Marek's disease virus oncogene Meq expression in infected cells in vaccinated and unvaccinated hosts.

Marek's disease (MD) vaccines are unique in their capability to prevent MD lymphomas as early as a few days after vaccination, despite the fact that they do not eliminate virulent viruses from the host. To help understand the mechanism behind this unique MD vaccine effect, we compared the expression of MDV oncoprotein Meq among CD4+ T cells between vaccinated and unvaccinated birds. Chickens were vaccinated by an MD vaccine, herpesvirus of turkeys, and then challenged by a recombinant virulent MDV that expresses green fluorescent protein simultaneously with Meq. We found significantly fewer Meq-expressing CD4+ T cells appeared in peripheral blood mononuclear cells (PBMC) of the vaccinated birds compared to the unvaccinated birds as early as one week after the virulent virus challenge. In contrast, the quantity of virulent MDV genome remained similar in Meq- PBMC in both vaccinated and unvaccinated birds. Our results suggest that MD vaccination affects the dynamics of Meq-expressing, possibly transformed, cells while impact on the overall infection in the Meq- cells was not significant.

Marek's disease vaccines-induced differential expression of known and novel microRNAs in primary lymphoid organ bursae of White Leghorn.

Marek's disease (MD) is a contagious disease of domestic chickens caused by MD viruses. MD has been controlled primarily by vaccinations, yet sporadic outbreaks of MD take place worldwide. Commonly used MD vaccines include HVT, SB-1 and CVI988/Rispens and their efficacies are reportedly dependent of multiple factors including host genetics. Our previous studies showed protective efficacy of a MD vaccine can differ drastically from one chicken line to the next. Advanced understanding on the underlying genetic and epigenetic factors that modulate vaccine efficacy would greatly improve the strategy in design and development of more potent vaccines. Two highly inbred lines of White Leghorn were inoculated with HVT and CVI988/Rispens. Bursa samples were taken 26 days post-vaccination and subjected to small RNA sequencing analysis to profile microRNAs (miRNA). A total of 589 and 519 miRNAs was identified in one line, known as line 63, 490 and 630 miRNAs were identified in the other, known as line 72, in response to HVT or CVI988/Rispens inoculation, respectively. HVT and CVI988/Rispens induced mutually exclusive 4 and 13 differentially expressed (DE) miRNAs in line 63 birds in contrast to a non-vaccinated group of the same line. HVT failed to induce any DE miRNA and CVI988/Rispens induced a single DE miRNA in line 72 birds. Thousands of target genes for the DE miRNAs were predicted, which were enriched in a variety of gene ontology terms and pathways. This finding suggests the epigenetic factor, microRNA, is highly likely involved in modulating vaccine protective efficacy in chicken.

Towards a mechanistic understanding of the synergistic response induced by bivalent Marek's disease vaccines to prevent lymphomas.

BACKGROUND: Marek's disease (MD) is a lymphoproliferative disease of chickens caused by Marek's disease virus (MDV), an oncogenic α-herpesvirus. Since 1970, MD has been controlled by widespread vaccination; however, more effective MD vaccines are needed to counter more virulent MDV strains. The bivalent vaccine combination of SB-1 and herpesvirus of turkey (HVT) strain FC126 has been widely used. Nonetheless, the mechanism(s) underlying this synergistic effect has not been investigated. METHODS: Three experiments were conducted where SB-1 or HVT were administered as monovalent or bivalent vaccines to newly hatched chickens, then challenged five days later with MDV. In Experiment 1, levels of MDV replication in PBMCs were measured over time, and tumor incidence and vaccinal protection determined. In Experiment 2, MDV and vaccine strains replication levels in lymphoid organs were measured at 1, 5, 10, and 14 days post-challenge (DPC). In Experiment 3, to verify that the bursa was necessary for HVT protection, a subset of chicks were bursectomized and these birds plus controls were similarly vaccinated and challenged, and the levels of vaccinal protection determined. RESULTS: The efficacy of bivalent SB-1 + HVT surpasses that of either SB-1 or HVT monovalent vaccines in controlling the level of pathogenic MDV in PBMCs until the end of the study, and this correlated with the ability to inhibit tumor formation. SB-1 replication in the spleen increased from 1 to 14 DPC, while HVT replicated only in the bursa at 1 DPC. The bursa was necessary for immune protection induced by HVT vaccine. CONCLUSION: Synergy of SB-1 and HVT vaccines is due to additive influences of the individual vaccines acting at different times and target organs. And the bursa is vital for HVT to replicate and induce immune protection.

