feathers and NDV antibody titers over time. Interestingly, no HVT DNA was detected in the spleens of four controllers. This is the first description of chickens that durably cleared MD vaccine infection of feathers suggesting that control of Mardivirus shedding is achievable by the host.

Study of Efficacy and Replication of Recombinant Vector Vaccines by Using Turkey Herpesvirus Combined with Other Marek's Disease Vaccines.

Several recombinant turkey herpesviruses (rHVTs) have been developed within the past decades, and they are now used commercially worldwide. In broiler chickens, rHVTs are usually administered alone, but in long-living birds they are used in combination with Marek's disease (MD) vaccines of other serotypes (i.e., CVI988). The objectives of this work were to 1) evaluate protection against MD conferred by HVT and two rHVTs when combined with CVI988 and 2) optimize the use of rHVT in combination with CVI988 to maximize replication of rHVT without compromising MD protection. Various vaccine protocols, all using rHVT or HVT at the recommended dose (RD), were evaluated. Protocols evaluated included in ovo vaccination with HVT+CVI988 or rHVT+CVI988 (using either the double dose [DD] or the RD of CVI988), day of age vaccination of rHVT+CVI988 at DD, and revaccination protocols using rHVT in ovo followed by CVI988 at DD at day of age. Our results show that, when combined with CVI988, HVT and rHVTs confer a similar level of protection against MD (>90%) regardless of whether CVI988 was used at RD or at DD. However, the combination of rHVT with CVI988 at DD resulted in reduced replication rates of rHVT (60%-76% vs. 95%-100%). Our results show that such a negative effect could be avoided without jeopardizing MD protection by administering CVI988 at RD (if combined in ovo with rHVT) or administered rHVT first in ovo followed by CVI988 at DD at day of age.

Estudio de la eficacia y replicación de vacunas con vectores recombinantes mediante el uso del virus del herpes del pavo combinado con otras vacunas contra la enfermedad de Marek. Varios herpesvirus de pavo recombinantes (rHVT) se han desarrollado en las últimas décadas y ahora se utilizan comercialmente en todo el mundo. En pollos de engorde, los rHVT generalmente se administran solos, pero en aves de vida larga se usan en combinación con vacunas contra la enfermedad de Marek (MD) de otros serotipos (especialmente, CVI988). Los objetivos de este trabajo fueron 1) evaluar la protección contra la enfermedad de Marek conferida por herpesvirus de pavo (HVT9 y por dos rHVT cuando se combinan con la cepa CVI988 y 2) optimizar el uso de rHVT en combinación con la cepa CVI988 para maximizar la replicación de rHVT sin comprometer la protección contra la enfermedad de Marek. Se evaluaron varios protocolos de vacunas, todos con rHVT o con HVT a la dosis recomendada (RD). Los protocolos evaluados incluyeron la vacunación in ovo con HVT + CVI988 o rHVT + CVI988 (usando la dosis doble o la dosis recomendada de la cepa CVI988), la vacunación al día de la edad con rHVT + CVI988 con dosis doble, y los protocolos de revacunación usando rHVT seguido por la cepa CVI988 con dosis doble al día de edad. Los resultados muestran que cuando se combinan con CVI988, HVT y rHVT confieren un nivel de protección similar contra la enfermedad de Marek (> 90%) independientemente de que la cepa CVI988 se haya usado a la dosis recomendada o con dosis doble. Sin embargo, la combinación de rHVT con la cepa CVI988 con doble dosis produjo una reducción en las tasas de replicación de rHVT (60% ­76% vs. 95% ­100%). Estos resultados muestran que dicho efecto negativo podría evitarse sin poner en peligro la protección contra la enfermedad de Marek administrando la cepa CVI988 a la dosis recomendada (si se combina in ovo con rHVT) o administrando rHVT primero in ovo, seguido de CVI988 con dosis doble al día de la edad.

Vaccination and Host Marek's Disease-Resistance Genotype Significantly Reduce Oncogenic Gallid alphaherpesvirus 2 Telomere Integration in Host Birds.

