Marek's Disease Vaccine Research Articles

Effect of Diluting Marek's Disease Vaccines on the Outcomes of Marek's Disease Virus Infection When Challenged with Highly Virulent Marek's Disease Viruses

Dilution of Marek's disease (MD) vaccines is a common practice in the field to reduce the cost associated with vaccination. In this study we have evaluated the effect of diluting MD vaccines on the protection against MD, vaccine and challenge MD virus (MDV) kinetics, and body weight when challenged with strains Md5 (very virulent MDV) and 648A (very virulent plus MDV) by contact at day of age. The following four vaccination protocols were evaluated in meat-type chickens: turkey herpesvirus (HVT) at manufacturer-recommended full dose; HVT diluted 1:10; HVT + SB-1 at the manufacturer-recommended full dose; and HVT + SB-1 diluted 1:10 for HVT and 1:5 for SB-1. Vaccine was administered at hatch subcutaneously. One-day-old chickens were placed in floor pens and housed together with ten 15-day-old chickens that had been previously inoculated with 500 PFU of either Md5 or 648A MDV strains. Chickens were individually identified with wing bands, and for each chicken samples of feather pulp and blood were collected at 1, 3, and 8 wk posthatch. Body weights were recorded at 8 wk for every chicken. Viral DNA load of wild-type MDV, SB-1, and HVT were evaluated by real time-PCR. Our results showed that dilution of MD vaccines can lead to reduced MD protection, reduced relative body weights, reduced vaccine DNA during the first 3 wk, and increased MDV DNA load. The detrimental effect of vaccine dilution was more evident in females than in males and was more evident when the challenge virus was 648A. However, lower relative body weights and higher MDV DNA load could be detected in chickens challenged with strain Md5, even in the absence of obvious differences in protection.

Comparison of Blood and Feather Pulp Samples for the Diagnosis of Marek's Disease and for Monitoring Marek's Disease Vaccination by Real Time-PCR

Comparison of blood and feather pulp (FP) samples for the diagnosis of Marek's disease (MD) and for monitoring Marek's diseases vaccination in chickens (serotypes 2 and 3 vaccines) by real time-PCR was evaluated. For diagnosis of MD, quantification of serotype 1 Marek's disease virus (MDV) DNA load was evaluated in 21 chickens suffering from MD. For each chicken, samples of blood and FP were collected and MDV DNA load was quantified. Solid tumors are the sample of choice for MD diagnosis by real time-PCR and, hence, 14 solid tumors were included in the study as positive controls. Load of MDV DNA in FP was equivalent to that detected in solid tumors (threshold cycle [Ct] ratio above 1.7). MDV DNA load in blood samples was lower than in solid tumors and FP samples. Nonetheless, there was a statistically significant correlation of the results obtained from FP and blood (r = 0.92). Results of the Pearson correlation test showed that Ct ratio values of 1.7 in FP correspond to Ct ratio values of 1.2 in peripheral blood. For monitoring vaccines, serotypes 2 and 3 MDV DNA load was evaluated in blood and FP samples of vaccinated chickens. Serotype 2 MDV DNA load was evaluated in samples of blood and FP from 34 chickens vaccinated with SB-1 strain. Serotype 3 MDV DNA load was evaluated in blood and FP samples from 53 chickens vaccinated with HVT strain. For both serotypes, frequency of positive samples and load of vaccine DNA was higher in FP than in blood samples. There was not a statistically significant correlation between the load of SB-1 DNA (r = 0.17) or HVT DNA (r = -0.04) in FP and blood. Our results show that the load of serotypes 1, 2, and 3 DNA is higher in FP than in blood. Diagnosis of MD could be done using both FP and blood samples. Monitoring of MD vaccination by real time-PCR required the use of FP samples. There were a high percentage of false negative samples when using blood to detect serotypes 2 and 3 MDV by real time-PCR.

