Experimental infection with avian leukosis virus isolated from Marek's disease vaccines

Recently, avian leukosis virus (ALV) was isolated from four lots of Marek's disease vaccine produced by two laboratories. The ALVs isolated were characterized by examination of their interactions with cells of two phenotypes (C/E and C/A,E), subgroup-specific polymerase chain reaction (PCR), virus neutralization, envelope gene sequencing, and phylogenetic analysis. All four ALVs are exogenous, belong to subgroup A, and appear to be virtually identical to each other based on PCR and envelope gene nucleotide sequences. We describe herein the characterization of the contaminant viruses in vivo by means of experimental infection in chickens. The contaminant viruses established transient viremia in specified pathogen-free (SPF) Leghorn chickens and elicited a robust and lasting antibody response detectable by enzyme-linked immunosorbent assay. None of the contaminant ALVs induced tumors up to 31 wk of age, and mortality was insignificant. Despite a strong antibody response against the contaminant ALVs, vertical (congenital) transmission to the progeny of experimentally infected SPF chickens took place, albeit at a very low rate (< or = 1.6%). Experimental infection in meat-type chicken embryos resulted in viremia at hatch, suggesting that some meat-type chickens are susceptible to infection and support virus replication.     

An acute form of transient paralysis induced by highly virulent strains of Marek's disease virus

A novel syndrome was observed after inoculation of 3-wk-old chickens with highly virulent Marek's disease virus (MDV) strains. This syndrome was characterized by the acute onset of neurologic signs including flaccid paralysis of neck and limbs 9-10 days postinoculation, typically resulting in death 1-3 days after the onset of clinical signs. Most affected birds died, and spontaneous recovery was rare. Few if any gross tissue changes were found. Histologic brain lesions included acute vasculitis with vasogenic edema and perivascular cuffing. The syndrome was influenced by the virus strain and dose and by chicken strain and B haplotype and was prevented by vaccination with turkey herpesvirus. Chickens up to 18 wk of age were susceptible. On the basis of clinical signs and histopathology, the syndrome was determined to be an acute form of transient paralysis (TP); its more acute nature and virtual lack of spontaneous recovery differentiated this syndrome from classical TP. Affected birds were viremic, and brains were positive for viral DNA by polymerase chain reaction assays, but these tests were also positive in inoculated chickens without clinical signs and may have limited value for diagnosis. Although acute TP should occur only rarely in Marek's disease-vaccinated commercial flocks, this syndrome may be important in laboratory studies, where it could interfere with pathogenesis trials. Finally, acute TP appears to be one component in the pathogenesis of the early mortality syndrome, a previously described immunodepressive disease induced by inoculation of 1-day-old chicks with highly virulent MDV.     

Persistent infection with chicken anaemia virus and some effects of highly virulent infectious bursal disease virus infection on its persistency

Chicken anaemia virus (CAV) infectivity and the effect of highly virulent infectious bursal disease virus (hv IBDV) infection on CAV's infectivity were examined in chickens inoculated with CAV or inoculated dually with CAV and hv IBDV. Five chickens inoculated dually with hv IBDV at 35 days old and then with CAV at 40 days old exhibited no clinical signs of disease, but showed atrophic bursae of Fabricius when necropsied 4 weeks later. Upon examining the chickens at 7 days postinoculation (dpi) with CAV, it was found that hv IBDV infection had inhibited production of virus neutralising (VN) antibody to CAV, and that it was possible to recover CAV from plasma of these chickens. Although VN antibody to CAV appeared after 14 dpi, CAV was recovered from blood cells (BC s) at high titres ranging from 10(2.5)to 10(5.5)TCID(50)/0.1 ml, 7 to 28 dpi in IBDV -induced immunosuppressed chickens. In addition, CAV was sporadically recovered, using rectal swabs, from the dually inoculated chickens at low titers, ranging from 10(1.0)to 10(2. 0)TCID(50)/0.1 ml). In contrast, although CAV was recovered from BC s in most of the chickens inoculated with CAV alone, the titers were lower (10(1.0)to 10(2.5)TCID(50)/0.1 ml). No CAV was detected from the rectal swabs of these chickens. The results of virus recovery were confirmed by polymerase chain reaction. This study first examined the persistency of CAV in BC s and the effective enhancement of primary CAV infection as a result of immunosuppression caused by hv IBDV infection.     

