Search

Understanding the pathogenesis of herpesvirus of turkeys (HVT) in its natural host is necessary before recombinant HVT (rHVT) can be used efficiently in turkey flocks. The objectives of this study were to evaluate when commercial turkey flocks get infected with wild type HVT, to study replication of HVT (conventional and recombinant rHVT-Newcastle disease, rHVT-ND) and other Marek's disease (MD) vaccines (SB-1 and CVI988) in turkey embryonic tissues, and to evaluate the expression of TLR-3 and IFN-γ in the lung and spleen of one-day-old turkeys after in ovo vaccination with MD vaccines. Our results demonstrated that commercial turkeys got exposed to wild type HVT within the first days of life; therefore, there is a potential of interaction between wild type HVT and rHVT when administered at day of age. On the other hand, all evaluated vaccines (especially HVT and rHVT-ND) replicated very well in turkey embryonic tissues. In ovo vaccination with HVT and CVI988 increased transcription of TLR-3 in the spleen of one-day-old turkeys. However, no effect on the transcription of TLR-3 or IFN-γ in the lungs and IFN-γ in the spleen in newly hatched turkeys was detected in the present study. Because of the limitations of evaluated genes, timepoints, and studied tissues, future studies are warranted to better understand the effect of MD vaccines on the turkey embryo immune responses.RESEARCH HIGHLIGHTS Commercial turkey flocks get infected with wild type HVT within the first days of life.HVT and rHVT replicates readily in turkey embryonic tissues.SB-1 and CVI988 also replicate in turkey embryonic tissues, but at lower rates than HVT and rHVT.HVT and CVI988 increase transcription of TLR-3 in the spleen.

Md5-BAC-REV-LTR is a recombinant Marek's disease virus (MDV), with an insertion of the long terminal repeat (LTR) of reticuloendotheliosis virus (REV) into the genome of the highly virulent MDV strain rMd5. It has been shown that Md5-BAC-REV-LTR does not induce tumours and confers high protection against challenge with MDV in 15 × 7 chickens. The objective of the present study was to evaluate the protection and safety (in terms of oncogenicity and immunosuppression) of Md5-BAC-REV-LTR in commercial meat-type chickens bearing maternal antibodies against MDV. Our results show that sub-cutaneous administration of Md5-BAC-REV-LTR at 1 day of age conferred high protection (protection index PI = 84.2) against an early challenge (1 day) by contact exposure to shedder birds infected with the vv+ MDV 648A strain. In such stringent challenge conditions, Md5-BAC-REV-LTR was more protective than a commercial CVI988 (PI = 12.4) and similar to the experimental vaccine Md5-BACΔmeq (PI = 92.4). Furthermore, Md5-BAC-REV-LTR did not induce either tumours or immunosuppression in this study. Immunosuppression was evaluated by the relative lymphoid organ weights and also by the ability of the vaccine to induce late-MDV-induced immunosuppression associated with reactivation of the virus. This study shows that Md5-BAC-REV-LTR has the potential to be used as a MD vaccine and is highly protective against early challenge with vv+ MDV. RESEARCH HIGHLIGHTSMd5-BAC-REV-LTR is highly protective against early challenge with vv+ MDV in commercial meat-type chickens.Md5-BAC-REV-LTR does not cause early immunosuppression.Md5-BAC-REV-LTR does not cause late immunosuppression.Unlike other serotype 1 vaccines, Md5-BAC-REV-LTR is not detected in feather pulp at 7 days post vaccination.

Cytokine transcripts were evaluated chronologically in the brain and in the eye of chickens infected with the very virulent plus Marek's disease virus (vv + MDV) strain 648A. Brain and eye samples were collected from chickens that were either suffering from transient paralysis (TP) (11 days post inoculation, dpi) or had completely recovered from TP but started developing clinical signs of persistent neurological disease (PND) (18-31 dpi). Results obtained from samples collected at 11 dpi are referred as EL (early lesions) and results obtained from samples collected at later times (18-31 dpi) are referred as LL (late lesions). Marked differences were found in the cytokine transcripts in brain and eye. While proinflammatory cytokines (IL-1β, IL-8, IL-18), iNOS, IFN-α, IFN-γ, and IL-15 were upregulated in the brain during EL and LL, only IL-8 and IFN-γ were upregulated in the eye at both times (EL and LL). The two evaluated viral transcripts (gB and meq) were found in both eye and brain during EL and LL. Levels of the two viral transcripts evaluated were higher at LL than at EL in both brain and eye. No differences were found in any of the viral transcripts between eye and brain during EL. However, during the LL, the levels of meq transcripts were higher in the eye than in the brain. Our results suggest that MDV elicits different immune responses in the brain and in the eye of infected chickens. Because immune responses in the eye of chickens have been poorly studied, further studies on the pathogenesis of MDV in the eye could greatly contribute to our knowledge on the chicken eye immunity.

Marek’s disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes T cell lymphoma in chickens. MDV-encoded Meq and vIL8 proteins play important roles in transformation and early cytolytic infection, respectively. Previous studies identified a spliced transcript, meq-vIL8, formed by alternative splicing of meq and vIL8 genes in MDV lymphoblastoid tumour cells. To determine the role of Meq-vIL8 in MDV pathogenesis, we generated a recombinant MDV (MDV-meqΔSD) by mutating the splice donor site in the meq gene to abrogate the expression of Meq-vIL8. As expected, our results show that MDV-meqΔSD virus grows similarly to the parental and revertant viruses in cell culture, suggesting that Meq-vIL8 is dispensable for MDV growth in vitro. We further characterized the pathogenic properties of MDV-meqΔSD virus in chickens. Our results show that lack of Meq-vIL8 did not affect virus replication during the early cytolytic phase, as determined by immunohistochemistry analysis and/or viral genome copy number, but significantly enhanced viral DNA load in the late phase of infection in the spleen and brain of infected chickens. In addition, we observed that abrogation of Meq-vIL8 expression reduced the mean death time and increased the prevalence of persistent neurological disease, common features of highly virulent strains of MDV, in inoculated chickens. In conclusion, our study shows that Meq-vIL8 is an important virulence factor of MDV.

In ovo vaccination with herpesvirus of turkey (HVT) or recombinant HVT (rHVT) is commonly used in meat-type chickens. Previous studies showed that in ovo vaccination with HVT enhances innate, cellular, and humoral immune responses in egg-type chicken embryos. This study evaluated if in ovo vaccination with HVT hastens immunocompetence of commercial meat-type chickens and optimized vaccination variables (dose and strain of HVT) to accelerate immunocompetence. A conventional HVT vaccine was given at recommended dose (RD), HVT-RD = 6080 plaque forming units (PFU), double-dose (2x), half-dose (1/2), or quarter-dose (1/4). Two rHVTs were given at RD: rHVT-A = 7380 PFU, rHVT-B = 8993 PFU. Most, if not all, treatments enhanced splenic lymphoproliferation with Concanavalin A and increased the percentage of granulocytes at day of age. Dose had an effect and HVT-RD was ideal. An increase of wing-web thickness after exposure to phytohemagglutinin-L was only detected after vaccination with HVT-RD. Furthermore, compared to sham-inoculated chickens, chickens in the HVT-RD had an increased percentage of CD3+ T cells and CD4+ T-helper cells, and increased expression of major histocompatibility complex (MHC)-II on most cell subsets (CD45+ cells, non-T leukocytes, T cells and the CD8+ and T cell receptor γδ T-cell subsets). Other treatments (HVT-1/2 and rHVT-B) share some of these features but differences were not as remarkable as in the HVT-RD group. Expression of MHC-I was reduced, compared to sham-inoculated chickens, in most of the cell phenotypes evaluated in the HVT-RD, HVT-2x and rHVT-A groups, while no effect was observed in other treatments. The effect of in ovo HVT on humoral immune responses (antibody responses to keyhole limpet hemocyanin and to a live infectious bronchitis/Newcastle disease vaccine) was minimal. Our study demonstrates in ovo vaccination with HVT in meat-type chickens can accelerate innate and adaptive immunity and we could optimize such effect by modifying the vaccine dose.

