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

Dilution of Marek's 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.     

Protective efficacy of Marek's disease virus (MDV) CVI-988 CEF65 clone C against challenge infection with three very virulent MDV strains

Comparative 50% protective dose (PD50) assays were performed using a plaque-purified preparation of Marek's disease virus (MDV) strain CVI-988 at the 65th chicken embryo fibroblast (CEF) passage level (MDV CVI-988 CEF65 clone C) and three commercial MD vaccines: herpesvirus of turkeys (HVT) FC126, MDV CVI-988 CEF35, and a bivalent vaccine composed of HVT FC126 and MDV SB-1. In addition, comparative PD50 assays were performed in groups of chickens with maternal antibody to each of the three vaccines. Three representatives of the newly emerged biovariant very virulent (vv) MDV strains-RB/1B, Tun, and Md5-were employed as challenge virus. The experiments made feasible the differentiation between virulent MDV and vvMDV strains, within serotype 1. Vaccination with CVI-988 clone C vaccine resulted in PD50 estimates of about 5 plaque-forming units (PFUs) against challenge infection with each of the three vvMDV strains. The PD50 estimate of CVI-988 clone C vaccine was 12-fold below the PD50 of HVT FC126. The protective synergism of bivalent vaccine, composed of HVT and SB-1, was confirmed by groups given the lowest vaccine doses. The bivalent vaccine, however, resulted in incomplete protection in groups given the highest vaccine doses. Homologous maternal antibodies to serotype 1 caused a fivefold increase in the PD50 estimate of CVI-988 clone C. Heterologous maternal antibodies against HVT did not interfere with efficacy of CVI-988 clone C vaccination. However, the combination of maternal antibodies against both HVT and SB-1 (serotypes 2 and 3) showed a strong adverse effect on CVI-988 clone C vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The efficacy of recombinant fowlpox vaccine protection against Marek's disease: its dependence on chicken line and B haplotype

Earlier studies have shown that the B haplotype has a significant influence on the protective efficacy of vaccines against Marek's disease (MD) and that the level of protection varies dependent on the serotype of MD virus (MDV) used in the vaccine. To determine if the protective glycoprotein gene gB is a basis for this association, we compared recombinant fowlpox virus (rFPV) containing a single gB gene from three serotypes of MDV. The rFPV were used to vaccinate 15.B congenic lines. Nonvaccinated chickens from all three haplotypes had 84%-97% MD after challenge. The rFPV containing gB1 provides better protection than rFPV containing gB2 or gB3 in all three B genotypes. Moreover, the gB proteins were critical, since the B*21/*21 chickens had better protection than chickens with B*13/*13 or B*5/*5 using rFPV with gB1, gB2, or gB3. A newly described combined rFPV/gB1gEgIUL32 + HVT vaccine was analyzed in chickens of lines 15 x 7 (B*2/*15) and N (B*21/*21) challenged with two vv+ strains of MDV. There were line differences in protection by the vaccines and line N had better protection with the rFPV/gB1gEgIUL32 + HVT vaccines (92%-100%) following either MDV challenge, but protection was significantly lower in 15 X 7 chickens (35%) when compared with the vaccine CVI988/Rispens (94%) and 301B1 + HVT (65%). Another experiment used four lines of chickens receiving the new rFPV + HVT vaccine or CVI988/Rispens and challenge with 648A MDV. The CVI 988/Rispens generally provided better protection in lines P and 15 X 7 and in one replicate with line TK. The combined rFPV/gB1gEgIUL32 + HVT vaccines protected line N chickens (90%) better than did CVI988/Rispens (73%). These data indicate that rFPV + HVT vaccines may provide protection against MD that is equivalent to or superior to CVI988/ Rispens in some chicken strains. It is not clear whether the rFPV/gB1gEgIUL32 + HVT vaccine will offer high levels of protection to commercial strains, but this vaccine, when used in line N chickens, may be a useful model to study interactions between vaccines and chicken genotypes and may thereby improve future MD vaccines.     

