Development of TaqMan Real-Time Polymerase Chain Reaction Assay for Detection of Chicken Anemia Virus in Newcastle Disease Vaccines.

Exogenous chicken anemia virus (CAV) has been detected in commercial poultry vaccines in various countries of the world. The presence of unwanted CAV in vaccines not only influences the epidemiology of chicken infectious anemia disease, but may also lead to vaccine failure and confusing results when vaccine responses are monitored. To detect CAV in contaminated vaccines, nucleic acid testing (unlike conventional testing) has a shorter processing time and does not require cell culture or live animals. The aim of the current study was to develop a TaqMan real-time polymerase chain reaction (PCR) assay to detect and quantify CAV in poultry vaccines and investigate CAV contamination in Razi live Newcastle disease vaccines. The TaqMan real-time PCR assay was set up, optimized, and validated in successive experiments. A standard plasmid pUC-VP2 containing viral protein 2 of CAV was constructed and used in the assay to generate a standard curve to quantify CAV genomes. A clear linear correlation was observed between threshold cycle (Ct) values and plasmid copy numbers in the amplification plots of 10-fold serial dilution of the plasmid. Total DNA of three samples of each of four different Razi live Newcastle disease vaccines, namely LaSota, B1, clone.12IR, and thermo-resistant strains, were extracted and subjected to real-time PCR assay. No CAV contamination was detected in the Razi Live Newcastle vaccines. The developed TaqMan real-time PCR assay provides a quick, specific, and sensitive method for use in detecting CAV in quality control vaccine testing and viral load studies.

Comparison of Cattle Serum Antibody Responses to Five Different Mycobacterial Antigens in an ELISA System.

The presence of common zoonosis diseases caused by Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM), such as Johne's and Crohn's diseases, poses a public health threat and economic losses to Iranian livestock. Therefore, the early detection of mycobacteria is of paramount importance. In this regard, enzyme-linked immunosorbent assay (ELISA) is a new, simple to use, rapid, and useful diagnostic tool. This study was performed to evaluate different crude antigens obtained from Mycobacterium species using an indirect ELISA test to identify the mycobacterial infection in infected livestock. Five different strains of Mycobacteria including M. tuberculosis, M. phlei, M. bovis, M. aviumsubspecies paratuberculosis, and M. bovis AN5 were cultured. The crude antigens in the samples were precipitated with trichloroacetic acid 4%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude antigens isolated from different Mycobacterium species was reported. The total level of protein was determined by the Lowry protein assay. After the crude antigen preparation, the ELISA test was performed and the results were compared with the purified protein derivative skin test. Data analysis was performed using SPSS software version 25. All five strains were detected in more than 92% of healthy animals. The highest sensitivity of ELISA tests was in M. bovis AN5 antigen which was greater than 83%. The highest diagnostic specificity and efficiency of assays were in M. avium subspecies paratuberculosis which was 95.83% and over 83%, respectively. Regarding the results, M. avium subspecies paratuberculosis and M. bovis AN5 antigens were promising candidates for the design of diagnostic ELISA due to their sensitivity, specificity, and efficiency.

Designing a polytopic complex vaccine candidate against Gallibacterium anatis: an In-silico study.

The haemolytic biovar of Gallibacterium anatis (G. anatis) is responsible for urogenital, gastrointestinal, and respiratory diseases in chickens. There are numerous reports on the resistance of G. anatis to antibiotics and recurrence of the disease, which raise concerns about antimicrobial treatment efficiency. Vaccination has been considered as the most feasible procedure of prevention in high risk farms. Subunit vaccines containing immunogenic components can have practical protective value in preventive measures regarding the infection. The present study aimed to introduce a polytopic vaccine candidate based on epitope detection. All registered sequences of four immunogenic proteins, includig Flfa, GTxA, Gab_1309, and Gab_2348 were retrieved and directed for variational analysis. A vaccine isolate was selected for each protein and tested for B-cell epitope mapping using different tools. Furthermore, consensus selected immunogenic regions with special patterns fused together by flexible linkers were integrated into two constructs and checked for the best status of proteasomal cleavage sites, as well as hydropathy plot. Moreover, back translations, along with codon optimization were performed, and then some tags were added to the constructs. The selected consensus B-cell immunogenic epitopes were for 12656: AA114-181, 7990: AA114-181, Avicor: AA42-77, 134-197, and IPDH: 61-155 for Flfa protein, AA185-235, AA372-457, and AA807-941 for GtxA-N, AA260-305, AA340-400, and AA110-146 for Gab-1309, and AA125-AA175 for Gab-2348. Two suitable patterns of attachment were selected from the different fusion patterns of epitopes in B-cell polytopic vaccinal constructs. Finally, the examination of these constructs showed their effect and efficacy for immune system stimulation. Based on bioinformatics results, these immunogens could be utilized as potential candidates to develop polytopic protective vaccines and design diagnostic kits.