An enzyme-linked immunosorbent assay (ELISA) for detection of Marek's disease virus-specific antibodies and its application in an experimental vaccine trial

An enzyme-linked immunosorbent assay (ELISA) for the detection of Marek's disease virus (MDV)-specific antibodies was developed. Chicken embryo cells (CEC) or chicken kidney cells (CKC) were infected with MDV vaccine strain CVI988/Rispens, and infected-cell lysates were prepared at day 5 post-infection by freeze-thawing. Uninfected-cell lysates served as negative controls. Sera were used at a 1 : 100 dilution and were added in parallel to wells containing the infected and uninfected cell lysates. The optical densities at 492 nm (OD(492 nm)) were measured after detection of bound chicken antibodies with anti-chicken IgG peroxidase conjugate and colour reactions using o-phenylenediamine (OPD) as a substrate. The best results concerning the signal-to-noise ratio were obtained by using CKC cells rather than CEC for antigen preparation. The OD(492 nm) of plasma or serum samples with infected CKC was <0.02 when samples of unvaccinated and unchallenged maternal antibody-negative white leghorn chickens were tested. Sera and plasma samples of positive control birds exhibited OD(492 nm) of <0.01 when tested with uninfected CKC. The assay was used to monitor a trial that compared experimental BAC DNA vaccines and a commercial vaccine. Sustained seroconversion and antibody titers that were constantly rising until day 84 after vaccination (71 days after challenge) was observed only when chickens did not develop Marek's disease. In contrast, chickens developing the disease mounted marginal and short-lived antibody titers only. We conclude that the developed ELISA may be a valuable tool for the evaluation of the efficacy of MDV vaccination under experimental but possibly also under field conditions.     

Cell-mediated immunity in Marek's disease: cytotoxic responses in resistant and susceptible chickens and relation to disease

Two experiments were conducted to study the cell-mediated cytotoxicity of peripheral blood leukocytes (PBL) from chickens inoculated with Marek's disease virus (MDV) against a Marek's disease-derived lymphoblastoid cell line (MSB-1) and to associate the cytotoxicity with incidence of disease. In experiment I, moderately susceptible random-bred, specific-pathogen-free chickens were inoculated with MDV (group 1), vaccinated with a herpesvirus of turkeys (HVT) and inoculated with MDV (group 2), vaccinated with HVT and inoculated with chicken kidney cells (CKC; group 3), and inoculated with CKC only (group 4). Cytotoxic activity in the PBL was detected initially during the first week after MDV inoculation and periodically throughout the observation period (groups 1, 2, and 3). Throughout the observation period, the magnitude of cytotoxic activity was similar in PBL from groups 1 and 2 chickens. The PBL from both surviving and fatally infected chickens (groups 1 and 2) were similarly cytotoxic when sampled during the first 16 days after MDV inoculation. In experiment II, inbred genetically susceptible (line 7) and resistant (line 6) chickens were used. Cytotoxic activity of PBL of significantly greater magnitude was associated with a lower mortality or incidence of gross lesions (or both) in MDV-inoculated line 6 (group B) and HVT-vaccinated and MDV-inoculated line 7 (group C) chickens compared with activity of PBL from MDV-inoculated line 7 (group A) chickens. The cytotoxic activity of PBL from individual inbred chickens did not correlate with the outcome of the infection.     

Molecular characteristics of Polish field strains of Marek's disease herpesvirus isolated from vaccinated chickens

Background

Twenty-nine Marek''s disease virus (MDV) strains were isolated during a 3 year period (2007-2010) from vaccinated and infected chicken flocks in Poland. These strains had caused severe clinical symptoms and lesions. In spite of proper vaccination with mono- or bivalent vaccines against Marek''s disease (MD), the chickens developed symptoms of MD with paralysis.Because of this we decided to investigate possible changes and mutations in the field strains that could potentially increase their virulence. We supposed that such mutations may have been caused by recombination with retroviruses of poultry - especially reticuloendotheliosis virus (REV).