Embryo vaccination against Marek's disease with serotypes 1, 2 and 3 vaccines administered singly or in combination

Marek's disease virus (MDV) vaccines of serotypes 1 and 2 administered in 18-day-old embryonated eggs induced better protection against post-hatch challenge at 3 days with virulent MDV than vaccines given at hatch. Embryonal vaccination with a polyvalent vaccine containing equal quantities of serotypes 1 and 2 of MDV and serotype 3 virus (turkey herpesvirus, HVT) was also significantly more effective than post-hatch vaccination. These and earlier results indicate that protective efficacy of single or combined Marek's disease vaccine serotypes against post-hatch challenge at 3 days can be substantially improved if the vaccines are injected into 18-day embryos rather than at hatch. Injection of vaccines of serotypes 1 or 2 into embryonated eggs or hatched chicks did not cause detectable gross or microscopic lesions in chickens. Vaccine viruses of serotypes 1 and 2 could be isolated from spleen cells of chickens 1 week post-vaccination, and the titer of recoverable viruses was higher in chickens that received the vaccines at the 18th day of embryonation than in chickens vaccinated at hatch. Although embryo vaccination with HVT usually provided better protection than post-hatch vaccination against early post-hatch challenge with variant pathotypes of MDV, the protection was poor regardless of vaccination protocol. If challenge with variant pathotypes of MDV was delayed until embryonally or post-hatch HVT-vaccinated chickens were 21 days of age, protection of chickens by HVT was not enhanced. Thus, resistance induced by embryonal vaccination with HVT was qualitatively similar to that induced by post-hatch vaccination with this virus.     

A comparative evaluation of the protective efficacy of rMd5deltaMeq and CVI988/ Rispens against a vv+ strain of Marek's disease virus infection in a series of recombinant congenic strains of White Leghorn chickens

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a highly infectious, oncogenic alpha-herpesvirus known as Marek's disease virus (MDV). MD is presently controlled by vaccination. Current MD vaccines include attenuated serotype 1 strains (e.g., CVI988/Rispens), avirulent serotype 2 (SB-1), and serotype 3 (HVT) MDV strains. In addition, recombinant MDV strains have been developed as potential new and more efficient vaccines to sustain the success of MD control in poultry. One of the candidate recombinant MDV strains, named rMd5deltaMeq, was derived from Md5, a very virulent strain of MDV lacking the MDV oncogene Meq. Our earlier reports suggest that rMd5deltaMeq provided protection equally well or better than commonly used MD vaccines in experimental and commercial lines of chickens challenged with very virulent plus (vv+) strains of MDV. In this study, maternal antibody-positive (trial 1) and negative (trial 2) chickens from a series of relatively MD resistant lines were either vaccinated with the rMd5deltaMeq or CVI988/Rispens followed by infection of a vv+ strain of MDV, 648A, passage 10. This report presents experimental evidence that the rMd5deltaMeq protected significantly better than the CVI988/Rispens (P < 0.01) in the relatively resistant experimental lines of chickens challenged with the vv+ strain of MDV. Together with early reports, the rMd5deltaMeq appeared to provide better protection, comparing with the most efficacious commercially available vaccine, CVI988/Rispens, for control of MD in lines of chickens regardless of their genetic background.     

鸡马立克氏病三价疫苗的安全性及免疫效力研究

将MD三价疫苗以25000PFU／只的剂量颈部皮下接种1日龄SPF雏鸡，结果表明，疫苗的接种不影响鸡体重的增加；不引起鸡法氏囊、脾脏等组织器官发生MD组织病理学变化，对鸡安全无毒性。用SPF鸡评价MD三价疫苗的免疫效力，三批疫苗对RB1B超强毒株攻击的平均保护效力为95．99％，而且无论是用RB1B超强毒株还是用BJMDV-1血毒攻击，MD三价疫苗的保护效力明显高于HVT＋SB1二价疫苗及HVT冻干苗，好于CV1988疫苗；接种MD三价疫苗的4个品系的商品鸡群，抗RB1B超强毒株攻毒的平均保护效力为94．60％；在模拟MD强毒自然传染的试验中，MD三价疫苗的保护效力达到95．65％。上述效力试验的结果说明：MD三价疫苗的免疫接种可使鸡形成抗MD强毒攻击的坚强免疫力。     

Influence of a reovirus-antibody complex vaccine on efficacy of Marek's disease vaccine administered in ovo

