Characterization of four very virulent Argentinian strains of Marek's disease virus and the influence of one of those isolates on synergism between Marek's disease vaccine viruses.

Isolates of Marek's disease virus (MDV) from vaccinated flocks in Argentina were characterized as very virulent (vv) and very virulent plus (vv+) strains. Experimental infection with these viruses caused a high incidence of Marek's disease in both resistant N-2a line and susceptible P-2a line birds. Vaccine viruses from each of the three Marek's disease viral serotypes were evaluated alone and in various combinations for protection against challenge with a vvMDV called NULP-1. Vaccination of P-2a birds with HVT did not protect satisfactorily against any of the vv and vv+MDV strains isolated. However, CVI988/Rispens vaccine alone or combined with serotype 2 and/or serotype 3 vaccine strains enhanced protection significantly against NULP-1. Serotype 2 plus serotype 3 vaccines also provided significant protection when challenged with this strain. This is one the first reports of the occurrence of vvMDV and vv+MDV in Argentina and Latin America. It is also a preliminary evaluation of the synergistic protective effect of different vaccine viruses with local MDV strains. However, further studies are needed to evaluate the real role of these and other Marek's disease isolates in 'vaccination failures' and the influence of serotype and virus strain on synergism between Marek's disease vaccine viruses.     

Protection of chickens against very virulent infectious bursal disease virus (IBDV) and Marek's disease virus (MDV) with a recombinant MDV expressing IBDV VP2.

To develop a herpes virus vaccine that can induce immunity for an extended period, a recombinant Marek's disease (MD) virus (MDV) CVI-988 strain expressing infectious bursal disease virus (IBDV) host-protective antigen VP2 at the US2 site (rMDV) was developed under the control of an SV40 early promoter. Chickens vaccinated with the rMDV showed no clinical signs and no mortality and 55% of the chickens were considered protected histopathologically after challenge with very virulent IBDV (vvIBDV), whereas all of the chickens vaccinated with the conventional IBDV vaccine showed no clinical signs and were protected. Chickens vaccinated with the CVI-988 or chickens in the challenge control showed severe clinical signs and high mortality (70-75%) and none of them were protected. Also, the rMDV conferred full protection to chickens against vvMDV just as the CVI-988 strain did, whereas 90% of the challenge control chickens died of MD. Antibody levels against IBDV and MDV following the vaccination increased continuously for at least 10 weeks. No histopathological lesions in the rMDV-vaccinated chickens and no contact transmission of the rMDV to their penmates were confirmed. These results demonstrate that an effective and safe recombinant herpesvirus-based IBD vaccine could be constructed by expressing the VP2 antigen at the US2 site of the CVI-988 vaccine strain.     

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.     

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.     

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.     

Attenuation of lymphoid leukosis enhancement by serotype 2 Marek's disease virus

Serotype 2 Marek's disease virus (MDV), already known to augment the protective efficacy of turkey herpes virus (HVT) vaccine against MD, may also enhance the frequency of lymphoid leukosis (LL) in retrovirus-infected chickens of certain strains. LL enhancement refers to an increase in frequency and decrease in latent period for development of LL, and is measured by comparison of tumour responses in retro-virus-infected, MD-vaccinated chickens to those of retrovirus-infected control chickens. LL enhancement has been documented with both of the serotype 2 MDV strains tested thus far. These experiments were conducted to investigate whether LL enhancement ability varied within a larger collection of serotype 2 strains or was influenced by serial passage in cell culture. Each of seven low-passage serotype 2 MDV strains enhanced LL responses. The HN-1 strain was competent for LL enhancement at passage 19, but LL enhancement ability was absent at passages 26, 27 and 40. Enhancement of LL by strains 471B/1, 281MI/1, 287C/1 and 298B/1 was reduced by 40 to 64 serial passages in chicken embryo fibroblast cultures. Strains SB-1 and 301B/1 continued to demonstrate enhancement of LL through 66 and 40 passages, respectively. Thus, LL enhancement appeared to be a general property of serotype 2 MDV, but was susceptible in five of seven strains to reduction (attenuation) by cell culture passage. Enhancement of LL by serotype 2 MDV was attenuated more rapidly by cell culture passage than was the ability of such viruses to protect against virulent MDV challenge either alone or in combination with HVT. One such LL enhancement-attenuated strain, 471B/1 (passages 33 and 40), when combined with HVT, induced protection against MD challenge (76 and 78%) that was comparable with that induced by SB-1 + HVT (82%) or 301B/1 + HVT (89%). This or similar serotype 2 viruses could provide suitable protection against MD with a reduced risk of LL enhancement.     

