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1.
Molecular characterization of three recombinant isolates of avian leukosis virus obtained from contaminated Marek's disease vaccines 总被引：2，自引：0，他引：2
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. 相似文献
2.
An outbreak of subcutaneous sarcomas in commercial White Leghorn egg layers was observed in the northeastern United States during late 2004. Subcutaneous tumors were confined to three flocks distributed in two locations and belonging to the same company. The tumors were first observed grossly by farm personnel at approximately 7 wk of age and persisted throughout the economic life of the flocks. Most of the tumors observed during the growing period were present on the facial region or around the head, wings, and legs. There was no gross evidence of bursal or visceral involvement. Microscopically, most tumors were undifferentiated sarcomas and myxomas. There was no microscopic evidence of Marek's disease or lymphoid leukosis. Reticuloendotheliosis virus proviral DNA was not detected by polymerase chain reaction either in tumors or in cell cultures. Egg production and mortality rates were within normal limits in the affected flocks and many of the chickens exhibiting tumors seemed healthy otherwise, albeit approximately one-half of the daily mortality exhibited tumors. Avian myeloblastosis-associated virus type 1 (MAV-1) was isolated from tumors, plasma, and serum. Upon initial virus neutralization, the viruses isolated seemed at least partially related antigenically to avian leukosis virus (ALV) subgroups A and B but not to subgroup J (ALV-J). Sequencing of the variable and hypervariable regions of gp85 in the envelope gene revealed that the viruses involved are closely related to MAV-1. Attempts to reproduce subcutaneous sarcomas with MAV-1 isolated from White Leghorn chickens in the case herein reported produced exclusively myelocytomas indistinguishable histologically from those induced by ALV-J in meat type chickens. 相似文献
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Formalin-fixed suspect tumors were submitted to the Poultry Diagnostic and Research Center at the University of Georgia (Athens, GA) for diagnosis. Samples were from fancy breed chickens with a history of increased tumor prevalence in both hens and roosters. Microscopically, in all the samples, there were neoplastic proliferations of spindle-shaped cells. The matrix surrounding tumor cells stained positively with Alcian blue at pH 2.5, but neoplastic cells did not stain with periodic acid-Schiff. Immunohistochemistry stains were positive for vimentin and neuron-specific enolase and negative for desmin, smooth muscle actin, and S-100 protein. Tumors were determined to be myxosarcomas. All samples were positive for PCR targeting the gp85 avian leukosis virus (ALV) envelope protein. However, analysis of the predicted amino acid sequences in the envelope gene from three separate samples showed high similarity between them and to ALV subgroup A. 相似文献
5.
Development and characterization of monoclonal antibodies to subgroup J avian leukosis virus. 总被引：6，自引：0，他引：6
In an attempt to develop a specific diagnostic test for avian leukosis virus (ALV) subgroup J (ALV-J) strain Hc1, four monoclonal antibodies (MAbs), JE9, G2, 145, and J47, were generated that are specific for ALV-J envelope glycoprotein, gp85. Polymerase chain reaction (PCR) was used to amplify genomic pro-viral DNA of Avian Disease and Oncology Laboratory (ADOL)-Hc1 and ADOL-4817 envelope genes. Both open reading frames encoding glycoproteins gp85 and gp37 were cloned into baculoviruses. Abundant expression of gp85 and gp37 was detected in the recombinant viruses with specific antibody to Hc1 strain of the ALV-J. The expressed proteins were used for immunization of mice to produce hybridoma cell lines secreting MAbs specific to ALV-J envelope protein. A panel of MAbs was generated by fusing NS1 myeloma cells and spleen cells from mice immunized with the recombinant baculoviruses. With the use of an immunofluorescence assay, three MAbs (JE9, G2, 145) reacted with ALV-J but not with subgroups A, B, C, D, or E of ALV. MAb J47 reacted with all exogenous subgroups of ALV including A, B, C, D, and J but not with endogenous subgroup E viruses. Western blot analysis was performed with all four MAbs against recombinant baculovirus and Hc1-infected chicken embryo fibroblast (CEF) lysates. A major band with a molecular weight about 90 kD corresponding to the size of ALV-J envelope was consistently obtained. With these MAbs, we detected the Hc1 antigen in CEFs infected with several ALV-J viruses isolated in the United States and also in tissue sections from chickens infected with Hc1 strain of ALV-J. These MAbs will be useful reagents for the diagnosis of ALV-J infection because they recognize a common antigenic epitope in six isolates tested thus far. 相似文献
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Avian leukosis virus subgroup A (ALV‐A) is a retrovirus which infects egg‐type chickens and is the main pathogen of lymphoid leukosis (LL) and myeloid leukosis (ML). In order to greatly enhance the diagnosis and treatment of clinical avian leukemia, two monoclonal antibodies (MAbs) to ALV‐A were developed by fusion between SP2/0 and spleen cells from mice immunized with expressed ALV‐A env‐gp85 protein. Using immunofluorescence assay (IFA), two MAbs reacted with ALV‐A, but not with subgroups B and J of ALV. Western blot tests showed that molecular weight of ALV‐A envelope glycoprotein recognized by MAbs was about 53 kD. Isotyping test revealed that two MAbs (A5C1 and A4C8) were IgG1 isotypes. These MAbs can be used for diagnosis and epidemiology of ALV‐A. 相似文献
7.