A healthy immune system is a cornerstone for poultry production. Any factor diminishing the immune responses will affect production parameters and increase cost. There are numerous factors, infectious and noninfectious, causing immunosuppression (IS) in chickens. This paper reviews the three viral diseases that most commonly induce IS or subclinical IS in chickens: Marek's disease virus (MDV), chicken infectious anemia virus (CIAV), and infectious bursal disease virus (IBDV), as well as the interactions among them. MDV-induced IS (MDV-IS) affects both humoral and cellular immune responses. It is very complex, poorly understood, and in many cases underdiagnosed. Vaccination protects against some but not all aspects of MDV-IS. CIAV induces apoptosis of the hemocytoblasts resulting in anemia, hemorrhages, and increased susceptibility to bacterial infections. It also causes apoptosis of thymocytes and dividing T lymphocytes, affecting T helper functions, which are essential for antibody production and cytotoxic T lymphocyte (CTL) functions. Control of CIAV is based on vaccination of breeders and maternal antibodies (MAbs). However, subclinical IS can occur after MAbs wane. IBDV infection affects the innate immune responses during virus replication and humoral immune responses as a consequence of the destruction of B-cell populations. Vaccines with various levels of attenuation are used to control IBDV. Interactions with MAbs and residual virulence of the vaccines need to be considered when designing vaccination plans. The interaction between IBDV, CIAV, and MDV is critical although underestimated in many cases. A proper control of IBDV is a must to have proper humoral immune responses needed to control CIAV. Equally, long-term control of MDV is not possible if chickens are coinfected with CIAV, as CIAV jeopardizes CTL functions critical for MDV control.

The effect of two Marek's disease (MD) vaccines on the chicken embryo immune responses were evaluated. Transcription of interferon (IFN-α, IFN-β, IFN-λ, and IFN-γ) and interferon-I receptors (IFN-AR1 and IFN-AR2), as well as transcription of toll like receptors (TLR-3, TLR-7, and TLR-21) were evaluated in the bursa, thymus, spleen and lung of 1-day-old chickens that had been vaccinated with HVT, CVI988, or sham inoculated at embryonic day 18 (ED18). Each vaccine had a unique effect on the transcription of the evaluated genes and it differs among tissues. HVT increased IFN-γ and TLR-3 transcripts in the spleen and lung and IFN-β in the bursa. The immune responses elicited by CVI988 differed from that observed in the HVT inoculated group. CVI988 downregulated several of the studied genes and only upregulated IFN-β and TLR-21 in spleen. Differences in vaccine replication (53% of spleens and lungs of HVT-vaccinated embryos but only 22% of spleens of CVI988-vaccinated embryos had detectable viral gB transcripts) were detected. Previously, we have shown that intra-amniotic vaccination at ED18 with HVT but not with CVI988 rendered chickens more immunocompetent at hatch. The role of increased transcription of TLR-3 and IFN-γ in such positive effect warrant further investigations.

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.

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.

Marek's disease (MD) vaccines are cell-associated and require special handling and care during administration. Vaccine dose is evaluated by plaque assay and is indicated as the number of plaque-forming units (PFUs) per dose. The objectives of this study were to evaluate the dose variability within each vial of MD vaccines and to assess those factors (from both manufacturing and handling and administration of the vaccine) that could affect vaccine dose variability. Three experiments were conducted. Experiment 1 was to evaluate dose variability in 36 MD vaccine vials and the effect of manufacturing factors on dose variability. Vaccines were titrated 10 times. Dose variability was measured as the coefficient of variability (CV) calculated as standard deviation divided by average PFU and multiplied by 100. Our results showed that all evaluated vaccines had levels of CV ranging from 10% to 34%. Variability existed regardless of manufacturer, vaccine serotype, and batch. Experiment 2 was conducted to evaluate the effect of infectivity rate (IR) on CV. IR was artificially reduced by adding noninfected chicken embryo fibroblast to the reconstituted vaccine before titration. Our results showed that decreased IR results in higher CV. Experiment 3 was to evaluate the handling and administration factors (time and mixing during administration) on CV. Our results showed that CV tends to increase with time and that this effect is more remarkable if vaccines were not mixed. Our study emphasizes the relevance of proper handling of MD vaccines and shows that dose variability can jeopardize the uniformity of vaccination in a flock and therefore the success of vaccination.Evaluación de factores que influyen en la variabilidad de las dosis de las vacunas contra la enfermedad de Marek. Las vacunas contra la enfermedad de Marek (MD) están asociadas a células y requieren un manejo y cuidado especiales durante la administración. La dosis de la vacuna se evalúa mediante un ensayo de placa y se indica como el número de unidades formadoras de placa (UFP) por dosis. Los objetivos de este estudio fueron evaluar la variabilidad de la dosis dentro de cada vial de vacunas contra la enfermedad de Marek y evaluar los factores (tanto de fabricación como de manipulación/administración de la vacuna) que podrían afectar la variabilidad de la dosis de la vacuna. Se realizaron tres experimentos. El experimento número 1 consistió en evaluar la variabilidad de la dosis en 36 viales de vacuna de Marek y el efecto de los factores de fabricación en la variabilidad de la dosis. Las vacunas fueron tituladas 10 veces. La variabilidad de la dosis se midió como el coeficiente de variación (CV) calculado como desviación estándar dividido por las UFP promedio y multiplicado por 100. Nuestros resultados mostraron que todas las vacunas evaluadas tenían coeficientes de variación que variaban del 10% al 34%. La variabilidad existía independientemente del fabricante, el serotipo de la vacuna y el lote. El experimento número 2 se realizó para evaluar el efecto de la tasa de infectividad (IR) en el coeficiente de variación. La tasa de infectividad se redujo artificialmente mediante la adición de fibroblastos de embrión de pollo no infectados a la vacuna reconstituida antes de la valoración. Los resultados mostraron que la disminución en la tasa de infectividad resulta en mayores coeficientes de variación. El experimento número 3 consistió en evaluar los factores de manipulación y administración (tiempo y mezclado durante la administración) sobre los coeficientes de variación. Nuestros resultados mostraron que el coeficiente de variación tiende a aumentar con el tiempo y que este efecto es más notable si las vacunas no se mezclan. Este estudio enfatiza la relevancia del manejo adecuado de las vacunas contra la enfermedad de Marek y muestra que la variabilidad de la dosis puede poner en peligro la uniformidad de la vacunación en una parvada y por lo tanto el éxito de la vacunación.