Marek's disease (MD) is an infectious disease characterized by lymphomas and high mortality in susceptible chickens. The causative and ubiquitous alpha-herpesvirus known as MD virus (MDV) integrates into host telomeres during early infection through latency, known to be an important phase for oncogenic transformation. Herein, we sought to determine the influence of vaccination and host genetics on the temporal dynamics of MDV-host genome interactions. We studied integration profiles using 2 MD vaccines that vary in protective efficacy in 2 genetic lines that differ in MD resistance/susceptibility. Virus integration of both oncogenic MDV and vaccine strains was observed in both MD susceptible and resistant birds, however, the lines differed in their dynamic telomere-integration profiles. Notably, the resistant host genotype exhibited a smaller percentage of replicating cells with the virus telomere-integrated only phenotype as compared to the susceptible genotype. Vaccination with Rispens, the most protective MD vaccine, also reduced the establishment of the virus telomere-integrated only phenotype, suggesting a significant role of the phenotype in MD lymphoma development. The effect of Rispens vaccination was most dramatic in the susceptible genotype. These results suggest important connections between vaccinal immunity, MDV telomere integration, virus-induced oncogenesis, and virus-host genome interactions in the context of host genetics and disease susceptibility.

In ovo evaluation of FloraMax®-B11 on Marek's disease HVT vaccine protective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis infection in broiler chickens.

Three experiments were conducted to evaluate the effect of in ovo administration of FloraMax®-B11 (FM) on Marek's disease (MD) herpesvirus of turkeys (HVT) vaccine protective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis (SE) infection in chickens. Experiment 1 consisted of 3 trials. In trials 1 and 2, d 18 White Leghorn 15I5x71 embryos were randomly distributed in 4 groups: 1) HVT vaccinated in ovo and no Marek's disease virus (MDV) challenge; 2), HVT + FM vaccinated in ovo and no MDV challenge; 3) HVT vaccinated in ovo and challenge with virulent MDV (vMDV; strain 583A); and 4), HVT + FM vaccinated in ovo and challenge with vMDV. Trial 3 was designed exactly the same as Experiment 1 but chicks were challenged with very virulent MDV (vvMDV; strains Md5 and 612). Birds were monitored until 8 wk of age, and tested for MD incidence. Experiment 2 consisted of 3 trials. In each trial, d 18 broiler embryos were injected in ovo with either saline or FM to measure hatchability and gastrointestinal bacterial composition. In Experiment 3, d 18 broiler embryos were injected in ovo with either saline or FM. All chickens that hatched were orally gavaged with SE at hatch and kept for 7 d to monitor post-hatch BW. No significant difference (P > 0.05) between MD percentage in birds vaccinated with HVT alone or HVT + FM were observed in Experiment 1. In Experiment 2, probiotic did not negatively affect hatchability, but did reduce lactose positive Gram-negative bacteria. Further, increase in BW was associated with higher villi surface area in the ileum in chickens that received the probiotic as well as a significant reduction in the SE incidence in Experiment 3. These results suggest that in ovo administration of FM does not negatively impact the ability of HVT to protect against MD or hatchability of chickens, but improves BW during the first 7 d of life and decreases SE recovery in chickens.

Efficacy of Various HVT Vaccines (Conventional and Recombinant) Against Marek's Disease in Broiler Chickens: Effect of Dose and Age of Vaccination.

Herpesvirus of turkeys (HVT) has been successfully used as a Marek's disease (MD) vaccine for more than 40 yr. Either alone (broiler chickens) or in combination with vaccines of other serotypes (broilers, broiler breeders, and layers), HVT is used worldwide. In recent years, several vector vaccines based on HVT (rHVT) have been developed. At present, there are both conventional HVT and rHVTs in the market, and it is unknown if all of them confer the same level of protection against MD. The objective of this study was to further characterize the protection conferred by two conventional HVTs (HVT-A and HVT-B) and three recombinant HVTs (rHVT-B, rHVT-C, and rHVT-D) against MD in broiler chickens. In a first study we evaluated the efficacy of two conventional HVTs (HVT-A and HVT-B) administered at different doses (475, 1500, and 4000 PFU) at day of age on the ability to protect against an early challenge with very virulent plus strain 645. In a second experiment we evaluated the protection ability of several HVTs (both conventional and recombinant) when administered in ovo at a dose of 1500 PFU using the same challenge model. Our results show that each HVT product is unique, regardless of being conventional or recombinant, in their ability to protect against MD and might require different PFUs to achieve its maximum efficacy. In Experiment 1, HVT-A at 4000 PFU conferred higher protection (protection index [PI] = 63) than any of the other vaccine protocols (PI ranging from 36 to 47). In Experiment 2, significant differences were found among vaccine protocols with PI varying from 66 (HVT-A) to 15 (rHVT-D). Our results show that each HVT is unique and age at vaccination and vaccine dose greatly affected vaccine efficacy. Furthermore, they highlight the need of following manufacturer's recommendations.