Methods

In order to detect the possible reasons of recent changes in virulence of MDV strains, polymerase chain reaction (PCR) analyses for meq oncogene and for long-terminal repeat (LTR) region of REV were conducted. The obtained PCR products were sequenced and compared with other MDV and REV strains isolated worldwide and accessible in the GeneBank database.

Results

Sequencing of the meq oncogene showed a 68 basepair insertion and frame shift within 12 of 24 field strains. Interestingly, the analyses also showed 0.78, 0.8, 0.82, 1.6 kb and other random LTR-REV insertions into the MDV genome in 28 of 29 of strains. These genetic inserts were present after passage in chicken embryo kidney cells suggesting LTR integration into a non-functional region of the MDV genome.

Conclusion

The results indicate the presence of a recombination between MDV and REV under field conditions in Polish chicken farms. The genetic changes within the MDV genome may influence the virus replication and its features in vivo. However, there is no evidence that meq alteration and REV insertions are related to the strains'' virulence.     

鸡传染性贫血病毒与马立克氏病病毒共感染SPF鸡群免疫抑制协同作用的研究

将鸡传染性贫血病毒（Chicken infectious anemia virus,CIAV or CAV）和马立克氏病病毒（Marek s dis-ease virus,MDV）人工单一和共同感染1日龄的SPF鸡,感染后分别于14、21、28、35日龄检测鸡体红细胞压积的变化,并检测鸡群疫苗免疫3周后的抗体反应,以探讨CAV与MDV共感染对鸡体的免疫抑制是否有协同作用。结果表明,在血液分析方面,CAV与MDV共感染组较病毒单一感染组与对照组差异极显著,共感染不仅加重了鸡群贫血现象,而且延长了贫血的病理症状;而在禽流感病毒（Avian influenza virus,AIV）H5/H9疫苗、新城疫病毒（Newcastle disease virus,NDV）疫苗和传染性法氏囊病毒（Infectious bursal disease virus,IBDV）疫苗免疫后3周的抗体检测中,CAV与MDV共感染组较其它各实验组差异极显著,抗体滴度大大低于其它实验组;此外,CAV与MDV共感染组,鸡体生长状况明显差于实验各组,有6只鸡只死亡（6/25）,比病毒单一感染时的死亡率大大增加。综上研究证明,CAV与MDV共感染在免疫抑制作用上有协同作用。     

Marek's disease in turkeys. II. Characterization of the viral glycoprotein B gene and antigen of a turkey strain of Marek's disease virus

Marek's disease virus (MDV) causes immunosuppression and tumors in chickens. As sporadic cases of Marek's disease (MD) were recorded in turkeys, the antigenic and genomic characteristics of the MDV glycoprotein B (gB) gene and antigen of turkeys were compared to the chicken MDV gB. The whole chicken and turkey gB genes were sequenced and found identical. By immunoblotting of infected-cell culture lysates using chicken convalescent and gB monoclonal antibodies, the antigenic epitopes of the chicken and turkey viruses were found to differ. The turkey MDV had a unique epitope, compared to the chicken MDV and compared with our previous findings. While the chicken MDV had two epitope types, heat-labile but dithiothreitol (DTT)-stable and heat-stable but DTT-labile, the turkey MDV gB epitope is both heat and DTT-labile.     

The correlation of protective effects and antibody production in immunized chickens with recombinant R7 vaccine against Leucocytozoon caulleryi

Protective effects of recombinant R7 (rR7) vaccine against Leucocytozoon caulleryi in chickens were studied. After injection of oil-adjuvanted rR7 vaccine into chickens, antibody titers against second-generation schizonts (2GS) antigen of L. caulleryi (anti-2GS antibody) rapidly rose in all the immunized chickens, reached to a peak value 2 weeks after injection, and the titers persisted through 4 or 5 months after injection. Chickens having high levels of the anti-2GS antibody titers (> or = 102,400 ) at pre-challenge completely protected against sporozoites challenge of L. caulleryi. After the challenge inoculation, relatively high parasitemia of L. caulleryi was observed in all the inadequately immunized chickens having low levels of the antibody titers (< or = 3,200) at pre-challenge, although some of them seemed to be clinically normal. Correlation of protective effects in the immunized chickens was observed between both prevention of appearance of clinical signs and parasitemia after parasites challenge and anti-2GS antibody titers of the chickens at pre-challenge. The present study shows that chicken leucocytozoonosis can be prevented by vaccination, and humoral immunity may play an important role in the control of chicken leucocytozoonosis.     

