Novel Mycoplasma Agalactiae with new P 30 Protein Pattern by Major Change in 17 Amino Acids

M. agalactiae (Mycoplasma agalactiae) is the main agent of CA (contagious agalactia) that is primarily disease of dairy sheep and goats. In Iran, a three-valent Agalactia inactivated vaccine were prepared based on three M. agalactiae isolated from milk secretion of sheep and goat of Taleghan, Shiraz and Lorestan and preventive policies against CA disease founded in Razi Institute since 1966. The P30 is a specific and stable lipoprotein of M. agalactiae. In this study, complete coding sequence of P30 gene of three Iranian vaccine strains and ten recently field isolates were analyzed in nucleotide and protein levels by bioinformatics tools. Interestingly, results observed a novel protein pattern K106VLKTKEIRLSQERKLS122 in P30 protein of vaccine strains compared to field isolates and the other available sequences in GenBank. Our findings demonstrated different B and T cell epitope patterns in Iranian vaccine strains. We suggested that the major change in P30 protein pattern may be occurred due to mutation during adaptation process in PPLO (Pleuropneumonia-Like Organisms) broth media.


Introduction
Contagious agalactia (CA) is primarily a disease of dairy sheep and goats characterized by mastitis, arthritis and keratoconjunctivitis (Amores et al., 2010, Nicolas et al., 2008, Stear, 2005).This syndrome is produced by Mycoplasma agalactiaeand causes the reduction and the suppression of milk production and death in many young animals (Katarína et al., 2009, Madanat et al., 2001, Tola et al., 1997).
CA is found across several countries, including mainly North America, western Asia, North Africa, Europe and is endemic in most Mediterranean countries (Zendulkova et al., 2007, Bergonier et al., 1997).Borry and Entessar (1963) reported the presence of Agalactia disease in small ruminant for the first time in Iran and also Sotoodehnia and Aarabi (1986) identified CA as cause of economic losses in most part of Iran.
Phase variation of surface lipoproteins is a common mechanism in mycoplasma species and is probably a major adaptive strategy for these minimal pathogens (Nouvel et al., 2010).Ability of mycoplasma to evade the immune response of the hosts, leading to facilitate chronic infection (Browning et al., 2011).Most importantly, prevention and eradication of CA can be obtained through better diagnostic tests and through a more efficient vaccine (Greco et al., 2002).
Nowadays, attention has been paid to the interaction betweenlipoproteins and pathogenicity of M. agalactiae videlicet the lipoproteins appear to play a prominent role in adherence, in enzymatic interactions, in transport of nutrients and finally in facilitate chronic infection with both inducing the hostimmune response and evasion of thisresponse (Browning et al., 2011, Bergonier et al., 1996).Zavagli (1951) had succeeded to prepared Formalin-inactivated Agalactia vaccine in Italy.Effective Agalactia vaccines were prepared using washed cultures inactivated with phenol-saponin by Tola et al. (1999).Greco et al. (2002), indicated a good efficacy of the vaccine which inactivated by oil-emulsion in eliciting protection against M. agalactiae infection.In Iran, three-valent Agalactia vaccine which inactivated with saponin were prepared based on threeM.agalactiae isolated from milk secretion of sheep and goats of different geographic areas in Razi Institute since 1971(Naseri Radet al., 2007).
With due attention to production of Agalactia vaccine against CA in Iran since 46 years ago and with the aim to raise of efficiency of current vaccine, it seems necessary to evaluated specific and stable lipoproteins of M. agalactiae.
The P30 gene is a stable, specific andstrongly immunogenic antigen for M. agalactiae as the immune response against P30 is strong and persistent.Immunoblot analysis using the monospecific polyclonalanti-P30-His serum indicated that P30 is specific to this pathogen means presence this gene in all M. agalactiae strainstested and absence in the other mycoplasma species (Fleury et al., 2001).
It is important to understand the properties of protein sequences that are important for antigenicity and to identify peptide epitopes whose utilization result in the development and optimization of vaccines (Fleri et al., 2017, Jones et al., 2014, Schlehuber et al., 2011, Leroux-Roels., 2010).
In this study the P30 gene of three M. agalactiae vaccine strains of Razi Vaccine and Serum Research Institute were analyzed and compared to new field isolated from different provinces of Iran and all other sequences available in GenBank.

