Differential amplification of Marek’s disease CVI988 vaccine and of wild-type isolates from organs of commercial chickens using single or duplexed probes in real-time PCR

The differentiation of Marek's disease virus (MDV)-infected and vaccinated animal (DIVA) test, based on the MDV pp38 gene was described by Baigent et al. [(2016). Real-time PCR for differential quantification of CVI988 vaccine and virulent MDV strains. Journal of Virological Methods, 233, 23-36], using similar primers and alternate probes for virulent MDV-1 and the vaccine CVI988 virus. We explored the assay’s applicability for commercial vaccines and commercial chickens, as the above-mentioned study employed tissue-cultured MDV strains and tissues from experimental trials. DNA of visceral organs and feathers of vaccinated or naturally infected chickens was used. Further, the applicability of the DIVA assay was evaluated using single or duplexed probes for the two viruses in the same amplification tube. Due to the high viral content in the commercial vaccines and in the clinical cases of MDV-1 infected commercial chickens, their examination by the MDV-1 DIVA real-time PCR was performed in one step. However, for the feather DNAs of commercially vaccinated birds, a step of pre-amplification was required. The MDV-1 DIVA real-time PCR performed as single probe in separate tubes using the Vir3 probe was very sensitive for virulent MDV-1 strains, but not very specific, as it also gave a clear signal with CVI988 vaccine virus. In contrast, the CVI vaccine probe was specific for CVI988, and did not recognize the MDV-1 strains. When both probes were present in one tube, the CVI probe showed a greater sensitivity for CV1988, while the Vir3 probe showed a much better specificity for virulent MDV-1.     

A recombinant fowlpox virus vaccine expressing glycoprotein B gene from CVI988/Rispens strain of MDV: protection studies in different chickens

Recombinant fowlpox virus (rFPV) was constructed to express glycoprotein B (gB) gene from CVI988/Rispens strain of Marek's disease virus (MDV). The rFPV-gB/R alone and in combination with herpesvirus of turkey (HVT) preparations were evaluated for their protective efficacy against challenge with very virulent MDV strains Md5 and RB1B in different chickens. The rFPV-gB/R alone induced protection comparable to that by HVT vaccines in both Ab- SPF chickens and Ab+ production chickens. Significant protective synergism was observed in one of these two types of commercial production chickens when rFPV-gB/R was combined with HVT of either cell-associated or cell-free preparations. Immunogenesis studies showed that rFPV-gB/R, just like conventional vaccines, significantly reduced the level of viremia, splenocytes infection and feather follicle shedding of challenge virus in vaccinated chickens.     

The use of serotype 1- and serotype 3-specific polymerase chain reaction for the detection of Marek's disease virus in chickens

A serotype 1- and serotype 3-specific detection of Marek's disease virus (MDV) by polymerase chain reaction (PCR) was developed. The sensitivity of the method when applied to cell culture grown virus was comparable with that of cultivation. The method was applied to various tissue samples from chickens experimentally inoculated with serotype 1 or serotype 3 MDV.The serotype 1 strains CVI988 and RB-1B could be detected in feather follicle epithelium up to 56 and 84 days post-inoculation (p.i.), respectively, while the MDV-3 serotype was detected until 42 days p.i. The purpose of this study was to develop and evaluate a reliable and easy-to-handle method for surveillance of the occurrence of MDV in chicken flocks. We emphasize the development of a method, which can be applied to types of samples conveniently collected in the field, e.g. feather tips and blood samples. In addition, the PCR was applied to samples collected from four commercial table egg layer flocks of young stock or pullets vaccinated with either serotype 1 (CVI988) or serotype 3 (HVT) vaccine. These flocks had various clinical signs of Marek's disease. MDV-1 was detected in buffy-coat cells, spleen, liver, skin, feather tips and ovaries. The detection of MDV in feather tips appeared to be as sensitive as co-cultivation of buffy-coat cells, although an inhibiting factor was observed in extracts from feather tips of non-white chickens. This inhibition could be overcome in most extracts by applying a bovine serum albumen pretreatment. The PCR proved to be a convenient tool for the monitoring of MDV in the poultry population, and feather tips were the most convenient and sensitive samples.     