Immunosuppressive effects of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) in broiler chickens and the protective effect of HVT vaccination against MDV challenge

Much of the impact of Marek's disease in broiler chickens is considered to be due to immunosuppression induced by Marek's disease virus (MDV). The present study evaluates the effects of an Australian isolate of pathogenic MDV (strain MPF 57) and a non-pathogenic vaccinal strain of herpesvirus of turkeys (HVT) (strain FC 126) on the immune system of commercial broiler chickens for 35 days following challenge at days 0 or 3 of age. It also investigates the extent of protection provided by HVT vaccine against MDV-induced immunosuppression. Immune system variables, including relative lymphoid organ weight, blood lymphocyte phenotype (CD45+/CD3+, putatively T, and CD45+/LC+, putatively B) and antibody production following vaccination against infectious bronchitis (IB) at hatch, were used to assess the immune status of chickens. Immunosuppression was also assessed by susceptibility to secondary challenge with pathogenic Escherichia coli on day 29 post-MDV challenge. MDV infection reduced the weight of the thymus and bursa of Fabricius, the numbers of circulating T lymphocytes and B lymphocytes, and IB antibody titre. The timing of these effects varied. MDV infection greatly increased susceptibility to E. coli infection. HVT alone caused mild depletion of T and B lymphocytes but no effect on immune organ weight or IB titre. Vaccination with HVT provided good protection against most of the immunosuppressive effects of MDV but not against MDV-induced growth impairment and reduced responsiveness to IB vaccination, suggesting that recent Australian strains of MDV may be evolving in virulence to overcome the protective effects of HVT.     

Protection by attenuated and polyvalent vaccines against highly virulent strains of Marek's disease virus

Tests confirmed that turkey herpesvirus (HVT) vaccine protected chickens poorly against challenge with the highly virulent Md5 strain of Marek's disease (MD) virus, especially in chickens with homologous HVT antibodies. The naturally avirulent SB-1 vaccine virus was likewise poorly protective against challenge with the Md5 strain. Homologous antibodies reduced the protective efficacy of both vaccines, but SB-1 was not affected by HVT antibodies. In order to provide better protection against strains of MD virus poorly protected against by HVT, such as Md5, the Md11 strain of MD virus was attenuated by 75 cell culture passages and evaluated for protective efficacy. This vaccine virus, designated Mdl 1/75C, provided good protection against challenge with Md5 and most other highly virulent MD viruses tested, but was less efficacious against challenge with the JM/102W strain, a prototype MD virus protected against well by HVT and SB-1 vaccines. Furthermore, its efficacy was consistently lower in chicks with HVT antibody. Thus, although HVT, SB-1, and Md11/75C were all efficacious against certain MD viruses, none of these vaccines protected optimally against all MD challenge viruses in all chickens. A polyvalent vaccine composed of Md11/75C, HVT and SB-1 viruses protected chickens better against a battery of five highly virulent MD challenge viruses, including three strains poorly protected against by HVT, than any single vaccine and was not influenced by HVT antibody. These data suggest that vaccinal immunity may be partially viral strain specific.     

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.     

Highly virulent Marek’s disease virus strains affect T lymphocyte function and viability of splenocytes in commercial meat-type chickens

ABSTRACT

In previous studies, we have demonstrated that very virulent plus Marek’s disease viruses (vv+MDV) are highly immunosuppressive in commercial meat-type chickens. The specific objectives of this work were to evaluate if vv+MDV immunosuppression (MDV-IS) is induced by reduction of lymphocyte responsiveness and/or viability. Three experiments were conducted to (i) compare vv+MDV 686 with a partially attenuated 686-BAC; (ii) compare vv+MDV strains (648A and 686) with vMDV (GA) and vvMDV (Md5); and (iii) compare chickens vaccinated with Md5-BACΔMEQ and with CVI988?+?HVT. In each experiment, spleens were collected at 28–30 days post infection and lymphocytes were isolated and investigated in three ways: their proliferative response to Concanavalin A (ConA) was analysed by MTT proliferation assay; cell death, and expression of CD45 and MHC-I was studied by flow cytometry; and MHC-IA and β-2 microglobulin (B2M) expression was evaluated by real time RT-PCR. Splenocytes of chickens inoculated with vv+MDV were severely impaired to proliferate when exposed to ConA. Furthermore, vv+MDV induced severe splenocyte death that did not occur after infection with v or vvMDV strains. Vaccination with CVI988?+?HVT, and at less level with Md5-BACΔMEQ reduced these negative effects. This is in contrast to our previous results in which Md5-BACΔMEQ but not CVI988?+?HVT protected against MDV-IS suggesting that although cell death and decrease lymphocyte function seem to be related to MDV virulence and certainly will be associated with immunosuppression, they might not fully explain the previously reported MDV-IS.