Sampling and Culture Methods
In this study, three vaccine strains of M. agalactiae were taken from Department of Aerobic Bacterial Vaccine Production Laboratory of Razi Institute and cultured with ten new field isolates which were isolated from different provinces of Iran (Table 1).Samples were collected from eye and milk secretion of sheep and goat herds with clinical signs of infection of mycoplasma, transported in transport medium and transmitted on ice to the mycoplasma Reference Laboratory of Razi Institute, Karaj, Iran.The specimens were diluted and filtered into the fresh Pleuropneumonia-Like Organisms (PPLO) broth media and then inoculated on to PPLO agar medium (BBL, Becton Dickinson and company, Cockeyville, Sparks, MD).Inoculated agar and broth were incubated at 37°C in 5% Co2 and 98% humid atmosphere.The broths were observed daily for signs of growth and the plates were considered for the typical appearance of mycoplasma colonies.M. agalactiae reference strain (NCTC 10123) and uncultured PPLO broth have used as a positive and negative controls respectively.

DNA Extraction and PCR Amplification
DNA was extracted from samples using phenol-chlorform method developed by Tola et al. (1997).
Identification of genus and species of isolates were performed with published primers set based on 16SrRNA and P80 lipoprotein genes (van Kuppeveld et al., 1992;Tola et al., 1997) respectively according to protocol described by Tola et al. (1996).
The thermal program was conducted in a Gradient Mastercycler (Eppendorf, Germany) as follows: 3 min at 95°C, followed by 38cycles of 1min at 93°C, 30 s at 54°C and 40s at 72°C, with a final extension cycle of 5 min at 72°C.The PCR bands were visualized by UV transilluminator after electrophoresis (1% agarose gel in 1×Tris-acetic acid-EDTA (TAE) buffer) and ethidium bromide staining.

Sequencing and Phylogenetic Analysis
Complete coding sequences of P30 gene of thirteen Iranian M. agalactiae isolates were sequenced in both directions by Sanger method (Macrogen Company).Nucleotide and protein sequences of P30 were compared and percentage of divergence was calculated by MegAlign software.

Epitopes Prediction
B and T-cells epitopes were predicted in P30 protein by online IEDB (Immune Epitope Database and Analysis Resource) software with consensus method and consider cutoff ≤ 1% for all lengths of H-2-Kd, H-2-Dd, H-2-Ld alleles of MHC-Iand ≤ 8.6 for all lengths of H2-IAd, H2-IEd alleles of MHC-II in mouse.http://www.iedb.org/

Database Submission
P30 sequences of three vaccine strains and ten field isolates of Iranian M. agalactiae were submitted to the GenBank databases under accession numbers MF380352 to MF380364 which were released to the public database on Jan 1, 2018.https://www.ncbi.nlm.nih.gov/WebSub/?tool=genbank

Culture and PCR
Ten field isolates of M. agalactiae which were isolated from 5 provinces of Iran and also three vaccine strains were proved by culture and PCR methods.The results of culture proved the presence of mycoplasma in all the samples that weregrown in PPLO broth media and also observation colonies on PPLO agar media.Genus and species were detected by observation of PCR amplicons with 163 bp and 375 bp length respectively with published primers set were used for the specific detection of M.agalactiae and we success to detected this pathogen with both methods.
The complete coding sequence of P30 gene with 800bp length was amplified in all field isolates andthree Iranian vaccine strains of M. agalactiaesuccessfully.Briefly, in all studied field isolates and vaccine strains, the DNA fragment of Mycoplasma genus with approximately 163 bp of 16SrRNA and the M. agalactiae species in 375bp and 800 bp of P80 and P30 lipoprotein genes were amplified respectively by Polymerase Chain Reaction (Figure 1).

InsilicoAnalysis of P30 Protein Pattern in Vaccine and Field Isolates
Alignment of P30 protein sequences by MegAlign software showed some important differences between vaccine strains and all field isolates.The most importance differences caused by the variation happened in the position 348-401 of nucleotide alignment in three vaccine strains of group No.I that caused two frame shifts in nucleotide sequence of P30 gene which were substitute17 amino acids in residues 106-122 (Figure 4).2).(Southwoodet al., 1998).

MHC class I binding Epitopes Prediction in polymorphic region of P30 protein of M. agalactiae
For MHC class I epitope predictions, selection of predicted binders can be done based on the percentile rank or MHC binding affinity.The IEDB currently recommends making selections based on a percentile rank of <= 1% for each (MHC allele, length) combination to cover most of the immune responses.