Absence of Marek's disease virus antigen in the feather follicle epithelium of vaccinated chicks demonstrated by immunofluorescence

In chickens vaccinated with MDV strain CVI 988, HVT strain FC 126 or PB-THV 1, no MDV-specific antigens could be demonstrated in the feather follicle epithelium by immunofluorescence (IF). In chickens given virulent MDV strains, the epithelium of the feather follicle was positive in IF. In an experiment where chickens were vaccinated with strain CVI 988, positive IF was observed in the lung, bursa and pancreas, but not in the feather follicle epithelium, kidney, cloaca, or caecal tonsils. Absence of IF antigen in the skin may be indicative of avirulence of the strain of MDV.     

Re-isolation of Marek’s disease virus from T cell subsets of vaccinated and non-vaccinated chickens

Summary.  To know the effect of Marek’s disease (MD) vaccines, we analyzed the distribution of MD virus (MDV) among T cell subsets from chickens vaccinated or non-vaccinated with MD vaccine and subsequently challenged with a virulent MDV. The challenged MDV was reisolated preferentially from CD4⁺ T cells, and the average titers of challenged MDV rescued were significantly lower in vaccinated chickens compared to that of non-vaccinated chickens. In addition, it was also shown that different serotypes of MDV, CVI988 and SB-1, have remarkable difference in recovery rates of viruses from CD4+ and CD8+ T cells, though both CVI988 and SB-1 can reduce the infection rates of virulent MDV to splenocytes. Received April 14, 1998 Accepted August 15, 1998     

Correlation of Marek's disease herpesvirus vaccine virus genome load in feather tips with protection, using an experimental challenge model.

We previously developed a real-time polymerase chain reaction (PCR) assay for absolute quantitation of serotype 1 Marek's disease virus in feather tips of chickens, and this has been used clinically to monitor a flock's response following vaccination with CVI988, an attenuated serotype 1 strain. The level of vaccine virus in feather tips associated with protection against challenge by virulent virus is not known. Here, we used an experimental challenge model, in which one dose of vaccine gives over 90% protection against mortality, to investigate correlation between the CVI988 level in feathers and protection. One-day-old chickens were vaccinated with 1, 0.1 or 0.01 commercial dose of CVI988 vaccine, and were then challenged with a virulent strain (RB-1B) 14, 21 or 28 days later. Replication of CVI988 virus was followed in each bird by real-time PCR analysis of feather DNA samples. Since the PCR does not differentiate between CVI988 and RB-1B, samples were taken only prior to challenge to ensure that the virus being measured was CVI988. Administration of one dose of vaccine ensured a uniform, rapid and high replication amongst birds, while replication following administration of the 0.1 or 0.01 dose was very variable. However, given time, a low early level of vaccine virus eventually replicated to high levels in some birds. Both the dose of vaccine virus administered and the level of vaccine virus in feather tips at 13 days post vaccination showed significant correlation with protection against challenge. A level of CVI988 vaccine virus of 132 genome copies/10000 feather tip cells was calculated to be the level required for 90% protection in this experimental model. The potential of this assay, and its limitations for monitoring protection in the field, are discussed.     

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.     

Protection provided by Rispens CVI988 vaccine against Marek's disease virus isolates of different pathotypes and early prediction of vaccine take and MD outcome

We tested the level of protection provided by the Rispens CVI988 (Rispens) vaccine against challenge with a virulent Marek's disease virus (MDV) pathotype (vMDV) and a very virulent pathotype (vvMDV) and the accuracy of a range of predictive measures of Marek's disease (MD) incidence and vaccine take. Commercial layer chicks (n?=?236) were vaccinated (or not) with 4000?plaque-forming units (pfu) of Rispens vaccine at hatch and challenged (or not) with 500?pfu of each challenge virus five days post vaccination. The vvMDV pathotype FT158 induced higher MD incidence (65%) and mortality (33%) when compared with the vMDV pathotype MPF57 (39% and 8%, respectively). The protective index provided by the Rispens vaccine against FT158 (61%) did not differ significantly from that against MPF57 (66%). This provides additional evidence that protection provided by the Rispens vaccine is not influenced by pathotype determined in studies using vaccines of other Mardivirus species. The challenge viruses did not differ in MDV or Rispens viral load in spleen at 14?dpc (days post challenge) determined by specific quantitative polymerase chain reaction test. MDV load in peripheral blood leucocytes at 7 and 14?dpc, splenocytes at 14?dpc, feather cells at 14 and 21?dpc and isolator dust at 21?dpc were significant early indicators of subsequent MD incidence to 56?dpc. These are potentially useful as the sampling can be carried out well before the onset of MD and some measures are non-invasive. The Rispens viral load in both invasive and non-invasive samples was more useful as a measure of vaccine take.     

