Efficacy of a bivalent vaccine against Marek's disease

A bivalent vaccine was prepared by combining inactivated Marek's disease virus and turkey herpesvirus. The efficacy of this vaccine, compared to turkey herpesvirus and inactivated Marek's disease virus separately, was studied in unsexed White Leghorn chicks which were vaccinated at one day old and then challenged at 21 days old with fowl blood infected with virulent Marek's disease virus. The bivalent vaccine appreciably delayed mortality resulting from Marek's disease and elicited the highest protective efficacy as judged on the basis of Marek's disease-specific mortality and percentage occurrence of lesions. The occurrence, extent and severity of gross lymphomas and microscopic lymphoproliferative lesions in various organs of the bivalent vaccinated birds were less than in the other challenged groups. In addition, the level of viraemia remained consistently and significantly lower in the bivalent vaccinated birds.     

Isolation and Identification of a Field Strain of Marek's Disease Virus and Analysis of Major Pathogenic Genes

In a certain area of Shandong province, Marek's disease (MD) occurred in diseased chickens that had been vaccinated by turkey herpesvirus.In order to isolate the virus strain and detect the virus pathogenicity, agar diffusion test, cell culture and indirect immunofluorescence assay (IFA) were used to isolate the Marek's virus from chicken's blood and feather marrow.The isolated strain was adapted to grow in chick embryo fibroblasts (CEF).Genes involved in pathogenesis of MDV, such as meq, pp38 and 132 bp repeat sequence were amplified by PCR.The obtained sequences were compared with that of standard strains published in GenBank by DNAStar software.The results showed that pp38 gene of the SDAU-1 shared homology from 100% with standard virulent sequence.Analysis of 132 bp repeat sequence and meq gene sequences of the viral genome showed that the isolated virus belongs to the highly virulent MDV strains.     

一株鸡马立克氏病病毒的分离鉴定及主要致病基因分析

山东省某地区鸡马立克氏病疫苗免疫鸡群暴发马立克氏病(MD),为分离得到致病毒株,检测其致病性,采用琼脂扩散试验、细胞培养和间接免疫荧光试验(IFA)等方法从发病鸡的血液及羽髓中分离到一株适应鸡胚成纤维细胞(CEF)生长的马立克氏病病毒。采用PCR方法扩增分离毒株的meq、pp38、132bp重复序列等病毒致病相关基因,所得序列用DNAStar软件与GenBank上登录的参考毒株进行比对分析。结果显示,该分离株SDAU-1的pp38基因与标准强毒序列同源性为100%,132bp重复序列的拷贝数及meq基因的变异均符合MDV强毒株的序列特征。     

Effect of aflatoxin B1 on the efficacy of turkey herpesvirus vaccine against Marek's disease

The effect of feeding aflatoxin B1 (AFB1) (0.5 ppm) was studied in young chicks. The frequency and the severity of gross and microscopic lesions of Marek's disease were significantly higher in those birds which had been vaccinated with turkey herpesvirus (HVI) and birds challenged with Marek's disease virus which had been given AFB1 in the feed than in those given normal feed. The protective efficacy of HVT vaccine, as judged on the basis of gross and histopathological lesions, was 86.1 and 77.3 per cent in normally fed birds in comparison to 37.6 and 8 per cent in AFB1 fed birds.     

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.     

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.     

鸡马立克氏病双价疫苗的研究 Ⅰ.马立克氏病SB-1病毒株在鸡胚皮肤细胞中的培养试验

以简便技术制备鸡胚皮肤(CES)细胞,进而进行了鸡马立克氏病(MD)SB-1株病毒适应于CES细胞和致CES细胞病变效应(CPE)的研究.试验表明,在形成CPE的细胞中可检出核内包涵体,MD的SB-1株病毒的CES细胞毒的蚀斑形成单位(pfu)为105.7201-7.2122/m L.本研究为以后用CES细胞增殖SB-1株病毒以代替鸡胚成纤维细胞(CEF)制造疫苗提供了依据.     

Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes.

A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells.     

Efficacy of acyclovir against herpesvirus infection in Quaker parakeets.