Marek’s disease virus (MDV) is a herpesvirus that induces lymphoma and a variety of non-neoplastic syndromes in chickens. Furthermore, very virulent plus (vv+) MDVs induce a form of immunosuppression (late-MDV-IS) that might involve both neoplastic and non-neoplastic mechanisms. The objective of this study was to evaluate whether the attenuation of MDV-induced tumours and late-MDV-IS occurs simultaneously or can be dissociated. The immunosuppressive ability of three viruses derived from vv+ MDV strain 686 (wild-type 686, the somewhat attenuated molecular clone 686-BAC, and the nononcogenic molecular clone lacking the two copies of the oncogene meq 686-BACΔMEQ) was evaluated. Late-MDV-IS was evaluated indirectly by assessing the negative effect of MDV strains on the protection conferred by infectious laryngotracheitis (ILT) vaccines. Our results showed that the ability to induce late-MDV-IS was attenuated before the ability to induce tumours. Strain 686 induced both tumours and late-MDV-IS, 686-BAC induced tumours but did not induce late-MDV-IS and 686-BACΔMEQ did not induce either tumours or late-MDV-IS. Further comparison of strains 686 and 686-BAC revealed that strain 686 reduced the humoral immune responses to ILTV (1132 vs 2167) more severely, showed higher levels of meq transcripts (2.1E+09 vs 4.98E+8) and higher expression of MDV microRNAs (mdv1-miR-M4-5p and mdv1-miR-M2-3p) in the spleen, and further reduced the percentage of CD45+-MHC-I+splenocytes (13 vs32 %) compared to molecular clone 686-BAC. This study suggests that the immunosuppressive ability of MDV might follow a continuous spectrum and only the most virulent MDVs can overcome a certain threshold level and induce clinical MDV-IS in the ILT model.

This article reports nine cases of neurological disease in brown layer pullets that occured in various European countries between 2015 and 2018. In all cases, the onset of neurological clinical signs was at 4-8 weeks of age and they lasted up to 22 weeks of age. Enlargement of peripheral nerves was the main lesion observed in all cases. Histopathological evaluation of nerves revealed oedema with moderate to severe infiltration of plasma cells. Marek's disease (MD) was ruled out by real-time PCR as none of the evaluated tissues had a high load of oncogenic MD virus (MDV) DNA, characteristics of MD. Based on the epidemiological data (layers with clinical signs starting at 5-8 weeks of age), gross lesions (peripheral nerve enlargement with a lack of tumours in other organs), histopathological lesions (oedema and infiltration of plasma cells), and no evidence of high load of MDV DNA, we concluded that those cases were due to peripheral neuropathy (PN). PN is an autoimmune disease easily misdiagnosed as MD, leading to a costly enforcement of the vaccination protocol. Additional vaccination against MD does not protect against PN and could worsen the clinical signs by over-stimulating the immune system. Differential diagnosis between PN and MD should always be considered in cases of neurological disease with enlargement of peripheral nerves as the only gross lesion. This case report shows for the first time how real-time PCR to detect oncogenic MDV is a very valuable tool in the differential diagnosis of PN and MD.

Laryngotracheitis (LT) is a highly contagious respiratory disease of chickens that produces significant economic losses to the poultry industry. Traditionally, LT has been controlled by administration of modified live vaccines. In recent years, the use of recombinant DNA-derived vaccines using turkey herpesvirus (HVT) and fowlpox virus has expanded, as they protect not only against the vector used but also against LT. However, HVT-based vaccines confer limited protection against challenge, with emergent very virulent plus Marek's disease virus (vv+MDV). Serotype 1 vaccines have been proven to be the most efficient against vv+MDV. In particular, deletion of oncogene MEQ from the oncogenic vvMDV strain Md5 (BACδMEQ) resulted in a very efficient vaccine against vv+MDV. In this work, we have developed two recombinant vaccines against MD and LT by using BACδMEQ as a vector that carries either the LT virus (LTV) gene glycoprotein B (gB; BACΔMEQ-gB) or LTV gene glycoprotein J (gJ; BACδMEQ-gJ). We have evaluated the protection that these recombinant vaccines confer against MD and LT challenge when administered alone or in combination. Our results demonstrated that both bivalent vaccines (BACΔMEQ-gB and BACδMEQ-gJ) replicated in chickens and were safe to use in commercial meat-type chickens bearing maternal antibodies against MDV. BACΔMEQ-gB protected as well as a commercial recombinant (r)HVT-LT vaccine against challenge with LTV. However, BACδMEQ-gJ did not protect adequately against LT challenge or increase protection conferred by BACΔMEQ-gB when administered in combination. On the other hand, both BACΔMEQ-gB and BACδMEQ-gJ, administered alone or in combination, protected better against an early challenge with vv+MDV strain 648A than commercial strains of rHVT-LT or CVI988. Our results open a new avenue in the development of recombinant vaccines by using serotype 1 MDV as vectors.

Marek's disease virus (MDV) is a herpesvirus that induces lymphomas and immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is divided into two phases: early-MDV-IS occurring mainly in chickens lacking maternal antibodies (MAb) against MDV and associated with lymphoid organ atrophy; and late-MDV-IS occurring once MDV enters latency and during tumour development. Our objectives were to document the impact of late-MDV-IS on commercial poultry (meat-type chickens bearing MAb against MDV and that were vaccinated or unvaccinated against MD) and to optimize a model to study late-MDV-IS under laboratory conditions. The impact of late-MDV-IS was evaluated by assessing the effect of early infection (day of age) with a very virulent plus MDV (vv+MDV) on the efficacy of chicken-embryo-origin (CEO) infectious laryngotracheitis (ILT) virus vaccine against ILT challenge. The CEO ILT vaccine was administered in water at 14 days of age and ILT virus (ILTV) challenge was done intratracheally at 30 days of age. Development of ILT was monitored by daily evaluation of clinical signs, development of gross and histological lesions in trachea, and quantification of ILTV transcripts in trachea. Infection with vv+MDV strain 648A resulted in total abrogation of protection conferred by the CEO vaccine against ILTV challenge even in chickens vaccinated at 1 day of age with either HVT, HVT+SB-1, or CVI988. Chickens exposed to vv+MDV prior to vaccination with CEO ILTV vaccine had similar (P

A questionnaire was widely distributed in 2011 to estimate the global prevalence of Marek's disease (MD) and gain a better understanding of current control strategies and future concerns. A total of 112 questionnaires were returned representing 116 countries from sources including national branch secretaries of the World Veterinary Poultry Association, vaccine, breeder, and production companies, as well as MD researchers from various backgrounds. Each country listed on a questionnaire was recorded as an individual entry, and on average there were 2.0 entries per country (median 1; range 1-13). All flock types were listed as having increased MD incidence during the last 10 yr in close to 50% of countries by at least one respondent, with the majority of these countries located within French-speaking Africa, Eastern Europe, East Asia, and South America. Only 18 countries (16%) indicated increasing MD incidence was likely due to higher virulent strains, while the presence of other immunosuppressive diseases was a more common explanation. Increased use of CVI988/Rispens was cited as the most likely reason for decreasing MD incidence in 49 countries (42%). In the United States, MD incidence has continued to decrease during the last 10 yr, reaching a record low in 2007 (0.0008%) as measured by leukosis condemnation rates in broilers at slaughter. However, a recent increase of leukosis condemnations in North Carolina and Pennsylvania needs to be closely monitored.

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.

