Detection Of Avian Leukosis Virus Research Articles

Rapid and sensitive visual detection of avian leukosis virus by reverse transcription loop-mediated isothermal amplification combined with a lateral flow immunochromatographic strip assay.

Considering that avian leukosis virus (ALV) infection has inflicted massive economic losses on the poultry breeding industry in most countries, its early diagnosis remains an important measure for timely treatment and control of the disease, for which a rapid and sensitive point-of-care test is required. We established a user-friendly, economical, and rapid visualization method for ALV amplification products based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with an immunochromatographic strip in a lateral flow device (LFD). Using the ALVp27 gene as the target, five RT-LAMP primers and one fluorescein-isothiocyanate-labeled probe were designed. After 60 min of RT-LAMP amplification at 64 °C, the products could be visualized directly using the LFD. The detection limit of this assay for ALV detection was 102 RNA copies/μL, and the sensitivity was 100 times that of reverse transcription polymerase chain reaction (RT-PCR), showing high specificity and sensitivity. To verify the clinical practicality of this assay for detecting ALV, the gold standard RT-PCR method was used for comparison, and consistent results were obtained with both assays. Thus, the assay described here can be used for rapid detection of ALV in resource-limited environments.

Rapid detection of avian leukosis virus using a fluorescent microsphere immunochromatographic test strip assay

We developed a rapid fluorescent microsphere immunochromatographic test strip (FM-ICTS) assay for the quantitative detection of avian leukosis virus (ALV). A monoclonal antibody specific for the ALV major capsid protein encoded by the gag gene was coupled to label fluorescent microspheres. ALV antibodies were coated on a nitrocellulose membrane to prepare a test line for sample detection. The fluorescence signals of the test and control lines can be read either visually by exposure to UV light or using a fluorescence analyzer. ALV could be detected quantitatively using the ratio of fluorescence signals of the test and control lines (T/C). The assay threshold was determined as a T/C value of 0.0606. The fitting curve equation was established between 1 and 2,048 ng/mL P27 protein with an r2 value of 0.9998. The assay showed no cross reactivity with Newcastle disease virus, infectious laryngotracheitis virus, infectious bronchitis virus, Marek's disease virus, infectious bursal disease, Reoviridae virus, or avian influenza virus. The repeatability was satisfactory with an overall average CV of 8.65%. The Kappa coefficient between a commercial ELISA kit was 0.7031 using clinical chicken meconium samples. Thus, a simple, rapid, sensitive, and specific fluorescent microsphere immunochromatographic test strip was developed based on specific anti-capsid protein p27 monoclonal antibodies.

Development and application of a colloidal gold test strip for detection of avian leukosis virus.

Avian leukosis virus (ALV) is an avian oncogenic retrovirus that induces leukemia-like proliferative diseases in chickens. ALV infection can result in the development of immunological tolerance and persistent viremia. Since effective vaccines against ALV are not yet available, its current prevention primarily depends on detection and eradication to establish exogenous ALV-free poultry flocks. In this study, a rapid and simple colloidal gold test strip method, specific for the group-specific antigen, p27 protein, was developed and systematically evaluated for the detection of ALV from different samples. The detection limit of this assay was as low as 6.25ng/ml for p27 protein and 80 TCID50/ml for different subgroups of ALV. Besides, the test strip showed high specificity in the detection of different subgroups of ALV, including ALV-A, ALV-B, ALV-J, and ALV-K, with no cross-reaction with other avian pathogens. Furthermore, we artificially infected specific pathogen-free (SPF) chickens with ALV-J, collected cloacal swabs, and examined viral shedding using both test strips and ELISA. Results from the test strip were highly consistent with that from ELISA. In addition, 1104 virus isolates from anti-coagulant blood samples, 645 albumen samples, and 4312 meconium samples were tested, and the test strip results agreed with those of ELISA kit up to 97.1%. All the results indicated that the colloidal gold test strip could serve as a simple, rapid, sensitive, and specific diagnostic method for eradication of ALV in poultry farms.

Development of a novel immuno-PCR for detection of avian leukosis virus

Avian leukosis virus (ALV) is an important pathogen for various neoplasms, including lymphoid, myeloid, and erythroid neoplasms, and it causes significant economic loss in the poultry industry. Several efficient methods for the detection of ALV have been reported. However, these previously developed approaches are based on either PCR or immunoassays. Here, we used a proximity ligation technique and combined PCR with the immunoassay to develop a novel immuno-PCR (Im-PCR) approach for the detection of ALV. Our data showed that the Im-PCR had high specificity and sensitivity to ALV. The Im-PCR method selectively reacted to ALV but not to the other avian viruses tested. The limit of detection of Im-PCR could reach 0.5 TCID50. Moreover, the results of Im-PCR were in agreement with results from commercial ELISA when the clinical cloaca samples were used for ALV detection. The present results demonstrate that the novel Im-PCR method can be efficiently applied to detect ALV in a clinical setting. Our data also highlight that Im-PCR may have promising applications in the diagnosis of pathogens.