We evaluated the efficacy of acyclovir against experimentally induced herpesvirus infection (Pacheco's parrot disease) in Quaker parakeets. Thirty-two of 40 birds were challenge-exposed with 0.1 ml of a suspension of herpesvirus (10(4) median cell culture infective doses [CCID50]) given IM. Treatment with acyclovir was started 24 hours later and was continued for 7 days. The birds were allotted to 5 groups of 8 birds each. There was a considerable difference in mortality between groups 1-5. Of 8 bird in each group, 6 died in group 1 (control), 1 died in group 2 (gavage), 3 died in group 3 (low dose, IM), 4 died in group 4 (high dose, IM), and none died in group 5 (contact controls). There was a significant (P = 0.023) difference in mortality between groups 1 and 2, thus the oral form of acyclovir administered by gavage was the most efficacious therapeutic regimen. Clinical signs and death occurred after discontinuation of acyclovir in groups 2 and 3, whereas the mean time of death for the control group was 6 days after challenge exposure. Herpesvirus was recovered by inoculation of chick embryo cell culture with pooled tissue suspensions from all birds that died. Histologic evidence of herpesvirus infection was found in most birds that died, with the control group having the most severe lesions. Surviving Quaker parakeets were transferred to cages with seronegative Quaker parakeets with no known exposure to herpesvirus. There have been no deaths attributable to herpesvirus infection in a period exceeding 2 years.     

Comparative pathogenesis studies with oncogenic and nononcogenic Marek's disease viruses and turkey herpesvirus.

An apparently nononcogenic Marek's disease virus (SB-1) and turkey herpesvirus could be readily isolated from spleen, bursa of Fabricius, thymus, and peripheral blood lymphocytes of chickens beginning 4 to 6 days after inoculation, but unlike infections with two isolates of oncogenic Marek's disease virus (JM-10 and CU-2), virus replication in these cells was rare, and necrosis in the organs was essentially absent. Splenic enlargement was observed regularly during the first 4 to 11 days after inoculation, and Marek's disease tumor-associated surface antigen was observed on splenic and other lymphocytes in the four viral inoculation groups. Cellular cytotoxicity of splenic lymphocytes was demonstrated in vitro with cultured Marek's disease tumor cells (MSB-1 lymphoblastoid cell line) as the target in a chromium-release assay. The four viral infections induced sensitized lymphocytes.     

Development of a polymerase chain reaction to detect Vietnamese isolates of duck virus enteritis.

A polymerase chain reaction (PCR) method for the detection of duck virus enteritis (DVE) virus in tissues of infected and affected ducks, and in cell culture was developed. This required us to obtain specific nucleotide sequence information as we could not find any specific data about the genome of the virus. We found the assay to be highly effective in detecting the virus under experimental conditions and to be easily transferred to laboratories in Vietnam where it is being used in studies on the epidemiology of the disease. We have applied this simple and rapid diagnostic method to the detection of DVE isolates grown in cell culture and tissues from infected birds. The assay was also able to differentiate DVE from other avian herpesviruses, such as Marek's disease, infectious laryngotracheitis virus and goose herpesvirus.     

Dose-dependent inhibition of virus rescue from lymphocytes latently infected with turkey herpesvirus or Marek's disease virus

The number of plaque-forming units (PFU) of turkey herpesvirus (HVT) isolated per 10(6) latently infected splenic lymphocytes was determined by co-cultivation on permissive monolayer cultures in 35-mm-diameter Petri dishes. Doses of 1 x 10(6) spleen cells or less per culture gave uniform dose-related titers, whereas doses of 8 x 10(6) cells often yielded less than 1-2% of the expected number of PFU. Intermediate doses gave proportionally reduced virus yields. This dose-dependent inhibition was observed with spleen cells from birds within a week after infection and became more marked with time. A similar phenomenon occurred with a non-oncogenic Marek's disease virus (MDV) isolate (SB-1) but not with oncogenic MDV isolates (CU-2, JM-10, GA-5), except in genetically resistant birds. High numbers of uninfected spleen cells mixed with low numbers of HVT-infected cells during assay reduced titers only slightly. Immunosuppression by combined neonatal thymectomy and cyclophosphamide treatment before HVT infection prevented the inhibition, but embryonal bursectomy had no effect.     