Detection of antibodies against serotypes 1 and 2 infectious bursal disease virus by commercial ELISA kits

Two distinct serotypes of infectious bursal disease virus (IBDV) are recognized in chicken and turkey flocks in the United States. Serologic testing of chicken flocks for serotype 1 viruses is routinely performed to monitor disease status and vaccination. Earlier studies indicated that enzyme-linked immunosorbent assay (ELISA) test detects antibodies to both serotypes of the virus, while the virus neutralization (VN) test is serotype specific. It is useful to evaluate currently available commercial ELISA kits for their ability to differentiate between antibodies elicited by the two serotypes. Three trials were performed in which chickens were orally inoculated with either a high or a low dose of serotype 1 STC or serotype 2 OH strains of IBDV. Sera collected at 0, 7, 14, and 21 days from these chickens and antisera procured from naturally infected broiler (n=20) and layer (n=30) flocks were tested with five different commercial ELISA kits and by VN. All ELISA kits detected different levels of antibodies elicited against serotype 1 of the virus and moderate and high levels of antibodies against serotype 2 virus. A correlation existed between the ELISA and the VN titers of experimentally infected chickens. All serum samples tested from the commercial layer flocks and 65% of the broiler flocks had antibodies against the OH strain. However, no correlation between the VN titers and ELISA titers was observed for the commercial broilers and layers sera by the majority of the kits. The results indicated that currently available commercial ELISA kits detect antibodies elicited by the two serotypes of IBDV. Hence, the prevalence of serotype 2 antibodies in the flocks should be considered while determining antibody profiles of the flocks against serotype 1 viruses.     

Distinct expression pattern of miRNAs in Marek's disease virus infected-chicken splenic tumors and non-tumorous spleen tissues

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression. Emerging evidence suggests that differential miRNA expression is associated with viral infection and tumorigenesis. Recently discovered microRNAs in the Marek's disease virus (MDV) genome have been suggested to have regulatory roles during MDV oncogenesis. To gain more insight into the molecular mechanisms of the tumorigenesis of MDV, we used microarrays to screen host and viral miRNAs that were sensitive to infection by MDV. Microarray analysis showed significant differential expression of 79 miRNAs, which was confirmed by qRT-PCR analysis. These data suggest that differentially expressed miRNAs may have major roles in MDV-induced tumorigenesis. In addition, we found two clades of chicken miRNAs had increased expression in splenic tumors and non-tumorous spleen tissues from GA-infected chickens. Thus, the expression of these miRNAs can be considered signatures for MDV infection and tumorigenesis.     

Pathogenesis of Marek's disease virus-induced local lesions. 1. Lesion characterization and cell line establishment

Subcutaneous (wing-web) or intramuscular inoculation of chickens with allogeneic normal or Marek's disease virus (MDV)-infected chicken kidney cells induced local lesions visible by 3-4 days postinoculation (PI). Lesions were slightly larger (P less than 0.05) in infected than uninfected chickens 5 and 8 days PI. They persisted and grew past 9 days PI only when infected. Infiltrating lymphocytes in infected and uninfected early lesions were similar; they included B-cells and also T-cells with and without Ia antigen. Up to 42% of lymphocytes from infected or uninfected lesions had the surface antigen MATSA. At 3 to 6 days PI, infected lesions contained lymphocytes with viral internal antigen, especially in Ia-bearing cells and MATSA-bearing cells, but thereafter infection was latent. Cells harvested daily from local lesions induced with allogeneic MDV-infected cells were cultured; MD tumor cell lines were established from lesions as early as 4 days PI, with a total success rate of about 50% thereafter. Either transformed tumor cells were already present during the early cytolytic infection period or else appropriate target cells were present that became infected in vivo and/or in vitro and then became transformed in vitro.     

