Clinicopathomorphological studies on murine toxoplasmosis and evaluation of PCR based diagnosis with special reference to molecular characterization of Toxoplasma gondii SAG2 gene

The present study was undertaken to investigate the clinicopathomorpholy, evaluate the PCR of partial sequence of SAG2 gene to diagnose of Toxoplasmagondii(RH strain) from tissue and sequencing and characterization of SAG2 gene in experimental murine toxoplasmosis by inoculating 1.5 × 10⁴tachyzoites through intraperitoneal route.The infected mice showed the clinical manifestations such as less food and water intake; lethargic with in-coordination in movement; body hairs erected; ascites; marked tachypnea; loss of body weight. Some of the mice developed subcutaneous petechial hemorrhage bilaterally on the abdomen and ears. All mice died within 4–5 days post inoculation (DPI).Necropsy findings revealed ascites and accumulation of sero-sanguinous fluid in thoracic cavity. Liver, kidney, spleen were enlarged with congestion and presence of necrotic foci. Nodules in the sub serosa were also observed. Heart and brain did not reveal any significant lesions. Histopathologicalobservation revealed presence of tahyzoites in all the organs except in brain. Inflammatory reaction, degenerative and necrotic changes were observed in all the organs with varying magnitude of severity. All the organs were positive to immonoperoxidase against T. gondiitachyzoites except brain that was non-reactive to immunohistochemical assay.DNA was extracted from host cell free tachyzoites for standardization of PCR.A single band of 457bp ampliconwas observed after amplification of partial sequence SAG2 gene of T. gondii from all the vital organs including brain of infected mice.PCR product was purified and purified amplicon was sent for sequencing.The sequence was analysed by NCBI BLAST search, DNAStar and Gene tool lite Software Version 7.Accession Nos. KC607825 and AGK83126 were generated by NCBI GenBank for nucleotideand putative amino acid sequences, respectively.Sequence homology study with tachyzoites of RH strain revealed the nucleotide sequence accession no. KC607825 is 99.8% similar with accession no. AF108751 and 99.3% similar with ME49 strain accession no. XM_002369614.Sequence divergence was observed amongst the information obtained from SAG2 gene of bradyzoites.Phylogenetic study showed that SAG2 gene obtained from tachyzoites and bradyzoites formed two separate clades. Among the SAG2 gene of tachyzoites, accession no. KC607825 formed a single clade whereas accession no. AF108751 and accession no. XM_002369614 formed a separate clade.The computer simulated SAG2 revealed the monomeric nature of the protein having a single domain and the conformation consisted of beta sheets, with intermittent alpha chains.Antigenic index revealed five highly antigenic determinant sites.Computer gel simulation for RE of sequence of SAG2 gene reveals KpnI RE is a good marker enzyme for PCR restriction fragment length polymorphism (PCR-RFLP) analysis.No tachyzoite was detected in brain in acute murine toxoplasmosis either by histopathological or immunohistochemical studies whereas PCR could detect it. From these findings it is concluded that the amplification of SAG2 gene of T. gondii by using the customized primers designed in the laboratory are very useful for sensitive and specific diagnosis of T. gondii.