The potential for inoculating Lactobacillus animalis and Enterococcus faecium alone or in combination using commercial in ovo technology without negatively impacting hatch and post-hatch performance.

The poultry industry has recently undergone transitions into antibiotic free production, and viable antibiotic alternatives, such as probiotics, are necessary. Through in ovo probiotic inoculation, beneficial microflora development in the gastrointestinal tract may occur prior to hatch without negatively impacting chick performance. Therefore, the objective of the present study was to observe the impacts of the injection of probiotic bacteria individually or combined into fertile broiler hatching eggs on hatch and live performance characteristics. A total of 2,080 fertile broiler hatching eggs were obtained from a commercial source. On day 18 of incubation, 4 in ovo injected treatments were applied: 1.) Marek's Disease (HVT) vaccination, 2.) L. animalis (∼106 cfu/50µl), 3.) E. faecium (∼106 cfu/50µl), and 4.) L. animalis + E. faecium (∼106 cfu & ∼106 cfu/50µl each). On day of hatch, hatchability and hatch residue data were recorded. A portion of male chicks from each treatment were placed in a grow-out facility for a 21 d grow-out (18 chicks/pen × 10 pens/treatment = 720 male chicks) with a corn and soy bean meal-based diet without antibiotics or antibiotic alternatives. Performance data and gastrointestinal samples were collected on days 0, 7, 14, and 21. Results indicated no differences in all hatch parameters between treatments (P > 0.05) except for % pipped, where the L. animalis treatment had lower % pipped eggs compared to the HVT control and E. faecium treatments (P = 0.04). No differences were observed in body weight gain or mortality (P > 0.05). Probiotic treatments altered gastrointestinal tissue length, weight, and pH. This resulted in all in ovo injected probiotic treatments increasing feed conversion ratio (FCR) from days 7 to 14 as compared to the control (P = 0.01). Differences in FCR were not observed in any other week of data collection (days 0 to 7, 14 to 21, or 0 to 21; P > 0.05). Although probiotics altered live performance from days 7 to 14, these data suggest that in ovo inoculations of L. animalis and E. faecium in combination are viable probiotic administration practices that potentially improve hatch characteristics and gastrointestinal tract development.

Comparative effects of in ovo versus subcutaneous administration of the Marek's disease vaccine and pre-placement holding time on the intestinal villus to crypt ratios of Ross 708 broilers during early post-hatch development1,2,3.

Villus to crypt ratio (VCR) is used to quantify the microanatomical response of the intestine to various treatments. In early age chickens, comparative effects of the in ovo (i.o.) and s.c. methods of administration (moa) of the Marek's disease (MD) vaccine on 2 types of measurement of small intestinal VCR at 0 and 4 h post-hatch (poh) were investigated. The effects of moa and 4 and 18 h pre-placement holding times (pht) on the VCR measurements at 168 h (7 d) poh were also investigated. In the jejunum of the small intestine, a standard method for VCR determination, based on 10 villus and crypt length measurements, was utilized for the calculation of villus to crypt length ratio (VCLR). In that same region, a single histomorphometric determination of the crypt and total mucosa areas using image analysis software was also used. Subtraction of the crypt area from the total mucosa area provided the villus area, allowing for calculation of the villus to crypt area ratio (VCAR). Across 0, 4, and 18 h of poh bird age, the VCLR of birds that received an s.c. vaccination was higher in comparison to that of those that received an i.o. vaccination. The highest and lowest VCAR values were observed in the s.c. treatment at 0 h poh and in the i.o. treatment at 4 h poh, respectively. Furthermore, at 168 h poh, VCLR values in the 18 h pht and s.c. vaccination group were higher than those in the 4 h pht and s.c. vaccination or 18 h and i.o. vaccination groups. In conclusion, the effects of pht and MD vaccine moa on VCR were dependent on the use of either the VCLR or VCAR method of measurement. However, regardless of method, s.c. injection overall led to a higher VCR through 4 h poh in Ross 708 broilers, and the effects of moa on VCLR at 168 h were influenced by pht.

Vaccination and Host Marek's Disease-Resistance Genotype Significantly Reduce Oncogenic Gallid alphaherpesvirus 2 Telomere Integration in Host Birds.