Detection of Avian Retroviruses in Vaccines by Amplification on DF-1 Cells with Immunostaining and Fluorescent Product-Enhanced Reverse Transcriptase Endpoint Methods

In order to ensure the safety of vaccines produced on avian cells, rigorous testing for the absence of avian retroviruses must be performed. Current methods used to detect avian retroviruses often exhibit a high invalid-test/false-positive rate, rely on hard-to-secure reagents, and/or have readouts that are difficult to standardize. Herein, we describe the development and validation of two consistent and sensitive methods for the detection of avian retroviruses in vaccines: viral amplification on DF-1 cells followed by immunostaining for the detection of avian leukosis virus (ALV) and viral amplification on DF-1 cells followed by fluorescent product-enhanced reverse transcriptase (F-PERT) for the detection of all avian retroviruses. Both assays share an infectivity stage on DF-1 cells followed by a different endpoint readout depending on the retrovirus to be detected. Validation studies demonstrated a limit of detection of one 50% cell culture infectious dose (CCID(50))/ml for retrovirus in a 30-ml test inoculum volume for both methods, which was as sensitive as a classical method used in the vaccine industry, namely, viral amplification on primary chicken embryo fibroblasts followed by the complement fixation test for avian leukosis virus (COFAL). Furthermore, viral amplification on DF-1 cells followed by either immunostaining or F-PERT demonstrated a sensitivity that exceeds the regulatory requirements for detection of ALV strains. A head-to-head comparison of the two endpoint methods showed that viral amplification on DF-1 cells followed by F-PERT is a suitable method to be used as a stand-alone test to ensure that vaccine preparations are free from infectious avian retroviruses.

Detection of avian leukosis virus (ALV) in albumen of Shiraz commercial and local layer flocks using ELISA and RT-PCR

Summary Avian leukosis viruses (ALVS) cause different types of tumours in poultry and can affect the health and egg production of the birds. To investigate the presence of the virus in chicken layer flocks in Shiraz, 222 egg albumen from local layer breeder (25 eggs), local layer grand parent (30 eggs), broiler breeder (60 eggs), commercial layer (46 eggs) and broiler grand parent (61 eggs) were tested by antigen-capture enzyme-linked immunosorbent assay (AC-ELISA). The results showed that 3.33, 76 and 80% of commercial broiler breeder, local layer breeder and local layer grand parent were positive, respectively. Thirty-five albumen samples were randomly selected and tested by RT-PCR using PU1/PU2 and PA1/PA2 primer sets. The samples with ELISA S/P ratio equal or more than 0.17 were positive by RT-PCR using PA1/PA2 primers. This is the first report of the presence of the ALV in egg albumen samples of chicken layer flocks in Shiraz.

Detection of avian leukosis virus in albumen of chicken eggs using reverse transcription polymerase chain reaction

A reverse transcriptase polymerase chain (RT-PCR) assay was developed to detect avian leukosis retrovirus (ALV) in egg albumen. Eggs of Single Comb White Leghorns were from a commercial breeder (stock F) and from a pathogen-free flock (stock N). RT-PCR was undertaken on isolated RNA from 20 unfertilized egg samples using seven sets of primers that correspond to the ALV gp85 envelope glycoprotein which determines the ALV subgroup classification. An ELISA assay for ALV gs antigen of egg albumen was positive for all stock F birds tested and negative for all stock N birds. Virus isolation was undertaken by inoculating egg albumen, feather pulp, or blood from five stock F chickens onto cultures of chicken embryo fibroblasts (C/E). IFA analysis of the inoculated C/E cultures indicated that all stock F birds tested contained infectious ALV. For the virus-positive stock F chickens, RT-PCR analyses using primers designed to detect all ALV subgroups detected ALV in 15/15 (100%) egg albumen samples, while primers designed to detect subgroup A ALV were positive for 12/15 (80%) egg albumen samples. RT-PCR products were not detected from five egg albumen samples from five stock N chickens by any primer sets. Direct sequencing using primers specific for subgroup A ALV verified the viral subgroup in the RT-PCR amplification products. The combined use of RT-PCR and direct sequencing of the RT-PCR product provides a new approach for identifying ALV-infected poultry.

Detection of avian leukosis virus: comparison of five techniques

Five techniques were compared for their ability to detect decreasing dilutions of RAV-I, an avian leukosis sarcoma virus, in serially passaged chick embryo fibroblast cell cultures. The indirect fluorescent antibody test, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase assay were equally sensitive in detecting the virus. The indirect immunoperoxidase and complement fixation avian leukosis tests were less sensitive. It is recommended that the sandwich ELISA be used for routine detection of the avian leukosis sarcoma virus group as possible vaccine contaminants because it is rapid and simple to perform and may be carried out on a large number of samples conveniently.

Detection of avian leukosis virus with the elisa system: Evaluation of conjugation methodology and comparison of sensitivity with the phenotypic mixing test in commercial layer flocks

Conjugates of horseradish peroxidase with rabbit IgG antibody against gs antigen (p27) of avian myeloblastosis virus were prepared using glutaraldehyde and periodate methods of coupling. Conjugates were evaluated for the detection of gs antigen of avian leukosis and sarcoma viruses in the ELISA system and the periodate conjugate was found to be superior. With an ELISA based on a periodate conjugate it was possible to detect 5 x 10(3) IU of leukosis virus propagated in cell culture. Comparisons between the PM test and ELISA on biological samples showed the PM test to be 2 to 2,000 times more sensitive than ELISA for vaginal swabs and embryo extracts. ELISA was 16 to 64 times more sensitive than the CF test when comparisons were made on albumens and embryo extracts. When the ELISA was used for testing vaginal swabs for the prevalence of LLV infection in commercial laying flocks, it appeared that the reliability of ELISA varied. In all five flocks studied, ELISA detected a higher percentage of gs+ [virus+] hens than did the PM test, the proportion of ELISA(+) PM(-) samples ranging from 3 to 53%. In some flocks ELISA failed to detect a low proportion [ 1 to 5%] of infected hens when vaginal swabs were used for screening. The suitability of ELISA for use in screening of commercial flocks for LLV infection is discussed.