Marek's disease virus (MDV) induces tumors and severe immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is very complex and difficult to study. In particular, the late MDV-IS (late-MDV-IS) is of great concern since it can occur in the absence of lymphoid organ atrophy or gross tumors. We have recently developed a model to reproduce late-MDV-IS under laboratory conditions. This model measures MDV-IS indirectly by assessing the effect of MDV infection on the efficacy of infectious laryngotracheitis (ILT) vaccination; hence the name late-MDV-IS ILT model. In this study, we have used the late-MDV-IS ILT model to evaluate if MD vaccination can protect against late-MDV-IS. One experiment was conducted to determine whether serotype 1 MD vaccines (CVI988 and Md5ΔMEQ) could induce late-MDV-IS by themselves. Three additional experiments were conducted to evaluate efficacy of different MD vaccines (HVT, HVT+SB-1, CVI988, and Md5ΔMEQ) and different vaccine protocols (day-old vaccination, in ovo vaccination, and double vaccination) against late-MDV-IS. Our results show that none of the currently used vaccine protocols (HVT, HVT+SB-1, or CVI988 administered at day of age, in ovo, or in double vaccination protocols) protected against late-MDV-IS induced by vv+MDV strains 648A and 686. Experimental vaccine Md5ΔMEQ administered subcutaneously at one day of age was the only vaccine protocol that significantly reduced late-MDV-IS induced by vv+MDV strain 686. This study demonstrates that currently used vaccine protocols confer high levels of protection against MDV-induced tumors (protection index=100), but do not protect against late-MDV-IS; thus, commercial poultry flocks could suffer late-MDV-IS even in complete absence of tumors. Our results suggest that MDV-IS might not be related to the development of tumors and novel control methods are needed. Further evaluation of the experimental vaccine Md5ΔMEQ might shed light on protective mechanisms against late-MDV-IS.

Cells expressing CD4, CD8, major histocompatibility complex (MHC) Class II, and macrophage biomarkers in lungs of chickens were quantified by measuring total area of antigen expressed using imageJ, a software program developed at the National Institutes of Health and available at no cost. The procedures reported here were rapid, and reproducible. Total area of antigen expressed had positive correlation with manual counts of cells expressing CD4 and CD8 biomarkers after inoculation with serotype 1 Marek's disease virus (MDV) vaccines. Visual inspection and overlays prepared from outlines of cells counted by imageJ confirmed agreement between antigen expression and area measured. Total area measured was not dependent on time of image acquisition from randomly selected fields from the same slides. Total area values were not computer specific, but acquisition of the original images required standardization of microscope used and camera setup. All steps in the process from sample collection through sectioning, staining, and image acquisition must be standardized as much as possible. Chickens infected with a very virulent+ (vv + ) isolate of MDV (648A) had increased CD4, CD8, MHC Class II, and macrophage biomarker expression compared to noninfected control chickens at 10 days post infection, but variable responses depending on the specific biomarker measured at 3 and 5 days post infection. The procedure described here is faster and more reproducible than manual counting in cases (CD4 and CD8) where the number of positive cells is low enough for manual counts. Manual counting is not possible with MHC Class II and macrophage antigens nor when CD4 + cells are present in large numbers following proliferation to tumors, thus subjective systems are used for scoring in these conditions. Using imageJ as described eliminates the need for subjective and less reproducible methods for measuring expression of these antigens.

The present work is a chronological study of the pathogenesis of three attenuated serotype 1 Marek's disease (MD) virus strains (RM1, CVI988 and 648A80) that provide high protection against MD but have been attenuated by different procedures and induce different degrees of lymphoid organ atrophy. All studied strains replicated in the lymphoid organs (bursa,x thymus and spleen) and a peak of replication was detected at 6 days post inoculation (d.p.i.). Differences, however, were observed among vaccine strains. RM1 strain replicates more in all lymphoid organs compared with CVI988 and 648A80 strains. In addition, replication of RM1 in the thymus did not decrease after 6 d.p.i. but continued at high levels at 14 d.p.i. and until the thymus was completely destroyed. Lung infection occurred very early after infection with all of the three vaccines and the level of replication was similar to that found in the lymphoid organs. Infected cells were very large and appeared scattered in the lung parenchyma and in the parabronchial lining. The study of the target cells for the early infection in cell suspensions of blood and spleen showed that both non-adherent cell populations (enriched in lymphoid cells) and adherent cells (enriched in monocytes/macrophages) supported MD virus infection. Infection in adherent cells was especially high at very early stages of the infection (3 to 6 d.p.i.). Atrophy of lymphoid organs is a major drawback in the production of highly protective vaccines against MD. A better understanding of the mechanisms associated with lymphoid organ atrophy will aid in overcoming this problem.

Replication of a recombinant herpesvirus of turkey vaccine expressing infectious laryngotracheitis virus genes (rHVT-LT) was evaluated in specific pathogen free (SPF) and commercial broiler chickens after various vaccination protocols (amniotic route at embryonation day [ED] 18; intra-embryonic route at ED 19; and subcutaneous at 1 day of age [s.c.]). Three experiments were conducted: in the first experiment, replication of rHVT-LT vaccine was chronologically evaluated and compared with the replication of herpesvirus of turkey (HVT) in SPF chickens; in the second experiment, the effect of different in ovo vaccination procedures on rHVT-LT vaccine replication was evaluated in SPF chickens; and in the third experiment, the effect of different in ovo vaccination procedures on rHVT-LT vaccine replication was evaluated in commercial broiler chickens with maternal antibodies against HVT and infectious laryngotracheitis virus (LTV). rHVT-LT vaccine replicated in chickens after in ovo (ED 18 and ED 19) or s.c. administration at a similar level. In vivo replication of rHVT-LT vaccine was slower than HVT vaccine. However, in vivo both rHVT-LT and HVT vaccines replicated at similar levels. Both vaccines were consistently detected in the spleen and feather pulp and at lower frequency in the lung. The frequency of samples with detectable levels of rHVT-LT DNA was lower in broiler chickens than in SPF chickens, probably due to interactions with maternal antibodies. Differences between SPF chickens and broiler chickens were found also in the transcription of the LTV glycoprotein I gene (gI). In SPF chickens, in ovo inoculation resulted in a higher number of spleen samples with detectable gI transcripts than s.c. inoculation. In broiler chickens, however, no differences in the level of gI transcripts in spleen samples were found between chickens vaccinated in ovo and those vaccinated by the s.c. route. Transcription of LTV gI gene in lung samples was very low in both SPF and broiler chickens. Further studies to evaluate the mucosal immune response elicited by rHVT-LT in chicken with and without maternal antibodies are warranted.

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 5 dpv but decreased transcripts of IL-6, IL-8, and IL-18 at 10 dpv in the lung. HP vaccinated chickens had increased IFN-γ in the spleen at 3 and at 10 dpv. 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.

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.