Avian influenza adenovirus-vectored in ovo vaccination: target embryo tissues and combination with Marek's disease vaccine

We investigated embryo tissues targeted by replication competent adenovirus (Ad)-free recombinant Ad expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdH5) when injected into 18-day embryonated eggs. We also evaluated the effects of concurrent in ovo vaccination with the experimental AdH5 vaccine and commercially available Marek's disease virus (MDV) vaccine combinations Rispens/turkey herpesvirus (HVT) or HVT/SB-1. Computed tomography indicates that in ovo injection on day 18 of incubation places the solution in the amnion cavity, allantoic cavity, or both. Ad DNA was consistently detected in the chorioallantoic membranes as well as in the embryonic bursa of Fabricius, esophagus, and thymus 3 days postinoculation. H5 expression in these tissues also was detected by immunofluorescence assay. These results indicate possible swallowing of vaccine virus contained in the amnion. In contrast, vaccine localization in the allantoic fluid would have allowed bursal exposure through the cloaca. When the AdH5 vaccine was used in combination with MDV, chickens responding to the AdH5 vaccine had similar AI antibody levels compared with AdH5-only-vaccinated birds. However, combined vaccinated groups showed reduced vaccine coverage to AI, suggesting some level of interference. The combination of AdH5 with MDV Rispens/HVT affected the vaccine coverage to AI more severely. This result suggests that the replication rate of the more aggressive Rispens strain of serotype 1 may have interfered with the Ad-vectored vaccine. Increasing the Ad concentration produced similar AI antibody titers and AI vaccine coverage when applied alone or in combination with the HVT/SB-1 vaccine. Ad DNA was detected in hatched chickens 2 days after hatch but was undetectable on day 9 after hatch. MDV DNA was detected in feather follicles of all vaccinated birds at 12 days of age. Thus, Ad-vector vaccination does not interfere with the efficacy of MDV vaccination by using any of the commonly used vaccine strains.     

Difference between influences of homologous and heterologous maternal antibodies on response to serotype-2 and serotype-3 Marek's disease vaccines

Marek's disease (MD) vaccines representing serotypes 2 (SB-1 strain of MD virus) and 3 (FC-126 strain of turkey herpesvirus) were administered to 1-day-old MD-susceptible chicks that either were free of antibodies or carried maternally derived antibodies against SB-1, FC-126, or a serotype-1 MD virus, CU-2 strain. Homologous antibodies delayed the development of vaccine virus viremias and inhibited vaccinal immunity, as judged by protection against challenge with the virulent JM-10 strain of MD virus 7 days postvaccination. Heterologous antibodies had little effect on vaccine responses. Antibodies were shown to interfere with both cell-associated and cell-free vaccine virus.     

Replication of turkey herpesvirus and Marek's disease virus in chick embryo skin organ culture.

Turkey herpesvirus (HVT) and an attenuated Marek's disease virus (MDV) replicated in organ cultures of chick embryo skin as assessed by immunofluorescence and/or electron microscopy. HVT-specific immunofluorescent antigen was detected in the feather follicle epithelium (FFE) and in the surface layer of the skin epidermis. Electron microscopy of infected explants revealed herpes-type cytopathology. Immature particles of both viruses appeared first in the nucleus. Oval or horseshoe-shaped non-enveloped particles of HVT and enveloped virions of MDV were seen in the cytoplasm of some transitional cells. The difference in the ability of HVT and MDV to form an envelope was believed to account for the difference in their transmissibility in chickens. The results indicated that HVT replicated in the FFE and in the epidermis of the skin. However, attempts to localise the site(s) of MDV replication by electron microscopy were unsuccessful.     

