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

Document Type: Original Articles

Authors

1 Mycoplasma Reference Laboratory, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

2 Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran

3 Central Laboratory Department, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

4 Proteomics and Biochemistry Department, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

5 Department of Microbiology, School of Specialized Veterinary Science, Science and Research Branch, Islamic Azad University, Tehran, Iran.

Abstract

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.

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Main Subjects


Article Title [French]

Variation des Gènes Intraspécifiques dans les Épitopes Putatifs de la Protéine P48 Immunodominante de Mycoplasma Agalactiae

Abstract [French]

La protéine P48 de Mycoplasma agalactiae est un candidat vaccin potentiel et est utilisée dans le diagnostic de l'infection. Compte tenu de la nature génétique de Mycoplasma et de sa sensibilité variable dans les tests de diagnostic sérologique basés sur P48, la présente étude visait à étudier la variation intraspécifique de la séquence de nucléotides P48 dans 13 isolats de terrain obtenus de différentes provinces d’Iran, ainsi que de trois souches de vaccin. Après avoir recueilli des échantillons de moutons et de chèvres, ils ont été cultivés dans un bouillon de PPLO modifié. L'identification du genre et de l'espèce des isolats a été réalisée à l'aide de deux paires d'amorces; en outre, une paire d'amorces a été développée pour amplifier le gène P48. Les résultats de séquençage des produits de réaction en chaîne de la polymérase ont été alignés et analysés sur la base des séquences publiées dans GenBank. Les épitopes des cellules T et B et l'antigénicité de la séquence ont été informatiquement prédits. Selon les résultats, les séquences de nucléotides P48 étaient identiques à 99,9% dans les isolats de terrain et la souche vaccinale d’Iran. Néanmoins, l'analyse des séquences publiées par GenBank a démontré des taux de divergence allant jusqu'à 5,3% et 4,9% aux niveaux de nucléotide et de protéine des séquences P48, respectivement. Un polymorphisme mononucléotidique existait dans 89 positions, et des acides aminés variables ont été observés à 25 résidus. Sur la base de l'analyse phylogénétique, les isolats de Mycoplasma agalactiae sont répartis en trois groupes principaux basés sur les séquences de nucléotides P48. L'analyse immuno-informatique de toutes les séquences de nucléotides P48 disponibles a révélé que la variation du gène conduisait à des différences de propriétés immunologiques. Cependant, les gènes dans les isolats iraniens étaient conservateurs et stables. La variation de séquence d'épitopes peut être la source d'hétérogénéité antigénique, affectant ainsi la précision des tests sérologiques. En raison du niveau élevé de divergence dans les isolats du monde entier et du degré élevé de similitude de la protéine P48 dans les isolats iraniens, la conception de la protéine P48 recombinante basée sur un schéma local peut augmenter la sensibilité et la cohérence du test sérologique.

Keywords [French]

  • Mycoplasme
  • Agalactie Contagieuse
  • P48
  • Variation
  • Hétérogénéité Antigénique
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