Isolation and characterization of an adventitious avian leukosis virus isolated from commercial Marek's disease vaccines 总被引：1，自引：0，他引：1
Commercial Marek's disease (MD) vaccines produced by two manufacturers were tested for possible contamination with avian leukosis virus (ALV). Samples of MD vaccines manufactured by two companies (A and B) were received from a breeder company; samples were also received directly from vaccine company B. Using virus isolation tests, samples initially tested positive for subgroup E (endogenous) ALV. However, upon repassage, the vaccines also tested positive for exogenous ALV. The isolated exogenous ALV proved to be a subgroup A virus, as determined by flow cytometry using polyclonal chicken antibodies specific for various subgroups of ALV, and by DNA sequencing of the envelope glygoprotein (gp85). The exogenous ALV isolated from MD vaccines was inoculated in chickens from ADOL lines 15I(5) x 7(1) and 0 to determine its pathogenicity and compare it with that of Rous-associated-virus-1 (RAV-1), the prototype strain of ALV-A. Each chicken from each line was inoculated with approximately 10,000 infectious units of RAV-1 or the ALV-A isolated from vaccines termed B-39 virus at 7th day of embryonation. At hatch, and at 4, 8, and 16 wk of age, chickens were tested for viremia and cloacal shedding; chickens were also observed for ALV-induced tumors within 16 wk of age. Viremia and cloacal shedding results suggest that chickens from both lines were susceptible to infection with either virus. Within 16 wk of age, the proportion of ALV tumors induced by strain B-39 in line 0 and line 15I5 x 7(1) chickens was 0% and 12%, respectively, compared with 62% and 67% in chickens inoculated with RAV-1. The data indicate that commercial MD vaccines produced by two manufacturers were contaminated with endogenous subgroup E and an exogenous subgroup A ALV. Further, data from biological characterization suggest that the ALV-A isolated from commercial MD vaccines is of low oncogenicity, compared with that of RAV-1. GenBank accession numbers: The gp85 gene sequences of ALV isolated from commercial Marek's disease vaccines have been deposited in GenBank and assigned the following accession numbers: A46 subgroup A, DQ412726 ; B53 subgroup A, DQ412727; A46 subgroup E, DQ412728; B53 subgroup E, DQ412729. 相似文献
8.
Avian leukosis viruses (ALVs) are common in many poultry flocks and can be detected using an enzyme-linked immunosorbent assay or any other test designed to identify p27, the group-specific antigen located in gag. However, endogenous retroviruses expressing p27 are often present and can be confused with exogenous ALVs. A more specific and informative assay involves targeting the variable envelope glycoprotein gene (gp85) that is the basis for dividing ALVs into their different subgroups. We designed polymerase chain reaction (PCR) primers that would specifically detect and amplify viruses from each of the six ALV subgroups: A, B, C, D, E, and J. Subgroup B and D envelopes are related, and our B-specific primers also amplified subgroup D viruses. We also designed a set of common primers to amplify any ALV subgroup virus. To demonstrate the usefulness of these primers, we obtained from the Center for Veterinary Biologics in Iowa culture supernatant from chicken embryo fibroblasts infected with an ALV that was found to be a contaminant in two commercial Marek's disease vaccines. Using our PCR primers, we demonstrate that the contaminant was a subgroup A ALV. We cloned and sequenced a portion of the envelope gene and confirmed that the ALV was a subgroup A virus. Unlike typical subgroup A viruses, the contaminant ALV grew very slowly in cell culture. We also cloned and sequenced a portion of the long terminal repeat (LTR) from the contaminant virus. The LTR was found to be similar to those LTRs found in endogenous ALVs (subgroup E) and very dissimilar to LTRs normally found in subgroup A viruses. The E-like LTR probably explains why the contaminant grew so poorly in cell culture. 相似文献
9.