Detection and characterization of avian leukosis virus in Marek's disease vaccines

Avian leukosis virus (ALV) infection in chickens is known to induce increased mortality, tumors, delayed growth, and suboptimal egg production. Countries importing specified pathogen-free eggs, vaccines, and poultry breeding stock require freedom of infection or contamination with ALV in such products among other avian pathogens. Recently, ALV was found as a contaminant in a limited number of commercial poultry vaccines, even after routine quality assurance procedures cleared the vaccines for commercialization. The contaminated vaccines were promptly withdrawn from the market, and no direct detrimental effects were reported in poultry vaccinated with such vaccines. We describe herein the characterization in vitro of the contaminant viruses. All exogenous viruses detected in four vaccine lots belong to subgroup A of ALV based on cell receptor interaction, subgroup-specific polymerase chain reaction (PCR), envelope gene sequencing, and virus neutralization. A combination of thermal treatment and serial dilutions of the contaminated vaccines facilitated detection of contaminating ALVs in cell culture coupled with antigen-capture enzyme-linked immunosorbent assay. Subgroup-specific PCR readily detected ALV-A directly in the contaminated vaccines but not in naive vaccines or cell controls. Our methods are proposed as complementary procedures to the currently required complement fixation for avian leukosis test for detection of ALV in commercial poultry vaccines.     

超强马立克氏病病毒的鉴定及病毒Meq基因序列的比较

对1例感染超强马立克氏病病毒的病例进行确诊,对感染病毒的Meq基因进行比较分析。采用病理解剖、PCR检测、病毒分离、动物试验和Meq基因序列分析,对感染病毒进行研究。病理解剖结果为病死鸡的肝脏、脾脏、腺胃、肌胃、十二指肠表现为肿瘤病变。PCR检测结果为病死鸡的组织病料感染马立克氏病病毒。病毒分离和动物试验结果证明该感染病毒是1株马立克氏病超强毒株,该病毒可以引起免疫过CVI988疫苗的鸡发病。Meq基因序列分析表明该病毒与7株马立克氏病病毒参考毒株的同源性为98.8%~99.6%,该病毒在Meq的第115、119和176位氨基酸突变同国内流行株,该检测病毒在Meq的第217位氨基酸突变同超超强马立克氏病毒株。结果表明,通过病理解剖、PCR检测、基因序列分析、病毒分离和动物试验,确诊病鸡感染超强马立克氏病病毒。     

Derivation, safety and efficacy of a Marek's disease vaccine developed from an Australian isolate of very virulent Marek's disease virus

Objective To develop a serotype 1 Marek's disease (MD) vaccine from a very virulent MDV (vvMDV) pathotype and demonstrate safety and efficacy against early challenge with very virulent field strains in the presence of maternal antibody.
Study design Strain BH 16 was isolated and attenuated by serial cell culture passage. One of two cloned passages was selected for vaccine development following early laboratory-scale protection trials in commercial birds. Comparative protection trials were carried out on the BH 16 vaccine and on a CVI 988 Rispens vaccine using commercial and SPF chickens. Challenge viruses used were either a low passage strain BH 16 virus, the Woodlands No. 1 strain or MPF 57 strain of MDV. The BH 16 vaccine was back-passaged in SPF chickens six times and virus recovered from the final passage and the original vaccine virus were tested for safety. The immunosuppressive potential of the BH 16 and Rispens vaccines was also assessed in parallel.
Results The BH 16 and Rispens vaccines induced comparable levels of protection when used as monovalent or multi-valent vaccines, although protection achieved with the mono-valent vaccines was lower. No gross tumour formation was evident in any birds receiving the BH 16 vaccine or bird-passaged virus, although microscopic lesions were present in 2/12 birds that received the bird-passaged virus. In tests for immunosuppression, there was no histological evidence of damage to either the bursa of Fabricius or the thymus.
Conclusion The BH 16 vaccine was shown to be safe and at least as protective as the Rispens vaccine against three highly virulent MD challenge viruses.     