Two experiments determined the influence of an experimental reovirus-antibody complex vaccine on Mareks disease virus (MDV) vaccine when used in ovo. Designs were the same except that specific-pathogen-free (SPF) broiler eggs were used in Experiment 1 and commercial broiler eggs with maternal antibodies against reovirus were used in Experiment 2. At 18 days of incubation, embryos were separated into four groups and inoculated with either diluent, MDV vaccine, reovirus-antibody complex vaccine, or a combination of reovirus-antibody complex and MDV vaccine. At 5 days of age, half the chickens in each group were challenged with MDV. At 7 wk old, all were euthanatized, weighed, and examined. At 7 days of age, remaining chickens in each group were challenged with reovirus. At 21 days old, chickens were euthanatized and weighed. No vaccine adversely affected hatchability or posthatch mortality in SPF or commercial chickens. There were no significant differences in protection against reovirus challenge when vaccines were used separately or in combination, and lesion scores were nearly identical in all vaccinated groups in both experiments. However, percentage of protection against reovirus was lower in Experiment 2, indicating an adverse effect of maternal immunity on efficacy of the reovirus vaccine. There were no significant differences in protection against MDV when the vaccines were used separately or combined. Severity of MDV lesions was nearly identical in all vaccinated groups in both experiments. However, the combination of vaccines gave numerically lower protection against MDV than MDV vaccine alone. Use of a larger number of birds, as in field conditions, may result in statistically lower protection for the vaccine combination. Large field trials are needed to determine the potential of the reovirus-antibody complex vaccine.     

Susceptibility of adult chickens, with and without prior vaccination, to challenge with Marek's disease virus

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 vv+) 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 = 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 = 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 > or =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.     

Load of challenge Marek's disease virus DNA in blood as a criterion for early diagnosis of Marek's disease 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 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.     

Pathogenicity studies with plaque-purified preparations of Marek's disease virus strain CVI-988

The pathogenicity of Marek's disease (MD) strain CVI-988 vaccine, eight plaque-purified preparations originating from this strain, and the vaccine HVT FC126 (based on herpesvirus of turkeys) was determined by intramuscular administration of high virus doses to day-old specific-pathogen-free Rhode Island Red (RIR) chickens, which are extremely MD-susceptible. Paralysis and neuritis were observed in 88% of RIR chickens inoculated with MDV CVI-988 at the cell-passage level of the commercial vaccine. HVT FC126 caused paralysis in two of 39 RIR chickens tested, of which one had an endoneural lymphoma, and another three had endoneural inflammation. Five plaque-purified MDV CVI-988 virus preparations at various cell-culture-passage levels caused no lesions. Of another three clones, two caused inflammatory B-type lesions in the nerves of 1/10 chickens, and the third clone caused inflammatory nonneoplastic MD lesions in the liver of 1/11 chickens.     

Recent increase of Marek's disease in Korea related to the virulence increase of the virus

The incidence of Marek's disease (MD), an important neoplastic disease of chickens, suddenly increased in 1997 in Korea. Most MD cases of this country were detected in chickens over 20 wk of age. Five MD viruses were isolated from field flocks in which severe MD losses had occurred, and one of the viruses was studied to compare its pathotype with the prototype JM strain. The isolate KOMD-IC induced severe depression not only in body weight but also in relative bursal weight, and the depression by KOMD-IC was more severe than that induced by JM strain. In addition, the incidence of MD tumor caused by KOMD-IC was higher than that caused by the JM strain. The protective capacity of several MD vaccines was studied against challenge with KOMD-IC. The protective levels of several MD vaccines such as herpesvirus of turkeys (HVT), HVT plus SB1, and Rispens were usually lower against challenge with KOMD-IC than those challenged with JM strain, even if the chickens vaccinated with serotype 1 were not completely protected against challenge with KOMD-IC. The above results indicate that the virulence of KOMD-IC isolated recently was increased, and the increase of MD outbreak in Korea may be related to the virulence increase of the virus. Various MD vaccine programs were applied to reduce MD loss to a broiler breeder farm where severe MD loss had occurred. Serotype 1 vaccine could dramatically decrease the mortality due to MD, and the best results were obtained from the flocks vaccinated with bivalent vaccine of Rispens and HVT.     

Antibody response of chickens to serotype 1, 2, or 3 Marek's disease vaccines based on ELISA with infected cells as antigen

An enzyme-linked immunosorbent assay (ELISA) was applied to evaluate the antibody response of commercial White Leghorn chickens to vaccination against Marek's disease (MD) at hatch (day 0) with serotype-1 (Rispens), -2 (SB-1), or -3 (turkey herpesvirus, HVT) vaccine virus and to challenge on day 21 with MD virus. Antigens for the test were whole chicken embryo fibroblast cells infected with Rispens, SB-1, or HVT. The chickens were progeny of stock that had been vaccinated with HVT, and on day 21 the nonvaccinated group had higher levels of maternal antibodies to HVT than to other antigens (P < 0.05). Only SB-1 vaccine had induced antibodies by day 21, and this was detected only against homologous antigens. On day 49, all three vaccines had induced higher levels of antibodies to homologous than to heterologous antigens. Marek's Disease virus (MDV) induced antibodies to all three antigens, but challenging vaccinated chicks did not significantly increase levels of antibodies on day 81 to any of the three antigens. It was concluded that an ELISA using whole cells as antigens would have potential value for monitoring the antibody response induced by MD vaccines and virulent MDV.     