Marek's disease (MD) is an infectious disease characterized by lymphomas and high mortality in susceptible chickens. The causative and ubiquitous alpha-herpesvirus known as MD virus (MDV) integrates into host telomeres during early infection through latency, known to be an important phase for oncogenic transformation. Herein, we sought to determine the influence of vaccination and host genetics on the temporal dynamics of MDV-host genome interactions. We studied integration profiles using 2 MD vaccines that vary in protective efficacy in 2 genetic lines that differ in MD resistance/susceptibility. Virus integration of both oncogenic MDV and vaccine strains was observed in both MD susceptible and resistant birds, however, the lines differed in their dynamic telomere-integration profiles. Notably, the resistant host genotype exhibited a smaller percentage of replicating cells with the virus telomere-integrated only phenotype as compared to the susceptible genotype. Vaccination with Rispens, the most protective MD vaccine, also reduced the establishment of the virus telomere-integrated only phenotype, suggesting a significant role of the phenotype in MD lymphoma development. The effect of Rispens vaccination was most dramatic in the susceptible genotype. These results suggest important connections between vaccinal immunity, MDV telomere integration, virus-induced oncogenesis, and virus-host genome interactions in the context of host genetics and disease susceptibility.

Off-label use of ceftiofur in one-day chicks triggers a short-term increase of ESBL-producing E. coli in the gut.

This trial was designed to evaluate the off-label use of ceftiofur with Marek's vaccine in one-day-old broiler chicks, a prophylactic treatment that has been done in some commercial hatcheries, on the emergence of extended-spectrum beta-lactamase producing Escherichia coli (ESBL-E. coli). A total of 168 chicks (Cobb500®) were used in a completely randomized design. Birds were assigned to two treatments (Marek's vaccine plus saline vs Marek's vaccine plus ceftiofur) and six repetitions, with 14 animals each. Cloacal swabs were collected from 1 to 14 days post-hatch. The majority (86%; p<0.0001) of the ESBL-producing isolates harboring blaCTX-M and blaSHV genes originated from animals receiving the antimicrobial. None of the isolates were positive for plasmid-mediated AmpC betalactamase genes (blaACC, blaCMY-2, blaDHA, blaFOX, blaMOX and blaMIR). These findings indicate that the off-label use of ceftiofur with Marek's vaccine is associated with the short-term increase in ESBL-producing Escherichia coli in the gut of chicks.

Effect of Marek's disease vaccines on interferon and toll like receptors when administered in ovo.

The effect of two Marek's disease (MD) vaccines on the chicken embryo immune responses were evaluated. Transcription of interferon (IFN-α, IFN-ß, 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-ß 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-ß 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.

Comparative effects of in ovo versus subcutaneous administration of the Marek's disease vaccine and pre-placement holding time on the processing yield of Ross 708 broilers.

Effects of the in ovo (i.o.) or subcutaneous (s.c.) method of administration (moa) of the Marek's disease (MD) vaccine and 4 or 18 h pre-placement holding time (pht) on the processing yield of male broilers through 49 d of age (doa) were investigated. Ross 708 broiler hatching eggs (3,900) were either i.o.-vaccinated at 18 d of incubation or chicks from eggs that were not i.o.-vaccinated were s.c.-vaccinated at hatch. The i.o. injections (50 µL) were delivered by a commercial multi-egg injector and s.c. injections (200 µL) were delivered by an automatic pneumatic s.c. injector. The pht was imposed on chicks after vaccination. Sixteen birds were initially assigned to each of 15 replicate floor pens belonging to each of the moa and pht combination groups and were grown out through 48 doa. At 48 doa, 6 birds were randomly selected from each replicate pen and were weighed and fasted for 16 h before being processed. At 49 doa, whole carcass, fat pad, breast muscle, and tenders muscles weights were recorded. Whole carcass weight as a percentage of live BW, and fat pad, breast muscle, and tenders muscles weights as percentages of both live and whole carcass weights were calculated. Upon subjection of the data to a 2 × 2 factorial analysis, only a main effect due to moa was observed for tenders muscles weight as a percentage of live and whole carcass weights. Tenders muscles weight as a percentage of both live (P ≤ 0.010) and whole carcass (P ≤ 0.004) weight was higher in birds hatched from eggs that received i.o. rather than s.c. vaccinations. In conclusion, in comparison to s.c. vaccination, i.o. vaccination increased relative tenders weight yield, whether or not broilers were held for 4 or 18 h prior to placement. Therefore, with regard to broiler processing yield, i.o. and s.c. vaccinations were safe for the administration of the MD vaccine, with i.o. vaccination displaying a slight potential advantage.