Derivation,safety and efficacy of a Marek's disease vaccine developed from an Australian isolate of very virulent Marek's disease virus

OBJECTIVE: To develop a serotype 1 Marek's disease (MD) vaccine from a very virulent MDV (vvMDV) pathotype and demonstrate safety and efficacy against early challenge with very virulent field strains in the presence of maternal antibody. STUDY DESIGN: Strain BH 16 was isolated and attenuated by serial cell culture passage. One of two cloned passages was selected for vaccine development following early laboratory-scale protection trials in commercial birds. Comparative protection trials were carded out on the BH 16 vaccine and on a CVI 988 Rispens vaccine using commercial and SPF chickens. Challenge viruses used were either a low passage strain BH 16 virus, the Woodlands No. 1 strain or MPF 57 strain of MDV. The BH 16 vaccine was back-passaged in SPF chickens six times and virus recovered from the final passage and the original vaccine virus were tested for safety. The immunosuppressive potential of the BH 16 and Rispens vaccines was also assessed in parallel. RESULTS: The BH 16 and Rispens vaccines induced comparable levels of protection when used as monovalent or multivalent vaccines, although protection achieved with the monovalent vaccines was lower. No gross tumour formation was evident in any birds receiving the BH 16 vaccine or bird-passaged virus, although microscopic lesions were present in 2/12 birds that received the bird-passaged virus. In tests for immunosuppression, there was no histological evidence of damage to either the bursa of Fabricius or the thymus. CONCLUSION: The BH 16 vaccine was shown to be safe and at least as protective as the Rispens vaccine against three highly virulent MD challenge viruses.     

鸡马立克氏病病毒感染对鸡神经系统损伤的研究

为研究不同毒力的鸡马立克氏病毒(MDV)在鸡神经系统中的感染规律及其对神经系统的损伤进程,本研究选用不同毒力的血清1型MDV (MDV-1)病毒株感染4日龄的SPF鸡,在接毒后1 d、3 d、5 d、7 d、10 d、14 d、17 d、21 d、25 d、28 d和35 d动态检测病毒载量的变化,并对感染后5 d和21 d的不同病毒株感染鸡的脑部和坐骨神经进行组织病理学观察。结果显示,MDV-1强毒株在SPF鸡脑部的复制能力显著高于弱毒株(p<0.05);特超强病毒株BS在脑组织中出现的时间最早,早期复制最快。但不同毒力的MDV-1株在坐骨神经处的复制能力与其毒力无直接的关系。组织病理学观察显示,在感染早期MDV-1强毒株对SPF鸡脑组织的损伤强于弱毒株;在感染后21 d,强毒株和弱毒株造成的脑部损伤存在明显的不同;而在坐骨神经处,强毒株造成的损伤明显强于弱毒株。本研究揭示了不同MDV病毒株在SPF鸡脑和坐骨神经的复制动力学特征和组织病理学特征,为MDV-1在宿主神经系统中的感染、增殖及造成的损伤提供了实验依据。     

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)     

Effect of acyclovir on the replication of turkey herpesvirus and Marek's disease virus

The toxicity of acyclovir for chick embryo fibroblasts and its effect on the replication of turkey herpesvirus (strain FC 126) and Marek's disease virus (strain HPRS 16) multiplied on fibroblast culture was studied. The influence of using acyclovir on the development of the tumour process in birds infected with a virulent Marek's disease virus was also determined. Acyclovir used in doses below 12.5 micrograms ml-1 proved to be nontoxic for chick embryo fibroblast culture. It inhibited in vitro replication of turkey herpesvirus and Marek's disease virus. It was also shown to diminish the development of tumours in birds infected with Marek's disease virus.     

Effects of reticuloendotheliosis virus and Marek's disease virus infection and co-infection on IFN-gamma production in SPF chickens

Two experiments were used to examine the potential role of IFN-gamma in chickens infected with reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). First, chickens were infected with REV and/or MDV at 5 days of age and examined from 3 to 50 days post-infection (dpi). In REV+MDV co-infection chickens, IFN-gamma ELISA demonstrated a 3-fold increase at 7 dpi compared to the controls, while REV alone caused a 5-fold increase, the IFN-gamma levels peaked, and then gradually decreased. IFN-gamma levels significantly decreased in MDV infection at 3 dpi and 15 dpi. Second, experiments were designed to determine the effects of different viruses and ConA on IFN-gamma production. For REV- or MDV-infected chickens, the IFN-gamma levels decreased slightly after adding ConA. This is the first report of IFN-gamma production in SPF chickens infected with REV and MDV measured by directly quantitative method.     