Comparison of Blood and Feather Pulp Samples for the Diagnosis of Marek's Disease and for Monitoring Marek's Disease Vaccination by Real Time-PCR

Comparison of Blood and Feather Pulp Samples for the Diagnosis of Marek's Disease and for Monitoring Marek's Disease Vaccination by Real Time-PCR

Evaluation of Factors Affecting Vaccine Efficacy of Recombinant Marek's Disease Virus Lacking the Meq Oncogene in Chickens

We previously reported that deletion of the Meq gene from the oncogenic rMd5 virus rendered it apathogenic for chickens. Here we examined multiple factors affecting Marek's disease vaccine efficacy of this nonpathogenic recombinant Meq null rMd5 virus (rMd5deltaMeq). These factors included host genetics (MHC haplotype), strain or dose of challenge virus, vaccine challenge intervals, and maternal antibody status of the vaccinated chicks. Studies on host genetics were carried out in five chicken lines comprising four different MHC B-haplotypes. Results showed that chicken lines tested were highly protected, with protective indexes of 100% (B*2/*15), 94% (B*2/*2), 87% (B*19/*19), and 83% (B*21/*21). At a challenge dose above 8000 plaque-forming units, differences in protection were observed between the two highly virulent strains examined (648A and 686). The interval between vaccination and challenge indicated a protective efficacy from 0 to 2 days varied greatly (12%-82%) after challenge with vv+686, the most virulent virus. Less variation and significant protection began at 3 days post vaccination and reached a maximum at 5 days post vaccination with about 80%-100% protection. Taken together, our results indicate that the factors examined in this study are important for vaccine efficacy and need to be considered in comparative evaluations of vaccines.

Effect of Diluting Marek's Disease Vaccines on the Outcomes of Marek's Disease Virus Infection When Challenged with Highly Virulent Marek's Disease Viruses

Effect of Diluting Marek's Disease Vaccines on the Outcomes of Marek's Disease Virus Infection When Challenged with Highly Virulent Marek's Disease Viruses

Serial Transfer of a Transplantable Tumor: Implications for Marek's Vaccine Mechanisms

The mechanism of Marek's disease (MD) vaccination to prevent the lymphoproliferative disease in chickens is not well understood. It is generally recognized that vaccination prevents disease, including the induction of T-cell tumors, but it does not prevent the pathogenic virus from infecting and replicating in the vaccinated host, nor does it prevent bird to bird spread of the oncogenic virus. The stage at which the vaccinated immune system intervenes in the process from infection to the induction of tumors remains obscure. Using a transplantable tumor induced by the Md5 strain of MD virus (MDV), we show that CVI988 vaccination does not prevent the induction of transplantable tumors in the 15I(5) x 7(1) chicken line. A monoclonal tumor with a V beta 1 T-cell receptor spectratype of 207 base pairs was used to follow the transplantable tumor in serial passages in vivo. This transplantable tumor could be passed in vaccinated birds. The length of time between vaccination and challenge (5 to 12 days) had little or no influence on the ability to transfer the tumor. There was variability in the manifestation of the disease produced by the transplanted tumor. Some chickens presented as normal but were still capable of transmitting the transplanted tumor to newly vaccinated recipients via their blood. This indicates that some chickens can control, but not eliminate, the tumor. The variables inducing health or disease in the challenged chickens remain obscure, but environmental or other factors likely depress the immune system allowing the tumor to overwhelm the immune system.

Allele-specific expression analysis reveals CD79B has a cis-acting regulatory element that responds to Marek's disease virus infection in chickens

Marek's disease (MD) is a T cell lymphoma disease of domestic chickens induced by the Marek's disease virus (MDV), a highly infectious and naturally oncogenic alphaherpesvirus. Enhancing genetic resistance to MD in poultry is an attractive method to augment MD vaccines, which protect against MD but do not prevent MDV replication and horizontal spread. Previous work integrating QTL scans, transcript profiling, and MDV-chicken protein-protein interaction screens revealed 3 MD resistance genes; however, a major challenge continues to be the identification of the other contributing genes. To aid in this search, we screened for allele-specific expression (ASE) in response to MDV infection, a simple and novel method for identifying polymorphic cis-acting regulatory elements, which may contain strong candidate genes with specific alleles that confer MD genetic resistance. In this initial study, we focused on immunoglobulin β (CD79B) because it plays a critical role in the immune response and, more important, is transcriptionally coupled with growth hormone (GH1), one of the previously identified MD resistance genes. Using a coding SNP in CD79B and pyrosequencing to track the relative expression of each allele, we monitored ASE in uninfected and MDV-infected F(1) progeny from reciprocal intermatings of highly inbred chicken lines 6(3) (MD resistant) and 7(2) (MD susceptible). Upon screening 3 tissues (bursa, thymus, and spleen) at 5 time points (1, 4, 7, 11, and 15 d postinfection), we observed that MDV infection alters the CD79B allelic ratios in bursa and thymus tissues at 4 and 15 d postinfection in both mating directions. Our results suggest that CD79B has a cis-acting regulatory element that responds to MDV infection and probably cooperates with GH1 in conferring genetic resistance to MD. This result helps validates the use of ASE screens to identify specific candidate genes for complex traits such as genetic resistance to MD.