Recombinant Gallid herpesvirus 2 with interrupted meq genes confers safe and efficacious protection against virulent field strains.

Gallid herpesvirus 2 (GaHV-2) continuously evolves, which reduces the effectiveness of existing vaccines. To construct new GaHV-2 candidate vaccines, LMS, which is a virulent GaHV-2 field strain isolated from diseased chicken flocks in Southwest China in 2007, was modified such that both copies of its meq oncogene were partially deleted. The resulting virus, i.e., rMSΔmeq, was characterized using PCR and sequencing. To evaluate the safety and protective efficacy of rMSΔmeq, specific pathogen-free (SPF) chickens were inoculated with 2000 plaque forming units (pfu) and 20,000pfu of rMSΔmeq immediately after hatching. All birds grew well during the experimental period, and none of the challenged chickens developed Marek's disease-associated lymphoma. In addition, the rMSΔmeq- and CVI988/Rispens-vaccinated SPF chickens were challenged with 1000 pfu and 5000 pfu of the representative virulent GaHV-2 Md5 strain and 1000 pfu of the variant GaHV-2 strains LCC or LTS. The results showed that the rMSΔmeq strain provided complete protection, which was similar to that provided by the CVI988/Rispens vaccine (protective index (PI) of 95.5) when challenged with a conventional dose of the Md5 strain. However, rMSΔmeq provided a PI of 90.9 when challenged with 5000 pfu of the Md5 strain, which was significantly higher than that provided by the CVI988/Rispens vaccine (54.5). rMSΔmeq provided a PI of 86.4 against LCC, which was equal to that provided by the CVI988/Rispens vaccine (81.8). In addition, rMSΔmeq provided a PI of 100 against LTS, which was significantly higher than that provided by the CVI988/Rispens vaccine (68.2). Altogether, the rMSΔmeq virus provided efficient protection against representative and variant GaHV-2 strains. In conclusion, the rMSΔmeq virus is a safe and effective vaccine candidate for the prevention of Marek's disease and is effective against the Chinese variant GaHV-2 strains.

Protective efficacy of a novel recombinant Marek's disease virus vector vaccine against infectious bursal disease in chickens with or without maternal antibodies.

Infectious bursal disease (IBD) causes significant clinical and economic losses to the poultry industry worldwide. Current vaccine programs using live attenuated and inactivated vaccines have numerous drawbacks. As an alternative solution to control IBD, a Marek's disease virus (MDV) vector vaccine (rMDV-VP2) expressing the VP2 gene of infectious bursal disease virus (IBDV) has been developed. In this study, the protective efficacy of rMDV-VP2 was evaluated in a dose-related experiment which showed that a single dose of 1000 PFU was sufficient to fully protect chickens against IBDV infection. Chickens inoculated with lower doses of rMDV-VP2 (250 or 500 PFU) conferred 80 and 90% protection against IBDV. Next, rMDV-VP2 vaccine provided 90% protection against IBDV in commercial layer chickens with maternal antibodies, which was higher than the protective efficacy using the B87 live vaccine of IBDV. Additionally, rMDV-VP2 conferred effective protection against very virulent MDV challenge in chickens (95% for chickens vaccinated with 250 or 500 PFU and 100% for chickens vaccinated with 1000 or 2000 PFU). These results demonstrated that rMDV-VP2 may be a novel bivalent vaccine against IBD and Marek's disease in chickens.

Early Immune Responses to Marek's Disease Vaccines.

