Molecular Survey of Brucella melitensis Field Isolates using Sequence-Based PCR of Outer Membrane Protein 31.

Sequence-based Polymerase Chain Reaction (PCR) has been introduced as an effective and reliable method for bacterial strain typing, which could provide a reliable typing approach for clinical laboratories. This study aimed to describe the reproducibility and performance of the Outer Membrane Protein 31 (Omp31)-based PCR, as a molecular genotyping tool for Brucella melitensis (B. melitensis) typing. The 31 KD outer-membrane protein of Brucella, which encodes the Omp31 gene, can be applied as an antigen to diagnose brucellosis. For this purpose, 146 samples were taken from human blood samples, bovine and camel lymph nodes, as well as sheep and goat aborted fetuses, including fetal kidney, abomasum, liver, lung, spleen, and heart for bacteriological investigation. The molecular detection of the Omp31 and IS711 genes was performed using the isolated B. melitensis (n=14). The sequencing of the Omp31 gene of B. melitensis in the Iranian field isolates was also performed for the whole gene sequencing. The homology of all sequences was then checked with the reported National Center for Biotechnology Information sequences using a basic local alignment search tool for the nucleotide diversity evaluation. The findings revealed that B. melitensis isolates were recovered from 14 examined cases and confirmed by the IS711-based PCR with a PCR product of 731 bp. Moreover, 14 Iranian B. melitensis sequences clustered together as a monophyletic grouping with bootstrap support of 63, and they were closely related to the B. melitensis reference isolates. This Omp31-based phylogenetic placement strongly indicates the monophyletic origin of the Iranian B. melitensis in different animals and human hosts.

Flow Cytometric Evaluation of CD4+ and CD8+ T-cell Immune Response in SPF Chickens Induced by Fowlpox Vaccine.

Fowlpox (FP) is a viral disease that is widely distributed throughout the world. The disease has an economic impact on the poultry industry, and its prevalence has even been reported in vaccinated flocks. The present study used flow cytometry to evaluate the CD4+ and CD8+ T-cell immune response of chicks induced by FP vaccine. 120 specific pathogen-free (SPF) 21-day-old chicks were randomly divided into three groups of 40. One group was used as negative control with PBS inoculation, the other two groups were inoculated with the local fowlpox vaccine produced by Razi Institute and commercial FP vaccines, and they were kept for five weeks. Peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque density gradients and the percentages of CD3+, CD3+, CD4+, and CD3+CD8+ T lymphocytes were analyzed with flow cytometry. Seven days post-immunization, a maximum (90-100%) swelling formation ("take") on the vaccination site was observed. The ratios of CD4+ to CD8+ T-lymphocytes in both vaccinated groups were significantly higher (p < 0.05) than the control group inoculated with PBS. The percentages of CD3+, CD3+CD4+, and CD3+CD8+ T-lymphocytes were increased in chickens vaccinated with commercial and local FP vaccines. There were no significant differences between the groups receiving commercial and local fowl pox vaccines. The present study showed that protective immunity could be associated with increased cellular immune responses, which has been interpreted as enhancing T-cell proliferation and increasing CD4+ to CD8+ ratios through vaccination with the FP vaccine. This study further suggests that the induction of enhanced immune responses is due mainly to the Th1-type response.