Discussion
Protein pattern of an immunodominant antigen, P30, was evaluated from three vaccine strains and compared belong to Iranianrecentlyfield isolates of M. agalactiae.
The three vaccine strains of Razi vaccine and Serum Research Institute which were used in three-valent Agalactia inactivated vaccine production were introduced as a new strain with novel P30 protein pattern.
Three effective monovalent Agalactia vaccines which inactivated with formalin, phenol-saponin and oil-emulsion were prepared by Zavagli et al. (1951), Tola et al. (1999) and Greco et al. (2002) respectively.Vaccines which inactivated with formalin, heat or sodium hypochlorite and then adjuvanted with aluminum hydroxide did not provided significant protection (Tola et al., 1999).The mineral-oil, phenol or saponin-inactivated vaccine were highly immunogenic which produced a progressive increase in antibody production and enable to induced a fully protective preventing not only the clinical signs of CA but also the infection by M. agalactiae even at 8 months post-vaccination (Tola et al., 1999, De la Fe et al., 2007, Buonavoglia et al., 2007).
In Iran, an inactivated and saponinized vaccine has been prepared with different M. agalactiae isolates against CA in 1971(Naseri Rad et al., 2007).Baharsefat et al. (1971) announced the three-valent saponinized vaccine with good protectivity remain immune for 6 -9 months post-vaccination.
Prevalence evaluated of CA syndrome in sheep and goat herds of Iran based on previous studies between years 1986-2011 showed approximately 20% outbreak from 1986 to 2007 but a high Ascending Course was occurred and rate of disease had significant increased until 33.3% in 2011 (Kheirkhah et al., 2011, PiraliKheirabadi & Ebrahimi., 2007, Sotoodehnia & Aarabi., 1986).
Evidence related to an increase in prevalence has led to the conclusion that development of effective Agalactia vaccines through knowledge of the stable, specific and main immunogenic antigens of M. agalactiae is the most promising approach to control M. agalactiae infections in small ruminant.
Previous studied showed that P30 is a stable, specific and strongly immunodominant protein of M. agalactiae thus this gene presented as a suitable candidate for determined ELISA (Enzyme-Linked ImmunoSorbent Assay) kit manufactured and recombinant vaccine against CA (Fleury et al., 2001, Fleury et al., 2002).
The results of this study were in agreement of Fleury et al (2001) report that P30 is specific gene and detected in all of M. agalactiae which were isolated from Iran.Nucleotide sequences of P30 gene proved conservative of this gene (98% to 100% identity) among available sequences in GenBank and 77% of Iranian samples (Table 1).
Comprehensive bioinformatics analysis of P30 gene of M. agalactiae showed some importance differences in the ORF region that caused a considerable variation among two groups of Iranian isolates.
For the first time, unlike to previous studies, in three Iranian vaccine strains a new P30 protein pattern was identified.These novel M. agalactiae was located in new distinct group namely group No.I with 6-9% divergency in nucleotide sequences and 6-11% in amino acid levels respectively (Table 1).Bergonier et al. (1996) studied 245 field isolates originating from ten different countries to find an antigenic variability of M. agalactiae with species-specific monoclonal antibodies (MAbs) were developed against M. agalactiae reference strain PG2.Their results indicated that the antigenic variability of M. agalactiae partially related to the geographic origin of the isolates (Bergonier et al., 1997, Bergonier et al., 1996).This study was agree with them to indicated antigenic variability in isolates which were isolated from different region of Iran based on P30 protein.
Many of the researchers studied on various gene of M.agalactiae to detected genetic diversity and variations but concentration on the P30 gene close to the Fleury et al (2001) that they studied on the P30 gene of 15 samples of M.agalactiae.They detected a similar pattern in ORF region of P30 gene while, in the present study 13 samples which were isolated from 8 provinces of Iran were considered and some importance differences were detected in the P30 ORF region of M.agalactiae(Table 1).
The most importance differences were the variation happened in the position 348-401 and insertion sequences (G 349 , A 350 and T 402 ) of nucleotide alignment in three vaccine strains of group No.I that caused two frame shifts in nucleotide sequence of P30 gene which were substitute17 amino acids in residues 106-122 (Figure 2, Figure 3,Figure 4).Further analysis of amino acid alignment, indicated a Lysine (L 108 ) insertion in the Iranian isolates of group No.I (Taleghan, Shiraz and Lorestan).This pattern only showed in three Iranian vaccine strains and no detected in the other sequences of Iran and available in Database.
Strategy of mycoplasmas for avoiding genome erosion is Sharing genetic resources via horizontal gene transfer that may provide this pathogen for adapting to new niche or host (Sirand-Pugnet et al., 2007).Adaptation of M. agalactiae isolates to new environment needed the time and when adapted to laboratory conditions, these grow easily in commonly used media for mycoplasma growth freshly (Srivastava., 1982, Kumar et al., 2014).
Date of sample collection of these three old isolates back to 1966 whereas the other recently samples were collected from 2010 to 2012.Old isolates are routinely used as a master seed for production of contagious agalactia vaccine since 1966 in Razi Vaccine and Serum Research Institute of Iran.Based on scientifically evidence this genetic diversity occurred in vaccine isolates, maybe due to mutation during adaptation process and highly serial passages in PPLO broth media.
Furthermore, evaluation of P30 gene showed several differences between the active geneand submmited sequences of pseudogene in the GenBank.For example there was a cytosine (CCA) in residue 275 of nucleotide sequences of pseudo genes (AF327860-AF327861-AF327866-AF327872) while in the same position of all the genes was thymine (CTA) that caused to convert genetic code of proline to leucine.Also, in the nucleotide sequences of pseudogene some other mutations were detected: C 382 →A, T 397 →A, deletion of T 399 , T 401 →C, insertion of T 402 , A 428 →T, G 432 →A.Besides, stop codon (TGA) in the position 562 situated in nucleotide sequences of pseudogenes while Tryptophan codon (TGG) in the same positions has been observed in the genes.
Clinical and basic biomedical researchers are interested to epitopes investigation for vaccine design, disease prevention, diagnosis, and treatment (Ruthet al., 2015, Wang et al., 2008).Recombinant DNA can be a proper alternative way against whole pathogen-formulations in vaccination, capable of stimulating a specific immune response (Soria-Guerra et al., 2015).In the present study, epitopes prediction were done for variable area of P30 lipoprotein (position 106-122 of amino acid alignment) of M. agalactiae in two groups of Iranian isolates with the aim ofrecombinant vaccine design or specific antibodies to Differential Diagnosis of field isolates and vaccine strains.All the mouse alleles of B and T cells were analyzed in IEDB analysis resource and determined differential epitope patterns in this region.
Increased prevalence of CA syndrome up to 13% in recent years in Iran, might be occurred due to major change in antigenic region of immunodominant antigens of M.agalactiae likeness P30 protein that caused the possible reduction of protectivity of three-valent vaccine which made 46 years ago.
It seams that the polyvalentrecombinantvaccinecontaining two patterns of specific and immunodominant P30 gene can be better protects against several strains of M.agalactiae in Iran.