Chronological study of cytokine transcription in the spleen and lung of chickens after vaccination with serotype 1 Marek's disease vaccines

Transcription of cytokine genes was evaluated in the lung and spleen of chickens vaccinated with various serotype 1 Marek's disease (MD) vaccines. Three vaccine pairs/series, each consisting of one or two high protective (HP) and one low protective (LP) vaccine, were used. Vaccinated chickens had increased transcripts of IFN-γ in the lung and spleen at 3, 5, and 10 days post vaccination (dpv) compared to unvaccinated control chickens. In addition, transient increase of cytokine transcripts (iNOS, IL-1β, IL-18, IL-8, and IL-6 in the lung and iNOS, IL-18, and IL-6 in the spleen) was detected. Compared to chickens vaccinated with LP vaccines, HP vaccinated chickens had increased transcripts of iNOS at 5dpv but decreased transcripts of IL-6, IL-8, and IL-18 at 10dpv in the lung. HP vaccinated chickens had increased IFN-γ in the spleen at 3 and at 10dpv. This study demonstrated that MD vaccines administered subcutaneously elicit a pulmonary immune response and identified differences in the cytokine gene expression between HP and LP vaccinated chickens.

Genetics and vaccine efficacy: Host genetic variation affecting Marek’s disease vaccine efficacy in White Leghorn chickens

Marek's disease (MD) is a T-cell lymphoma disease of domestic chickens induced by MD virus (MDV), a naturally oncogenic and highly contagious cell-associated α-herpesvirus. Earlier reports have shown that the MHC haplotype as well as non-MHC genes are responsible for genetic resistance to MD. The MHC was also shown to affect efficiency of vaccine response. Using specific-pathogen-free chickens from a series of 19 recombinant congenic strains and their 2 progenitor lines (lines 6(3) and 7(2)), vaccine challenge experiments were conducted to examine the effect of host genetic variation on vaccine efficacy. The 21 inbred lines of White Leghorns share the same B*2 MHC haplotype and the genome of each recombinant congenic strain differs by a random 1/8 sample of the susceptible donor line (7(2)) genome. Chickens from each of the lines were divided into 2 groups. One was vaccinated with turkey herpesvirus strain FC126 at the day of hatch and the other was treated as a nonvaccinated control. Chickens of both groups were inoculated with a very virulent plus strain of MDV on the fifth day posthatch. Analyses of the MD data showed that the genetic line significantly influenced MD incidence and days of survival post-MDV infection after vaccination of chickens (P<0.01). The protective indices against MD varied greatly among the lines with a range of 0 up to 84%. This is the first evidence that non-MHC host genetic variation significantly affects MD vaccine efficacy in chickens in a designed prospective study.

Vaccine-induced host responses against very virulent Marek's disease virus infection in the lungs of chickens

The aim of this study was to investigate the kinetics of virus replication and cellular responses in the lungs following infection with Marek's disease virus (MDV) and/or vaccination with herpesvirus of turkey (HVT) via the respiratory route. Chickens vaccinated with HVT and challenged with MDV had a higher accumulation of MDV and HVT genomes in the lungs compared to the chickens that received HVT or MDV alone. This increase in virus load was associated with augmented expression of interferon (IFN)-γ and interleukin (IL)-10, and increased T cell infiltration. These findings shed more light on the immunological events that occur in the lungs after vaccination or infection with MDV.