Extent and duration of virulent virus excretion upon challenge of pigs vaccinated with different glycoprotein-deleted Aujeszky's disease vaccines

Different deleted Aujeszky's disease vaccines were compared for their ability to induce an immunity which suppresses virus excretion optimally upon infection. Groups of pigs were vaccinated once with attenuated deleted Aujeszky's disease vaccine (gI, gX or gp63 negative), suspended in phosphate buffered saline. Two additional groups were vaccinated with a gI deleted vaccine virus suspended in an oil-in-water emulsion. Other groups were vaccinated twice with gI deleted inactivated vaccines. The three control groups included were: pigs immune after infection, unvaccinated pigs and pigs receiving vaccine without known deletion in the envelope. Experimental challenge took place 3 or 4 weeks after the only or the last vaccination. The number of excreting pigs, the duration of excretion and the virus titers excreted, were determined for all the groups. All the pigs vaccinated with glycoprotein deletion vaccines suspended in phosphate buffered saline, excreted virus for 2 to 6 days after challenge. A 100 to 1000 fold reduction in excreted virus titers was obtained in vaccinated pigs compared to unvaccinated ones. Some vaccines suppressed virus excretion better than others, but no correlation could be made between the type of deletion (gI, gX or gp63) and the degree of reduction in virus excretion. Similar results were obtained with two applications of inactivated vaccines. The lowest number of excreting pigs, the lowest duration of excretion and the lowest titers were obtained in groups vaccinated with the attenuated vaccine suspended in an oil-in-water emulsion. No vaccine suppressed virus excretion totally.     

Molecular characterization of three recombinant isolates of avian leukosis virus obtained from contaminated Marek's disease vaccines

Three natural recombinant avian leukosis viruses (ALV; PDRC-1039, PDRC-3246, and PDRC-3249) expressing a subgroup A gp85 envelope protein and containing long terminal repeats (LTR) of endogenous ALV-E viruses were isolated from contaminated commercial Marek's disease vaccines, cloned, and completely sequenced. Their full genomes were analyzed and compared with representative strains of ALV. The proviral DNA of all three isolates displayed 99.3% identity to each other, suggesting a possible common ancestor, even though the contaminating viruses were obtained from three separate vaccine serials produced by two different vaccine manufacturing companies. The contaminating viruses have a genetic organization typical of replication-competent alpharetroviruses. The proviral genomes of PDRC-1039 and PDRC-3246 are 7497 bp long, and the PDRC-3249 is three base pairs shorter because of a deletion of a threonine residue within the gp85 coding region. The LTR, gag, pol, and the transmembrane (TM) region (gp37) of the env gene of all three viruses displayed high identity to endogenous counterpart sequences (>98%). Only the surface (SU) region (gp85) of the env gene displayed high identity with exogenous ALV-A (98.7%). Locus-specific polymerase chain reaction (PCR) analysis for ALV endogenous sequences (ev loci) in the chicken embryo fibroblasts used to produce the original vaccine vials identified the presence of ev-1, ev-2, ev-3, ev-4, and ev-6 in all three vaccines. Homologous recombination most likely took place to involve the SU region of the env gene because the recombinant viruses only differ in this particular region from the consensus ALV-E. These results suggest that the contaminating ALV isolates probably emerged by recombination of ALV-A with endogenous virus sequences before vaccine preparation.     

Protective efficacy of commercial Newcastle disease vaccines against challenge of goose origin virulent Newcastle disease virus in geese

Since 1997, severe outbreaks of Newcastle disease (ND) in geese in many regions throughout China have resulted in high morbidity and mortality, and great economic loss to farmers; however, no licensed, specific vaccine is yet available for this disease in China. In this study, goslings were immunized with different combinations and dosages of several commercial ND vaccines including La Sota vaccine, Mukteswar vaccine, recombinant live vaccine against avian influenza (AI) and ND (rL-H5 strain), and inactivated ND oil-emulsion vaccine (La Sota strain). The protective effects were evaluated based upon the level of antibody response and the degree of protection against the goose-origin virulent NDV strain. The result showed that two doses (i.e., one more than that for chicken) of La Sota vaccine priming, followed by 2-5 doses of Mukteswar vaccine boosting 2-3 weeks later, not only induced higher HI antibody levels, but also conferred longer-lasting protection. This immunization procedure can be recommended for prevention of ND in geese.