Conclusion
In this study, three novel M. agalactiae was identified based on P30 protein pattern.These three vaccinal strains showed new pattern and different B and T-cells epitopes in P30 protein compare to field isolates and the other available sequences in NCBI DATA bases.Additional studies are needed for better understanding about P30 protein structure of M. agalactiae and also immune study is required to promote the effectiveness of current CA vaccine for preventing infections.

Figure 2 .
Figure 2. Phylogenetic tree with Neighbor joining clustering method, IR(1-3) are vaccine strains of Iran, IR(4-13) are field isolates of Iran and the other samples are submitted in the GenBank Two different patterns including K 106 VLKTKEIRLSQERKLS 122 (Pattern A, belong to group No.I) and G 106 S-KNEGDKVIAGKEAI 121 (Pattern B, belong to group No.II) was detected between two P30 groups(Figure 4).Furthermore a unique amino acid insertion (Lysine108) was observed in group No.I and also for the first time a unique amino acid substitute (S 63 → Y 63 ) in more than 92% of Iranian M. agalactia isolates was detected.Considerable, that all of the Iranian mycoplasma isolates in group No.II showed similar pattern with available sequences in GenBank based on P30 gene.But P30 protein pattern of vaccine strains in group No.I was unique among available data in DATA bases (2017 May 21).

Figure 4 .
Figure 4. Amino acid alignment, IR(1-3) are vaccine strains of Iran, IR(4-13) are field isolates of Iran and the other samples are submitted in the GenBank

Table 1 .
M. agalactiae samples which were used in this study

Table 2 .
B & T Cells Epitopes Prediction in polymorphic region of P30 protein of M. agalactiae MHC class II binding Epitopes Prediction in polymorphic region of P30 protein of M. agalactiae -For MHC class II epitope predictions, selection of predicted binders can be done based on the percentile rank or MHC binding affinity.The IEDB currently recommends making selections based on a consensus percentile rank of the top 10%.Alternatively, selecting peptides predicted to bind at 1,000nM is also supported by experimental data