Marek's disease virus (MDV), a highly cell-associated lymphotropic α-herpesvirus, is the causative agent of Marek's disease (MD) in domestic chickens. MDV replicates in chicken cells and establishes a latent infection within CD4+ T cells. Although MD vaccines have been in use for several decades, the exact mechanism of vaccine-induced protection is unclear. It is believed that the innate immune system plays a role in vaccine-induced immunity against pathogenic strains of MDV. To shed light on the possible function of the innate immunity in vaccine-mediated protection, we investigated the effect of vaccination, Rispens/CVI988, on the activation of cellular components of the innate immune system by analyzing the expression pattern of select immune-related genes in the cecal tonsils (CT) and duodenum of two MD-susceptible and MD-resistant chicken lines at 3, 5, and 10 days postvaccination (dpv). The differential expression patterns of the tested genes within the CT and duodenum of vaccinated birds revealed the activation of the innate immune system in both the susceptible and resistant lines. Stronger innate immune response was induced within the CT of the vaccinated birds of the susceptible line at 5 dpv. Upregulation of some of the tested genes at 10 dpv was likely due to the activation and response of the adaptive immune system to vaccination. Immunohistochemical analysis showed no increase in the number of CD3+ T cells in the CT and duodenum of the vaccinated birds of either line at 5 dpv. There was, however, an increase in the macrophage populations within the duodenum of the vaccinated birds of both the susceptible and resistant lines at 5 dpv. The vaccine strain antigen was detected in the CT and duodenum of the susceptible line, but not the resistant line at 5 dpv.

In ovo evaluation of FloraMax®-B11 on Marek's disease HVT vaccine protective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis infection in broiler chickens.

Three experiments were conducted to evaluate the effect of in ovo administration of FloraMax®-B11 (FM) on Marek's disease (MD) herpesvirus of turkeys (HVT) vaccine protective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis (SE) infection in chickens. Experiment 1 consisted of 3 trials. In trials 1 and 2, d 18 White Leghorn 15I5x71 embryos were randomly distributed in 4 groups: 1) HVT vaccinated in ovo and no Marek's disease virus (MDV) challenge; 2), HVT + FM vaccinated in ovo and no MDV challenge; 3) HVT vaccinated in ovo and challenge with virulent MDV (vMDV; strain 583A); and 4), HVT + FM vaccinated in ovo and challenge with vMDV. Trial 3 was designed exactly the same as Experiment 1 but chicks were challenged with very virulent MDV (vvMDV; strains Md5 and 612). Birds were monitored until 8 wk of age, and tested for MD incidence. Experiment 2 consisted of 3 trials. In each trial, d 18 broiler embryos were injected in ovo with either saline or FM to measure hatchability and gastrointestinal bacterial composition. In Experiment 3, d 18 broiler embryos were injected in ovo with either saline or FM. All chickens that hatched were orally gavaged with SE at hatch and kept for 7 d to monitor post-hatch BW. No significant difference (P > 0.05) between MD percentage in birds vaccinated with HVT alone or HVT + FM were observed in Experiment 1. In Experiment 2, probiotic did not negatively affect hatchability, but did reduce lactose positive Gram-negative bacteria. Further, increase in BW was associated with higher villi surface area in the ileum in chickens that received the probiotic as well as a significant reduction in the SE incidence in Experiment 3. These results suggest that in ovo administration of FM does not negatively impact the ability of HVT to protect against MD or hatchability of chickens, but improves BW during the first 7 d of life and decreases SE recovery in chickens.

Comparative effects of in ovo versus subcutaneous administration of the Marek's disease vaccine and pre-placement holding time on the post-hatch performance of Ross 708 broilers1,2,3.

Effects of 2 types of methods of administration (moa; in ovo or s.c.) of the Marek's disease (MD) vaccine and 4 and 18 h pre-placement holding times (pht) on the performance of male broilers through 48 d of age were investigated. Ross 708 broiler hatching eggs (3,900) were either in ovo-vaccinated at 18 d of incubation or chicks from eggs that were not in ovo-injected were vaccinated s.c. at hatch, and chicks from each moa group were held for one of the 2 pht. In ovo injections (50 µL) were delivered by a commercial multi-egg injector and s.c. injections (0.2 mL) were delivered by an automatic pneumatic s.c. injector. Sixteen birds were assigned to each of 15 replicate floor pens belonging to each of the 4 moa and pht combination groups. Mortality and BW gain were determined at weekly intervals, and feed consumption and conversion were determined in the zero to 14, 14 to 28, 28 to 42, and 42 to 48 d age intervals. No interactive effects between moa and pht were observed for any variable, and mortality was not significantly affected by moa or pht. The 14 to 28 d feed consumption and 14 to 21 d BW gain of s.c.-vaccinated birds were lower than that of in ovo-vaccinated birds, and the increase in pht from 4 to 18 h decreased feed consumption through 28 d post hatch and BW gain through 35 d post hatch. Overall, the performances of male Ross 708 broilers through 48 d of age in response to in ovo and s.c. injections of the MD vaccine were comparable, and delays in hatchling placement should be less that 18 h in duration. Furthermore, despite the decrease in BW gain through 35 d associated with the reduction in feed consumption through 28 d in response to the 14 h increase in pht, in ovo injection did not exacerbate the effect of the increase in pht.