Bovine leukaemia proviral DNA detection in cattle using the polymerase chain reaction 总被引：3，自引：0，他引：3
Bovine leukaemia virus (BLV) is the causative agent in enzootic bovine leukosis a disease occurring worldwide. This virus is normally detected by the agar gel immunodiffusion or ELISA assays which rely on the appearance of antibodies to a major surface protein of the virus, gp51, present in the serum of infected cattle. We have used the polymerase chain reaction, which depends on the amplification of specific DNA sequences as a sensitive assay for the detection of BLV. It was possible to detect proviral DNA in 100 pg of tumour DNA from an infected host using agarose gel electrophoresis followed by ethidium bromide staining. The sensitivity of the assay was increased by two log orders when hybridization analysis, using a BLV proviral DNA probe, was used in combination with amplification of the DNA. Proviral DNA was detected in both lymphocytic and tumour DNA and at all stages of infection in cattle. 相似文献
10.
禽白血病病毒囊膜基因gp85片段的克隆与鉴定 总被引：11，自引：0，他引：11
用ＰＣＲ从禽白血病病毒（Avian Leukosis Virus,ALV)RAV-1,RAV-2感染或未感染的ＳＰＦ鸡胚成纤维细胞（ＣＥＦ）分别扩增出１．２kb囊膜基因片段，分别将上述ＰＣＲ产物的KpnⅠ／SaⅡ酶切片段克隆到质粒pGEM-3zf( )中得到３个重组子即pGEM-3zf-RAV-1env、pGEM-3zf-RAV-2env和pGEM-3zf-Eenv。酶切分析和序列测定结果表明克隆的ＰＣＲ片段分别来自ＡＬＶ ＲＡＶ－１，ＲＡＶ－２和内源生Ｅ亚群病毒，这为病毒囊膜基因gp85的表达及个体鸡遗传抗性的鉴定打下基础。 相似文献
11.
Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica 总被引：11，自引：1，他引：11 
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下载免费PDF全文 Qiang Liu Li Wang Philip Willson Brendan O'Connor Julia Keenliside Manuel Chirino-Trejo Ronald Melndez Lorne Babiuk 《Canadian journal of veterinary research》2002,66(4):225-231
Porcine circovirus (PCV) was recently divided into 2 antigenically distinct types that differ (65% amino acid identity) in the protein encoded by open reading frame 2 (ORF2). Porcine circovirus 1 is apparently non-pathogenic and, in contrast, PCV2 is associated with porcine multisystemic wasting syndrome (PMWS). Our objective was to determine the extent of exposure of normal pigs in Canada and Costa Rica to PCV2. Recombinant DNA techniques were used to produce an antigen from ORF2 of PCV2 that was suitable for the detection of antibody in swine sera. The presence of PCV2 nucleotide sequences was detected using polymerase chain reaction (PCR) techniques. Using these tests, specific antibody and nucleotide sequences were demonstrated in sera from a cohort of pigs during a PMWS outbreak. Antibody was detected in normal, healthy hogs slaughtered in Canada (82.4% of 386) and in Costa Rica (14.6% of 322). This is the first report indicating the presence of PCV2 in Latin America. More than 50% of these sera also contained PCV2 nucleotide sequence. Although these hogs were healthy when slaughtered, they were infected with PCV2 and may have previously been ill. The widespread occurrence of PCV2 in swine suggests that this virus is adapted to replication in porcine tissue. 相似文献
12.
V. L. Sanger T. N. Frederickson C. C. Morrill 《Canadian journal of veterinary research》1964,28(10):236-238
Day-old chicks were injected with 0.2 ml. of a suspension of lymphomatosis virus which was known to cause osteopetrosis in a high percentage of susceptible birds. A comparable number of uninoculated chicks were kept for controls. Alizarin red S was administered for the purpose of marking the bones in order to measure the rate of growth of normal bone and the osteopetrotic bone. The dye was given at 27, 41 and 55 days of age. In this experiment it was found that a layer of normal bone was formed on the surface of the tibia at a rate of 0.25 mm. per week but in the largest osteopetrotic lesion that was found in any chicken spongy bone was formed at a rate greater than 1 mm. per week.
