Molecular Identification of Mycoplasma agalactiae in Iran Based on P30 Gene.

Mycoplasma agalactiae (M. agalactiae) is known as the main etiological agent of contagious agalactia (CA). The CA is a disease affecting dairy sheep and goats, the main characteristics of which include keratoconjunctivitis, arthritis, and mastitis. This pathogen results in milk production reduction and suppression, thereby leading to serious economic loss. In the present study, 125 sheep and goat samples were collected from 15 provinces of Iran. Cultural and molecular methods were used for sample characterization. After extracting genomic DNAs using the phenol/chloroform method, the PCR technique was employed to detect Mycoplasma genus in 163bp fragment of 16S rRNA gene (M-PCR) and M. agalactiae in 800bp fragment of conserve and specific P30 lipoprotein gene (P30-PCR) in cultural and clinical samples. Finally, to validate the experimental approach, a 375 bp amplicon of P80 lipoprotein was amplified using the MA-PCR. Out of 125 samples under investigation, 43 cases were positive, and Mycoplasma colonies were observed in the pleuropneumonia-like organisms agar culture. Based on the results of the M-PCR method, 61 specimens (out of 125 samples) were scored positive for Mycoplasma presence. Furthermore, 20 samples were positive according to the P30-PCR data. It should be mentioned that the MA-PCR was performed based on the P80 gene on 125 total samples to furtherverify the results for M.agalactiae detection. Based on the obtained data, P30 and P80 genes were presented and amplified in all Iranian M. agalactiae isolates (n=20). Our results indicated that the P30 gene was conserved and specific to all Iranian M. agalactiae isolates and this new P30-PCR method (as an MA-PCR technique) might be useful in the detection of this pathogen.

Development of New Modified Simple Polymerase Chain Reaction and Real-time Polymerase Chain Reaction for the Identification of Iranian Brucella abortus Strains.

Brucellosis is primarily a worldwide zoonotic disease caused by Brucella species. The genus Brucella contains highly infectious species that are classified as biological threat agents. In this regard, the identification of Brucella can be a time-consuming and labor-intensive process posing a real risk of laboratory-acquired infection to the laboratory staff. This study aimed to present a novel conventional and real-time polymerase chain reaction (PCR) assay for the identification of Brucella abortus strains. Regarding this, two primers (bru ab2) were designed based on the unique loci encoding autotransporter-associated beta strand repeat-containing protein (ID:YP00113760). A total of 56 Brucella strains (e.g., reference, vaccinal, and field isolates) and Yersinia enterocolitica, as a non-Brucella isolate, were evaluated in conventional and real-time PCR systems. The results of the study indicated that 0.4 ng and 400 FG of genomic DNA of B. abortus strains can be detected by conventional and real-time PCR, respectively. The primers, bru ab2, were suitable for both PCR methods. Both methods were specific for the detection of all strains of the bacterium; however, real-time PCR assay was 1000-fold more sensitive than the conventional PCR method. Therefore, this new detection system could be a suitable selective modified method for the accurate identification of all B. abortus strains.

Intraspecies Gene Variation within Putative Epitopes of Immunodominant Protein P48 of Mycoplasma agalactiae.

P48 protein of Mycoplasma agalactiae is used to diagnose infection and was identified as potential vaccine candidate. According to the genetic nature of mycoplasma and variable sensitivity in P48-based serological diagnosis tests, intra species variation of P48 nucleotide sequence investigated in 13 field isolates of difference province of Iran along with three vaccine strains. Samples were collected from sheep and goat and were cultured in modified PPLO broth. Two pair of primer employed to confirm genus and species of isolates and a pair of primer has developed to amplify the P48 gene. The sequencing results of PCR products were aligned and analyzed besides published sequences in GenBank. T-Cell and B-Cell epitopes and antigenicity of sequence were computationally predicted. The results have shown P48 nucleotide sequences are 99.9% identical in field isolates and vaccine strain of Iran, but analysis of GenBank published sequences have shown divergence up to 5.3% at the nucleotide level and up to 4.9% divergence in protein level of P48 sequences of Iran isolates and other available sequences in GenBank. Single nucleotide polymorphism exists in 89 positions and variable amino acid was observed at 25 residues. Phylogenetic analyses have shown that Mycoplasma agalactiae isolates fall into three main groups based on P48 nucleotide sequences. Immunoinformatics analysis of all available P48 nucleotide sequences have revealed that gene variation lead to differences in immunological properties, but the gene in Iranian isolates are conservative and stable. The sequence variation in epitopes can be underlying source of antigen heterogeneity as a result, affect serological tests accuracy. Due to the high level of divergence in worldwide isolates and high degree of similarity in P48 protein of Iranian isolates, designing recombinant P48 protein based on local pattern can increase the sensitivity and consistency of serological test.

Identification of novel E347Q and G362K amino acid substitutions in HN neutralization epitope and major antigenic difference in novel sub-genotype VIIj isolates.

Hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in induction of immune response against Newcastle disease infection. Mutation in residues 347 and 362 of HN linear antigenic site has been identified to be responsible for antigenic variations. Hence we studied antigenic difference between sub-genotype VIIj isolates and vaccine strains by the use of polyclonal serum against LaSota strain in hemagglutination inhibition test. Furthermore, epitope patterns of the isolates under study were analyzed and compared to HN sequences in GenBank. The results demonstrated that new Newcastle disease isolates (Mazandaran and Behshahr) had hemagglutination inhibition (HI) titer three and five, respectively while LaSota strain titer was eight. In addition, observation of sequences and epitope patterns revealed three unique amino acid substitutions (D144N, E347Q and G362K) in HN protein. E347Q and G362K mutations were located in neutralization antigenic site. Thus, we suggest that, these two novel amino acid substitutions in major linear epitope might be responsible for antigenic variation and decrease of HI activity.

Comparative analysis of sialidase protein in velogenic and lentogenic strains of Newcastle disease virus.

The sialidase protein is a  major part of hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV), which is an important multifunctional envelope protein. This protein plays key roles in virus attachment to cells and virus-cell fusion. In this study, we compared the sialidase protein of Iranian virulent velogenic field strains with that of avirulent lentogenic strains. Six of seventeen variations in amino acid 395, 523, 550 432, 479 and 540 were observed near the catalytic and glycosylation sites in the sialidase protein. The obtained results showed fundamental differences in various biological parameters such as post-translational modification, antigenic index and electrostatic potential of tertiary structure of the sialidase protein. We suggest these six amino acids might play an effective role in the pathogenesis of NDV.