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.     

Detection of specific antibodies against deflagellated Salmonella Enteritidis and S. Enteritidis FliC-specific 9kDa polypeptide

Specific antibody levels of laying hens and young chickens experimentally infected with Salmonella Enteritidis and vaccinated farm flocks were evaluated by enzyme-linked immunosorbent assays (ELISAs) with two different antigens, deflagellated S. Enteritidis whole cell (DEWC) and S. Enteritidis FliC-specific 9kDa polypeptide (SEP9). Infected laying hens excreted S. Enteritidis throughout the experimental period, and the specific antibody titers in DEWC-ELISA, were significantly higher than the uninfected group. It suggests that this DEWC-specific antibody will serve as an effective indicator of S. Enteritidis infection, especially for non-vaccinated laying flocks. SEP9-specific antibodies were detected in spray-inoculated young chickens but not in oral-inoculated young chickens. Compared with greatly high SEP9-specific antibody levels of vaccinated farm flocks, no response was observed in orally infected hens. These results indicate that S. Enteritidis discontinues expressing SEP9 once it has crossed the intestinal barrier, and that SEP9-ELISA will serve as a valuable monitoring tool for the status of S. Enteritidis vaccination on a flockwide basis, independent of stable S. Enteritidis infections.     

Effects of chemically or virus-induced immunodepression on response of chickens to avian leukosis virus

The effects of chemically or virus-induced immunodepression on the infection profile (development of viremia and antibody) and shedding of avian leukosis virus (ALV) were studied in progeny chickens of experimental or commercial breeder flocks. Chickens were infected with ALV subgroup A by contact at hatching and by oral inoculation at 4-5 weeks of age. In the first experiment, chickens were inoculated with a virulent strain of infectious bursal disease virus (IBDV) at 1 day or 6 weeks of age. In the second experiment, chickens were neonatally treated with cyclophosphamide (CY), or were inoculated with strain T of reticuloendotheliosis virus (REV) at hatching, or were inoculated with strain JM of Marek's disease virus (MDV) at 2 weeks of age. The infection profile and cloacal shedding of ALV in chickens exposed to ALV and inoculated with immunodepressive viruses or CY were compared with those in hatchmates exposed only to ALV. In two of four chicken lines tested in the first experiment, shedding of ALV, as determined by virological assays of cloacal swabs at 22 weeks of age, was significantly higher in chickens infected with IBDV at 1 day of age than in uninfected hatchmates. The rate of shedding of ALV in one of these two lines was also significantly higher in chickens infected with IBDV at 6 weeks of age than in uninfected chickens. Further, the frequency of ALV-antibody detection at 22 weeks of age was significantly lower in chickens of these two lines infected with IBDV at 1 day of age than in uninfected chickens. In the second experiment, neonatal treatment with CY significantly increased the frequency of viremic chickens of both experimental and commercial flocks. The frequency of ALV-viremic chickens at 22 weeks of age was considerably higher in the REV- and MDV-inoculated groups (54% and 44%, respectively) than in control hatchmates (29%), but only in chickens of the commercial line. These findings suggest that chemically or virus-induced immunodepression may lead to an increase in rates of viremia and shedding of ALV in chickens infected with virus after hatching, especially in certain genetic lines.     