Efficacy of a combination vaccine containing MDV CVI 988 strain and HVT against challenge with very virulent MDV

With the emergence of very virulent Marek's disease virus (MDV) strains, vaccines based on herpesvirus of turkeys (HVT) appear to be not powerful enough to confer full protection, whereas in chicken flocks vaccinated with MDV CVI 988 strain protective immunity sometimes is generated not early enough for full protection. For this reason combination vaccines containing HVT as well as CVI 988 have been developed. In this paper the beneficial effect of combining both types of virus strains in one vaccine for early protection is shown in a vaccination challenge experiment, in which one-day-old chickens were vaccinated with suboptimal dosages of the monovalent vaccines and the same dosages in a combination vaccine. After 5 days the chickens were challenged with a very virulent MDV strain and subsequently observed for a period of approx. 50 days. It appeared that the combination vaccine provided better early protection than the monovalent vaccines. In addition, the combination vaccine was tested as vaccine administered in ovo. It appeared that after in ovo vaccination the vaccine conferred adequate protection against challenge with a very virulent MDV strain, 5 days after hatch, and that protection after in ovo vaccination was similar to that obtained after subcutaneous vaccination with the same combination vaccine.     

Kinetics of Marek's disease virus (MDV) infection in broiler chickens 1: effect of varying vaccination to challenge interval on vaccinal protection and load of MDV and herpesvirus of turkey in the spleen and feather dander over time

Two experiments in commercial broiler chickens vaccinated with herpesvirus of turkeys (HVT) and challenged with Marek's disease virus (MDV) investigated the effects of the vaccination-to-challenge interval (VCI) on vaccinal protection against Marek's disease, and the kinetics of MDV and HVT load in the spleen and feather dander determined using real-time quantitative polymerase chain reaction. Experiment 1 in isolators tested VCI of 2, 4 and 7 days, while Experiment 2 in floor pens tested VCI of 0, 2, 4, 7 and 10 days. MDV challenge induced gross Marek's disease lesions in 14% to 74% of chickens by 56 days post-challenge. Vaccinal protection increased from ～40% to ～80% with increasing VCI between days 2 and 7 in both experiments, but not thereafter. MDV was detected in both the spleen and dander at 7 days post-challenge and increased rapidly to approximately 21 days post-challenge, after which levels plateaued, rose or fell gradually depending on treatment. HVT was also shed in significant amounts, 1 to 2 logs lower than for MDV, with a clear peak around 14 to 21 days post-vaccination. Vaccination significantly reduced the log₁₀MDV load in the spleen (vaccinated, 2.99±0.20/10⁶ spleen cells; unvaccinated, 4.60±0.23/10⁶ spleen cells) and dander (vaccinated, 5.28±0.13/mg; unvaccinated, 6.00±0.18/mg) from infected chickens. The MDV load had a significant negative association with the VCI and the level of vaccinal protection. Measurement of dander production in Experiment 1 and the dust content of air in Experiment 2, combined with determination of the MDV load in these, enabled estimation of total daily shedding rates of MDV per chicken and of the MDV load in air for the first time.     

Suspension culture of Marek’s disease virus and evaluation of its immunological effects

Marek’s disease virus (MDV) is a cell-associated α-herpesvirus of chickens. It is difficult to grow MDV in suspension culture. Therefore, MDV vaccines are currently produced using adherent primary chicken embryo fibroblasts, and on a large scale this is labour-intensive and costly. In this study, the CVI988 strain of MDV was inoculated into chicken fibroblast cell line UMNSAH/DF-1 (DF-1) cultured by microcarrier suspension for the proliferation experiment. Moreover, the effects of culture conditions, such as inoculation method, multiplicity of infection (MOI), microcarrier concentration, and pH value, on the proliferation of MDV were investigated. The results demonstrated that the maximum viral load of 64.76?±?2.64?×?10⁶ PFU/flask in a working volume of 100?ml could be obtained using synchronous cell seeding and inoculation method at an MOI of 0.02 and a microcarrier concentration of 5?g/l at pH 7.2. At the same time, the CVI988/DF-1 vaccines prepared by the microcarrier culture process and the traditional adherent cell culture process (CVI988/Rispens) were compared through bird experiments. We found a protective rate of 94.4% using the CVI988/DF-1 vaccine with specific pathogen-free chickens that was equivalent to that of the commercial vaccine CVI988/Rispens (protection rate of 94.1%). In this study, the MDV CVI988/DF-1 vaccine prepared by the microcarrier suspension culture of DF-1 cells could provide effective immune protection for specific pathogen-free chickens, providing a reference for the prevention and control of MD and further development of a large-scale bioreactor for producing the MD vaccine.     

