Aneg L. Cortes

Load of Challenge Marek's Disease Virus DNA in Blood as a Criterion for Early Diagnosis of Marek's Disease Tumors

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

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

Abstract Comparison of blood and feather pulp (FP) samples for the diagnosis of Mareks disease (MD) and for monitoring Mareks diseases vaccination in chickens (serotypes 2 and 3 vaccines) by real time-PCR was evaluated. For diagnosis of MD, quantification of serotype 1 Mareks 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.

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

Abstract The use of Flinders Technology Associates (FTA®) filter cards to quantify Mareks disease virus (MDV) DNA for the diagnosis of Mareks disease (MD) and to monitor MD vaccines was evaluated. Samples of blood (43), solid tumors (14), and feather pulp (FP; 36) collected fresh and in FTA cards were analyzed. MDV DNA load was quantified by real-time PCR. Threshold cycle (Ct) ratios were calculated for each sample by dividing the Ct value of the internal control gene (glyceraldehyde-3-phosphate dehydrogenase) by the Ct value of the MDV gene. Statistically significant correlation (P < 0.05) within Ct ratios was detected between samples collected fresh and in FTA cards by using Pearsons 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 (Pearsons 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.

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

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

Detection and Differentiation of CVI988 (Rispens Vaccine) from Other Serotype 1 Marek's Disease Viruses

SUMMARY The serotype 1 Mareks disease virus (MDV) is the causative agent for Mareks 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 sets were 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 × 101) and CVI988 plasmid was very high (1 × 106). Specificity of CVI988 primers was not as high because they could amplify oncogenic plasmids when the concentration of CVI988 plasmid was 1 × 103 and the concentration of oncogenic 1 × 102. 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. RESUMEN Detección y diferenciación de la cepa CVI988 (Rispens Vacuna) de otras cepas del virus de la enfermedad de Marek pertenecientes al serotipo 1. El serotipo 1 del virus de la enfermedad de Marek (MDV) es el agente causal de la enfermedad de Marek (MD), que es una enfermedad linfoproliferativa de los pollos de gran importancia para la industria avícola. La cepa CVI988 (vacuna Rispens), una vacuna atenuada del serotipo 1 del virus de Marek, es actualmente la vacuna más eficaz disponible comercialmente para prevenir la enfermedad de Marek. Sin embargo, es difícil de detectar y diferenciar la cepa CVI988 cuando otros virus de Marek del serotipo 1 están presentes. Para facilitar la detección de la cepa CVI988, se desarrollaron dos conjuntos de iniciadores para un ensayo de PCR para la amplificación de mutaciones por discrepancia de nucleótidos (con las siglas en inglés MAMA), dirigido al polimorfismo de nucleótido simple asociado con el gene del epítope H19 de la proteína fosforilada (PP) 38. El método de PCR fue muy específico. Un conjunto de iniciadores (primers oncogénicos) amplificó el ADN de 15 virus diferentes pertenecientes al serotipo 1 excepto la cepa CVI988. El otro conjunto de iniciadores (primers CVI988) amplificó el ADN de la cepa CVI988 pero ninguno de los otros 15 virus incluidos en el serotipo 1. Un ensayo de PCR en tiempo real fue desarrollado usando los iniciadores del ensayo MAMA y se evaluó la especificidad y sensibilidad in vitro e in vivo. Se usaron mezclas de plásmidos (plásmido CVI988 y el plásmido oncogénico) con varias concentraciones para evaluar la sensibilidad/especificidad de los iniciadores MAMA in vitro. Ambos conjuntos de iniciadores fueron capaces de amplificar hasta una copia de su respectivo plásmido. Los iniciadores oncogénicos fueron altamente específicos y solo amplificaron al plásmido CVI988 cuando la concentración del plásmido oncogénico fue muy baja (1 × 101) y la concentración del plásmido CVI988 era muy alta (1 × 106). La especificidad de los iniciadores para la cepa CVI988 no fue muy alta, ya que pudieron amplificar plásmidos oncogénicos cuando la concentración de plásmido CVI988 fue de 1 × 103 y la concentración de los plásmidos oncogénico fue de 1 × 102. La validación de los iniciadores MAMA en muestras in vivo demostró que los iniciadores oncogénicos se pueden utilizar tanto para el diagnóstico precoz de la enfermedad de Marek en muestras de pulpa de la pluma (FP) recolectadas a las tres semanas de edad y para el diagnóstico confirmatorio de la enfermedad de Marek en tumores. Los iniciadores CVI988 podrían utilizarse para el seguimiento de la vacunación CVI988 en muestras con una baja carga de ADN del virus oncogénico del virus de Marek (muestras infectadas de forma latente o negativas), pero no en las muestras con una alta carga de ADN del virus oncogénico de Marek (tumores). Estos resultados sugieren que el seguimiento de la vacunación con la cepa CVI988 en muestras de pulpa de la pluma recolectadas en la primera semana de edad asegura la especificidad de los iniciadores CVI988.

