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.

Diagnostic potential of recombinant protein of hexahistidine tag and infectious bursal disease virus VPX expressed in Escherichia coli.

The current method to detect antibody titre against infectious bursal disease virus (IBDV) in chickens is based on enzyme-linked immunosorbent assay (ELISA) using whole virus as coating antigen. Coating the ELISA plates requires a purified or at least semi-purified preparation of virus as antigen, which needs special skills and techniques. In this study, instead of using whole virus, recombinant protein of hexahistidine tag (His 6 tag) and VPX protein of IBDV expressed in E. coli was used as an alternative antigen to coat the ELISA plates. There was a good correlation coefficient (R2 = 0.972) between the results of the ELISA using plates coated with monoclonal antibody against His 6 tag and those of the commercial IBDV ELISA kit. Hence, His 6 tag and VPX recombinant protein expressed in E. coli has the potential for the development of ELISA for the measurement of IBDV-specific antibody.

Molecular characterization of an Infectious bursal disease virus isolate from Iran.

The segment A of an Infectious bursal disease virus (IBDV) isolate from Iran was amplified by reverse transcription-polymerase chain reaction (RT-PCR), sequenced and compared with published sequences of 26 IBDV isolates from other parts of the world. The Iranian isolate showed 8 unique amino acid differences. In addition, 9 common amino acid differences, namely 3 in VP2, (222 Ala, 256 lIe and 294 lIe), 3 in VP4 (685 Asn/Ser, 715 Ser and 751 Asp), 2 in VP3 (990 Val and 1005 Ala), and 1 in VP5 (49 Arg) were found. Phylogenetic analysis indicated that the Iranian isolate is closely related to highly virulent (hv) IBDV isolates from Asian countries. Nevertheless, it may share a common origin with hv isolates from other parts of the world.