Host responses are induced in feathers of chickens infected with Marek's disease virus

Control measures are ineffective in curtailing Marek's disease virus (MDV) infection and replication in the feather follicle epithelium (FFE). Therefore, vaccinated birds which subsequently become infected with MDV, shed the virulent virus although they remain protected against disease. The present study investigated host responses generated against MDV infection in the feather. We observed that in parallel with an increase in viral genome load and viral replication in the feather, there was a gradual but progressive increase in infiltration of CD4+ and CD8+ T cells into the feather pulp of MDV-infected chickens, starting on day 4 and peaking by day 10 post-infection. Concomitant with infiltration of T cells, the expression of interleukin (IL)-18, IL-6, interferon (IFN)-gamma and major histocompatibility complex class I genes was significantly enhanced in the feather pulp of MDV-infected chickens. The finding that host responses are generated in the feather may be exploited for developing strategies to control MDV infection in the FFE, thus preventing horizontal virus transmission.     

Effect of Cryptosporidium baileyi in specific pathogen free chickens vaccinated (CVI988/Rispens) and challenged with HPRS-16 strain of Marek's

This study was performed to examine the effect of Marek's disease virus (MDV) serotype 1 vaccine (CVI988/Rispens) on the pathogenicity of Cryptosporidium baileyi , and to determine whether C. baileyi infection could prevent the development of vaccinal Marek's disease (MD) immunity in specific pathogen free (SPF) chickens. Sixty-eight SPF homozygous B13 White Leghorn chickens were divided into seven groups. C. baileyi was orally administered at 5 days of age (day 4) in chickens infected with Rispens vaccine at day 0 or at day 8 and challenged with HPRS—16 strain of oncogenic MDV at day 15. Relevant control groups were constituted. The chickens were kept in isolators until the end of the experiment at day 62. The parameters evaluated were clinical signs, kinetics of oocyst shedding, mortality, macroscopic and microscopic lesions, cryptosporidia location in various organs and serum anti- C. baileyi antibodies at days 42 and 62. Our results show that C. baileyi , which is considered to be non-pathogenic when inoculated orally, may become highly pathogenic. It induced severe mortality and developed in organs other than classical target sites when chickens were vaccinated with Rispens vaccine and challenged with the HPRS—16 strain of MDV.However,parasite infection does not prevent the induction of vaccinal immunity for MD. Our results also show that vaccination of B13 chickens at hatching induces higher protection against challenge with HPRS—16 MDV at day 15 than vaccination at day 8.     

Protective efficacy of a recombinant bacterial artificial chromosome clone of a very virulent Marek’s disease virus containing a reticuloendotheliosis virus long terminal repeat

Marek’s disease virus (MDV), an alphaherpesvirus, causes Marek’s disease (MD), a lymphoproliferative disease in poultry characterized by T-cell lymphomas, nerve lesions, and mortality. Vaccination is used worldwide to control MD, but increasingly virulent field strains can overcome this protection, driving a need to create new vaccines. Previous studies revealed that insertion of reticuloendotheliosis virus (REV) long terminal repeat (LTR) into a bacterial artificial chromosome (BAC) clone of a very virulent strain of MDV, Md5, rendered the resultant recombinant virus, rMd5 REV-LTR BAC, fully attenuated in maternal antibody positive (Mab+) chickens at passage 40. In the current study, the protective efficacy of rMd5 REV-LTR BAC was evaluated. First, passage 70 was identified as being fully attenuated in maternal antibody negative chickens and chosen as the optimal passage level for use in protective efficacy studies. Second, three protective efficacy trials were conducted comparing the rMd5 REV-LTR p70 BAC to the CVI988/Rispens vaccine. Groups of Mab+ and Mab? 15I₅?×?7₁ chickens were vaccinated in ovo at 18 days of embryonation or intra-abdominally at day of hatch, and challenged at 5 days post-hatch with the vv+MDV strain 686. Vaccination at day of hatch and in ovo with rMd5 REV-LTR p70 BAC protected chickens against MDV-induced bursa and thymic atrophy, but did not provide the same level of protection against MD tumours as that afforded by the commercial vaccine, CVI988/Rispens.     