Replication of recombinant herpesvirus of turkey expressing genes of infectious laryngotracheitis virus in specific pathogen free and broiler chickens following in ovo and subcutaneous vaccination

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.

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

Transcription of cytokine genes was evaluated in the lung and spleen of chickens vaccinated with various serotype 1 Mareks 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.

Optimization of the Protocols for Double Vaccination Against Marek's Disease by Using Commercially Available Vaccines: Evaluation of Protection, Vaccine Replication, and Activation of T Cells

SUMMARY. Revaccination against Mareks 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 Mareks 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 Mareks 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. RESUMEN. Optimización de los protocolos de vacunación doble contra la enfermedad de Marek mediante el uso de vacunas comercialmente disponibles: Evaluación de la protección, replicación de la vacuna y activación de células T. La revacunación contra la enfermedad de Marek es una práctica muy extendida en algunos países. La justificación de esta práctica es desconocida, y no hay un consenso en los protocolos. Recientemente, se demostró que la administración de la primera vacuna a los 18 días de desarrollo embrionario (DE) seguido por la aplicación de una segunda vacuna más protectora a la eclosión (18DE/1d), reproduce de manera sistemática los beneficios de la revacunación bajo condiciones de laboratorio. En este trabajo, se utilizó el mismo modelo para optimizar los protocolos de la revacunación con las vacunas actualmente disponibles y para determinar si dos aspectos asociados con protección inducida por la vacuna contra la enfermedad de Marek (la activación de las células T y la replicación de virus vacunal) están involucrados en los protocolos de la revacunación. La protección conferida por tres protocolos de la revacunación (virus herpes de pavo [HVT] a los18DE/HVT + SB-1 al día 1; HVT 18DE/CVI988 al día 1; y HVT + SB-1 18DE/CVI988 al día 1) se evaluaron. Los protocolos de revacunación también se compararon con los protocolos de vacunación individuales (HVT 18ED, HVT + SB-1 18ED, HVT + SB-1 día 1, CVI988 18ED, y CVI988 día 1). Los resultados observados demostraron que es posible mejorar la eficacia de las vacunas actualmente disponibles mediante su aplicación dentro de programas de revacunación. La administración de HVT 18ED/CVI988 día 1 y HVT + SB-1 18ED/CVI988 día 1, fueron los dos protocolos que le confirieron la máxima protección contra a un desafío muy temprano (dos días de edad) con una cepa muy virulenta del virus de la enfermedad de Marek, la cepa 648A. En un experimento por separado, se evaluaron la replicación del virus vacunal y la activación de células T en los protocolos individuales y de revacunación. Los resultados demuestran que la replicación de la segunda vacuna, aunque disminuida en comparación con una sola vacunación, se pudo detectar a los tres días (HVT, CVI988) o a los seis días (SB-1). La administración de la primera vacuna (HVT) a los 18 días de desarrollo embrionario, resultó en un alto porcentaje de células T activadas. La administración de una segunda vacuna (ya sea HVT-SB-1 o CVI988) al primer día de edad resultó en un aumento de la intensidad de la detección de moléculas MHC-II en las células T activadas.

Replication ability of three highly protective Marek's disease vaccines: implications in lymphoid organ atrophy and protection

The present work is a chronological study of the pathogenesis of three attenuated serotype 1 Mareks 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.

Standardization of a model to study revaccination against Marek's disease under laboratory conditions

Revaccination, the practice of administering Mareks 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.