The role of subgroup J avian leukosis virus (ALV J) infection profile in the development of histiocytic sarcomatosis (HS) in chickens was evaluated using retrospective analysis of 2 experiments involving in ovo and at-hatch inoculation of commercial meat-type and ADOL line 0 chickens with 100 or 10,000 TCID50of various strains ALV J. HS was observed only in persistently viremic, meat-type chickens that were inoculated at hatch, but not in immunotolerized (persistently viremic, with no antibodies), in ovo inoculated chickens. However, the immunotolerized, in ovo inoculated chickens developed a high incidence of myeloid tumors. HS appeared to arise from the splenic ellipsoids and red pulp, and metastasized to liver, kidney, and other organs. The neoplastic cells were diffusely positive for ChL5, CD45, and MHC class II with multifocal infiltration of T and B lymphocytes. Expression of viral antigen gp85 within HS was very low compared with that noted in ALV J-induced myelocytomas. The above observations suggest that the mechanisms of oncogenesis of HS might be different from that of other ALV J-induced 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 with75% 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.

Marek's disease is an economically important disease of poultry and is caused by an alphaherpesvirus, Marek's disease virus (MDV). The predicted protein product of the MDV UL41 open reading frame has significant protein sequence identity with the virion host shutoff (vhs) protein gene in other alphaherpesviruses. To determine whether the MDV UL41 gene functions as a vhs protein, we utilized a transient co-transfection assay and demonstrated that the MDV UL41 protein was as active in degrading RNA as the vhs protein of herpes simplex virus type 1. To evaluate whether the MDV UL41 gene was involved in pathogenesis, we deleted the MDV UL41 gene. The UL41 deletion mutant replicated in cell culture as well as the parental MDV. The deletion mutant was inoculated into susceptible day-old chicks. The pattern and degree of tumor lesions and neurovirulence produced by the deletion mutant was same as the pattern of lesions induced by the parental virus. The only observable difference between the inoculation of MDV and the MDV deletion mutant was that the early in vivo cytolytic infection with the deletion mutant was of a longer duration than in the non-mutant MDV.

Immunohistochemistry and polymerase chain reaction (PCR) were used to test for the presence of avian leukosis virus (ALV) J viral antigen gp85 and proviral DNA, respectively, in various tissues (adrenal gland, bone marrow, gonad, heart, kidney, liver, lung, pancreas, proventriculus, sciatic nerve, spleen, and thymus). Tissues were collected from 32-week-old commercial meat-type and Avian Disease and Oncology Laboratory experimental White Leghorn Line 0 chickens with the following different infection profiles: tV + A-, included in ovo-tolerized viraemic chickens with no neutralizing antibodies (NAbs) on any sampling; ntV + A-, included chickens that were viraemic and NAb-negative at the time of termination at 32 weeks post hatch, but had NAbs on up to two occasions; V+ A+, included chickens that were viraemic and NAb-positive at the time of termination at 32 weeks post hatch, and had NAbs on more than two occasions; V - A+, included chickens that were negative for viraemia and NAb-positive at the time of termination at 32 weeks post hatch, and had antibody on more than two occasions; V - A-, included chickens that were never exposed to ALV J virus. There was a direct correlation between viraemia and tissue distribution of gp85, regardless of the NAb status and strain of chickens, as expression of ALV J gp85 was noted in only viraemic chickens (tV + A-, ntV + A-, V+ A+), but not in non-viraemic seroconverted chickens (V - A+). Of the four oligonucleotide primers pairs used in PCR to identify ALV J provirus, only one primer set termed H5/H7 was useful in demonstrating ALV J proviral DNA in the majority of the tissues tested from non-viraemic, antibody-positive chickens (V - A+). The results suggest that PCR using primer pair H5/H7 is more sensitive than immunohistochemistry in identifying ALV J in chickens that have been exposed to virus, but are not actively viraemic.

Marek's disease virus (MDV) can be attenuated by serially passing the virus in cell culture. During cell culture passage, two copies of a 132 bp repeat are expanded to over 30 copies. We deleted the two copies of the 132 bp repeat region in a pathogenic MDV and demonstrated that the virus was still pathogenic. The pattern and frequency of tumors in the parental and mutant virus were the same. Early virus replication, and the appearance of persistent neurological disease were also similar between the parental and deleted virus. Nevertheless, wild-type MDV and the deletion virus could be attenuated by serial in vitro cell culture passages. Based upon analyzing the passage 40 viruses, attenuation of the MDV lacking the 132 bp repeats appears to occur in a manner that is analogous to the process occurring wild-type MDV attenuation. Whatever process is involved in the cell culture attenuation of MDV, the mechanism does not involve the 132 bp repeat region.

Marek's disease (MD) strain CVI988 is the most-protective commercially available vaccine against very virulent plus (vv+) Marek's disease virus (MDV). However, its use in meat-type chickens has been controversial. While several countries have been using CVI988 for more than 40 yr, others do not authorize its use or it is restricted mainly to layers. The use of CVI988 in meat-type chickens will be necessary in the future in areas where other vaccine protocols fail. The objective of this study was to evaluate factors (vaccine dose, vaccine origin, chicken genetics, age and route of vaccination, and combination with other MD vaccines) influencing the efficacy of CVI988 against MD in meat-type chickens. Three animal experiments were conducted in which various vaccine protocols using CVI988 were tested for their protection against challenge with vv+ strain 648A by contact at day of age. Experiments 1 and 2 were to compare the efficacy of CVI988 vaccines from three different origins (CVI988-A, CVI988-B, and CVI988-C) and evaluate the effect of vaccine dose and chicken genetics. Experiment 3 was to evaluate the effect of adding CVI988 vaccine to various vaccine protocols using other MD vaccines of serotypes 2 (SB-1) and 3 (rHVT). Our results show that, regardless of the origin of the vaccine, protection against early challenge with 648A was good when vaccines were administered at a high dose (3000 plaque-forming units [PFU]). Differences among vaccines, however, were detected even when using a high dose in experiment 2 (vaccine CVI988-B conferred higher protection than did CVI988-C) but not in Experiment 1 (CVI988-B was compared to CVI988-A). The use of a fixed low dose (2000 PFU) of vaccine resulted in reduction in protection, and such reduction was more remarkable when using CV1988-A. No statistically significant differences were found when we compared the efficacy of CVI988 in two different genetic lines of broiler chickens (G1 and G2). Vaccination protocols that included CVI988 had better protection than protocols that only included MD vaccines of serotypes 2 and 3. This was true regardless of the vaccine protocol used (CVI988/rHVT+SB-1; CVI988+rHVT+SB-1/None; rHVT+SB-1/CVI988; wherein the vaccine before the slash (/) was administered in ovo at embryonation day 18 and the vaccine after the slash was administered at day of age, subcutaneously). When only vaccines of serotypes 2 and 3 were used, protection against early challenge with vv+MDV was higher when vaccines were administered in ovo (rHVT+SB-1/None) than if vaccines were administered at hatch (None/rHVT+SB-1). Monitoring vaccine DNA load in feather pulp (FP) samples at 1 wk was used to monitor vaccination, and results showed that differences in vaccine replication exist among vaccines but such differences were not necessarily related to protection (r = 0.41, P0.05). Monitoring load of challenge MDV DNA in FP at 21 days was conducted, and results correlated (r = 0.85, P0.05) with the percentage of chickens with MD lesions at the termination of the study, confirming that early diagnosis is a very powerful tool with which to evaluate protection.