Biocharacteristics shared by highly protective vaccines against Marek's disease

Attenuated serotype 1 Marek's disease virus strains vary widely in their protection properties. This study was conducted to elucidate which biocharacteristics of serotype 1 MDV strains are related with protection. Three pairs of vaccines, each one including a higher protective (HP) vaccine and a lower protective (LP) vaccine originating from the same MDV strain, were studied. Two other highly protective vaccines (RM1 and CVI988/BP5) were also included in the study. Comparison within pairs of vaccines showed that marked differences existed between the HP and the LP vaccines. Compared with LP vaccines, HP vaccines replicated better in vivo. Also, they induced a significant expansion of total T cells and of the helper and cytotoxic T cell lineages (CD45⁺CD3⁺, CD4⁺CD8^?, CD4^?CD8⁺) as well as a marked increase in the expression of the antigens of MhcI and MhcII on T cells. Thus, our results show that in vivo replication and early stimulation of the T-cell lineage are two characteristics shared by HP vaccines. However, comparison among the four HP vaccines that provided protection equal to that of CVI988 (RM1, CVI988/BP5, CVI988 and 648A80) revealed variability, especially regarding in vivo replication. Strains RM1 and CVI988/BP5 showed much stronger replication in vivo than the other two vaccine strains (CVI988 and 648A80). Thus, no single set of characteristics could be used to identify the most protective Marek's disease vaccines, implying, perhaps, that multiple mechanisms may be involved.     

Marek's disease in broiler chickens: Effect of route of infection and herpesvirus of turkey-vaccination status on detection of virus from blood or spleen by polymerase chain reaction,and on weights of birds,bursa and spleen

The polymerase chain reaction (PCR) has recently emerged as an additional tool for the monitoring and diagnosis of Marek's disease. We investigated a number of factors that may influence the interpretation of PCR results in commercial broiler chickens including the effects of route of infection and herpesvirus of turkeys (HVT)-vaccination status. We also investigated the suitability of peripheral blood lymphocytes (PBL) and spleen as tissues for Marek's disease virus (MDV) detection. HVT-vaccinated and unvaccinated commercial broiler chickens were challenged or not challenged with virulent MDV either by intraperitoneal injection or inhalation of feather dust containing the virus. Blood and spleen samples were collected at weekly intervals to day 35 post-infection for PCR of spleen or PBL. Live weight and lymphoid organ weights were also measured. Spleen and PBL were found to provide similar sensitivity of detection of MDV with a small advantage in favour of spleen. In terms of the timing of detection of MDV, intraperitoneal challenge broadly mimicked natural challenge via inhalation, although infection of birds by inhalation of infective feather dust resulted in slightly later but more complete detection of MDV in challenged birds. Vaccination with HVT delayed the detection of MDV by approximately 10 to 14 days and did not protect against the reduced growth observed in challenged chickens at day 35 post-challenge.     

Quantitative pathogenicity of Marek's disease virus and vaccine virus strains in chickens: macroscopical aspects

Two virulent strains (JM and K) and one vaccine strain (CVI 988) of Marek's disease virus (MDV), together with two vaccine strains of the herpesvirus of turkeys (HVT) (FC 126 and PB-THV 1), all in the cell-associated state, were administered intramuscularly at 3.7 log TCID50 per dose to day-old SPF White Leghorn chickens. A control group of chicks received uninfected cells. The pathological parameters studied were onset and duration of clinical symptoms, mortality, bird weight and macroscopical lesions of peripheral nerves and visceral organs. Data were obtained from females autopsied at the age of 3, 8 and 20 weeks, and from chickens which died. Virological and serological data were procured mainly from males taken at various ages. The results indicate a clear distinction between virulent and vaccine strains. MD vaccines had no significant influence on bird weight and caused no mortality or macroscopical lesions, whereas the virulent MDV strains produced all these effects. Macroscopical lesions caused by the virulent MDV strains were seen predominantly in nerves (in about 50% of birds succumbing to MD) and gonads (in 0% to 80% of such birds depending on sex and on strain of MDV). Differences between the two virulent strains could be demonstrated. Strain JM induced earlier incidence and shorter duration of clinical disease. With strain JM death occurred earlier in females than in males. Strain K caused significantly more macroscopical lesions in gonads, heart and liver. Under the conditions of the experiment, detection of macroscopical lesions after inoculation with a virulent MDV strain was possible 3 weeks after inoculation.