CAV与REV共感染SPF鸡对疫苗免疫反应的抑制作用

用1日龄SPF鸡人工感染鸡贫血病毒(CAV)和禽网状内皮增生病病毒(REV),探讨病毒感染对鸡体疫苗免疫反应的影响。结果表明,在用禽流感病毒(AIV,H5和H9)疫苗免疫后,CAV与REV单独感染均显著抑制了鸡体对H5和H9亚型禽流感病毒灭活疫苗的HI抗体反应,在CAV与REV共感染后,这种抑制作用更为明显。CAV单独感染后鸡体对新城疫病毒(NDV)和传染性法氏囊病病毒(IBDV)疫苗的免疫反应受到抑制,但与对照组在统计学上的差异不显著,然而,CAV可以显著加重REV感染对鸡体在NDV和IBDV疫苗免疫后抗体反应的抑制作用。从而证实CAV与REV共感染在疫苗免疫抑制上有协同作用。     

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.     

抗体滴度可作为鸡毒支原体药物疗效的评价指标

以泰乐菌素、硫氰酸红霉素、克拉霉素、强力霉素作为供试药物,通过鸡毒支原体人工感染鸡群的治疗试验,对其药效评价方法进行比较。结果显示,对鸡毒支原体敏感性高的泰乐菌素治疗组鸡群的抗体阳性率,特别是抗体滴度明显比低敏的强力霉素组低,且抗体滴度的高低与鸡群炎症变化率呈正相关。数据表明,抗体滴度可作为鸡毒支原体药物疗效的又一种评价指标。     

DNA Immunization Against Very Virulent Infectious Bursal Disease Virus with VP2‐4‐3 Gene and Chicken IL‐6 Gene

The present study was to investigate the feasibility and efficiency of the DNA vaccine to protect chickens against very virulent infectious bursal disease virus (vvIBDV) infection. A plasmid DNA carrying VP2‐4‐3 genes of vvIBDV SH95 and a plasmid DNA carrying chicken interleukin‐6 (ChIL‐6) genes were constructed and designated as pALTER‐MAX‐VP2‐4‐3 and pALTER‐MAX‐ChIL‐6 respectively. Several DNA vaccination experiments were performed: 1‐week‐old chickens were intramuscularly injected with only plasmid pcDNA3‐VP2, pALTER‐MAX‐VP2‐4‐3 or mixture with pALTER‐MAX‐ChIL‐6. The chickens at 4 weeks old were orally inoculated with vvIBDV SH95. The results showed that immunization with the mixture of pALTER‐MAX‐VP2‐4‐3 and pALTER‐MAX‐ChIL‐6 three times conferred protection for 90% of chickens. Enzyme‐linked immunosorbent assay (ELISA) antibody titres in chickens immunized together with pALTER‐MAX‐ChIL‐6 were higher than those immunized simply with plasmid pcDNA3‐VP2 or pALTER‐MAX‐VP2‐4‐3. IBDV was not detected in the bursa of the protected chickens at 8 days after challenge by RT‐PCR. The results indicate that protection against vvIBDV can be achieved by using the VP2‐4‐3 gene of vvIBDV as a DNA vaccine. Furthermore, the simultaneous injection of ChIL‐6 plasmid significantly increased the protection after challenge with the very virulent strain.     

Influence of in ovo administered Cryptosporidium baileyi oocyst extract on the course of homologous infection

In order to evaluate the effect of in ovo vaccination on avian cryptosporidiosis, two doses (1 and 10microg) of Cryptosporidium baileyi oocyst extract (OE) were injected into the amnionic sac of embryonated, specific pathogen-free chicken eggs. After hatching these birds as well as infected controls (IC) were inoculated with 8x10(5) C. baileyi oocysts at 10 days of age. Another group of chickens remained uninfected (UC). Faecal oocyst shedding was measured every second day, and weekly ELISAs were performed to monitor seroconversion. Those chickens that received OE during embryogenesis showed dose-dependent shift in their oocyst shedding, with higher oocyst output of OE1 and OE10 birds compared to IC ones. The patency was significantly longer in the OE10 group than in IC or OE1. ELISA results showed low seroconversion of OE1 and OE10 chickens prior to homologous challenge. Challenge infection resulted in antibody levels without significant difference between IC, OE1 and OE10 groups. These data suggest that in ovo vaccination with C. baileyi oocyst extract does not promote immune response, moreover, it may impair immunity and thus delay the clearance of cryptosporidia from chickens.