Administration of Marek's disease (MD) vaccines in ovo has become a common practice for the poultry industry. Efficacy of MD vaccines is very high, even though they are administered to chicken embryos that are immunologically immature. We have recently demonstrated that in ovo vaccination with turkey herpesvirus (HVT) results in increased activation of T cells at hatch. Our previous results suggested that in ovo vaccination with HVT might have a positive impact not only on MD protection but also on the overall maturity of the developing immune system of the chicken (Gallus gallus domesticus). The objective of this study was to evaluate the effect of administration of HVT at 18 days of embryonation (ED) on the maturation of the embryo immune system. Four experiments were conducted in Specific-Pathogen-Free Avian Supplies (SPAFAS) chickens to evaluate the effect of administration of HVT at 18 ED on the splenic cell phenotypes at day of age (experiment 1) and on the ability of 1-day-old chickens to respond to various antigens compared with older birds (experiments 2 and 3). In addition, a fourth experiment was conducted to elucidate whether administration of other serotype's MD vaccines (CVI988 and SB-1) at 18 ED had the same effect as HVT on the spleen cell phenotypes at day of age. Our results demonstrated that 1-day-old chickens that had received HVT in ovo (1-day HVT) had higher percentages of CD45+, MHC-I+, CD45+MHC-I+, CD3+, MHC-II+, CD3+MHC-II+, CD4+, CD8+, and CD4+CD8+ cells in the spleen than 1-day-old sham-inoculated chickens (1-day sham). Moreover, spleens of 1-day HVT chickens had greater percentages of CD45+MHC-I+ cells and equal or greater numbers of CD4+CD8- and CD4-CD8+ cells than older unvaccinated chickens. In addition, administration of HVT at 18 ED rendered chicks at hatch more responsive to unrelated antigens such as concavalin A, phytohemagglutinin-L, and keyhole limpet hemocyanin. Administration of MD vaccines of other serotypes had an effect, although less remarkable than HVT, on the spleen cell phenotypes at hatch. Vaccines of all three serotypes resulted in an increased percentage of MHC-I+, CD45-MHC-I+, CD4-CD8+, and CD8+ cells, but only HVT resulted in a higher percentage of CD45+, CD45+MHC-I+, CD3+MHC-II+, and CD4+CD8- cells. Results of this study show that it is possible to hasten maturation of the chicken embryo immune system by administering HVT in ovo and open new avenues to optimize the procedure to improve and strengthen the immunocompetency of commercial chickens at hatch.

SUMMARY. Marek’s disease (MD) outbreaks can occur in previously healthy adult layer or breeder flocks. However, it is not clear whether such outbreaks are caused by recent challenge with highly virulent (vv and vvþ) strains of MD virus (MDV; i. e., new infection hypothesis) or by exacerbation of an earlier MDV infection (i. e., old infection hypothesis). To discriminate between these hypotheses, adult White Leghorn chickens of laboratory strains or commercial crosses with or without prior vaccination or MDV exposure were challenged at 18–102 wk of age with highly virulent MDVs, and lesion responses were measured. Horizontal transmission was studied in one trial. Challenge of adult chickens, which were free from prior MDV vaccination or exposure, with highly virulent MDV strains induced transient paralysis or tumors in 60%–100% of 29 groups (mean ¼ 91%), and horizontal spread of virus was detected. The magnitude of the response was similar to that induced by challenge at 3 wk of age. In contrast, comparable challenge of adult chickens, which had been vaccinated or exposed to MDV early in life, induced transient paralysis or tumors in 0%–6% of 12 groups (mean ¼ 0. 5%), although some birds showed limited virologic evidence of infection and transmission of the virus to contacts. The MD responses were influenced by the virulence of the challenge virus strain, and to a lesser extent by virus dose and route of exposure. Strong inflammatory lesions were induced in the brain and nerves of adult specific pathogen-free (SPF) chickens at 9–15 days after infection. The low susceptibility of previously vaccinated and exposed groups to challenge at � 18 wk of age suggests that late outbreaks of MD in commercial flocks are not likely a result of recent challenge alone and that additional factors could be involved.

Marek's disease virus (MDV), a highly cell-associated oncogenic chicken herpesvirus, causes Marek's disease in domestic chickens. A unique phosphoprotein of MDV, pp38, has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. However, recently, the biological properties of a deletion mutant virus (rMd5Deltapp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. Thus, pp38 is important for early cytolytic infection and not for transformation. The pp38 protein of the MDV serotype 1 vaccine strain CVI988/Rispens differs by one amino acid when compared to the pathogenic strains of MDV. Monoclonal antibody, H19, recognizes all serotype 1 MDV strains except CVI988/Rispens. Previous studies have also shown that the unique pp38 epitope in CVI988/Rispens induced high antibody response. In order to study the role of this epitope in the protective properties of CVI988/Rispens, we generated a mutant rMd5 virus in which the wild type pp38 gene has been substituted with that of CVI988/Rispens (rMd5/pp38CVI). The replication properties of rMd5/pp38CVI, both in vitro and in vivo, and tumor induction were examined. We found that the biological properties of rMd5/pp38CVI were similar to the wild type rMd5 virus with regards to in vivo replication, antibody response and tumor induction. This shows that the pp38 derived from CVI988/Rispens is not involved in protective properties as was previously suggested.

Several novel criteria have been tested to assist in the differential diagnosis of tumours induced by Marek's disease virus (MDV) from those induced by avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). A collection of tumours induced by inoculation of specific strains of MDV, ALV and REV, alone or in combination, were tested for quantification of MDV DNA by real-time polymerase chain reaction, expression of the MDV oncogene Meq, expression of several cell markers associated with transformation (CD30, Marek's disease-associated surface antigen, and p53), and level of DNA methylation in the tumour cells. In addition, tissues latently infected with MDV and non-infected tissues were tested as controls. Tumours induced by MDV had about 10(2)-fold more copies of MDV DNA than either tissues latently infected by MDV or tumours induced by retrovirus in MDV-vaccinated chickens. Moreover, the MDV antigen Meq was consistently expressed in all MDV tumours but it could not be detected in tissues latently infected with MDV or in tumours induced by retrovirus in MDV-vaccinated chickens. Other markers studied were not specific for MDV and therefore had limited value for diagnosis. Nonetheless, some of these markers might have potential value in research as they will help to identify transformed cells.

Marek's disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in T cell lymphomas in visceral organs and peripheral nerves. Earlier studies have determined that the repeat regions of oncogenic serotype 1 MDV encode a basic leucine zipper protein, Meq, which structurally resembles the Jun/Fos family of transcriptional activators. Meq is consistently expressed in MDV-induced tumor cells and has been suggested as the MDV-associated oncogene. To study the function of Meq, we have generated an rMd5ΔMeq virus by deleting both copies of themeqgene from the genome of a very virulent strain of MDV. Growth curves in cultured fibroblasts indicated that Meq is dispensable forin vitrovirus replication.In vivoreplication in lymphoid organs and feather follicular epithelium was also not impaired, suggesting that Meq is dispensable for lytic infection in chickens. Reactivation of the rMd5ΔMeq virus from peripheral blood lymphocytes was reduced, suggesting that Meq is involved but not essential for latency. Pathogenesis experiments showed that the rMd5ΔMeq virus was fully attenuated in chickens because none of the infected chickens developed Marek's disease-associated lymphomas, suggesting that Meq is involved in lymphocyte transformation. A revertant virus that restored the expression of themeqgene, showed properties similar to those of the parental virus, confirming that Meq is involved in transformation but not in lytic replication in chickens.

