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.     

Trials with Marek's disease vaccines prepared from a turkey herpes virus and an attenuated Marek's disease virus.

In field trials involving over 224,000 fowls in 11 different commercial flocks, three vaccines were used, namely a freeze-dried vaccine prepared from a turkey herpes virus, a cell-associated virus vaccine prepared from the same isolate and a cell-associated vaccine prepared from a strain of Marek's disease virus isolated from a fowl. The mortality from Marek's disease was reduced by 80 per cent to 95 per cent in birds vaccinated with the freeze-dried vaccine. Cell associated vaccines gave slightly less protection.     

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.     

鸡马立克病毒流行强毒株的致弱研究

针对马立克病毒(MDV)毒力逐渐上升的现状,本研究对国内MDV流行强毒株进行了致弱研究。本实验采用近年来从东北、四川2地区免疫发病鸡场中分离的4株MDV流行强毒株(L-SY、L-MS、L-CZ、L-ZY),经噬斑纯化后,采用鸡胚成纤维细胞(CEF)传代培养至75代～85代,获得了4株高代次细胞毒株(L-SYp85C、L-MSp75C、L-CZp75C、L-ZYp75C)。并分析了L-SYp85C和L-MSp75C毒株的体外、体内生长特性和对SPF鸡的致病力。结果表明,L-SYp85C和L-MSp75C株在CEF适应性显著提高;以10倍感染剂量感染的SPF鸡,在12周内均未发生MD肿瘤,体重平均值与对照组体重平均值差异不显著;检测7d～45d羽髓中病毒载量,均低于106copies/106cell,显著低于亲本毒株。同时,4株传代毒株132bpr基因拷贝数显著增加。上述结果为MDV强毒株的致弱研究以及进一步筛选MDV弱毒疫苗株提供了实验依据。     

Influence of vaccination with avirulent herpesvirus on subsequent infection of chickens with virulent Marek's disease herpesvirus.

Vaccination of chickens with turkey herpesvirus (HVT) or attenuated Marek's disease herpesvirus (aMDHV) blocked infection with virulent MDHV (VMDHV) for approximately 5 weeks after contact exposure. However, there was no apparent blockage of infection when challenge virus was administered intraabdominally (IA). Evidence for infection with VMDHV was based on viral isolation by in vivo assay or by detecting precipitins to "A" antigen associated with virulent virus. The HVT stimulated production of neutralizing antibody against VMDHV in a high percentage of chickens, whereas the aMDHV was a comparatively poor inducer of such antibody. Despite this difference, both of the vaccinal viruses conferred protection against development of Marek's disease.     

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.     

Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication,disease, and mortality in chickens

Highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are two of the most important viruses affecting poultry worldwide and produce co-infections especially in areas of the world where both viruses are endemic; but little is known about the interactions between these two viruses. The objective of this study was to determine if co-infection with NDV affects HPAIV replication in chickens. Only infections with virulent NDV strains (mesogenic Pigeon/1984 or velogenic CA/2002), and not a lentogenic NDV strain (LaSota), interfered with the replication of HPAIV A/chicken/Queretaro/14588-19/95 (H5N2) when the H5N2 was given at a high dose (10^6.9 EID₅₀) two days after the NDV inoculation, but despite this interference, mortality was still observed. However, chickens infected with the less virulent mesogenic NDV Pigeon/1984 strain three days prior to being infected with a lower dose (10^5.3–5.5 EID₅₀) of the same or a different HPAIV, A/chicken/Jalisco/CPA-12283-12/2012 (H7N3), had reduced HPAIV replication and increased survival rates. In conclusion, previous infection of chickens with virulent NDV strains can reduce HPAIV replication, and consequently disease and mortality. This interference depends on the titer of the viruses used, the virulence of the NDV, and the timing of the infections. The information obtained from these studies helps to understand the possible interactions and outcomes of infection (disease and virus shedding) when HPAIV and NDV co-infect chickens in the field.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0237-5) contains supplementary material, which is available to authorized users.     

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.     

Acid alpha naphthyl acetate esterase reacting lymphocytes in the peripheral blood of chickens challenged with Marek's disease after vaccination with three different vaccines

The average percentage of acid alpha naphthyl acetate esterase reacting lymphocytes (APARL) was enumerated in the peripheral blood of chickens challenged with Marek's disease after vaccination with either turkey herpesvirus (HVT), inactivated Marek's disease virus (IMDV) or a mixture of the two (bivalent vaccine). A gradual increase in APARL value was noticed in the vaccinated chickens from day 7 to 70 after challenge with a virulent Marek's disease virus. The increase was consistent and significantly higher in bivalent (HVT plus IMDV) than in HVT-vaccinated chickens while the slight increase noticed in IMDV vaccinated-challenged birds was inconsistent.