A MEQ-Deleted Marek's Disease Virus Cloned as a Bacterial Artificial Chromosome Is a Highly Efficacious Vaccine

The Marek's disease virus (MDV) induces T-cell tumors in susceptible chickens. Of the 80 to 100 known MDV genes, only the MDV MEQ gene was shown to have transforming properties. Further evidence that MEQ is probably the principal oncogene in MDV came when researchers used overlapping cosmid clones of MDV and demonstrated that deleting MEQ resulted in a highly protective Marek's disease (MD) vaccine. We deleted both copies of MEQ from a bacterial artificial chromosome clone (BAC) of MDV. The virus, BACdelMEQ, was completely attenuated and did not appear to have any adverse effect on chicken body weight in MDV maternal-antibody-positive chickens, as measured at 8 wk of age. In two protection studies, BACdelMEQ efficiently protected susceptible chickens from a challenge by MDV strain 686, one of the most virulent MDV strains. In both protection studies, the BACdelMEQ protected chickens significantly better than the commercial MD vaccine, CVI988/Rispens. Only the protein-coding sequences of MEQ were deleted and all upstream and downstream regulatory sequences were left intact. Thus, BACdelMEQ has the potential to be a superior MD vaccine as well as a vector to deliver various foreign genes to poultry.

Evaluation of factors influencing replication of serotype 1 Marek's disease vaccines in the chicken lung

Factors influencing replication of serotype 1 Marek's disease vaccines in the lung of chickens within the first 10 days of age (doa) were evaluated. In particular, the effect of vaccine efficacy, age/route of vaccination, and vaccine dose were examined in three experiments. In the first experiment, three vaccine pairs, each pair consisting of a high protective (HP) and a low protective (LP) vaccine (CVI988/BP5 and CVI988-Clone C, 648A80 and 648A100, R2 and R2/23) were used to inoculate chickens subcutaneously (s.c.) with 2000 plaque-forming units (PFU) at hatch. DNA load in the lung was significantly higher in the HP vaccine group than the LP vaccine group at 5 and 10 doa in two of the three vaccine pairs. In the lung, at 5 doa, early MDV gene transcripts (ICP4 and pp38) were detected in most vaccine groups, whereas late MDV gene transcripts (gB and gI) were detected only in the HP vaccine group. In the second experiment, chickens were vaccinated in ovo or s.c. at hatch with 2000 PFU R2, R2/23, or CVI988/BP5. Compared with s.c. vaccination, in ovo vaccination resulted in higher MDV DNA load in the lung at 3 doa, lower or equal MDV DNA load at 5 doa, and lower MDV DNA load at 10 doa. In the third experiment, chickens were vaccinated s.c. at hatch with either 2000 or 10,000 PFU strain R2. There were no statistically significant differences in the load of MDV DNA in the lung after vaccination with R2 between the two doses. Our results showed that HP vaccines tend to replicate better than LP vaccines in the lung; and vaccine replication in the lung within the first 10 days of age was affected by the age/route of inoculation (in ovo versus s.c.) but not by the dose of vaccine administered.

Validation of Marek's Disease Diagnosis and Monitoring of Marek's Disease Vaccines from Samples Collected in FTA®Cards

Validation of Marek's Disease Diagnosis and Monitoring of Marek's Disease Vaccines from Samples Collected in FTA^®Cards

Validation of Marek's Disease Diagnosis and Monitoring of Marek's Disease Vaccines from Samples Collected in FTA® Cards