A statistical approach was used to establish a new classification system of Marek's disease virus (MDV) on the basis of neurologic responses. To develop the system, neurologic response data from 15x7 chickens inoculated with 30 strains of serotype 1 MDV were statistically analyzed by a cluster analysis. The goal was to identify a statistical system that would verify if three neurovirulence groups correlated with the three pathotypes previously described. The system was also validated in two additional strains of specific-pathogen-free (SPF) chickens, SPAFAS and line SC (Hy-Vac). The proposed system is based on analysis of three variables: 1) frequency of birds showing transient paralysis between 9 and 11 days postinoculation (dpi), (2) mortality before 15 dpi, and (3) frequency of birds showing persistent neurologic disease between 21 and 23 dpi. By use of this system, a MDV may be classified in one of three groups, designated neuropathotypes A, B, and C, which roughly correspond to the virulent, very virulent, and very virulent plus pathotypes, respectively. However, correlation between neuropathotype and pathotype was not absolute, and neuropathotyping is more a complement to the current pathotyping system than a replacement for it. Our results showed that neuropathotyping studies can be conducted in two types of commercial SPF chickens by the use of the same variables, although the system would first have to be standardized by the use of prototype viruses. Neuropathotypes can also be estimated with our statistical analysis with reasonable accuracy. By use of this analysis, we established that MDV strains within the very virulent pathotype may be subdivided into neuropathotypes B and C, thus establishing a previously unrecognized pathotypic classification. This finding illustrates how neuropathotyping may extend important information not identified by conventional pathotyping.

This report describes an outbreak of tuberculosis in a flock of 38,500 48-wk-old layer hens. Clinical characteristics of the process included a gradual drop in egg production and feed intake, as well as an increased mortality rate. Two well-defined clinical features were observed. On the one hand, a number of birds displayed good body condition and continued to lay but presented granulomatous nodular lesions, particularly in the infraorbital sinus, liver, and intestine. In contrast, other hens were emaciated and presented granulomatous lesions of various sizes throughout the internal organs. The lesions observed in the various organs of all the affected birds consisted of granulomas containing acid/alcohol-fast bacilli. The presence of Mycobacterium avium was confirmed through polymerase chain reaction techniques. This report describes the epidemiologic and histopathologic characteristics of the outbreak.

Marek's disease virus (MDV) genetics has lagged behind that of other herpesviruses because of the lack of tools for the introduction of site-specific mutations into the genome of highly cell-associated oncogenic strains. Overlapping cosmid clones have been successfully used for the introduction of mutations in other highly cell-associated herpesviruses. Here we describe the development of overlapping cosmid DNA clones from a very virulent oncogenic strain of MDV. Transfection of these cosmid clones into MDV-susceptible cells resulted in the generation of a recombinant MDV (rMd5) with biological properties similar to the parental strain. To demonstrate the applicability of this technology for elucidation of gene function of MDV, we have generated a mutant virus lacking an MDV unique phosphoprotein, pp38, which has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. Inoculation of Marek's disease-susceptible birds with the pp38 deletion mutant virus (rMd5Δpp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. This powerful technology will speed the characterization of MDV gene function, leading to a better understanding of the molecular mechanisms of MDV pathogenesis. In addition, because Marek's disease is a major oncogenic system, the knowledge obtained from these studies may shed light on the oncogenic mechanisms of other herpesviruses.

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.

The serotype 1 Marek's disease virus (MDV) is the causative agent for Marek's disease (MD), a lymphoproliferative disease of chickens of great concern to the poultry industry. CVI988 (Rispens vaccine), an attenuated serotype 1 MDV, is currently the most efficacious commercially available vaccine for preventing MD. However, it is difficult to detect and differentiate CVI988 when other serotype 1 MDVs are present. To facilitate the detection of CVI988, we developed two sets of primers for a mismatch amplification mutation assay (MAMA) PCR that targeted the single nulceotide polymorphism associated with the H19 epitope of the phosphorylated protein 38 gene. The PCR was very specific. One primer set (oncogenic primers) amplified DNA from 15 different serotype 1 MDVs except CVI988. The other primer set (CVI988 primers) amplified DNA from CVI988 but not from any of the other 15 serotype 1 MDVs. A real-time PCR assay was developed using MAMA primers, and specificity and sensitivity was evaluated in vitro and in vivo. Mixtures of plasmids (CVI988 plasmid and oncogenic plasmid) at various concentrations were used to evaluate the sensitivity/specificity of MAMA primers in vitro. Both primer setswere able to amplify as little as one copy of their respective plasmid. Oncogenic primers were highly specific and only amplified CVI988 plasmid when the concentration of oncogenic plasmid was very low (1 X 10(1)) and CVI988 plasmid was very high (1 X 10(6)). Specificity of CVI988 primers was not as high because they could amplify oncogenic plasmids when the concentration of CVI988 plasmid was 1 x 10(3) and the concentration of oncogenic 1 x 10(2). Validation of MAMA primers in in vivo samples demonstrated that oncogenic primers can be used for both early diagnosis of MD in feather pulp (FP) samples collected at 3 wk of age and confirmation of MD diagnosis in tumors. CVI988 primers could be used to monitor CVI988 vaccination in samples with a low load of oncogenic MDV DNA (latently infected samples or negative) but not in samples with a high load of oncogenic MDV DNA (tumors). Our results suggest that monitoring CVI988 vaccination in FP samples collected at 1 wk of age ensures the specificity of the CVI988 primers.

Marek's disease (MD) is a lymphoproliferative disease of chickens that causes great economic losses to the poultry industry. It is induced by Marek's disease virus (MDV), an alphaherpesvirus that continues increasing in virulence. Although vaccines have successfully protected against MD for 50 years, the increased virulence of MDV threaten the possibility of controlling MD in the future. In this article, the most relevant syndromes associated with MDV infection, methods of diagnosis, and control are discussed.

Chickens infected with subgroup J avian leukosis virus (ALV J) early in posthatch life develop viremia followed by a neutralizing antibody (Nab) response that may or may not be able to clear the viremia. Occasionally, chickens that do clear viremia by developing an efficient Nab response revert to viremia, and the factors responsible for this reversion are not clear. In this study, it was hypothesized that stress can cause seroconverted viremia-free chickens to revert to viremia. Adult (52-wk-old) male commercial meat-type chickens that were exposed to ALV J at hatch and had since cleared viremia and remained viremia-free for up to 40 wk, when subjected to chronic stress (for 14 days) induced by porcine adrenocorticotrophin (ACTH), reverted to viremia and cloacal shedding (2/6 [33%]). However, chickens that were contact-exposed to ALV J at 32 wk of age and had seroconverted failed to revert to viremia when subjected to similar chronic stress. Stress did not increase the susceptibility of adult meat-type chickens to ALV J infection by contact exposure. The lack of statistical significance due to the small sample size is a limitation of this study. However, in general, the results suggest that treatment of chickens with ACTH can cause reversion of viremia and cloacal shedding in ALV J-seroconverted adult male chickens that had been exposed to the virus at hatch, but not in chickens that were contact-exposed at 32 wk of age. The results warrant further studies with greater sample size to examine the role of stress in ALV J epidemiology.