RESEARCH HIGHLIGHTS

• vv+MDV induces extensive death in splenocytes in meat-type chickens 28–30?dpi.

• vv+MDV impairs lymphocyte function in meat-type chickens 28–30?dpi.

• Vaccination protects against splenocyte death and reduced lymphocyte function.

• Cell lysis and reduced lymphocyte function do not fully explain MDV-IS.

     

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.     

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.     

Evaluation of factors influencing the development of late Marek’s disease virus-induced immunosuppression: virus pathotype and host sex

Marek’s disease virus (MDV) is a herpesvirus that induces lymphoma and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is complex and can be divided into two phases: early-MDV-IS associated with cytolytic infection in the lymphoid organs in chickens lacking maternal antibodies against MDV (MAbs) and late-MDV-IS that appears later in the pathogenesis and occurs even in chickens bearing MAbs. We have recently developed a model to reproduce late-MDV-IS under laboratory conditions. This model evaluates late-MDV-IS indirectly by assessing the effect of MDV infection on the efficacy of infectious laryngotracheitis (ILT) vaccines against challenge with ILT virus. In the present study, we have used this model to investigate the role of two factors (MDV pathotype and host sex) on the development of late-MDV-IS. Five MDV strains representing three different pathotypes: virulent (vMDV; 617A, GA), very virulent (vvMDV; Md5), and very virulent plus (vv+MDV; 648A, 686), were evaluated. Only vv+ strains were able to induce late-MDV-IS. An immunosuppression rank (IS-rank) was established based on the ability of MDV to reduce the efficacy of chicken embryo origin vaccine (values go from 0 to 100, with 100 being the highest immunosuppressive ability). The IS-rank of the evaluated MDV strains ranged from 5.97 (GA) to 20.8 (617A) in the vMDV strains, 5.97 to 16.24 in the vvMDV strain Md5, and 39.08 to 68.2 in the vv+ strains 648A and 686. In this study both male and female chickens were equally susceptible to MDV-IS by vv+MDV 686. Our findings suggest that late-MDV-IS is a unique feature of vv+ strains.     

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.     

Cross-protection between JMV Marek's disease-derived tumour transplant, Marek's disease and turkey herpesviruses

Cross-protection tests were conducted using attenuated JMV (JMV-A) Marek's disease-derived lymphoblasts, glutaraldehyde-treated JMV tumour cells, attenuated Marek's disease virus (MDV, strain HPRS-16/att) and turkey herpesvirus (HVT, strain FC126) as vaccines, and virulent JMV and MDV (HPRS-16) for challenge. The JMV and JMV-A preparations were free of MDV, leukosis and reticuloendotheliosis viruses. Vaccination of chickens with attenuated MDV or with HVT provided good protection against both JMV lymphoblastosis and Marek's disease (MD). In one experiment HVT (cell-free) caused a better resistance to JMV than to MD. Inoculation of JMV-A always resulted in a 100% resistance to virulent JMV. However, JMV-A did not induce any appreciable resistance to MD, even when the birds were challenged with MDV by contact exposure. Control experiments revealed that high doses of normal lymphocytes from uninfected chickens also had a protective effect against JMV. The 50% protective dose varied from 10(7) to 10(8) lymphocytes. JMV tumour cells inactivated by glutaraldehyde were used in different experiments but rarely caused a clear-cut protection against virulent JMV. The results of this study suggested that a one-way relationship exists in vivo between HVT or MDV and JMV lymphoblastic leukaemia. However, resistance induced against JMV tumour cells appeared to be related to histocompatibility antigens at least as much as to tumour-specific cell surface antigens. The results obtained failed to provide clear evidence for or against vaccinal resistance to MDV being dependent on the action of a common Marek's disease tumour-associated surface antigen (MATSA) additional to the immune response to viral antigens.     