A comparison of the onset of protection induced by Newcastle disease virus strain B1 and a fowl poxvirus recombinant Newcastle disease vaccine to a viscerotropic velogenic Newcastle disease virus challenge

Four-week-old specific-pathogen-free white rock chickens were immunized with either a commercial recombinant fowl poxvirus-vectored Newcastle disease vaccine (FPN) expressing the hemagglutinin-neuraminidase and fusion protein genes of Newcastle disease virus (NDV) strain B1 or live NDV B1. Vaccinates and controls were challenged by eyedrop and intranasal (E/I) route with a viscerotropic velogenic NDV at 14 days postvaccination to determine the time of clearance of challenge virus. In a subsequent experiment, chickens were challenged at 3, 6, or 10 days postvaccination to determine the onset of immunity. Chickens that received a recommended field dose (1x) or a 0.01x dose of FP-N subcutaneously (s.c.) and were seropositive by hemagglutination-inhibition test at 14 days postvaccination cleared the challenge virus by 14 days postchallenge. Clinical Newcastle disease and high challenge virus titers in tissues were seen only in seronegative FP-N 0.01x dose vaccinates and controls. In a comparison of vaccination with FP-N (1x, 10(4,9) median tissue culture infective dose) s.c., B1 (10(6) median egg infective dose [EID50]) s.c., or B1 (10(6) EID50) E/I, chickens vaccinated at 6 or 10 days before challenge with all vaccines were protected against clinical disease, but only those vaccinated with B1 E/I 10 days before challenge were protected against infection with the challenge virus. Vaccination at 3 days before challenge with B1 E/I provided early protection, but severe nervous signs developed later and reduced overall protection to 60%, whereas disease in chickens vaccinated with B1 s.c. and FP-N s.c. 3 days before challenge was similar to the challenge controls.     

Evaluation of factors affecting vaccine efficacy of recombinant Marek's disease virus lacking the Meq oncogene in chickens

We previously reported that deletion of the Meq gene from the oncogenic rMd5 virus rendered it apathogenic for chickens. Here we examined multiple factors affecting Marek's disease vaccine efficacy of this nonpathogenic recombinant Meq null rMd5 virus (rMd5deltaMeq). These factors included host genetics (MHC haplotype), strain or dose of challenge virus, vaccine challenge intervals, and maternal antibody status of the vaccinated chicks. Studies on host genetics were carried out in five chicken lines comprising four different MHC B-haplotypes. Results showed that chicken lines tested were highly protected, with protective indexes of 100% (B*2/*15), 94% (B*2/*2), 87% (B*19/*19), and 83% (B*21/*21). At a challenge dose above 8000 plaque-forming units, differences in protection were observed between the two highly virulent strains examined (648A and 686). The interval between vaccination and challenge indicated a protective efficacy from 0 to 2 days varied greatly (12%-82%) after challenge with vv+686, the most virulent virus. Less variation and significant protection began at 3 days post vaccination and reached a maximum at 5 days post vaccination with about 80%-100% protection. Taken together, our results indicate that the factors examined in this study are important for vaccine efficacy and need to be considered in comparative evaluations of vaccines.     

Delayed replication of Marek's disease virus following in ovo inoculation during late stages of embryonal development

Several oncogenic and non-oncogenic isolates of Marek's disease virus (MDV) were inoculated into embryonated eggs on embryonation day (ED) 16 to 18, and embryos or chicks hatching from inoculated eggs were examined for infectious virus and viral internal antigen (VIA) in lymphoid organs. There was no evidence of extensive replication of MDV in any of the embryonic tissues examined. Levels of VIA peaked 4-5 days after chicks hatched. This indicated that MDV remained inactive during embryonation and did not initiate pathogenic events until chicks hatched. Because HVT replicated rapidly in the embryo but MDV did not, in ovo inoculation of HVT simultaneously with oncogenic MDV or several days after MDV resulted in significant protection (P less than 0.025) of hatched chicks against Marek's disease (MD). Little protection was obtained if HVT was given simultaneously with MDV or after MDV to chicks already hatched. The relative susceptibility of the embryo to extensive replication of the vaccine virus but not the challenge virus apparently accounted for protection against MD in chicks hatching from dually infected eggs.     