Alizarin red S was irritating to tissue and was toxic when given intravenously.
相似文献13.
In Experiment 1, a monoclonal antibody against the envelope glycoprotein (gp85) of subgroup J avian leukosis virus (ALV-J) was used to study the distribution of ALV-J in various tissues of White Leghorn chickens inoculated as embryos with the strain ADOL-Hcl of ALV-J. At 2 and 6 wk of age, various tissues from infected and control uninfected chickens were tested for the presence of ALV-J gp85 by immunohistochemistry. In Experiment 2, using the methyl green-pyronine (MGP) stain, sections of bursa of Fabricius (BF) from chickens of line 15I5 x 7(1), inoculated with ALV-J or Rous-associated virus-1 (RAV-1), a subgroup A ALV, at hatch were examined for transformation of bursal follicles at 4 and 10 wk of age. In Experiment 1, specific staining indicative of the presence of ALV-J gp85 was noted at both 2 and 6 wk of age in the adrenal gland, bursa, gonads, heart, kidney, liver, bone marrow, nerve, pancreas, proventriculus, spleen, and thymus. In Experiment 2, by 10 wk of age, transformed bursal follicles were detected in MGP-stained sections of BF in only one of five (20%) chickens inoculated with ALV-J at hatch, compared with five of five (100%) chickens inoculated with RAV-1. The data demonstrate distribution of ALV-J gp85 in various tissues of White Leghorn chickens experimentally inoculated as embryos with the virus. The data also confirm our previous observation that ALV-J is capable of inducing transformation of bursal follicles, albeit the incidence is less frequent than that induced by subgroup A ALV. 相似文献
14.
Clifford R. Berry DVM John K. House DVM Paul P. Poulos DVM PhD John E. Madigan DVM MS J. Carroll Woodard DVM PhD Roy R. Pool DVM PhD Timothy R. O'Brien DVM PhD Norman Ackerman DVM 《Veterinary radiology & ultrasound》1994,35(5):355-361
The radiographic and pathologic findings of two Peruvian Paso foals with osteopetrosis are described. Both foals, one male and one female, presented with respiratory difficulty, brachygnathia and failure to rise after birth. Both foals were mildly anemic, hypogammaglobulinemic and had elevations in serum alkaline phosphatase. Increased medullary bone opacity was noted on radiographs of the extremities, spine and skull in both foals. A lack of normal cortical:medullary bone distinction was evident radiographically. The medullary primary spongiosa appeared to run in parallel columns away from the physes of all long bones and the vertebrae. This created a distinctive hour glass appearance to the osteopetrotic bones. One foal developed a bacterial pneumonia. Both foals were euthanized due to failure to thrive. Histopathology and electron microscopy documented these foals to have normal osteoclastic numbers but lack normal ruffled borders, lack of a clear zone and normal lysosomal numbers indicative of cellular dysfunction. These clinical, radiographic and pathologic findings are similar to the juvenile, lethal autosomal recessive form of osteopetrosis described in humans. Osteopetrosis has not been previously described in a female foal. 相似文献
15.