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。     

Isolation of serotype 2 Marek's disease virus from birds belonging to genus Gallus in Japan

Serotype 2 of Marek's disease virus (MDV) was isolated from apparently healthy birds belonging to genus Gallus that had no history of vaccination with MDV or herpesvirus of turkeys (HVT). Buffy-coat cells from these birds were inoculated onto chicken embryo fibroblast (CEF) cultures for primary isolation. Thirteen isolates from one golden pheasant and three white silky fowls, three black silky fowls, three Japanese long crowers, and three Japanese bantams produced herpes-like cytopathic effects (CPE) in the CEF cultures. Using serotype-specific monoclonal antibodies to MDV and HVT, 11 isolates were identified as serotype 2 MDV by indirect fluorescent antibody tests. The other two isolates were complicated with serotypes 1 and 3 of MDV-related viruses. Of 13 isolates, three cloned by the limiting-dilution method were further characterized as serotype 2 MDV biologically, genetically, and serologically. The results showed that the birds of the genus Gallus were naturally infected with serotype 2 MDV. This is the first report ever published about the distribution of serotype 2 MDV among healthy birds of the genus Gallus.     

A simple in vitro differentiation between turkey herpesvirus and Marek's disease virus

The growth and plaque formation by turkey herpesvirus (HVT) amd Marek's disease herpesvirus (MDHV) were examined in QT35 cells, a continuous fibroblast cell line derived from chemically induced tumors of Japanese quail. HVT grew and formed plaques consistently in QT35 cells when inoculated with cell-culture-propagated virus or peripheral mononuclear leukocytes (PML) from chickens that had been inoculated with HVT. Both oncogenic and nononcogenic strains of MDHV, however, failed to grow and induced neither plaques nor cytopathic effects in QT35 cells, whether inoculated with cell-culture-grown virus or heavily infected PML. When PML from chickens infected with both HVT and MDHV were assayed, only HVT plaques had developed, despite the presence in the inocula of high levels of MDHV with less HVT. The QT35 cell line provides a simple in vitro system for differentiating between HVT and MDHV and for selective isolation and identification of HVT from chickens infected with both HVT and MDHV.     

Effect of in ovo vaccine delivery route on herpesvirus of turkeys/SB-1 efficacy and viremia

A study was designed to ascertain the influence of in ovo site of inoculation and embryonic fluid type on the development of Marek's disease (MD) vaccine viremia and efficacy against MD challenge. The experiments were divided into in vitro and in vivo phases. In the in vitro phase, herpesvirus of turkeys/SB-1 vaccine was combined with basal medium eagle (BME) medium (control), amniotic fluid, or allantoic fluid and subsequently titrated on secondary chick embryo fibroblast cultures. There were no significant differences in titer between the virus inoculum carried in BME and the virus inoculum combined with either the allantoic fluid or the amniotic fluid. In the in vivo phase, five routes of inoculation, amniotic, intraembryonic, allantoic, air cell, and subcutaneous at hatch, were compared for generation of protection against virulent MD challenge. Comparisons were made in both specific-pathogen-free and commercial broiler embryos/chicks and, for the amniotic and allantoic routes, injection at either day 17 or day 18 of embryonation. Reisolation of the vaccine virus at day 3 of age was also done for all routes with the exception of the air cell route. Vaccine virus was recovered from all birds tested that were injected in ovo via the amniotic and intraembryonic routes and the subcutaneously at hatch route but was isolated only sporadically from birds inoculated via the allantoic route. Vaccination protective efficacy against virulent MD for all birds vaccinated in ovo via the amniotic or intraembryonic routes and birds vaccinated subcutaneously at hatch was over 90% regardless of day of in ovo injection or bird type. Protective efficacy for vaccines delivered in ovo by either the allantoic or the air cell routes was less than 50% regardless of day of injection or bird type. Therefore, in ovo MD vaccines must be injected either via the amniotic route or the intraembryonic route for optimal performance.     

The incidence of herpesvirus hepatitis in parrots--Pacheco's disease--in Czechoslovakia

A highly fatal disease of parrots kept in zoological garden is described; it manifests itself as abrupt mass death of the birds. Deposits of necrotic changes with the occurrence of intranuclear inclusions of the Cowdry A type were found in the liver of dead parrots. The virus was isolated on chick embryos and tissue cultures of chick embryo cells. The bioassay was performed on Psittacus undulatus. The occurrence of herpesvirus nucleocapsids and complete virions in the infected tissue cultures was detected by means of electron microscopy.