Enrique Montiel

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.

Bilateral effects of vaccination against infectious bursal disease and Newcastle disease in specific-pathogen-free layers and commercial broiler chickens.

Abstract Different infectious bursal disease virus (IBDV) live vaccines (intermediate, intermediate plus) were compared for their immunosuppressive abilities in specific-pathogen-free (SPF) layer-type chickens or commercial broilers. The Newcastle disease virus (NDV) vaccination model was applied to determine not only IBDV-induced immunosuppression but also bilateral effects between IBDV and NDV. None of the IBDV vaccines abrogated NDV vaccine–induced protection. All NDV-vaccinated SPF layers and broilers were protected against NDV challenge independent of circulating NDV antibody levels. Sustained suppression of NDV antibody development was observed in SPF layers, which had received the intermediate plus IBDV vaccine. We observed a temporary suppression of NDV antibody development in broilers vaccinated with one of the intermediate, as well as the intermediate plus, IBDV vaccines. Different genetic backgrounds, ages, and residual maternal antibodies might have influenced the pathogenesis of IBDV in the different types of chickens. Temporary suppression of NDV antibody response in broilers was only seen if the NDV vaccine was administered before and not, as it was speculated previously, at the time the peak of IBDV-induced bursa lesions was detected. For the first time, we have demonstrated that the NDV vaccine had an interfering effect with the pathogenesis of the intermediate as well as the intermediate plus IBDV vaccine. NDV vaccination enhanced the incidence of IBDV bursa lesions and IBDV antibody development. This observation indicates that this bilateral effect of an IBDV and NDV vaccination should be considered in the field and could have consequences for the performance of broiler flocks.

Protection in specific pathogen free chickens with live avian metapneumovirus and Newcastle disease virus vaccines applied singly or in combination

This paper describes two experiments. In each experiment, 1-day-old specific pathogen free chicks were divided into three groups. In Experiment 1—[avian metapneumo virus (aMPV) challenge]—one group served as unvaccinated controls; the second group was vaccinated with live aMPV (subtype B) vaccine only, and the third group received the aMPV vaccine in combination with live Newcastle disease virus (NDV) vaccine (VG/GA strain). Oropharyngeal swabs, tissues and blood samples were collected before and after challenge with a virulent subtype aMPV at 21 days post vaccination. Chicks were monitored for post-challenge clinical signs. Swabs and tissues were examined for the detection of challenge aMPV by virus isolation and by reverse-transcriptase polymerase-chain reaction. Sera were assayed for antibodies against aMPV and NDV. The single and combined vaccinated chicks were all protected against clinical signs and no challenge virus was isolated from either of the vaccinated-challenged groups. In Experiment 2 (NDV challenge), as in Experiment 1, chicks were divided into three groups where one group remained as unvaccinated control and the other two groups were vaccinated as above, except that the second group received live NDV vaccine only, instead of aMPV. At 21 days post vaccination, 15 chicks from each of the three groups were removed to a different site and challenged with a virulent NDV (Texas GB strain). Re-isolation of the challenge virus was not attempted. All chicks in both NDV-vaccinated challenged groups were protected against clinical signs and mortality. These results show that, based on parameters monitored for the respective challenge virus, simultaneous application of live aMPV and NDV vaccines did not affect the efficacy of either vaccine.

Early infection with Marek’s disease virus can jeopardize protection conferred by laryngotracheitis vaccines: a method to study MDV-induced immunosuppression

ABSTRACT 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 < 0.05) clinical scores, gross lesions, histopathologic lesion scores, and load of ILTV transcripts in trachea after ILTV challenge, as chickens that were not vaccinated with CEO ILTV vaccine.

Repeated Challenge with Virulent Newcastle Disease Virus Does Not Decrease the Efficacy of Vaccines

SUMMARY Globally, poultry producers report that birds well-vaccinated for Newcastle disease (ND) often present clinical disease and mortality after infection with virulent strains of Newcastle disease (vNDV), which is contrary to what is observed in experimental settings. One hypothesis for this discrepancy is that the birds in the field may be exposed to multiple successive challenges with vNDV, rather than one challenge dose, and that the repeated infection may overwhelm the immune system and neutralizing antibodies available to prevent clinical disease. In this study, we evaluated this hypothesis under highly controlled conditions. We challenged well-vaccinated chickens with high doses of vNDV daily for 10 days, and looked for signs of clinical disease, changes in antibody titers, and mortality. All sham-vaccinated birds died by the fourth day postchallenge. No morbidity or mortality was observed in any of the NDV-vaccinated birds up to 14 days postchallenge; repeated high-dose challenges of vNDV was not sufficient to overcome vaccine immunity.

Protection Conferred by a Live Avian Metapneumovirus Vaccine when Co-Administered with Live La Sota Newcastle Disease Vaccine in Chicks

This paper examines the effects on specific pathogen-free (SPF) chicks when avian metapneumovirus (aMPV) and Newcastle disease virus (NDV) La Sota strain vaccines are co-administered. Day-old SPF chicks were divided into five groups. The first group was inoculated with sterile water (SW) and the rest of the groups were inoculated with live NDV vaccine VG/GA by the oculo-oral route. At 21 days-old, the unvaccinated chicks were again inoculated with SW. The four VG/GA-vaccinated groups were further inoculated with (i) SW, (ii) live aMPV vaccine, (iii) live NDV La Sota, or (iv) combined live NDV La Sota and live aMPV, respectively. Chicks were monitored for post-vaccination reactions and oropharyngeal swabs were collected for vaccines detection. Blood samples were collected to detect aMPV ELISA and NDV haemagglutination-inhibition antibodies. Twenty-one days following the second vaccination, six chicks from each group were challenged with virulent NDV or aMPV respectively. Chicks were monitored for clinical signs and mortality and oropharyngeal swabs collected for aMPV detection. Results showed that, when challenged with a virulent aMPV, both chicks previously vaccinated with VG/GA and subsequently given aMPV vaccine singly or in combination with La Sota were equally protected against clinical signs. Chicks that were vaccinated against NDV either once with VG/GA or followed by La Sota (singly or in combination with aMPV) were fully protected when challenged with velogenic NDV. We concluded that simultaneous administration of live aMPV and NDV La Sota vaccines have no adverse effects on protection conferred by either live vaccine.