History of the First-Generation Marek's Disease Vaccines: The Science and Little-Known Facts.

Shortly after the isolation of Marek's disease (MD) herpesvirus (MDV) in the late 1960s vaccines were developed in England, the United States, and The Netherlands. Biggs and associates at the Houghton Poultry Research Station (HPRS) in England attenuated HPRS-16, the first cell-culture-isolated MDV strain, by passaging HPRS-16 in chick kidney cells. Although HPRS-16/Att was the first commercially available vaccine, it never became widely used and was soon replaced by the FC126 strain of herpesvirus of turkeys (HVT) vaccine developed by Witter and associates at the Regional Poultry Research Laboratory (now Avian Disease and Oncology Laboratory [ADOL]) in East Lansing, MI. Ironically, Kawamura et al. isolated a herpesvirus from kidney cell cultures from turkeys in 1969 but never realized its potential as a vaccine against MD. Rispens of the Central Veterinary Institute (CVI) developed the third vaccine. His associate, Maas, had found commercial flocks of chickens with MDV antibodies but without MD. Subsequently, Rispens isolated a very low pathogenic strain from hen number 988 from his MD antibody-positive flock, which was free of avian leukosis virus and clinical MD. This isolate became the CVI-988 vaccine used mostly in The Netherlands. During the late 1970s, HVT was no longer fully protective against some new emerging field strains. The addition of SB-1, isolated by Schat and Calnek, to HVT improved protection against the emerging very virulent strains. In the 1990s CVI-988 became the worldwide vaccine gold standard. This review will present data from published papers and personal communications providing additional information about the exciting 15-yr period after the isolation of MDV to the development of the different vaccines.

Efficacy of Recombinant HVT-IBD Vaccines Administered to Broiler Chicks from a Single Breeder Flock at 30 and 60 Weeks of Age.

The efficacy of commercially available recombinant herpesvirus of turkeys-infectious bursal disease (rHVT-IBD) virus vaccines was studied in broiler chickens derived from an IBDV-vaccinated breeder flock at 30 wk of age (Trial 1) and 60 wk of age (Trial 2). In parallel, specific-pathogen-free (SPF) white leghorn chickens were used to evaluate vaccine efficacy to control for the effects of maternally derived antibodies (MDA) associated with the broiler chickens. Broilers and SPF leghorns were vaccinated subcutaneously in the neck at 1 day of age with Vaxxitek® HVT+IBD or Vectormune® HVT-IBD vaccines and were placed in isolators. On 10, 14, 18, 22, and 26 days postvaccination (DPV), vaccinated and nonvaccinated broilers and SPF leghorns were bled prior to challenge via the oral-nasal route with infectious bursal disease (IBD) reference strains ST-C, Delaware variant E (Del E), or contemporary field isolates DMV/5038/07 or FF6. Microscopic lesion assessment of the bursa was useful for assessing IBDV challenge in both rHVT-IBD-vaccinated broiler and SPF leghorn chickens. In general, rHVT-IBD vaccines induced greater protection as the time between vaccination and challenge increased. Based on incidence of microscopic lesions (IML) of bursa tissue, Vaxxitek HVT+IBD vaccination of SPF leghorns induced protection by 18 DPV and continued to protect 22 DPV and 26 DPV in Trials 1 and 2. Vectormune HVT-IBD vaccine induced protection of SPF leghorns by 18 or 22 DPV in Trial 1, depending upon the IBDV challenge strain. However, the onset of protection was delayed until 22 or 26 DPV in Trial 2. With either commercial vaccine, rHVT-IBD vaccination of broiler chickens was not as effective as was observed in SPF leghorns, based on IML of bursa tissue. However, Vaxxitek HVT+IBD vaccination protected broilers following challenge with ST-C in both Trial 1 (30-wk-old breeder progeny) and Trial 2 (60-wk-old breeder progeny). Partial protection against FF6 (Trial 1) and DMV/5038/07 (Trial 2) challenges was observed. Vectormune HVT-IBD vaccination protected broilers vs. FF6 challenge in Trial 1. In Trial 2, the vaccine did not offer protection on the basis of IML of bursa tissue. The results indicate that 1) bursa/body weight ratios were not consistently useful as a tool for assessing IBDV challenge in broiler chickens with anti-IBDV MDA compared to assessment by IML of bursa tissue, though were useful for assessing protection in SPF leghorns; and 2) both vaccines may offer some protection to older broilers; however, a window of susceptibility exists between the waning of MDA and the development of vaccine-induced antibodies. The SPF studies showed that some vaccinated chickens were not protected from an IBDV challenge earlier than 14 DPV while broiler studies showed that MDA was not fully protective beyond 10 DPV. Because these vaccines did not protect chickens from an IBDV challenge during this window of susceptibility, our data show that breeder vaccination programs for IBDV must aim to maximize anti-IBDV MDA in progeny to protect against early IBDV challenge.