Vaccination-challenge interval markedly influences protection provided by Rispens CVI988 vaccine against very virulent Marek's disease virus challenge.

The Rispens (CVI988) vaccine is widely used to vaccinate chickens worldwide. We tested the protective effects of the Rispens vaccine against challenge with very virulent Marek's disease virus (vvMDV) at various intervals at, before or after vaccination. The experiment used commercial ISA Brown layers and vvMDV isolate 02LAR. The protective index (PI) was measured for vaccination challenge intervals (VCI) of -10, -5, 0, 5 and 10 days, with the negative values indicating challenge prior to vaccination. Chickens were challenged by injection with 400 plaque-forming units (PFU) of 02LAR and/or vaccinated with 3200 PFU of the Rispens vaccine virus at days 0, 5 and 10 of age, with appropriate negative controls injected with diluent only. The presence of visible Marek's disease tumours was assessed up to 56 days post challenge. MDV challenge in unvaccinated chickens resulted in tumours in 52% of chickens. The Rispens vaccine provided no significant protection when challenge preceded vaccination, with PIs of -4 and 21% for VCI of -5 and -10 days respectively. On the other hand, it provided PIs of 60, 85 and 100% at VCI of 0, 5 and 10 days respectively. The study also revealed that the vvMDV load in peripheral blood lymphocytes or feather tips at 14 and 21 days post infection as determined by quantitative real-time polymerase chain reaction, which can distinguish pathogenic MDV from the Rispens vaccine strain, was an accurate early predictor of Marek's disease incidence at 56 days post challenge. The load of Rispens virus in peripheral blood lymphocytes or feathers at the same times post vaccination did not offer similar predictive power.

Influence of vaccination with CVI988/Rispens on load and replication of a very virulent Marek's disease virus strain in feathers of chickens.

Several highly efficacious vaccines are currently available for control of Marek's disease, a lymphoproliferative disease in chickens. However, these vaccines are unable to prevent infection with Marek's disease virus (MDV) in vaccinated birds. This leads to shedding of virulent MDV from feather follicle epithelium and skin epithelial cells of vaccinated and infected chickens. The objective of the present study was to study the interactions between a vaccine strain (CVI988/Rispens) and a very virulent strain of MDV (RB1B) in feathers. We examined genome load and replication of CVI988 and MDV-RB1B strains at various time points post infection. Moreover, we evaluated cytokine expression in feathers as indicators of immunity generated in response to vaccines against MDV. Analysis of feathers collected between 4 and 21 days post infection (d.p.i.) revealed a steady level of CVI988 genome load in the presence or absence of RB1B. Infection with MDV resulted in a significant increase in RB1B genome load peaking at 14 d.p.i. Importantly, vaccination with CVI988 resulted in a significant reduction in accumulation of MDV-RB1B in feathers. RB1B genome accumulation in feather tips was associated with increased expression of interferon-α at 14 d.p.i. and interferon-Sγ at earlier time points, 4 and 7 d.p.i. compared with 10 and 14 d.p.i. Interleukin-10 and interleukin-6 were up-regulated at 14 d.p.i. in the infected groups. This study expands our understanding of the dynamics of replication of vaccine and virulent MDV strains in the feathers and illuminates mechanisms associated with immunity to Marek's disease.

Transcriptome and proteome profiling of host responses to Marek's disease virus in chickens.

Marek's disease (MD) is an immunosuppressive and proliferative disease of domestic chickens caused by a highly oncogenic cell-associated alpha-herpesvirus, named Marek's disease virus (MDV). Despite the availability of highly efficacious vaccines for control of MD and existence of lines of chickens which display differential genetic susceptibility or resistance to this disease, little is known about the underlying mechanisms of MDV-host interactions. The recent advent of global or targeted gene and protein expression profiling has paved the way towards gaining a better understanding of host responses to MDV. The main objective of this review is to discuss some of the recent advancements made in relation to elucidating the mechanisms of MDV pathogenesis, host responses to MDV, genetic resistance/susceptibility to MD, and immunity conferred by vaccines. In this regard, particular emphasis has been placed on studies employing proteome and transcriptome profiling approaches. Finally, the utility of microRNA and RNA interference (RNAi) technologies for functional analysis of genes, proteins, and pathways that play a role in the complex interactions between MDV and its host is discussed.

