Abstract Mycoplasma agalactiae is an economically important pathogen and the main etiological agent of contagious agalactiae (CA) in small ruminants with a relatively high prevalence in sheep and goat, mainly characterized by mastitis, conjunctivitis and arthritis as predominant symptoms of a localized infection. Two genes encoding p48 and p40 major surface lipoprotein are played a fundamental role in the pathogenesis of Mycoplasma agalactiae. In this study a chimeric protein P48-40 of Mycoplasma agalactiae was designed, expressed, purified and evaluated in indirect ELISA. Coding sequence of antigenic regions of two p48 and p40 proteins was cloned into the expression vector Pet32a+ then expressed successfully into the E. coli BL21 (DE3) at optimal temperature 22oC after 4 hours with 0.1mM IPTG as an inducer. Affinity batch formation method was done for purification of recombinant P48-40 by using Nickel resin. The protein was highly expressed in soluble form and purified with yield of 33mg/L. Function of recombinant protein in indirect ELISA was evaluated and the results showed that 125ng of novel P48-40 protein could well detect specific antibodies with 100% sensitivity and Specificity by indirect ELISA. We concluded that the recombinant p48-40 protein could be applied for serodiagnosis of Mycoplasma agalactiae or evaluation of antibody levels in vaccinated animal.

Abstract Leptospirosis is a wide spread bacterial zoonosis that is common worldwide. The disease symptoms are mild or acute. Leptospira has pathogenic and non-pathogenic species; it has a lot of surface antigens. Adenylate Guanylate Cyclase (AGC) is a membrane protein that is found only in pathogenic species. In this study, the complete coding sequences of AGC protein of 242 pathogen serovars were investigated by bioinformatics tools. A Pattern was selected as a target sequence based on high prevalence pathogenic serovars in Iran Antigen sites; moreover, B-cell and T-cell epitopes were predicted by IEDB web server. An antigen site amino acid (D259-R462) in complete coding sequence of AGC protein was selected. This nucleotide related sequence was cloned into the pET32a+ expression vector. Expression of recombinant protein was optimized in E. coli strain Bl21-DE3 by 0.2mM IPTG after 16-hour incubation at 37 ͦ C and confirmed by 10% SDS-PAGE and western blotting. Antigenic peptide D259-R462 was highly expressed as Trx tag fusion protein. Recombinant peptide (rAcB) was purified by 6M urea from inclusion body with high extent yield 514.2 mg per 1000ml culture of E. coli. 20µg rAcB protein with montanide adjuvant was injected subcutaneously in BALB/c mice. Results showed that the recombinant peptide D259-R462 was produced significant antibody compared to adjuvant and PBS groups. The induced antibody in sera of immunized animal with Leptospira vaccine was detected by 250 ng of rAcB coated in ELISA microplate. This study demonstrated that antigenic region (D259-R462) of AGC protein might be useful for evaluation of antibody level in vaccinated animal.

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

The polypeptide Tams1 is an immunodominant major merozoite piroplasm surface antigen of the protozoan parasite Theileria annulata and is highly variable. In this study, the partial nucleotide (nt) sequence of the Tams1 (522 nt) gene of Iranian vaccine strain (Vaccine-ir68) recovered from an outbreak of disease in Iran was determined and compared with the corresponding sequences of eighteen previously published Tams1 genes. According to sequencing result, a novel amino acid substitution at the Tams1 region (K→Q) was found exclusively in isolate Vaccine-ir68. Genetic distance values, estimated from the sequence data, revealed striking sequence homology (approximately 99%) between Vaccine-ir68 isolate and Tunisian isolates, showing that they were same isolates of T. annulata which were spread in these areas. The phylogenetic tree constructed based on the sequence alignment of 19 Tams1 coding regions was distinctly divided into five lineages. There might be some unknown tick carrier birds immigrating to the different geographical regions. These birds have an effective role to distribute the T. annulata species in North Africa, Palestine and Iran.