Polyvalent Marek's disease vaccines: safety, efficacy and protective synergism in chickens with maternal antibodies

Three Marek's disease (MD) vaccines were evaluated for safety and protective efficacy in chickens with maternal antibodies against serotype 1, 2 and 3 MD viruses and in chickens with no maternal antibodies. The vaccines were: (1) Md11/75C, an attenuated serotype 1 MD virus, (2) a trivalent mixture of MD virus strains Md11/75C and SB-1, and turkey herpesvirus (HVT) strain FC 126, and (3) bivalent mixtures of these three viruses. These vaccines were compared with HVT or SB-1 vaccines in some trials. None of the vaccines was pathogenic or immunodepressive in susceptible chickens with or without maternal antibodies. No interference with in vivo HVT replication by additional viral components was noted, although some interference was demonstrated in vitro. Md11/75C replicated to limited titres in vivo and did not spread by contact, however, it acquired mild pathogenicity upon serial back-passage. Although Md11/75C provided good protection against highly virulent MD viruses in chickens without maternal antibodies, it was poorly protective in chickens with homologous maternal antibodies, and appeared more susceptible to in vivo neutralisation than did SB-1 or HVT. The trivalent vaccine, in contrast, was highly efficacious against very virulent MD virus challenge, even in chickens with maternal antibodies of all three serotypes, and was significantly more effective than monovalent vaccines. A bivalent vaccine composed of SB-1 and HVT was superior to the other two bivalent combinations of the three viruses. Protective synergism among all three vaccine viruses was confirmed. The efficacy of HVT was enhanced by as little as 80 PFU of SB-1, and even fractional doses of HVT and SB-1 together were superior to full doses of HVT alone.     

Comparative studies on Marek's disease virus and herpesvirus of turkey DNAs.

DNA of Marek's disease virus (MDV) was compared to that of herpes virus of turkey (HVT). Centrifugation of the two virus DNAs in neutral glycerol and CsCl density gradients showed that the MDV genome was slightly larger than that of HVT and that the buoyant density (1.705 g/ml) of MDV DNA in CsCl gradients was slightly lower than that (1.707 g/ml) of HVT DNA. MDV and HVT DNAs were digested with either EcoRI or HindIII restriction endonuclease and analysed by 0.5% agarose gel electrophoresis. The cleavage patterns of HindIII or EcoRI DNA digests of two strains of these two viruses showed general similarities between the strains, but not between MDV and HVT. However, a few fragments of EcoRI or HindIII digests of MDV DNA co-migrated with those of HVT DNA. DNA-DNA reassociation kinetics and DNA-RNA hybridization between the two viruses indicated that MDV and HVT DNAs share detectable homology, although it is less than 5%. The DNA of a HVT variant, which has lost the ability to protect chickens from Marek's disease, appeared similar to DNA of the vaccine strain in the size buoyant density and in its restriction endonuclease cleavage pattern.     

Relationship between Marek's disease virus load in peripheral blood lymphocytes at various stages of infection and clinical Marek's disease in broiler chickens.

Vaccination with herpesvirus of turkey (HVT) vaccine provides protection against clinical Marek's disease (MD) but does not preclude infection with wild-type MD virus (MDV). The quantity of MDV detected in circulating lymphocytes during the early period after infection may be a useful predictor of subsequent clinical MD later in the life. A study was designed to quantify MDV and HVT copy number in peripheral blood lymphocytes (PBL) using real-time polymerase chain reaction between days 5 and 35 post-challenge and to relate this to subsequent development of gross MD lesions. Female commercial broiler chickens were vaccinated with HVT or were sham-vaccinated at hatch, then challenged with MDV strain MPF-57 at day 2 post-vaccination and reared in positive-pressure isolators up to 56 days post-challenge, when all survivors were euthanized. All dead and euthanized chickens were examined post mortem for gross MD lesions. Birds were scored for MD lesions and mortality. MDV and HVT genome copy numbers were determined for each PBL sample. There was an increase in HVT load in PBL between days 7 and 37 post-vaccination, with marked increases between days 7 and 16 and again between days 30 and 37. There was a steady increase in MDV load to 35 days post-challenge. The mean MDV copy number (log(10)) was greater in chickens subsequently exhibiting gross MD lesions (5.05 +/- 0.21) than in those that did not (2.88 +/- 0.223), with the largest difference at 14 and 21 days post-challenge (P < 0.001). Quantification of MDV during early infection is therefore a potential tool for monitoring MD in broiler flocks.     

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.