Load of challenge Marek's disease virus DNA in blood as a criterion for early diagnosis of Marek's disease tumors.

Outbreaks of Marek's disease (MD) in vaccinated flocks still occur sporadically and lead to economic losses. Unfortunately, adequate methods to predict MD outbreaks are lacking. In the present study, we have evaluated whether high load of challenge MD virus (MDV) DNA in peripheral blood could aid in the early diagnosis of MD and in monitoring efficacy of vaccines against MD. One experiment was conducted to simulate field conditions by combining various vaccines (turkey herpesvirus [HVT] and HVT + MDV serotype 2 [SB1]) and challenge viruses (GA, Md5, and 648A). Vaccine efficacy among our experimental groups ranged from 13.3% to 94.2%. Each chicken was sampled three times during the length of the experiment (3, 5, and 15 wk postchallenge [wpc]), and gross lesions were evaluated in chickens that died and at termination of the experiment. DNA was extracted from whole blood and buffy coats from each sample, and the load of challenge MDV DNA and HVT DNA were quantified by real-time polymerase chain reaction. Chickens that developed MD by the end of the experiment had higher load of challenge MDV DNA (threshold cycle [Ct] glyceraldehyde-3-phosphate dehydrogenase [GAPDH]/Ct glycoprotein B [gB] ratios of 1.0, 1.04, and 1.05 at 3, 5, and 15 wpc, respectively) than those that did not develop MD (Ct GAPDH/Ct gB ratios of 0.7, 0.69, and 0.46 at 3, 5, and 15 wpc, respectively). However, load of HVT DNA in blood was not correlated with the development of tumors (Ct GAPDH/Ct HVT ratios from 0.04 to 0.10 in both groups). Vaccinated groups with >75% protection had statistically significant less challenge DNA virus (Ct GAPDH/Ct gB ratios of 0.76, 0.70, and 0.45 at 3, 5, and 15 wpc, respectively) than less protected groups (Ct GAPDH/Ct gB ratios of 0.92, 0.97, and 0.85 at 3, 5, and 15 wpc, respectively). No differences in the load of HVT DNA could be found between protected and nonprotected groups at any time point of the study (Ct GAPDH/Ct HVT from 0.05 to 0.09 in both groups). Our results showed that load of challenge MDV DNA but not load of HVT DNA in blood can be used as criterion for early diagnosis of MD.

Vaccinal control of Marek's disease: current challenges, and future strategies to maximize protection.

Marek's disease is an economically important lymphoid neoplasm of chickens, caused by oncogenic strains of Marek's disease herpesvirus. The disease can be successfully controlled by vaccination with attenuated or non-pathogenic MDV strains. However, vaccine failures do occur as field strains continue to evolve towards pathotypes of greater virulence, and this evolution is likely to be driven by the vaccines themselves. Two general strategies can be considered to improve protection by vaccination. Firstly by the development of novel vaccines, and secondly by maximizing the potential of existing vaccines. This second goal requires investigation of optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. Accurate quantitation of Marek's disease vaccine virus in vaccinated chicks will contribute significantly to our understanding of vaccinal protection. We recently developed a real-time polymerase chain reaction (PCR) assay for quantitation of CVI988 vaccine virus in the feather tips, a rich source of viral DNA which can easily be sampled in a non-invasive manner. This PCR test is now used commercially to confirm the successful vaccination of chicks. We have also used the PCR to examine various aspects of vaccination in experimental chicks and commercial chicks with a view to determining how vaccine level in feathers correlates with protection against challenge, and for identifying optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. In this article we review some aspects of the current vaccinal control of Marek's disease, before highlighting some of the problems associated with current vaccines and vaccination strategies, and the challenges for the future. We go on to discuss the development and use of our real-time PCR feather test, its current applications and potential opportunities in Marek's disease vaccine research.