Revaccination against Marek's disease is a widespread practice in some countries. The rationale of this practice is unknown, and there is no consensus in the protocols. Recently, we have demonstrated that administration of the first vaccine at 18 days of embryonation followed by a more protective second vaccine at hatch (18ED/1d) reproduced systematically the benefits of revaccination under laboratory conditions. Here, we have used the same model to optimize the revaccination protocols by using currently available vaccines and to determine whether two features associated with Marek's disease vaccine-induced protection (activation of T cells and replication of vaccine virus) are involved in the revaccination protocols. Protection conferred by three revaccination protocols (turkey herpesvirus [HVT] 18ED/HVT+SB-1 1d, HVT 18ED/CVI988 1d, and HVT+SB-1 18ED/ CVI988 1d) was evaluated. Revaccination protocols also were compared with single vaccination protocols (HVT 18ED, HVT+SB-1 18ED, HVT+SB-1 1d, CVI988 18ED, and CVI988 1d). Our results demonstrated that it is possible to improve efficacy of the currently available vaccines by using them in revaccination programs. Administration of HVT 18ED/CVI988 1d and HVT+SB-1 18ED/CVI988 1d were the two protocols that conferred the highest protection against a very early challenge (2 days of age) with very virulent plus Marek's disease virus strain 648A. In a separate experiment, we evaluated vaccine replication and activation of T cells in single and revaccination protocols. Our results demonstrated that replication of the second vaccine, although decreased compared with single vaccination, could be detected at 3 days (HVT, CVI988) or at 6 days (SB-1). Administration of the first vaccine (HVT) at 18ED resulted in a high percentage of activated T cells. Administration of a second vaccine (either HVT-SB-1 or CVI988) at 1d resulted in increased intensity of MHC-II stain in activated T cells.

Revaccination, the practice of administering Marek's disease (MD) vaccine a second time, has been used in commercial poultry flocks for many years. The rationale is largely anecdotal as the few published reports have failed to provide support for the value of the practice. In the present work, we have standardized a model to study MD revaccination under laboratory conditions. Nine bird experiments were conducted to evaluate homologous revaccination (same vaccine administered twice) and heterologous revaccination (administration of two different vaccines) with various challenge models. Our results demonstrated that heterologous revaccination (with a second vaccine more protective than the first vaccine) but not homologous revaccination provided a beneficial increase in protection. Administration of the first vaccine at 18 days of embryonation followed by a more protective second vaccine at hatch reproduced systematically the benefits of revaccination. In addition, our results show that revaccination protocols might aid in solving major drawbacks associated with various highly protective experimental MD vaccines; that is, lymphoid organ atrophy and residual virulence. Strain RM1 is one of the most protective vaccines against early challenge with highly virulent MD virus but it induces severe lymphoid atrophy in chickens lacking maternal antibodies against MD virus. In this study, strain RM1 did not induce lymphoid organ atrophy when administered as second vaccine in a revaccination protocol. Similarly, strain 648A100/BP5 maintains residual virulence in chickens lacking maternal antibodies against MD virus but did not induce any lesions when used as a second vaccine. Until now, arbitrary revaccination protocols have been occasionally proven useful to the poultry industry. The model developed in this study will allow for a better understanding of this phenomenon and its optimization. A more rational use of this practice will be of great help to control MD outbreaks until better vaccines are available.

Attenuation of Gallid herpesvirus-2 (GaHV-2), the causative agent of Marek’s disease, can occur through serial passage of a virulent field isolate in avian embryo fibroblasts. In order to gain a better understanding of the genes involved in attenuation and associate observed changes in phenotype with specific genetic variations, the genomic DNA sequence of a single GaHV-2 virulent strain (648A) was determined at defined passage intervals. Biological characterization of these “interval-isolates” in chickens previously indicated that the ability to induce transient paralysis was lost by passages 40 and the ability to induce persistent neurological disease was lost after passage 80, coincident with the loss of neoplastic lesion formation. Deep sequencing of the interval-isolates allowed for a detailed cataloguing of the mutations that exist within a single passage population and the frequency with which a given mutation occurs across passages. Gross genetic alterations were identified in both novel and well-characterized genes and cis-acting regions involved in replication and cleavage/packaging. Deletions in genes encoding the virulence factors vLipase, vIL8, and RLORF4, as well as a deletion in the promoter of ICP4, appeared between passages 61 and 101. Three mutations in the virus-encoded telomerase which predominated in late passages were also identified. Overall, the frequency of mutations fluctuated greatly during serial passage and few genetic changes were absolute. This indicates that serial passage of GaHV-2 results in the generation of a collection of genomes with limited sequence heterogeneity.

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 (P0.05) 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.

Since different biological characteristics of Marek's disease virus (MDV) are attenuated at different passage levels in cell culture, an analysis of attenuation times provides, in theory, a model for establishing the presence or absence of relationships between characteristics, thus providing a basis to link them to genetic changes in the causative virus. We have used this model to better understand the pathogenesis of the central nervous system infection as well as to evaluate the relationship of clinical neurological disease to various other parameters of MDV infection. Inoculation of 15 x7 crossbred chickens with strain 648A of very virulent plus MDV at different passage levels (between 10 and 100) showed that two neurological syndromes (transient paralysis (TP) and persistent neurological disease), were attenuated at different passage levels. While strain 648A lost the ability to induce TP between 30 and 40 passages in chicken embryo fibroblast cultures, an event closely related with all parameters of MDV infection involving viral replication (early cytolytic infection in lymphoid organs and viral replication in the feather follicle epithelium), the ability to induce persistent neurological disease was lost between 80 and 90 passages in chicken embryo fibroblasts, coincident with the loss of neoplastic lesions in peripheral nerves and other visceral organs. These data strongly suggest that transient paralysis and persistent neurological disease are unrelated and differently regulated. Moreover, comparison of brain changes induced by strain 648A at passage level 30 (TP) and at passage level 40 (no TP) also contributed to a better understanding of which brain alterations are associated with the onset of TP. The use of viruses at different passage levels with varying degrees of attenuation is presented as a useful tool for studying pathogenesis of MDV infection.

SUMMARY. Marek’s disease virus (MDV) infection in the eye was studied chronologically after inoculating 1-day-old chickens with a very virulent MDV strain, Md5. The ocular lesions could be classified as early lesions (6–11 days postinoculation [dpi]) and late lesions (26 and 56 dpi), based upon the location and severity of the lesions. The early lesions involved iris, ciliary body, and choroid layer, and were characterized by endothelial cell hypertrophy, vasculitis, and infiltration of lymphocytes (mainly CD8+), plasma cells, macrophages, and heterophils. Expression of early MDV-antigen pp38 in the cells infiltrating choroid layer was detected as early as 11 dpi. Late lesions consisted of severe lymphohistiocytic uveitis, keratitis, pectenitis, vitreitis, retinitis, and segmental to diffuse retinal necrosis. Cell infiltration included macrophages, granulocytes, plasma cells, and both CD4+ and CD8+ cells of various sizes. Expression of early MDV-antigen pp38 was readily found within the retina, uveal tract, and corneal epithelium. No expression of late-antigen gB or oncoprotein meq was detected in any of the eyes examined. A second experiment was conducted to study the effect of vaccination on the development of ocular lesions. Both HVT and CVI988 were able to protect against the development of early ocular lesions in chickens infected with very virulent plus strain MDV 648A. However, only CVI988 conferred complete protection against the development of late ocular lesions. HVT conferred partial protection, as it reduced the frequency and severity of the late ocular lesions. These results enhance our understanding of the nature and pattern of MDV infection in the eye.