الفهرس 
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Abstract
Tachyzoite to bradyzoite stage conversion is accompanied by the expression of different specific antigens. Studies on the T. gondii stage specific genes help to elucidate the overall mechanisms orchestrating stage conversion. As the diagnosis of T. gondii infection is fundamental for proper management and control of infection in humans and animals, development of a better diagnostic assay based on highly reactive antigens along with development of alive vaccine will also help in prevention and control strategies of toxoplasmosis.
The current study aimed to molecular characterization of a bradyzoite specific gene (T. gondii Ank1 gene), analysis of the role of lactate dehydrogenase (LDH) enzyme for stage conversion and evaluation of recombinant antigens in combination and single formula for diagnosis of feline toxoplasmosis.
In this study, RT-PCR, amplification and cloning were performed. Alternative splicing produced at least two forms of Ank1 transcripts designated Ank1a and Ank1b with a predicted molecular weight of proteins of 55 and 48 kDa, respectively. Ank1a has seven Ankyrin repeats, however, Ank1b has a deletion at the fourth and fifth ankyrin repeats. Recombinant TgANK1 protein and anti-TgANK1 antibody were produced.
Western blot analysis was carried out in T. gondii lysates of tachyzoites and in vitro-induced bradyzoites. A 55 kDa band corresponding to ANK1a protein was detected at day 3 and band intensity was stronger at day 5 after bradyzoite induction.
IFA was carried out to examine the expression and localization of ANK1 protein in in vitro-induced bradyzoites. In vitro differentiation of tachyzoites to bradyzoites of ME49 strain was performed and bradyzoite conversion was confirmed by cyst wall staining at day 5 after induction. In tachyzoites (day 0) and bradyzoites at day 1, almost all cells displayed a nuclear staining. At day 2 after induction, BAG1 positive and SAG1 negative parasites displayed in addition to a nuclear localization also a cytosolic staining. These signal intensities became stronger at day 3 and thereafter. IFAT using mouse brain section chronically infected with T. gondii was also performed. Both nuclear and cytosolic staining was observed in encysted bradyzoites and, of note, the intensity of a cytosolic signal was stronger than that of nuclear signal.
Using Co-Ip and Mass Spectrometry analysis,LDH was found to be interacting partner with ANK1.The role of LDH was investigated by production of antibodies using GST fused recombinant LDH1 and LDH2. LDH1 immunized mouse serum strongly reacted with V5-tagged LDH1 and weakly reacted with V5-tagged LDH2 produced in 293T cells. On the other hand, LDH2 immunized mouse serum almost equally reacted with V5-tagged LDH1 and LDH2. Thereafter, knockout parasites was generated by homologous recombination in Pru∆Ku80:HXGPRT parental strain parasites. Gene disruptions was confirmed by PCR, western blot analysis and IFA using anti-LDH1 and anti-LDH1/2 antibodies
Immunofluorescence staining by anti-LDH1/2 antibody was performed using parental and knockout strains. Dolichosbiflorus lectin (DBL) staining was also performed to see cyst wall formation. In parental strain, signal was apparent in tachyzoite and during bradyzoite induction, suggesting that at least one of LDH protein was present in all stages. The expression of LDH2 was below detectable level in tachyzoite of ∆ldh1 strain and gradually increased during three days after bradyzoite induction. The expression of LDH1 was apparent during time course and no clear evidence of suppression of LDH1 was obtained after bradyzoite induction. Three days after induction, all three strains showed DBL positive staining, suggesting that no impairment of cyst wall formation by LDH deletion.
In vitro growth kinetics of mutant strains was examined. Parasites were infected in HFF cells and parasite numbers per PV were counted at indicated time points. All four strains demonstrated similar growth kinetics
For in vivo studies, with a dose of 106tachyzoites, ∆LDH1 infected mice showed 100% survival rate. All deletion mutants had significant reduction in cyst number of infected mice. Using a dose of 104 and 103tachyzoites, the survival rate was significantly increased for ∆LDH1 and ∆LDH1/2 in comparison to parentral and ∆LDH2 strains with a significant reduction in cyst number. Then, the survived mice was challenged by a lethal dose of Pru strain (less virulent) and RH strain (highly virulent). Control group received only the challenge died. However, all mice injected previously with deletion mutants survived the subsequent challenge with both strains.
Cat serum samples were tested primarily by LAT as a reference test. Out of 419 cat samples, 73 were positive and 346 were negative. The sensitivity, specificity, concordance and kappa values obtained with the use of different recombinant antigens and TLA were variable. Among these antigens, TLA yielded a perfect concordance with LAT results, as evidenced by kappa values of (0.93) with a high sensitivity and specificity 97.29% and 93.62%, respectively. Recombinant SAG2 and GRA6 proteins showed greater performance than other recombinant proteins with substantial kappa values of 0.67 and 0.62, respectively. On the other hand, recombinant GRA2 and GRA7, GRA15, MIC10 and ANK1 proteins exhibited lesser performance in detection of T. gondii antibodies. These results indicate that TLA has a better performance than single recombinant proteins in diagnosing feline toxoplasmosis. Furthermore, different combination formula of recombinant proteins (M1: GRA6+GRA7; M2: GRA2+GRA7; M3: SAG2+GRA7; M4: SAG2+GRA6; M5: GRA2+GRA6+GRA7+GRA15; M6: SAG2+GRA2+GRA6+GRA7+GRA15) was used as ELISA antigen. The M1 and M5 had a moderate concordance as evidenced by kappa values of 0.58 and 0.5, respectively. Fair concordance was yielded by M2 with kappa value of 0.35. Substantial concordance was yielded by M4 and M3 with kappa values of 0.72 and 0.62, respectively. Noteworthy, M6 exhibited the highest concordance, as evidenced by kappa value of 0.81, sensitivity, 89.19% and specificity 95.36 %.