Efficacy of Various HVT Vaccines (Conventional and Recombinant) Against Marek's Disease in Broiler Chickens: Effect of Dose and Age of Vaccination.

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.

Efficacy of various Marek's disease vaccines protocols for prevention of Marek's disease virus-induced immunosuppression.

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.

Comparative effects of in ovo versus subcutaneous administration of the Marek's disease vaccine and pre-placement holding time on the early post-hatch quality of Ross × Ross 708 broiler chicks.

Effects of method of administration [moa; in ovo (i.o.) or s.c.] of the Marek's disease vaccine and pre-placement holding time (pht) on early post-hatch male broiler chick quality was investigated. Sixty-five Ross × Ross 708 broiler hatching eggs were randomly set in each of 15 replicate trays (blocks) belonging to each of 4 pre-assigned moa and pht treatment combinations (3,900 total eggs) in a single stage Jamesway incubator. Eggs that were i.o.-vaccinated received injections at 18 d of incubation and male chicks from eggs that were not i.o.-injected were vaccinated by s.c. injection at hatch. The i.o. injections (50 µL) were delivered by a commercial multi-egg injector and the s.c. injections (200 µL) were delivered by an automatic pneumatic s.c. injector. Male chicks from each moa group also were subjected to either a 4 or 18 h pht. At hatch and placement total and yolk-free BW; body length; body mass index; yolk sac weight; yolk-free body and yolk sac weights as percentages of total BW; and yolk-free body and yolk moisture concentrations were determined. Chick BW also was determined at 7 d of age. Hatchability of fertile eggs was not affected by i.o. injection. However, at hatch, body length was increased and body mass index was decreased in response to i.o. injection. No main effect of moa or an interactive effect with pht was observed for the above variables at placement. However, body length was longer and body mass was lower in the 18 h than in the 4 h pht chicks. Placement yolk sac and body weights, and the 7 d BW of 18 h pht chicks was also lower than that of 4 h pht chicks. In conclusion, prolonging pht for 14 h adversely affected early chick quality, whereas i.o. injection did not negatively affect the early post-hatch quality of Ross × Ross 708 broiler chicks whether or not they were held for 4 or 18 h prior to placement.

A recombinant field strain of Marek's disease (MD) virus with reticuloendotheliosis virus long terminal repeat insert lacking the meq gene as a vaccine against MD.

Marek's disease virus (MDV) GX0101, which is a field strain with a naturally occurring insertion of the reticuloendotheliosis virus (REV) long terminal repeat (LTR) fragment, shows distinct biological activities. Deletion of the meq gene in GX0101 contributes to its complete loss of pathogenicity and oncogenicity in SPF chickens, but this virus has a kanamycin resistance gene (kan(r)) residual at the site of meq gene. In the present study, the kan(r) was knocked out and a meq-null virus with a good replicative ability termed SC9-1 was selected. In vivo studies showed that SC9-1 had no pathogenicity or tumorigenicity to chickens. There were no obvious impacts on chicken weight, immune organ index or antibody levels induced by avian influenza virus (AIV)/newcastle disease virus (NDV) inactivated vaccines compared with the control group. The SC9-1 virus provided superior protection than CVI988/Rispens vaccine in both SPF chickens and Hy-line brown chickens when challenged with a very virulent MDV (rMd5 strain). There was no obvious change in SC9-1 protection against MDV rMd5 in SPF chickens after 20 passages in chicken embryonic fibroblast cells (CEFs). In conclusion, SC9-1 is a safe and effective vaccine candidate for the prevention of Marek's disease.