鸡马立克氏病活疫苗免疫效力比较试验

用ＨＶＴ冻干苗、ＨＶＴ细胞结合苗、ＣＶＩ９８８细胞结合苗、ＳＢ１＋ＦＣ１２６双价活疫苗、３０１Ｂ／１＋ＦＣ１２６双价活疫苗和Ｚ４＋ＦＣ１２６双价活疫苗等６种鸡马立克氏病（ＭＤ）疫苗免疫ＳＰＦ白来航鸡或普通伊莎鸡，用鸡马立克氏病病毒（ＭＤＶ）强毒ＧＡ株、京－１血毒以及鸡马立克氏病超强毒ｖｖＭＤＶ－Ｍｄ５毒株分别攻击进行免疫效力比较试验。试验表明，ＭＤ单价苗的免疫效力强弱顺序依次是ＣＶＩ９８８、ＨＶＴ细胞结合苗和ＨＶＴ冻干苗，这３种ＭＤ单价苗均能给免疫鸡群提供有效的免疫保护力。ＳＢ１＋ＦＣ１２６、Ｚ４＋ＦＣ１２６和３０１Ｂ／１＋ＦＣ１２６等３种ＭＤ双价苗免疫效力显著高于ＭＤ单价苗，均能给免疫鸡群提供较强的免疫保护力，并能有效地抵抗ｖｖＭＤＶ－Ｍｄ５毒株的致瘤作用。Ｚ４＋ＦＣ１２６和３０１Ｂ／１＋ＦＣ１２６ＭＤ双价苗免疫效力无显著差异     

The resistance of meat chickens vaccinated by aerosol with a live V4 Newcastle disease virus vaccine in the field to challenge with a velogenic Newcastle disease virus

Meat chickens housed on a commercial broiler farm in Australia were vaccinated once at 10 to 11 days-of-age by aerosol with live V4 Newcastle disease virus (NDV) vaccine. Groups of vaccinated and unvaccinated birds were flown to Malaysia, where they were challenged with a virulent strain of NDV. Survival rates in vaccinated chickens challenged 7, 14, 21 or 31 d after vaccination were 0.47, 0.77, 0.97 and 0.92, respectively. All unvaccinated chickens died due to Newcastle disease (ND) following challenge. Chickens in Australia and Malaysia were bled and the serums tested for haemagglutination-inhibiting (HI) antibody to NDV. Many vaccinated birds with no detectable antibody, and all birds with a log2 titre of 2 or greater, survived challenge. The results showed that this V4 vaccine induced protective immunity in a significant proportion of chickens within 7 d of mass aerosol vaccination. This early immunity occurred in the absence of detectable circulating HI antibody. Non-HI antibody mediated immunity continued to provide protection up to 31 d after vaccination. Almost all vaccinated birds were protected within 3 w of vaccination. It is concluded that the V4 vaccine is efficacious and could be useful during an outbreak of virulent ND in Australia.     

Effects of vaccine route and dosage on protection from rabies after intracerebral challenge in mice

OBJECTIVE: To evaluate the effect of various routes of administration and number of doses of 3 commercially produced rabies vaccines on serum antibody responses and protection in mice challenged by intracerebral injection with fixed-strain rabies virus. ANIMALS: 2,213 mice. PROCEDURE: Inactivated, adjuvanted rabies vaccines were administered to mice in either 2, 1, or 0 (control) doses via IP, IM, and SC routes, and mice were challenged intracerebrally with fixed-strain rabies virus. RESULTS: Vaccination route and dose number significantly influenced serum antibody responses and protection from rabies virus challenge, independent of vaccine strain origin and mouse strain, although mouse age significantly affected results. Extended challenge studies revealed that IM vaccination of mice resulted in the highest serum neutralizing antibody responses and protection levels equivalent to IP vaccination. Even multiple doses administered SC (a vaccination route used in dogs) resulted in poor serum anti-rabies neutralizing antibody responses in mice and were far less protective than other routes. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest possible improvements for the current rabies vaccine potency test in mice by using 1 dose, the IM route, and a delayed time of challenge. These modifications would more closely model vaccination practices in target species and yield more accurate information regarding primary immunogenicity of a vaccine.     

Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus

Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody-positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gl were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.     

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

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.