Infection of broiler chickens with subgroup J avian leukosis virus (ALV) results in the induction of myeloid tumors. However, although egg-type chickens are susceptible to infection with ALV-J, the tumor incidence is very low, and on rare occasions the tumors observed are of the myeloid lineage. We recently described the isolation of an ALV (AF115-4) from commercial egg-type chickens suffering from myeloid leukosis. AF115-4 was initially identified as an ALV-J isolate based on PCR analysis of the long terminal repeat (LTR). However, further characterization of the viral envelope indicated that the virus is recombinant with subgroups B envelope and J LTR. Here we further characterize this recombinant virus at both the molecular and biological levels. We show that the AF115-4 isolate expresses a recombinant envelope glycoprotein encoded by a subgroup B gp85 region and a subgroup E gp37 region. The host range ofAF115-4 was analyzed using cells resistant to infection by subgroups A/B, J, or E; this shows that no ALV-J was present in the isolates obtained from the affected chickens. Additional antigenic characterization of AF115-4 using chicken sera specific for subgroups B or J indicated that no ALV-J was present in the samples examined. Inoculation of AF 115-4 into ALV-susceptible 1515 X 71 chickens resulted in the induction of lymphoid leukosis but not the expected myeloid leukosis affecting the commercial chickens. These results suggest that differences in the genetic makeup of the chickens from which AF115-4 was isolated and the line 1515 X 71 used in the present experiments may be responsible for the observed differences in pathogenicity. In addition, the results suggest that ALV-J continues to evolve by recombination, generating new viruses with different pathological properties. 相似文献
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《Preventive veterinary medicine》1988,6(1):1-8
The temporal distribution of vesicular stomatitis in cattle in Costa Rica, from 1972 to 1986, was analysed using the time series analysis. Seasonal fluctuations were observed with the highest occurrence in the dry season. The possible existence of 3–4-year cycles was demonstrated. 相似文献
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A study was conducted in 2008 to determine the prevalence of Anaplasma and Babesia infections in cattle in the Puntarenas Province of Costa Rica. Blood samples were taken from a total of 449 cattle during the month of March at 30 farms in the region of Espiritu Santu, Costa Rica. Commercially available enzyme-linked immunosorbent assays (ELISA) were used to determine presence of antibodies to Babesia bigemina and Anaplasma marginale, and real-time PCR was used to determine the presence of DNA from the disease-causing organisms. The ELISA results indicated that 87.5% of the cattle sampled were positive for antibodies to A. marginale, while 59.1% were positive for antibodies to B. bigemina. The real-time PCR results showed that 235 cattle were carrying A. marginale DNA (56.9%), 6 with B. bigemina DNA (1.34%), and 2 with B. bovis DNA (0.45%). 相似文献
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Detection and localization of naturally transmitted avian leukosis subgroup J virus in egg-type chickens by in situ PCR hybridization 总被引：1，自引：0，他引：1
Li N Xu B Dong W Qiao S Lee LF Zhang HM Li M Du N 《Journal of veterinary medicine. A, Physiology, pathology, clinical medicine》2007,54(10):553-558
Avian leukosis virus (ALV) subgroup J (ALV-J) is an exogenous ALV and causes myeloid leukosis in meat-type chickens. We have previously reported the isolation and identification of ALV-J in commercial layer flocks from 12 farms in northern China. In this report, we further characterized this virus by in situ polymerase chain reaction (PCR) hybridization in various affected organs of chickens from six of the 12 farms. A routine method for hybridization of nucleic acid uses radioactive probe, such as a P32-labelled probe. We found that the non-radioactive digoxigenin (DIG) probe is sensitive enough to detect the nucleic acid of virus in chicken tissues. We used a pair of published primers (H5/H7) specific to the gp85 envelope gene and 3' region of pol gene of prototype ALV-J strain HPRS-103. The total RNA extracted from tumour, bone marrow, oviduct, liver and spleen of the diseased chickens from six commercial flocks, and cDNA was successfully amplified. Using the primers and cDNA, we obtained an ALV-J-specific cDNA probe of 545 bp in length by PCR. In situ PCR with H5/H7 primers was carried out in the paraffin sections from tissues of the diseased chickens, followed by in situ hybridization using the DIG-labelled cDNA probe. Positive hybridization signals were detected in the cytoplasm of paraffin sections of tumours and other organ tissues. The intensity of the signals was documented using an image analysis system measuring integral optical density (IOD). The IOD values for tissue sections treated by in situ PCR hybridization are significantly higher than that by in situ hybridization alone (P < 0.01). These data taken together suggest that in situ PCR hybridization is a more sensitive technique for detection of ALV-J in tissue sections. 相似文献
20.
The early diagnosis of bovine leukosis virus (BLV) infection, the aetiological agent in enzootic bovine leukosis, is important for the implementation of control measures. BLV infection is currently assessed by the detection of circulating antibodies against the viral envelope protein, gp51. However, this approach has shortcomings in the time taken to detect anti-BLV antibodies (three to four weeks after infection), and in the failure to detect antibodies in some animals. Clearly a technique such as the polymerase chain reaction (PCR), which directly detects the presence of viral DNA, has advantages over methods designed to measure host antibodies. The use of PCR for the detection of proviral DNA in an affected DNA sample with as little as 10(-5) micrograms of host DNA using agarose gel electrophoresis followed by ethidium bromide staining is described here. It was possible to improve the sensitivity of this assay by using hybridisation analysis with a BLV gene probe. PCR used in combination with hybridisation analysis will provide a sensitive diagnostic assay to detect BLV when antibody tests give weakly positive or equivocal results. 相似文献