The use of Flinders Technology Associates (FTA) filter cards to quantify Marek's disease virus (MDV) DNA for the diagnosis of Marek's disease (MD) and to monitor MD vaccines was evaluated. Samples of blood (43), solid tumors (14), and feather pulp (FP; 36) collected fresh and in FTA cards were analyzed. MDV DNA load was quantified by real-time PCR. Threshold cycle (Ct) ratios were calculated for each sample by dividing the Ct value of the internal control gene (glyceraldehyde-3-phosphate dehydrogenase) by the Ct value of the MDV gene. Statistically significant correlation (P < 0.05) within Ct ratios was detected between samples collected fresh and in FTA cards by using Pearson's correlation test. Load of serotype 1 MDV DNA was quantified in 24 FP, 14 solid tumor, and 43 blood samples. There was a statistically significant correlation between FP (r = 0.95), solid tumor (r = 0.94), and blood (r = 0.9) samples collected fresh and in FTA cards. Load of serotype 2 MDV DNA was quantified in 17 FP samples, and the correlation between samples collected fresh and in FTA cards was also statistically significant (Pearson's coefficient, r = 0.96); load of serotype 3 MDV DNA was quantified in 36 FP samples, and correlation between samples taken fresh and in FTA cards was also statistically significant (r = 0.84). MDV DNA samples extracted 3 days (t0) and 8 months after collection (t1) were used to evaluate the stability of MDV DNA in archived samples collected in FTA cards. A statistically significant correlation was found for serotype 1 (r = 0.96), serotype 2 (r = 1), and serotype 3 (r = 0.9). The results show that FTA cards are an excellent media to collect, transport, and archive samples for MD diagnosis and to monitor MD vaccines. In addition, FTA cards are widely available, inexpensive, and adequate for the shipment of samples nationally and internationally.

A Deletion Within Glycoprotein L of Marek's Disease Virus (MDV) Field Isolates Correlates with a Decrease in Bivalent MDV Vaccine Efficacy in Contact-Exposed Chickens

We examined the functional role of a naturally occurring deletion within the glycoprotein L (gL) gene of Marek's disease virus (MDV) field isolates. We previously showed that this mutation incrementally increased the virulence of an MDV in contact-exposed SPF leghorn chickens, when chickens shedding this virus were co-infected with herpesvirus of turkeys (HVT). In our present study, we examined this mutation using two stocks of the very virulent plus (vv+)MDV strain TK, one of which harbored this deletion (TK1a) while the other did not (TK2a). We report that TK1a replicating in vaccinated chickens overcame bivalent (HVT/SB1) vaccine protection in contact-exposed chickens. Treatment groups exposed to vaccinated chickens inoculated with a 1:1 mix of TK1a and TK2a showed decreased bivalent vaccine efficacy, and this decrease correlated with the prevalence of the gL deletion indicative of TK1a. These results were also found using quadruplicate treatment groups and bivalently vaccinated chickens obtained from a commercial hatchery. As this deletion was found in 25 out of 25 recent field isolates from Delaware, Maryland, North Carolina, Pennsylvania, and Virginia, we concluded that there is a strong selection for this mutation, which appears to have evolved in HVT or bivalently vaccinated chickens. This is the first report of a mutation in a vv+MDV field strain for which a putative biological phenotype has been discerned. Moreover, this mutation in gL has apparently been selected in MDV field isolates through Marek's disease vaccination.

Revaccination with Marek's disease vaccines induces productive infection and superior immunity.

The most common lymphoproliferative disease in chickens is Marek's disease (MD), which is caused by the oncogenic herpesvirus Marek's disease virus (MDV). The emergence of hypervirulent pathotypes of MDV has led to vaccine failures, which have become common and which have resulted in serious economic losses in some countries, and a revaccination strategy has been introduced in practice. The mechanism by which revaccination invokes superior immunity against MD is unknown. After field trials which showed that revaccination provided protection superior to that provided by a single vaccination were performed, experiments were conducted to explore the interaction between revaccinated chickens and MDV. The results showed that the chickens in the revaccination groups experienced two consecutive productive infections but that the chickens in the single-vaccination groups experienced one productive infection, demonstrating that revaccination of viruses caused the chickens to have productive and then latent infections. Revaccination of the virus induced in the chickens a higher and a longer temporary expansion of the CD8(+), CD4(+), and CD3(+) T-lymphocyte subpopulations, stronger peripheral blood lymphocyte proliferative activity; and higher levels of neutralizing antibody than single vaccination. These findings disagree with the postulate that MDV antigens persist, stimulate the immune system, and maintain a high level immunity after vaccination. The suppression of productive infection by maternal antibodies in chickens receiving the primary vaccination and a lower level of productive infection in the revaccination groups challenged with MDV were observed. The information obtained in this study suggests that the productive infection with revaccinated MDV in chickens plays a crucial role in the induction of superior immunity. This finding may be exploited for the development of a novel MD vaccine that results in the persistence of the antigen supply and that maintains a high level of immunity and may also have implications for other viral oncogenic diseases in humans and animals.

Marek's disease virus-induced expression of cytokine genes in feathers of genetically defined chickens

Marek's disease (MD) vaccines, although effective in reducing lymphoproliferation, cannot control infectious virus production in the feather follicle epithelium (FFE) which is the site of virus shedding. Therefore, we investigated Marek's disease virus (MDV) replication as well as the expression of cytokine genes in feathers of MDV-infected chickens belonging to genetically defined lines (N2a or B 21/B 21 haplotype-resistant and P2a or B 19/B 19 haplotype-susceptible). Though there was not a difference in MDV genome load and transcripts between feathers of these chicken lines at 4 and 10 days post-infection (d.p.i.), feathers of resistant chickens carried significantly lower viral genome load and transcripts at 21 d.p.i. Irrespective of genetic background of the chickens examined, MDV replication showed a significant positive correlation with the expression of IFN-γ gene. The results imply the usefulness of genetic control approach in reducing virulent MDV transmission.

Marek's disease vaccines: A solution for today but a worry for tomorrow?

Marek's disease (MD) is a lymphoproliferative disease of chickens that, in the absence of control measures, is capable of causing devastating losses in commercial poultry flocks. MD has been successfully controlled by vaccination since 1968. However, vaccine efficacy has decreased concomitantly with the increase in virulence of Marek's disease virus (MDV). The constant evolution of MDV has forced the development of new vaccines or vaccine strategies that control the more virulent emergent strains. However, this race between the introduction of new vaccines and the evolution of MDV represents a major threat for the poultry industry. In addition to vaccination, other factors might have contributed to the evolution of MDV (intensive methods of chicken production, early exposure of the chickens to MDV and administration of vaccines at very low doses). From all the possible factors influencing MDV evolution, the effect of vaccination has received the greatest attention. MD vaccines protect with great efficacy against the development of the disease but they do not prevent infection or transmission. Sterilizing immunity could be a solution to stop the evolution of the virus but it has been proven to be extremely difficult, if at all possible, to obtain with MDV or with other herpesviruses. Other solutions to improve vaccine-induced protection are discussed in this paper.

Molecular Characterization of Three Recombinant Isolates of Avian Leukosis Virus Obtained from Contaminated Marek's Disease Vaccines

Three natural recombinant avian leukosis viruses (ALV; PDRC-1039, PDRC-3246, and PDRC-3249) expressing a subgroup A gp85 envelope protein and containing long terminal repeats (LTR) of endogenous ALV-E viruses were isolated from contaminated commercial Marek's disease vaccines, cloned, and completely sequenced. Their full genomes were analyzed and compared with representative strains of ALV. The proviral DNA of all three isolates displayed 99.3% identity to each other, suggesting a possible common ancestor, even though the contaminating viruses were obtained from three separate vaccine serials produced by two different vaccine manufacturing companies. The contaminating viruses have a genetic organization typical of replication-competent alpharetroviruses. The proviral genomes of PDRC-1039 and PDRC-3246 are 7497 bp long, and the PDRC-3249 is three base pairs shorter because of a deletion of a threonine residue within the gp85 coding region. The LTR, gag, pol, and the transmembrane (TM) region (gp37) of the env gene of all three viruses displayed high identity to endogenous counterpart sequences (>98%). Only the surface (SU) region (gp85) of the env gene displayed high identity with exogenous ALV-A (98.7%). Locus-specific polymerase chain reaction (PCR) analysis for ALV endogenous sequences (ev loci) in the chicken embryo fibroblasts used to produce the original vaccine vials identified the presence of ev-1, ev-2, ev-3, ev-4, and ev-6 in all three vaccines. Homologous recombination most likely took place to involve the SU region of the env gene because the recombinant viruses only differ in this particular region from the consensus ALV-E. These results suggest that the contaminating ALV isolates probably emerged by recombination of ALV-A with endogenous virus sequences before vaccine preparation.

Molecular Characterization of Three Recombinant Isolates of Avian Leukosis Virus Obtained From Contaminated Marek's Disease Vaccines

Molecular Characterization of Three Recombinant Isolates of Avian Leukosis Virus Obtained From Contaminated Marek's Disease Vaccines